Thomas J.J. Blanck, Ph.D., M.D.

The N-methyl-d-aspartate-evoked cytoplasmic calcium increase in adult rat dorsal root ganglion neuronal somata was potentiated by substance P pretreatment in a protein kinase C-dependent manner
Castillo C; Norcini M; Baquero-Buitrago J; Levacic D; Medina R; Montoya-Gacharna JV; Blanck TJ; Dubois M; Recio-Pinto E
2011 Mar 17;177:308-320, Neuroscience
The involvement of substance P (SP) in neuronal sensitization through the activation of the neurokinin-1-receptor (NK1r) in postsynaptic dorsal horn neurons has been well established. In contrast, the role of SP and NK1r in primary sensory dorsal root ganglion (DRG) neurons, in particular in the soma, is not well understood. In this study, we evaluated whether SP modulated the NMDA-evoked transient increase in cytoplasmic Ca(2+) ([Ca(2+)](cyt)) in the soma of dissociated adult DRG neurons. Cultures were treated with nerve growth factor (NGF), prostaglandin E(2) (PGE(2)) or both NGF+PGE(2). Treatment with NGF+PGE(2) increased the percentage of N-methyl-d-aspartate (NMDA) responsive neurons. There was no correlation between the percentage of NMDA responsive neurons and the level of expression of the NR1 and NR2B subunits of the NMDA receptor or of the NK1r. Pretreatment with SP did not alter the percentage of NMDA responsive neurons; while it potentiated the NMDA-evoked [Ca(2+)](cyt) transient by increasing its magnitude and by prolonging the period during which small- and some medium-sized neurons remained NMDA responsive. The SP-mediated potentiation was blocked by the SP-antagonist ([D-Pro(4), D-Trp(7,9)]-SP (4-11)) and by the protein kinase C (PKC) blocker bisindolylmaleimide I (BIM); and correlated with the phosphorylation of PKCepsilon. The Nk1r agonist [Sar(9), Met(O(2))(11)]-SP (SarMet-SP) also potentiated the NMDA-evoked [Ca(2+)](cyt) transient. Exposure to SP or SarMet-SP produced a rapid increase in the labeling of phosphorylated-PKCepsilon. In none of the conditions we detected phosphorylation of the NR2B subunit at Ser-1303. Phosphorylation of the NR2B subunit at Tyr1472 was enhanced to a similar extent in cells exposed to NMDA, SP or NMDA+SP, and that enhancement was blocked by BIM. Our findings suggest that NGF and PGE(2) may contribute to the injury-evoked sensitization of DRG neurons in part by enhancing their NMDA-evoked [Ca(2+)](cyt) transient in all sized DRG neurons; and that SP may further contribute to the DRG sensitization by enhancing and prolonging the NMDA-evoked increase in [Ca(2+)](cyt) in small- and medium-sized DRG neurons
— id: 121314, year: 2011, vol: 177, page: 308, stat: Journal Article,

A single subanesthetic dose of ketamine relieves depression-like behaviors induced by neuropathic pain in rats
Wang, Jing; Goffer, Yossef; Xu, Duo; Tukey, David S; Shamir, D B; Eberle, Sarah E; Zou, Anthony H; Blanck, Thomas J J; Ziff, Edward B
2011 Oct;115(4):812-821, Anesthesiology
BACKGROUND: Chronic pain is associated with depression. In rodents, pain is often assessed by sensory hypersensitivity, which does not sufficiently measure affective responses. Low-dose ketamine has been used to treat both pain and depression, but it is not clear whether ketamine can relieve depression associated with chronic pain and whether this antidepressant effect depends on its antinociceptive properties. METHODS: The authors examined whether the spared nerve injury model of neuropathic pain induces depressive behavior in rats, using sucrose preference test and forced swim test, and tested whether a subanesthetic dose of ketamine treats spared nerve injury-induced depression. RESULTS: Spared nerve injury-treated rats, compared with control rats, showed decreased sucrose preference (0.719 +/- 0.068 (mean +/- SEM) vs. 0.946 +/- 0.010) and enhanced immobility in the forced swim test (107.3 +/- 14.6s vs. 56.2 +/- 12.5s). Further, sham-operated rats demonstrated depressive behaviors in the acute postoperative period (0.790 +/- 0.062 on postoperative day 2). A single subanesthetic dose of ketamine (10 mg/kg) did not alter spared nerve injury-induced hypersensitivity; however, it treated spared nerve injury-associated depression-like behaviors (0.896 +/- 0.020 for ketamine vs. 0.663 +/- 0.080 for control rats 1 day after administration; 0.858 +/- 0.017 for ketamine vs. 0.683 +/- 0.077 for control rats 5 days after administration). CONCLUSIONS: Chronic neuropathic pain leads to depression-like behaviors. The postoperative period also confers vulnerability to depression, possibly due to acute pain. Sucrose preference test and forced swim test may be used to compliment sensory tests for assessment of pain in animal studies. Low-dose ketamine can treat depression-like behaviors induced by chronic neuropathic pain
— id: 139733, year: 2011, vol: 115, page: 812, stat: Journal Article,

Transient Effects of Anesthetics on Dendritic Spines and Filopodia in the Living Mouse Cortex
Yang G; Chang PC; Bekker A; Blanck TJ; Gan WB
2011 Oct;115(4):718-726, Anesthesiology
BACKGROUND:: Anesthetics are widely used to induce unconsciousness, pain relief, and immobility during surgery. It remains unclear whether the use of anesthetics has significant and long-lasting effects on synapse development and plasticity in the brain. To address this question, the authors examined the formation and elimination of dendritic spines, postsynaptic sites of excitatory synapses, in the developing mouse cortex during and after anesthetics exposure. METHODS:: Transgenic mice expressing yellow fluorescence protein in layer 5 pyramidal neurons were used in this study. Mice at 1 month of age underwent ketamine-xylazine and isoflurane anesthesia over a period of hours. The elimination and formation rates of dendritic spines and filopodia, the precursors of spines, were followed over hours to days in the primary somatosensory cortex using transcranial two-photon microscopy. Four to five animals were examined under each experimental condition. Student t test and Mann-Whitney U test were used to analyze the data. RESULTS:: Administration of either ketamine-xylazine or isoflurane rapidly altered dendritic filopodial dynamics but had no significant effects on spine dynamics. Ketamine-xylazine increased filopodial formation whereas isoflurane decreased filopodial elimination during 4 h of anesthesia. Both effects were transient and disappeared within a day after the animals woke up. CONCLUSION:: Studies suggest that exposure to anesthetics transiently affects the dynamics of dendritic filopodia but has no significant effect on dendritic spine development and plasticity in the cortex of 1-month-old mice
— id: 137134, year: 2011, vol: 115, page: 718, stat: Journal Article,

Nimodipine prevents memory impairment caused by nitroglycerin-induced hypotension in adult mice
Bekker, Alex; Haile, Michael; Li, Yong-Sheng; Galoyan, Samuel; Garcia, Edwardo; Quartermain, David; Kamer, Angela; Blanck, Thomas
2009 Dec;109(6):1943-1948, Anesthesia & analgesia
BACKGROUND: Hypotension and a resultant decrease in cerebral blood flow have been implicated in the development of cognitive dysfunction. We tested the hypothesis that nimodipine (NIMO) administered at the onset of nitroglycerin (NTG)-induced hypotension would preserve long-term associative memory. METHODS: The passive avoidance (PA) paradigm was used to assess memory retention. For PA training, latencies (seconds) were recorded for entry from a suspended platform into a Plexiglas tube where a shock was automatically delivered. Latencies were recorded 48 h later for a testing trial. Ninety-six Swiss-Webster mice (30-35 g, 6-8 wk), were randomized into 6 groups 1) saline (control), 2) NTG immediately after learning, 3) NTG 3 h after learning, 4) NTG and NIMO, 5) vehicle, and 6) NIMO alone. The extent of hypotension and changes in brain tissue oxygenation (PbtO(2)) and in cerebral blood flow were studied in a separate group of animals. RESULTS: All groups exhibited similar training latencies (17.0 +/- 4.6 s). Mice subjected to hypotensive episodes showed a significant decrease in latency time (178 +/- 156 s) compared with those injected with saline, NTG + NIMO, or delayed NTG (580 +/- 81 s, 557 +/- 67 s, and 493 +/- 146 s, respectively). A Kruskal-Wallis 1-way analysis of variance indicated a significant difference among the 4 treatment groups (H = 15.34; P < 0.001). In a separate group of mice not subjected to behavioral studies, the same dose of NTG (n = 3) and NTG + NIMO (n = 3) caused mean arterial blood pressure to decrease from 85.9 +/- 3.8 mm Hg sem to 31.6 +/- 0.8 mm Hg sem and from 86.2 +/- 3.7 mm Hg sem to 32.6 +/- 0.2 mm Hg sem, respectively. Mean arterial blood pressure in mice treated with NIMO alone decreased from 88.1 +/- 3.8 mm Hg to 80.0 +/- 2.9 mm Hg. The intergroup difference was statistically significant (P < 0.05). PbtO(2) decreased from 51.7 +/- 4.5 mm Hg sem to 33.8 +/- 5.2 mm Hg sem in the NTG group and from 38.6 +/- 6.1 mm Hg sem to 25.4 +/- 2.0 mm Hg sem in the NTG + NIMO groups, respectively. There were no significant differences among groups. CONCLUSION: In a PA retention paradigm, the injection of NTG immediately after learning produced a significant impairment of long-term associative memory in mice, whereas delayed induced hypotension had no effect. NIMO attenuated the disruption in consolidation of long-term memory caused by NTG but did not improve latency in the absence of hypotension. The observed effect of NIMO may have been attributable to the preservation of calcium homeostasis during hypotension, because there were no differences in the PbtO(2) indices among groups
— id: 105366, year: 2009, vol: 109, page: 1943, stat: Journal Article,

Nimodipine prevents transient cognitive dysfunction after moderate hypoxia in adult mice
Haile, Michael; Limson, Fred; Gingrich, Kevin; Li, Yong-Sheng; Quartermain, David; Blanck, Thomas; Bekker, Alex
2009 Apr;21(2):140-144, Journal of neurosurgical anesthesiology
BACKGROUND: Cognitive changes associated with moderate hypoxia may be related to the elevation of cytosolic calcium (Ca) levels which may, in turn, affect neurotransmitter synthesis and metabolism. We tested whether treatment with nimodipine (NIMO), an L-type Ca channel blocker, would preserve working memory after hypoxic hypoxia. METHODS: We randomized 157 Swiss-Webster, 30 to 35 g mice (6 to 8 wk) to 6 groups, which were exposed to the following gas mixtures for 1 hour: (1) O2 21%; (2) O2 21% followed by 0.1 mg/kg of subcutaneous NIMO; (3) O2 21% followed by vehicle (60% polyethylene glycol/40% methanol); (4) O2 10%; (5) O2 10% then NIMO; (6) O2 10% then vehicle. The Object Recognition Test (ORT) was given once either on Day 1 or Day 7 to assess changes in short-term memory. ORT exploits the tendency of mice to prefer novel over familiar objects. Two identical objects were placed in an arena for 15 minutes of training. During the testing 1 hour later, one of the objects was replaced by a new object. Recognition Index (RI) was used to compare performance. It is defined as the time spent exploring the novel object divided by the time spent exploring both objects, the novel plus the familiar, and this ratio is converted to a percentage. RI was analyzed with analysis of variance. Tukey Honestly Significant Difference tests were used for post hoc comparisons when appropriate. P values <0.05 were considered significant. RESULTS: RI for the control group was 68.3% (SE+/-3.6%). RI was 53.7% (SE+/-3.8%) for the 10% O2 group on the first posttreatment day. O2 saturation (SpO2) for the hypoxic group was 71.7% (SE+/-0.5%). By Day 7, RI for the 10% O2 group increased to 64.2% (SE+/-4.7%), which was not significantly different from control. On Day 1, RI was 68.6% (SE+/-5.2%) for hypoxic rodents treated with NIMO. These results were statistically significant. Low RI indicates impaired working memory and high RI indicates intact working memory. These results suggest that NIMO prevented impairment of working memory after moderate hypoxia. CONCLUSIONS: NIMO reverses the disturbance of short-term working memory caused by moderate hypoxia in mice. The results may have implications for cognitive changes linked to Ca homeostasis in the postoperative period
— id: 132606, year: 2009, vol: 21, page: 140, stat: Journal Article,

Muscle-conditioned media and cAMP promote survival and neurite outgrowth of adult spinal cord motor neurons
Montoya G, Jose V; Sutachan, Jhon Jairo; Chan, Wai Si; Sideris, Alexandra; Blanck, Thomas J J; Recio-Pinto, Esperanza
2009 Dec;220(2):303-315, Experimental neurology
Embryonic spinal cord motor neurons (MNs) can be maintained in vitro for weeks with a cocktail of trophic factors and muscle-derived factors under serum-containing conditions. Here we investigated the beneficial effects of muscle-derived factors in the form of muscle-conditioned medium (MCM) on the survival and neurite outgrowth of adult rat spinal cord MNs under serum-free conditions. Ventral horn dissociated cell cultures from the cervical enlargement were maintained in the presence of one or more of the following factors: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), a cell permeant cyclic adenosine-3',5'-monophosphate (cAMP) analog and MCM. The cell cultures were immunostained with several antibodies recognizing a general neuronal marker the microtubule-associated protein 2 (MAP2) and either one or more motor neuronal markers: the non-phosphorylated neurofilament heavy isoform (SMI32), the transcription factors HB9 and Islet-1 and the choline acetyl transferase. We found that treatment with MCM together with the cAMP analog was sufficient to promote selective survival and neurite outgrowth of adult spinal cord MNs. These conditions can be used to maintain adult spinal cord MNs in dissociated cultures for several weeks and may have therapeutic potential following spinal cord injury or motor neuropathies. More studies are necessary to evaluate how MCM and the cAMP analog act in synergy to promote the survival and neurite outgrowth of adult MNs
— id: 102940, year: 2009, vol: 220, page: 303, stat: Journal Article,

Cytotoxicity of local anesthetics in human neuronal cells
Perez-Castro, Rosalia; Patel, Sohin; Garavito-Aguilar, Zayra V; Rosenberg, Andrew; Recio-Pinto, Esperanza; Zhang, Jin; Blanck, Thomas J J; Xu, Fang
2009 Mar;108(3):997-1007, Anesthesia & analgesia
BACKGROUND: In addition to inhibiting the excitation conduction process in peripheral nerves, local anesthetics (LAs) cause toxic effects on the central nervous system, cardiovascular system, neuromuscular junction, and cell metabolism. Different postoperative neurological complications are ascribed to the cytotoxicity of LAs, but the underlying mechanisms remain unclear. Because the clinical concentrations of LAs far exceed their EC(50) for inhibiting ion channel activity, ion channel block alone might not be sufficient to explain LA-induced cell death. However, it may contribute to cell death in combination with other actions. In this study, we compared the cytotoxicity of six frequently used LAs and will discuss the possible mechanism(s) underlying their toxicity. METHODS: In human SH-SY5Y neuroblastoma cells, viability upon exposure to six LAs (bupivacaine, ropivacaine, mepivacaine, lidocaine, procaine, and chloroprocaine) was quantitatively determined by the MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-odium bromide) colorimetry assay and qualitatively confirmed by fluorescence imaging, using the LIVE/DEAD assay reagents (calcein/AM and ethidium homodimer-1). In addition, apoptotic activity was assessed by measuring the activation of caspase-3/-7 by imaging using a fluorescent caspase inhibitor (FLICA). Furthermore, LA effects on depolarization- and carbachol-stimulated intracellular Ca(2+)-responses were also evaluated. RESULTS: 1) After a 10-min treatment, all six LAs decreased cell viability in a concentration-dependent fashion. Their killing potency was procaine < or = mepivacaine < lidocaine < chloroprocaine < ropivacaine < bupivacaine (based on LD(50), the concentration at which 50% of cells were dead). Among these six LAs, only bupivacaine and lidocaine killed all cells with increasing concentration. 2) Both bupivacaine and lidocaine activated caspase-3/-7. Caspase activation required higher levels of lidocaine than bupivacaine. Moreover, the caspase activation by bupivacaine was slower than by lidocaine. Lidocaine at high concentrations caused an immediate caspase activation, but did not cause significant caspase activation at concentrations lower than 10 mM. 3) Procaine and chloroprocaine concentration-dependently inhibited the cytosolic Ca(2+)-response evoked by depolarization or receptor-activation in a similar manner as a previous observation made with bupivacaine, ropivacaine, mepivacaine, and lidocaine. None of the LAs caused a significant increase in the basal and Ca(2+)-evoked cytosolic Ca(2+)-level. CONCLUSION: LAs can cause rapid cell death, which is primarily due to necrosis. Lidocaine and bupivacaine can trigger apoptosis with either increased time of exposure or increased concentration. These effects might be related to postoperative neurologic injury. Lidocaine, linked to the highest incidence of transient neurological symptoms, was not the most toxic LA, whereas bupivacaine, a drug causing a very low incidence of transient neurological symptoms, was the most toxic LA in our cell model. This suggests that cytotoxicity-induced nerve injury might have different mechanisms for different LAs and different target(s) other than neurons
— id: 94380, year: 2009, vol: 108, page: 997, stat: Journal Article,

Isoflurane inhibits cyclic adenosine monophosphate response element-binding protein phosphorylation and calmodulin translocation to the nucleus of SH-SY5Y cells
Zhang, Jin; Sutachan, Jhon-Jairo; Montoya-Gacharna, Jose; Xu, Chong-Feng; Xu, Fang; Neubert, Thomas A; Recio-Pinto, Esperanza; Blanck, Thomas J J
2009 Oct;109(4):1127-1134, Anesthesia & analgesia
BACKGROUND: Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively. RESULTS: P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS: Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels
— id: 102500, year: 2009, vol: 109, page: 1127, stat: Journal Article,

Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells
Masci, Alessandra; Mastronicola, Daniela; Arese, Marzia; Piane, Maria; De Amicis, Andrea; Blanck, Thomas J J; Chessa, Luciana; Sarti, Paolo
2008 Jan;1777(1):66-73, Biochimica & biophysica acta
Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P< or =0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients
— id: 95152, year: 2008, vol: 1777, page: 66, stat: Journal Article,

Pulses of extracellular K+ produce two cytosolic Ca2+ transients that display different temperature dependence and carbonyl cyanide m-chlorophenyl sensitivity in SH-SY5Y cells
Montoya G, Jose V; Sutachan, Jhon-Jairo; Corrales, Alexandra; Xu, Fang; Blanck, Thomas J J; Recio-Pinto, Esperanza
2008 Jun 5;1213:12-26, Brain research
In SH-SY5Y cells we have shown that stimulation with high extracellular K+ ([K+]e) evokes a transient increase in cytoplasmic Ca2+ ([Ca2+]cyt) (K+on) that is triggered by the opening of voltage-dependent Ca2+ channels and followed by Ca2+ -induced Ca2+ release from the endoplasmic reticulum (Xu, F., Zhang, J., Recio-Pinto, E. and Blanck, T.J., Halothane and isoflurane augment depolarization-induced cytosolic CA2+ transients and attenuate carbachol-stimulated CA2+ transients, Anesthesiology, 92 (2000) 1746-56). The removal of high-[K+]e results in a second transient increase in [Ca2+]cyt (K+off) that is independent of extracellular Ca2+ (Corrales, A., Montoya, G.J., Sutachan, J.J., Cornillez-Ty, G., Garavito-Aguilar, Z., Xu, F., Blanck, T.J. and Recio-Pinto, E., Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients, Brain Res., 1031 (2005) 174-184). In this study we further characterize the properties of K+off. We found that K+off was detectable at near physiological temperatures (34-36 degrees C) but, depending on the level of [K+]e, it was undetectable or highly diminished at room temperature. In contrast, K+on was increased by lowering the temperature. Extracellular Na+ -replacement with K+ did not affect K+off, indicating that K+off was not generated by osmolarity changes. Replacement of extracellular Na+ with choline+ did not affect K+off, indicating that K+off did not result from activity changes of the plasma membrane Na+/Ca2+ exchanger. Caffeine decreased K+on but not K+off. CCCP (carbonyl cyanide m-chlorophenyl), a protonophore uncoupler that decreases mitochondrial Ca2+ uptake, decreased K+on but not K+off. CGP37157, an inhibitor of the mitochondria Na+/Ca2+ exchanger, decreased K+off when added alone but not when added simultaneously with CCCP. Clonazepam had similar effects as CGP37157. These findings indicate that the generation of K+off is strongly temperature-dependent and its pharmacology is distinct from the [Ca2+]cyt changes observed previously at room temperature
— id: 86543, year: 2008, vol: 1213, page: 12, stat: Journal Article,

Physostigmine reverses cognitive dysfunction caused by moderate hypoxia in adult mice
Bekker, Alex; Haile, Michael; Gingrich, Kevin; Wenning, Leslie; Gorny, Alex; Quartermain, David; Blanck, Thomas
2007 Sep;105(3):739-743, Anesthesia & analgesia
BACKGROUND: Cognitive changes associated with moderate hypoxia in rodents may result from the diminished functioning of central cholinergic neurotransmission. We designed this study to examine whether treatment with physostigmine (PHY), an acetylcholinesterase inhibitor, could improve the impairment of working memory after hypoxic hypoxia. METHODS: We randomized 90 Swiss Webster, 30-35 g mice (6-8 wks) to three hypoxia groups at fraction of inspired oxygen, FiO2 = 0.10 (1. no treatment; 2. PHY 0.1 mg/kg intraperitoneally administered immediately before; or 3. after hypoxia), or to two room air groups (given either no treatment or PHY after an insult). An object recognition test was used to assess short-term memory function. The object recognition test exploits the tendency of mice to prefer exploring novel objects in an environment when a familiar object is also present. During the 15 min training trial, two identical objects were placed in two defined sites of the box. During the test trial performed 1 h later, one of the objects was replaced by a new object with a different shape. The time spent exploring the two objects was automatically recorded by a video camera and associated software. The performance was analyzed with ANOVA, followed by post hoc comparisons using the Newman-Keuls test when appropriate. P values <0.05 were considered significant. RESULTS: Untreated mice subjected to hypoxia at Fio2 = 0.1 spent significantly less time exploring a novel object on testing day 1 than did untreated mice breathing room air. Performance of the mice subjected to hypoxia, who received physostigmine after, but not before, the insult did not differ from the control group. CONCLUSION: Moderate hypoxia impairs rodents' performance in a working memory task. It appears that changes are transient, because the cognitive functioning of the mice returned to the baseline level 7 days after treatment. Postinsult administration of PHY prevented deterioration of cognitive function. An increased level of acetylcholine in the central nervous system may be responsible for the improved performance of the hypoxia-treated mice
— id: 86616, year: 2007, vol: 105, page: 739, stat: Journal Article,

Isoflurane inhibition of recombinant cardiac L-type Ca2+ channels
Gingrich, K; Blanck, T
2007 JAN ;108(11):100A-100A, Biophysical journal
— id: 71387, year: 2007, vol: 108, page: 100A, stat: Journal Article,

Isoflurane preserves spatial working memory in adult mice after moderate hypoxia
Bekker, Alex; Shah, Romin; Quartermain, David; Li, Yong-Sheng; Blanck, Thomas
2006 Apr;102(4):1134-1138, Anesthesia & analgesia
Perioperative hypoxia may contribute to postoperative cognitive impairment. It is unknown, however, whether anesthetics exacerbate or protect against hypoxia-related central nervous system impairment. We sought to determine whether hypoxia alone or in combination with isoflurane disrupts working memory in mice. To this extent, we assigned adult mice to one of four treatments for 1 h: oxygen 21%, oxygen 21% + isoflurane 1.2%, oxygen 8%, or oxygen 8% + isoflurane 1.2%. Mice breathed spontaneously throughout the experiment. Body temperature was maintained at 37 degrees C + 0.5 degrees C. Mice were allowed to recover for 24 h to avoid the confounding influence of residual anesthetics on neurobehavioral performance. Working memory was assessed by use of a Y maze modified for mice. For the training trial, entry to one arm was blocked and mice were permitted to run between the two open arms for 15 min and inspect the objects outside. For the test trial, carried out 1 h later, all arms were open. Time spent in each arm was automatically recorded by a camera and associated software. Mice were tested 1, 4, and 7 days after anesthesia. A different arm was used as the novel arm for each test. Performance was analyzed with repeated-measurements analysis of variance, followed by analysis of simple main effects and by post hoc comparison using Newman-Keuls test when appropriate. P values <0.05 were considered significant. Animals subjected to hypoxia (8% oxygen for 1 h) spent significantly less time in the novel arm 1 day after the insult. The impairment, however, was transient. Hypoxic mice performance improved to the level of the control animals on the fourth post-treatment day. Mice subjected to hypoxia plus isoflurane exhibited no impairment and were comparable to the control group at all time points. Hypoxia transiently impairs performance in a spatial memory task. It appears that isoflurane protects against this deleterious effect of hypoxia
— id: 63746, year: 2006, vol: 102, page: 1134, stat: Journal Article,

Advances in ultrasound guided regional anesthesia
Popovic J; Morimoto M; Blanck TJJ; Rosenberg AD
2006 ;58(2):40-46, NYSSA Sphere
— id: 67941, year: 2006, vol: 58, page: 40, stat: Journal Article,

Current practice of ultrasound-assisted regional anesthesia
Popovic, Jovan; Morimoto, Maki; Wambold, Daniel; Blanck, Thomas J J; Rosenberg, Andrew D
2006 Jun;6(2):127-134, Pain practice
— id: 71213, year: 2006, vol: 6, page: 127, stat: Journal Article,

Pluronic F-127 affects the regulation of cytoplasmic Ca2+ in neuronal cells
Sutachan, Jhon-Jairo; Montoya G, Jose V; Xu, Fang; Chen, Daniel; Blanck, Thomas J J; Recio-Pinto, Esperanza
2006 Jan 12;1068(1):131-137, Brain research
Fura-2 is one of the most widely used cytoplasmic Ca2+ ([Ca2+]cyt) sensors. In studies using isolated dorsal root ganglion (DRG) neurons, the loading of Fura-2 AM is often facilitated by the use of pluronic F-127. In preliminary studies, we detected that the use of pluronic F-127 appeared to be affecting the depolarization-evoked [Ca2+]cyt transient in DRG neurons. To determine whether this was the case, we conducted a systematic study. Adult rat DRG neurons were cultured, and their response to 50 mM KCl was measured in sister cultured cells (isolated on the same day) that were loaded with 5 microM Fura-2AM in the absence or in the presence of 0.02% pluronic F-127. In the absence of pluronic F-127, the KCl-evoked [Ca2+]cyt transient changed with time, being wider on day 1 than on day 2 after plating. On day 2, the KCl-evoked [Ca2+]cyt transient was wider in neurons Fura-2 loaded in the presence of pluronic F-127. These results indicate that pluronic F-127 significantly alters depolarization-evoked [Ca2+]cyt transients, which may reflect alteration in regulation of [Ca2+]cyt in neuronal cells
— id: 63600, year: 2006, vol: 1068, page: 131, stat: Journal Article,

Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients
Corrales, Alexandra; Montoya G, Jose V; Sutachan, Jhon-Jairo; Cornillez-Ty, Genoveve; Garavito-Aguilar, Zayra; Xu, Fang; Blanck, Thomas J J; Recio-Pinto, Esperanza
2005 Jan 21;1031(2):174-184, Brain research
Transient increases in extracellular K+ are observed under various conditions, including repetitive neuronal firing, anoxia, ischemia and hypoglycemic coma. We studied changes in cytoplasmic Ca2+ ([Ca2+]cyt) evoked by pulses of KCl in human neuroblastoma SH-SY5Y cells and rat dorsal root ganglia (DRG) neurons at 37 degrees C. A 'pulse' of KCl evoked two transient increases in [Ca2+]cyt, one upon addition of KCl (K+on) and the other upon removal of KCl (K+off). The K+on transient has been described in many cell types and is initiated by the activation of voltage-dependent Ca2+ channels followed by Ca2+-evoked Ca2+ release from intracellular Ca2+ stores. The level of KCl necessary to evoke the K+off transient depends on the type of neuron, in SH-SY5Y cells it required 100 mM KCl, in most (but not all) of dorsal root ganglia neurons it could be detected with 100-200 mM KCl and in a very few dorsal root ganglia neurons it was detectable at 20-50 mM KCl. In SH-SY5Y cells, reduction of extracellular Ca2+ inhibited the K+on more strongly than the K+off and slowed the decay of K+off. Isoflurane (1 mM) reduced the K+on)- but not the K+off-peak. However, isoflurane slowed the decay of K+off. The nonspecific cationic channel blocker La3+ (100 microM) had an effect similar to that of isoflurane. Treatment with thapsigargin (TG) at a concentration known to only deplete IP3-sensitive Ca2+ stores did not affect K+on or K+off, suggesting that Ca2+ release from the IP3-sensitive Ca2+ stores does not contribute to K+on and K+off transients and that the thapsigargin-sensitive Ca2+ ATPases do not contribute significantly to the rise or decay rates of these transients. These findings indicate that a pulse of extracellular K+ produces two distinct transient increases in [Ca2+]cyt
— id: 56139, year: 2005, vol: 1031, page: 174, stat: Journal Article,

Halothane inhibition of recombinant cardiac L-type Ca2+ channels expressed in HEK-293 cells
Gingrich, Kevin J; Tran, Son; Nikonorov, Igor M; Blanck, Thomas J
2005 Dec;103(6):1156-1166, Anesthesiology
BACKGROUND: Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. METHODS: HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. RESULTS: Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. CONCLUSIONS: The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer
— id: 61856, year: 2005, vol: 103, page: 1156, stat: Journal Article,

Pathophysiology of cardiovascular co-morbidities
Blanck TJJ; Muntyan I; Zayed-Moustafa H
Morbid obesity : peri-operative management Cambridge : Cambridge University Press, 2004,
— id: 3612, year: 2004, vol: , page: 69, stat: Chapter,

Isoflurane reduces the carbachol-evoked Ca2+ influx in neuronal cells
Corrales, Alexandra; Xu, Fang; Garavito-Aguilar, Zayra V; Blanck, Thomas J J; Recio-Pinto, Esperanza
2004 Oct;101(4):895-901, Anesthesiology
BACKGROUND: The authors previously reported that the isoflurane-caused reduction of the carbachol-evoked cytoplasmic Ca transient increase ([Ca]cyt) was eliminated by K or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca]cyt transient involved Ca influx through the plasma membrane. METHODS: Perfused attached human neuroblastoma SH-SY5Y cells were exposed to carbachol (1 mm, 2 min) in the absence and presence of isoflurane (1 mm) and in the absence and presence of extracellular Ca (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La (100 microm), of the L-type Ca channel blocker nitrendipine (10 microm), and of the N-type Ca channel blocker omega-conotoxin GVIA (0.1 microm) on isoflurane modulation of the carbachol-evoked [Ca]cyt transient. [Ca]cyt was detected with fura-2 and experiments were carried out at 37 degrees C. RESULTS: Isoflurane reduced the peak and area of the carbachol-evoked [Ca]cyt transient in the presence but not in the absence of extracellular Ca. La had a similar effect as the removal of extracellular Ca. Omega-conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response. CONCLUSIONS: The current data are consistent with previous observations in that the carbachol-evoked [Ca]cyt transient involves both Ca release from intracellular Ca stores and Ca entry through the plasma membrane. It was found that isoflurane attenuates the carbachol-evoked Ca entry. The isoflurane sensitive Ca entry involves a cationic channel different from the L- or N- type voltage-dependent Ca channels. These results indicate that isoflurane attenuates the carbachol-evoked [Ca]cyt transient at a site at the plasma membrane that is distal to the muscarinic receptor
— id: 45503, year: 2004, vol: 101, page: 895, stat: Journal Article,

Differential thapsigargin-sensitivities and interaction of Ca2+ stores in human SH-SY5Y neuroblastoma cells
Garavito-Aguilar, Zayra V; Recio-Pinto, Esperanza; Corrales, Alexandra V; Zhang, Jin; Blanck, Thomas J J; Xu, Fang
2004 Jun 18;1011(2):177-186, Brain research
In human SH-SY5Y neuroblastoma cells, two distinct intracellular Ca2+ stores, a KCl-/caffeine-sensitive and a carbachol-/IP3-sensitive store, were demonstrated previously. In this study, responses of these two intracellular Ca2+ stores to thapsigargin were characterized. Ca2+-release from these stores was evoked either by high K+ (100 mM KCl) or by 1 mM carbachol, and changes in the intracellular Ca2+ level were monitored using Fura-2 fluorimetry. A sequential stimulation protocol (KCl-->carbachol or vice versa) allowed evaluation of the individual contribution of different Ca2+ stores to the evoked intracellular Ca2+ ([Ca2+]i)-transients and the dynamic interaction between them. Thapsigargin (0.05 nM - 20 microM) alone induced a [Ca2+]i-transient. Both the carbachol- and the KCl-evoked [Ca2+]i-transients were inhibited by thapsigargin, but with very different sensitivities. Thapsigargin inhibited the carbachol-evoked [Ca2+]i-transients with (IC50 = 0.353 nM) or without (IC50 = 0.448 nM) a KCl-prestimulation, but an additional small component, with a much lower sensitivity (IC50=4814 nM), was observed in the absence of a KCl-prestimulation. In contrast, the KCl-evoked [Ca2+]i-transients displayed only one component with a very low sensitivity to thapsigargin in both absence (IC50=3343 nM) and presence (IC50=6858 nM) of a carbachol-prestimulation. These findings suggest that the sarco-/endoplasmic reticular Ca2+ ATPases associated with the KCl-/caffeine- and carbachol-/IP3-sensitive intracellular Ca2+ stores differ from each other, either in types or in their post-translational modification. Such difference might play important role in the regulation of neuronal Ca2+ homeostasis
— id: 44521, year: 2004, vol: 1011, page: 177, stat: Journal Article,

Angiotensin-converting enzyme activity: a novel way of assessing pulmonary changes during total knee arthroplasty
Jules-Elysee, Kethy; Blanck, Thomas J J; Catravas, John D; Chimento, George; Miric, Alexander; Kahn, Richard; Paroli, Leonardo; Sculco, Thomas
2004 Oct;99(4):1018-23, table of contents, Anesthesia & analgesia
Emboli after tourniquet release (TR) during total knee arthroplasty (TKA) occur in all patients. This may lead to fat embolism syndrome with lung injury. Angiotensin-converting enzyme (ACE) lines the pulmonary endothelium, and a decrease in ACE metabolism or hydrolysis of (3)HBPAP ((3)H-benzoyl-Phe-Ala-Pro; a substrate specific for ACE) has been associated with lung injury. We evaluated the association of this assay with pulmonary changes during TKA. Eleven consecutive patients undergoing bilateral TKA had the ACE assay performed perioperatively. We determined substrate hydrolysis and pulmonary capillary surface area (capillary perfusion index; CPI) and correlated it with pulmonary vascular resistance (PVR) and clinical outcome. Ten of the 11 patients demonstrated an increase in substrate hydrolysis and CPI along with a decrease in PVR after first or second TR when compared with baseline values (P < 0.05). In the other patient, PVR continued to increase even after TR, whereas CPI and substrate hydrolysis decreased after surgery. Whereas all others did well clinically, this patient developed confusion and hypoxemia. In previous studies, a decrease in PVR with an increase in CPI, as exhibited by the 10 patients, has been associated with pulmonary capillary recruitment. We believe this to be an important mechanism by which the lungs are able to accommodate the burden of emboli at the time of TR
— id: 45504, year: 2004, vol: 99, page: 1018, stat: Journal Article,

G-Protein Activation Decreases the Isoflurane Inhibition of N-type Ca2+ Currents. An Increase in the Isoflurane Blocking Potency of N-type Ca2+ Currents May Contribute to the Known Neuroprotection Action of Isoflurane During Ischemia
Recio-Pinto, Esperanza; Nikonorov, Igor M; Blanck, Thomas J J
2004 Jan;16(1):105-107, Journal of neurosurgical anesthesiology
— id: 41882, year: 2004, vol: 16, page: 105, stat: Journal Article,

Isoflurane reduction of carbachol-evoked cytoplasmic calcium transients is dependent on caffeine-sensitive calcium stores
Corrales, Alexandra; Xu, Fang; Garavito-Aguilar, Zayra; Blanck, Thomas J J; Recio-Pinto, Esperanza
2003 Oct;99(4):882-888, Anesthesiology
BACKGROUND: Many muscarinic functions are relevant to anesthesia, and alterations in muscarinic activity affect the anesthetic/analgesic potency of various drugs. Volatile anesthetics have been shown to depress muscarinic receptor function, and inhibition of the muscarinic signaling pathway alters the minimal alveolar anesthetic concentration of inhaled anesthetics. The purpose of this investigation was to determine in a neuronal cell which source of Ca2+ underlying the carbachol-evoked transient increase in cytoplasmic Ca2+ was reduced by isoflurane. METHODS: Experiments were performed at 37 degrees C on continuously perfused monolayers of human neuroblastoma SH-SY5Y cells using Fura-2 as the cytoplasmic Ca2+ indicator. Carbachol (1 mm) was applied to evoke a transient increase in cytoplasmic Ca2+. RESULTS: Isoflurane (1 mm) reduces the carbachol-evoked transient increase in cytoplasmic Ca2+, and this isoflurane action is eliminated when the cells are continuously stimulated with 200 mm KCl or pretreated with 10 mm caffeine or 200 microm ryanodine. CONCLUSIONS: Isoflurane reduction of the carbachol-evoked transient increase in cytoplasmic Ca2+ requires full caffeine-sensitive Ca2+ stores and Ca2+ release from the caffeine-sensitive stores through the ryanodine-sensitive Ca2+ release channels. The results indicate that isoflurane interferes with a muscarinic Ca2+ signaling through a mechanism downstream from the muscarinic receptors
— id: 39057, year: 2003, vol: 99, page: 882, stat: Journal Article,

G-protein activation decreases isoflurane inhibition of N-type Ba2+ currents
Nikonorov, Igor M; Blanck, Thomas J J; Recio-Pinto, Esperanza
2003 Aug;99(2):392-399, Anesthesiology
BACKGROUND: G-protein activation mediates inhibition of N-type Ca2+ currents. Volatile anesthetics affect G-protein pathways at various levels, and activation of G-proteins has been shown to increase the volatile anesthetic potency for inhibiting the electrical-induced contraction in ileum. The authors investigated whether isoflurane inhibition of N-type Ba2+ currents was mediated by G-protein activation. METHODS: N-type Ba2+ currents were measured in the human neuronal SH-SY5Y cell line by using the whole cell voltage-clamp method. RESULTS: Isoflurane was found to have two effects on N-type Ba2+ currents. First, isoflurane reduced the magnitude of N-type Ba2+ currents to a similar extent (IC50 approximately 0.28 mm) in the absence and presence of GDPbetaS (a nonhydrolyzable GDP analog). Interestingly, GTPgammaS (a nonhydrolyzable GTP analog and G-protein activator) in a dose-dependent manner reduced the isoflurane block; 120 microm GTPgammaS completely eliminated the block of 0.3 mm isoflurane and reduced the apparent isoflurane potency by approximately 2.4 times (IC50 approximately 0.68 mm). Pretreatment with pertussis toxin or cholera toxin did not eliminate the GTPgammaS-induced protection against the isoflurane block. Furthermore, isoflurane reduced the magnitude of voltage-dependent G-protein-mediated inhibition of N-type Ba2+ currents, and this effect was eliminated by pretreatment with pertussis toxin or cholera toxin. CONCLUSIONS: It was found that activation of G-proteins in a neuronal environment dramatically reduced the isoflurane potency for inhibiting N-type Ba2+ currents and, in turn, isoflurane affected the G-protein regulation of N-type Ba2+ currents
— id: 37575, year: 2003, vol: 99, page: 392, stat: Journal Article,

Phospholipid abnormalities in children with Barth syndrome
Schlame, Michael; Kelley, Richard I; Feigenbaum, Annette; Towbin, Jeffrey A; Heerdt, Paul M; Schieble, Thomas; Wanders, Ronald J A; DiMauro, Salvatore; Blanck, Thomas J J
2003 Dec 3;42(11):1994-1999, Journal of the American College of Cardiology
OBJECTIVES: We sought to identify characteristic lipid abnormalities in patients with Barth syndrome (BTHS) and to correlate the lipid profile to phenotype and genotype. BACKGROUND: Barth syndrome typically includes cardiomyopathy, skeletal myopathy, neutropenia, growth retardation, and 3-methylglutaconic aciduria, and it is commonly associated with mutations in the tafazzin (TAZ) gene, whose products are homologous to phospholipid acyltransferases. However, clinical features of BTHS have also been found in patients with normal TAZ gene. METHODS: We analyzed molecular species of phospholipids in left and right ventricle, skeletal muscle, platelets, lymphoblasts, and fibroblasts from 19 children with BTHS (positive TAZ mutation), 6 children with BTHS-like syndromes (wild-type TAZ), 4 children with isolated cardiomyopathy (wild-type TAZ), and various controls. RESULTS: Cardiolipin, the specific lipid found only in mitochondria, was decreased in all tissues from BTHS patients, whereas concentrations of other phospholipids were normal. The molecular composition of cardiolipin was altered in all tissues from BTHS patients. The molecular compositions of phosphatidylcholine and phosphatidylethanolamine were altered in the heart. Cardiolipin abnormalities were only found in children with true BTHS, not in children with BTHS-like disease or with isolated cardiomyopathy. The degree of cardiolipin deficiency was tissue-specific but did not correlate with severity or specific phenotypic expression of BTHS. CONCLUSIONS: Abnormal cardiolipin is a specific diagnostic marker of cardiomyopathies caused by TAZ mutations. These mutations lead to alterations in the fatty acid composition of several phospholipids, supporting the idea that TAZ encodes a human acyltransferase
— id: 45505, year: 2003, vol: 42, page: 1994, stat: Journal Article,

Actions of general anesthetics on voltage-gated ion channels
Topf N; Recio-Pinto E; Blanck TJJ; Hennings HCJr
Neural mechanisms of anesthesia Totowa NJ : Humana, 2003,
— id: 3141, year: 2003, vol: , page: 299, stat: Chapter,

Remodeling of cardiolipin by phospholipid transacylation
Xu, Yang; Kelley, Richard I; Blanck, Thomas J J; Schlame, Michael
2003 Dec 19;278(51):51380-51385, Journal of biological chemistry
Mitochondrial cardiolipin (CL) contains unique fatty acid patterns, but it is not known how the characteristic molecular species of CL are formed. We found a novel reaction that transfers acyl groups from phosphatidylcholine or phosphatidylethanolamine to CL in mitochondria of rat liver and human lymphoblasts. Acyl transfer was stimulated by ADP, ATP, and ATP gamma S, but not by other nucleotides. Coenzyme A stimulated the reaction only in the absence of adenine nucleotides. Free fatty acids were not incorporated into CL under the same incubation condition. The transacylation required addition of exogenous CL or monolyso-CL, whereas dilyso-CL was not a substrate. Transacylase activity was decreased in lymphoblasts from patients with Barth syndrome (tafazzin deletion), and this was accompanied by drastic changes in the molecular composition of CL. In rat liver, where linoleic acid was the most abundant residue of CL, only linoleoyl groups were transferred into CL, but not oleoyl or arachidonoyl groups. We demonstrated complete remodeling of tetraoleoyl-CL to tetralinoleoyl-CL in rat liver mitochondria and identified the intermediates linoleoyl-trioleoyl-CL, dilinoleoyl-dioleoyl-CL, and trilinoleoyl-oleoyl-CL by high-performance liquid chromatography. The data suggest that CL is remodeled by acyl specific phospholipid transacylation and that tafazzin is an acyltransferase involved in this mechanism
— id: 45506, year: 2003, vol: 278, page: 51380, stat: Journal Article,

Disease-specific remodeling of cardiac mitochondria after a left ventricular assist device
Heerdt, Paul M; Schlame, Michael; Jehle, Roswitha; Barbone, Alessandro; Burkhoff, Daniel; Blanck, Thomas J J
2002 Apr;73(4):1216-1221, Annals of thoracic surgery
BACKGROUND: Failing hearts can exhibit elements of structural and molecular 'reverse remodeling' after support with a left ventricular assist device (LVAD). The present study examined LVAD-induced remodeling of cardiac mitochondria. METHODS: Left ventricular tissue from 20 failing and 21 LVAD-supported hearts, catagorized as ischemic (ICM) or dilated (DCM) cardiomyopathy and four nonfailing hearts were studied. Myocyte mitochondrial ultrastructure was assessed by high-performance liquid chromatography determination of cardiolipin, a specific lipid component of the inner membrane, and its three major molecular species: L4, L3O, and L2O2. RESULTS: Both failing and LVAD-supported hearts exhibited a reduction in cardiolipin content that was independent of the type of cardiomyopathy. However, in failing/ICM hearts, there was a 25% increase in the L4/L3O ratio and a 70% increase in the L4/L2O2 ratio, indicating a change in cardiolipin composition. These alterations were normalized by LVAD support. In sharp contrast, molecular species ratios in DCM hearts were the same as those in nonfailing hearts regardless of whether LVAD support had been used or not. CONCLUSIONS: These data demonstrate LVAD-induced reverse remodeling of myocyte cardiolipin composition in ICM but not DCM hearts
— id: 45508, year: 2002, vol: 73, page: 1216, stat: Journal Article,

Assessing sedation with regional anesthesia: inter-rater agreement on a modified Wilson sedation scale
Nemethy, Maria; Paroli, Leonardo; Williams-Russo, Pamela G; Blanck, Thomas J J
2002 Mar;94(3):723-728, Anesthesia & analgesia
A valid and reliable means for measuring sedation during regional anesthesia would be valuable for both research and practice. Current methods of monitoring sedation include machine-, patient-, and observer-based assessment. The reliability of machine-based methods is limited at lower levels of sedation, whereas patient-based methods are impractical at higher levels. Observer-based methods offer the best alternative for assessing sedation during regional anesthesia; however, their reliability has not been adequately documented. We examined the interrater reliability of the Wilson sedation scale. Sedation was assessed by pairs of anesthesia care providers in 100 patients undergoing surgical procedures with regional anesthesia. On the basis of the findings, the scale was modified, and 50 additional patients were assessed. The study protocol called for a series of standardized stimuli administered by a research assistant. Raters were blinded to each other's ratings. Interrater reliability was assessed by using the kappa statistic, a measure of actual agreement beyond agreement by chance. When continuing checks on its operationalization and reliability are included, the modified Wilson scale provides a simple and reliable means by which to assess and monitor intraoperative sedation. IMPLICATIONS: We evaluated the interrater reliability of the Wilson scale for measuring sedation during regional anesthesia. Paired anesthesia care providers' ratings of patient sedation indicated very good interrater reliability in both the original scale and a modified version. The modified Wilson scale provides a quick noninvasive means of monitoring sedation during regional anesthesia
— id: 45509, year: 2002, vol: 94, page: 723, stat: Journal Article,

The neuronal lipid membrane permeability was markedly increased by bupivacaine and mildly affected by lidocaine and ropivacaine
Pardo, Luis; Blanck, Thomas J J; Recio-Pinto, Esperanza
2002 Nov 29;455(2-3):81-90, European journal of pharmacology
We investigated the local anesthetic action on ionic membrane conductance (membrane conductance) and selectivity in membranes formed with neuronal phospholipids in the absence and presence of cholesterol. In membranes without cholesterol, 1 mM bupivacaine and ropivacaine increased the membrane conductance approximately 4.5-fold; and 5 mM lidocaine, ropivacaine and bupivacaine increased the membrane conductance by 2.7-, 2.8- and 22.2-fold, respectively. In the presence of cholesterol, 5 mM ropivacaine had no effect, lidocaine decreased the membrane conductance by 2-fold, and bupivacaine increased the membrane conductance by 17-fold. Local anesthetics did not affect the ion selectivity in membranes without cholesterol, but they all decreased the Na(+) selectivity in membranes with cholesterol. Cholesterol reduced the lidocaine- and ropivacaine-induced membrane conductance increase by eliminating or reversing the Na(+) conductance increase and by lowering the Cl(-) conductance increase. In the absence of cholesterol, 5 mM bupivacaine increased both Na(+) conductance (38-fold) and Cl(-) conductance (19-fold), while in the presence of cholesterol it only increased Cl(-) conductance (26-fold). Of the local anesthetics studied, ropivacaine was the least membrane toxic while bupivacaine was the most toxic
— id: 37577, year: 2002, vol: 455, page: 81, stat: Journal Article,

Deficiency of tetralinoleoyl-cardiolipin in Barth syndrome
Schlame, Michael; Towbin, Jeffrey A; Heerdt, Paul M; Jehle, Roswitha; DiMauro, Salvatore; Blanck, Thomas J J
2002 May;51(5):634-637, Annals of neurology
Barth syndrome is an X-linked cardiac and skeletal mitochondrial myopathy. Barth syndrome may be due to lipid alterations because the product of the mutated gene is homologous to phospholipid acyltransferases. Here we document that a single mitochondrial phospholipid species, tetralinoleoyl-cardiolipin, was lacking in the skeletal muscle (n = 2), right ventricle (n = 2), left ventricle (n = 2), and platelets (n = 6) of 8 children with Barth syndrome. Tetralinoleoyl-cardiolipin is specifically enriched in normal skeletal muscle and the normal heart. These findings support the notion that Barth syndrome is caused by alterations of mitochondrial lipids
— id: 45507, year: 2002, vol: 51, page: 634, stat: Journal Article,

Effect of cardiolipin oxidation on solid-phase immunoassay for antiphospholipid antibodies
Schlame M; Haller I; Sammaritano LR; Blanck TJ
2001 Dec;86(6):1475-1482, Thrombosis & haemostasis
Diagnostic assays for antiphospholipid antibodies are routinely performed on microtitre plates coated with cardiolipin. Here we show that contact between cardiolipin and NUNC-Immuno plates leads to extensive oxidation, generating a series of peroxy-cardiolipins which were identified by electrospray ionization mass spectrometry. To investigate the impact of oxidation on the antibody assay. cardiolipin was resolved into 12 molecular species, including oxidized species and non-oxidized species with different degrees of unsaturation. All 12 species reacted under anaerobic conditions with serum from patients with primary antiphospholipid syndrome. Immune reactivity was similar for tetralinoleoyl-cardiolipin, trilinoleoyl-oleoyl-cardiolipin, and peroxycardiolipins, but somewhat lower for tristearoyl-oleoyl-cardiolipin. Oxidative treatment of cardiolipin with air, cytochrome c, or Cu2+/tert-butylhydroperoxide, either before or during the assay, did not enhance immune reactivity. Similar results were obtained with a monoclonal IgM from lupus-prone mice, that binds cardiolipin in the absence of protein cofactors. We conclude that the solid-phase assay for antiphospholipid antibodies can be supported by various oxidized and non-oxididized molecular species of cardiolipin
— id: 32233, year: 2001, vol: 86, page: 1475, stat: Journal Article,

Isoflurane pretreatment ameliorates postischemic neurologic dysfunction and preserves hippocampal Ca2+/calmodulin-dependent protein kinase in a canine cardiac arrest model
Blanck TJ; Haile M; Xu F; Zhang J; Heerdt P; Veselis RA; Beckman J; Kang R; Adamo A; Hemmings H
2000 Nov;93(5):1285-1293, Anesthesiology
BACKGROUND: Inhalational anesthetics are neuroprotective in rat models of global ischemia. To determine whether isoflurane at a clinically relevant concentration is neuroprotective in a canine model of cardiac arrest, we measured neurologic function and hippocampal Ca2+/calmodulin-dependent protein kinase II (CaMKII) content 20 h after cardiac arrest. METHODS: We tested the neuroprotective effect of 30 min of 1.5% isoflurane exposure before 8 min of global ischemia induced with ventricular fibrillation. Animals were randomized to four groups: control, isoflurane-control, ischemia, and isoflurane-ischemia. After resuscitation and 20 h of intensive care, each animal's neurologic deficit score was determined by two blinded evaluators. The hippocampal content of CaMKII, determined by immunoblotting, was measured by an individual blinded to the treatment groups. CaMKII activity was measured in samples from the cortex, hippocampus, and striatum of animals in each group. RESULTS: Isoflurane-ischemic animals had a median neurologic deficit score of 22.6% compared with 43.8% for the ischemic animals (P < 0.05). Hippocampal levels of the beta-subunit of CaMKII (CaMKIIbeta) were relatively preserved in isoflurane-ischemic animals (68 +/- 4% of control) compared with ischemic animals (48 +/- 2% of control; P < 0.001), although both groups were statistically significantly lower than control (P < 0. 001 ischemia vs. control and P < 0.05 isoflurane-ischemia vs. control). CONCLUSIONS: Isoflurane is an effective neuroprotective drug in a canine cardiac arrest model in terms of both functional and biochemical criteria
— id: 23985, year: 2000, vol: 93, page: 1285, stat: Journal Article,

CNS voltage-dependent Na(+) channel expression and distribution in an undifferentiated and differentiated CNS cell line
Castaneda-Castellanos DR; Cano M; Wang JK; Corbett A; Benson D; Blanck TJ; Thornhill WB; Recio-Pinto E
2000 Jun 2;866(1-2):281-285, Brain research
Upon serum removal, CAD-R1 cells undergo neurite outgrowth and an increase in voltage-dependent Na(+) current (VDNaC) density without changing their activation and inactivation properties. Insulin and endothelial cell growth supplement inhibited the increase in VDNaC density but not the neurite outgrowth. RI, RII, RIII Na(+) channel proteins were expressed in CAD-R1 cells. These proteins exhibited both similar and different distribution and clustering patterns which suggested the channel's structural differences play a role in channel distribution
— id: 37578, year: 2000, vol: 866, page: 281, stat: Journal Article,

Advances and strategies for spinal cord regeneration
Girardi FP; Khan SN; Cammisa FP Jr; Blanck TJ
2000 Jul;31(3):465-472, Orthopedic clinics of North America
Although a cure for spinal cord injuries does not currently exist, advances have been made in the field of spinal cord regeneration. This article discusses the pathophysiology of spinal cord injury, animal models, and strategies for restoration and regeneration of the spinal cord
— id: 23987, year: 2000, vol: 31, page: 465, stat: Journal Article,

Halothane and isoflurane augment depolarization-induced cytosolic CA2+ transients and attenuate carbachol-stimulated CA2+ transients
Xu F; Zhang J; Recio-Pinto E; Blanck TJ
2000 Jun;92(6):1746-1756, Anesthesiology
BACKGROUND: Neuronal excitability is in part determined by Ca2+ availability that is controlled by regulatory mechanisms of cytosolic Ca2+ ([Ca2+]cyt). Alteration of any of those mechanisms by volatile anesthetics (VAs) may lead to a change in presynaptic transmission and postsynaptic excitability. Using a human neuroblastoma cell line, the effects of halothane and isoflurane on cytosolic Ca2+ concentration ([Ca2+]cyt) in response to K+ and carbachol stimulation were investigated. METHODS: Volatile anesthetic (0.05-1 mm) action on stimulated [Ca2+]cyt transients were monitored in suspensions of SH-SY5Y cells loaded with fura-2. Potassium chloride (KCl; 100 mm) was used to depolarize and activate Ca2+ entry through voltage-dependent calcium channels; 1 mm carbachol was used to activate muscarinic receptor-mediated inositol triphosphate (IP3)-dependent intracellular Ca2+ release. Sequential stimulations, KCl followed by carbachol and vice versa, were used to investigate interactions between intracellular Ca2+ stores. RESULTS: Halothane and isoflurane in clinically relevant concentrations enhanced the K+-evoked [Ca2+]cyt transient whether intracellular Ca2+ stores were full or partially depleted. In contrast, halothane and isoflurane reduced the carbachol-evoked [Ca2+]cyt transient when the intracellular Ca2+ stores were full but had no effect when the Ca2+ stores were partially depleted by KCl stimulation. CONCLUSIONS: Volatile anesthetics acted on sites that differently affect the K+- and carbachol-evoked [Ca2+]cyt transients. These data suggest the involvement of an intracellular Ca2+ translocation from the caffeine-sensitive Ca2+ store to the inositol triphosphate-sensitive Ca2+ store that was altered by halothane and isoflurane
— id: 23988, year: 2000, vol: 92, page: 1746, stat: Journal Article,

Volatile anesthetic action on muscle Ca2+ homeostasis
Blanck TJ
1999 Dec;20(6):431-435, Italian journal of neurological sciences
It is proposed that volatile anesthetics act through the modification of Ca2+ homeostasis in excitable cells. To test this hypothesis, cardiac and skeletal muscles were used as models to examine Ca2+ response, and Ca2+ regulatory and delivery mechanisms. I found that halothane did not alter Ca2+ binding to cardiac troponin C. However, halothane and isoflurane reversibly decreased the Ca2+ affinity of calmodulin at low anesthetic concentration, and irreversibly increased the Ca2+ affinity of calmodulin at high anesthetic concentration. The volatile anesthetics also increased the permeability of light fraction of sarcoplasmic reticulum (SR) to Ca2+. I conclude that volatile anesthetics alter calcium homeostasis in cardiac and skeletal muscles. This work was in part performed in collaboration with Giovanni Salviati and the author benefited from Salviati's work in similar areas
— id: 23986, year: 1999, vol: 20, page: 431, stat: Journal Article,

Permeability of rat heart myocytes to cytochrome c
Sarti P; Silver RB; Paroli L; Nikonorov I; Blanck TJ
1999 Dec;56(11-12):1061-1069, Cellular & molecular life sciences: CMLS
Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellular vacuoles and plasma membrane blebbing. The damaged myocytes displayed impaired N,N'-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respiratory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluorescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 microM cytochrome c, establishing the threshold below which the turnover rate by cytochrome c oxidase in the cell depends on cytochrome concentration. Mildly damaged cardiac myocytes, as indicated by cell shortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeable to fluorescein-conjugated cytochrome c. The results, while consistent with an early cytochrome c release observed at the beginning of cell death, elucidate the role played by cytochrome c in the kinetic control of mitochondrial electron transfer under pathological conditions, particularly those involving the terminal part of the respiratory chain. These data are the first to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c
— id: 45510, year: 1999, vol: 56, page: 1061, stat: Journal Article,

Microanalysis of cardiolipin in small biopsies including skeletal muscle from patients with mitochondrial disease
Schlame M; Shanske S; Doty S; Konig T; Sculco T; DiMauro S; Blanck TJ
1999 Sep;40(9):1585-1592, Journal of lipid research
Cardiolipin is a specific mitochondrial phospholipid that is present in mammalian tissues in low concentration. To measure cardiolipin in small biopsies from patients with mitochondrial disease, we developed a new technique that can detect subnanomolar levels of well-resolved molecular species, the most abundant of which are tetralinoleoyl-cardiolipin (L(4)) and trilinoleoyl-oleoyl-cardiolipin (L(3)O). To this end, a fluorescence-labeled derivative of cardiolipin (2-[naphthyl-1'-acetyl]-cardiolipin dimethyl ester) was formed and analyzed by high performance liquid chromatography. Cardiolipin was measured in skeletal muscle biopsies from 8 patients with mitochondrial disease and in 17 control subjects. In 5 patients with mitochondrial disease, cardiolipin content was higher than normal (2. 4;-7.0 vs. 0.4;-2.2 nmol/mg protein). In 3 patients with mitochondrial disease, the L(4)/L(3)O ratio was lower than normal (2;-4 vs. 4;-6). Cardiolipin was also measured in various rat and dog muscle tissues. The L(4)/L(3)O ratio was higher in condensed 'muscle' type mitochondria (heart ventricle, skeletal muscle, ratios 4;-7) than in orthodox 'liver' type mitochondria (liver, smooth muscle, heart auricular appendage, H9c2 myoblasts, ratios 0.4;-3), suggesting that the L(4)/L(3)O proportion is important for cristae membrane structure. We concluded that the L(4)/L(3)O ratio is a tissue-specific variable that may change in the presence of mitochondrial disease. The new method is suitable to measure cardiolipin in muscle biopsies in order to estimate concentration of mitochondria
— id: 45511, year: 1999, vol: 40, page: 1585, stat: Journal Article,

The effects of halothane on single human neuronal L-type calcium channels
Nikonorov IM; Blanck TJ; Recio-Pinto E
1998 Apr;86(4):885-895, Anesthesia & analgesia
We investigated halothane's effects on the function of L-type Ca2+ channels in a human neuronal cell line, SH-SY5Y, by using the cell-attached patch voltage clamp configuration and Ba2+ as the charge carrier. In multiple-channel patches, halothane decreased the peak and persistent Ba2+ currents, accelerated the rate of inactivation, and slowed the rate of activation. Single-channel analysis showed that halothane (0.14-1.26 mM) increased the latency time for the first channel opening, increased the lifetime of nonconducting events, increased the proportion of short-lived open events, decreased the lifetime of the two open populations, and increased the percentage of current traces without channel activity. All of the observed halothane effects contribute to the halothane-induced decrease in macroscopic Ba2+ currents. The halothane concentration producing 50% reduction (IC50) of the peak Ba2+ current was 0.80 mM (approximately 1.9 hypothetical minimum alveolar anesthetic concentration [H-MAC] at 28 degrees C) and of the persistent Ba2+ current was 0.69 mM (approximately 1.7 H-MAC). The halothane effects did not always occur together, and the Hill slope of 1.6 suggested the presence of more than one interaction site or of more than one population of L-type Ca2+ channels. Halothane reduces L-type Ca2+ channel currents in human neuronal cells primarily through the stabilization of nonconducting states such as closed (before and after channel opening) and inactivated states. Implications: Calcium is a signaling molecule in neurons. We measured the effect of halothane on Ba2+ (a Ca2+ surrogate) movement into a human neuron-like cell electronically. Ba2+ entry through the L-type channel was depressed. Halothane decreased the likelihood of the channel opening and enhanced the rate at which the channel closed and inactivated. These actions of halothane are probably related to its anesthetic action
— id: 23990, year: 1998, vol: 86, page: 885, stat: Journal Article,

Halothane and isoflurane alter calcium dynamics in rat cerebrocortical synaptosomes
Xu F; Sarti P; Zhang J; Blanck TJ
1998 Sep;87(3):701-710, Anesthesia & analgesia
An increase in synaptosomal Ca2+ triggers neurotransmitter release and volatile anesthetics have been shown to inhibit neurotransmitter release by inhibition of Ca2+ entry. We have examined the effect of isoflurane and halothane on the kinetics of increase and decrease of Ca2+ in rat cerebrocortical synaptosomes ([Ca2+]in). We have also used specific Ca2+ antagonists to examine the role of L-, N-, and P-type Ca2+ channels. Synaptosomal [Ca2+]in was measured spectrofluorometrically using fura-2 as a Ca2+ reporter; Ca2+ transients were initiated by depolarization with 40 mM KCl. We found that < or = 1 minimum alveolar anesthetic concentration halothane and isoflurane decreased peak [Ca2+]in by approximately 40%, that both anesthetics decreased the rate of [Ca2+]in increase and decrease, that specific voltage-dependent calcium channel antagonists had little effect on peak or plateau [Ca2+]in, and that the volatile anesthetics increased the permeability of synaptosomal membranes to Ca2+. These results suggest that the volatile anesthetics, at clinically relevant concentrations, can alter Ca2+ homeostasis in the synapse. IMPLICATIONS: Clinically relevant concentrations of halothane and isoflurane markedly depress K+-evoked increases in rat cerebrocortical synaptosomal calcium (Ca2+) unrelated to L-, N-, and P-type voltage-dependent calcium channels and increase the Ca2+ permeability of the synaptosomal membrane. These changes in Ca2+ dynamics could have profound effects on Ca2+ signaling in the synapse
— id: 23989, year: 1998, vol: 87, page: 701, stat: Journal Article,

Volatile anaesthetic effects on calcium conductance of planar lipid bilayers formed with synthetic lipids or extracted lipids from sarcoplasmic reticulum
Andoh T; Blanck TJ; Nikonorov I; Recio-Pinto E
1997 Jan;78(1):66-74, British journal of anaesthesia
Volatile anaesthetics are known to increase leakage of calcium from the light fraction of skeletal sarcoplasmic reticulum (L-SR) which has no calcium release channels. To explore the role of the lipid environment, we have examined the effect of volatile anaesthetics on calcium conductance (gCa) of lipid membranes. Planar lipid bilayers were formed with a mixture of synthetic phospholipids and cholesterol, resembling the composition of SR membranes, or with lipids extracted from skeletal L-SR, gCa was estimated by calculating the calcium transference number (tCa) using diffusion potential measurements. Membranes formed with L-SR-extracted lipids had a higher gCa than membranes formed with synthetic lipids. Volatile anaesthetics increased total conductance and gCa in a dose-dependent manner, but did not affect tCa or membrane specific capacitance. In membranes formed with L-SR-extracted lipids, isoflurane induced the largest increase in gCa (1260 (SEM 304) % increase, n = 4, 0.94 mmol litre-1), followed by enflurane (264 (75)%, n = 5, 1.88 mmol litre-1) and halothane (53 (33)%, n = 5; 1.54 mmol litre-1). In membranes formed with synthetic lipids, volatile anaesthetic-induced increases in gCa followed the same trend but were larger. Volatile anaesthetics increased gCa without changing the ionic selectivity of membranes. However, the magnitude of the increase in gCa in the presence of volatile anaesthetics cannot account for the previously observed calcium leakage from L-SR vesicles. Therefore, the volatile anaesthetic-induced increase in calcium leakage in L-SR vesicles must be mediated via other pathways involving membrane proteins
— id: 23992, year: 1997, vol: 78, page: 66, stat: Journal Article,

Global ischemia increases the density of voltage-dependent calcium channels in porcine cardiac sarcolemma
Blanck TJ; Yasukochi S; Quigg M; Curtis WE; Gardner TJ
1997 May;84(5):972-975, Anesthesia & analgesia
The objective of this work was to determine whether normothermic global cardiac ischemia in a porcine model was associated with a change in the density (Bmax) of voltage-dependent calcium channels in myocardial sarcolemmal membranes. Pigs were anesthetized, a thoracotomy was performed, and samples were taken of the left and right ventricles from control and ischemic hearts. Dihydropyridine-binding sites were quantified using [3H]isradipine, and 5'-nucleotidase activity was measured by the liberation of inorganic phosphate from adenosine monophosphate. Bmax and dissociation constants and 5'-nucleotidase activity for control and ischemic tissues, respectively, were compared by using Student's t-test for unpaired samples. After normothermic global ischemia, the Bmax of [3H]isradipine binding increased in the left ventricle by 81% (299% +/- 1.7% to 540% +/- 11% fmoles/mg, P < 0.01) and in the right ventricle by 33% (387% +/- 9.9% to 515% +/- 38% fmoles/mg, P < 0.01) compared with control. 5'-nucleotidase activity increased by 48% in the left ventricle and by 96% in the right ventricle (p < 0.05). Fifteen minutes of normothermic ischemia in the pig is associated with marked sarcolemmal abnormalities, including increases in specific dihydropyridine binding and 5'-nucleotidase activity, which reflect global changes in membrane function, which might contribute to the increase in myoplasmic calcium during ischemia
— id: 23991, year: 1997, vol: 84, page: 972, stat: Journal Article,

Neuroprotection
Blanck, Thomas J
Baltimore MD : Williams & Wilkins, 1997,
— id: 2126, year: 1997, vol: , page: , stat: ,

Voltage-sensitive calcium channels and ischemia
Blanck TJ; Gardner TJ
1995 May;82(5):1308-1309, Anesthesiology
— id: 23994, year: 1995, vol: 82, page: 1308, stat: Journal Article,

Halothane and isoflurane alter the Ca2+ binding properties of calmodulin
Levin A; Blanck TJ
1995 Jul;83(1):120-126, Anesthesiology
BACKGROUND: Ca2+ plays an important role in signal transduction and anesthetic mechanisms. To date, no one has observed a direct effect of volatile anesthetics on a Ca(2+)-binding protein. We therefore examined the effects of halothane and isoflurane on the Ca(2+)-binding properties of bovine brain calmodulin. METHODS: The fluorescence emission of calmodulin was obtained over a range of Ca2+ concentrations (10(-7)-10(-4)M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KC1, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid at pH 7.0 and 37 degrees C. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography. RESULTS: The presence of volatile anesthetics altered the Ca(2+)-binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Ca2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Ca2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin-Ca2+ binding curve for Ca2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane. CONCLUSIONS: These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Ca(2+)-binding protein, undergoes a conformational shift when binding Ca2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act
— id: 23993, year: 1995, vol: 83, page: 120, stat: Journal Article,

Calcium-dependent translocation of sorcin to membranes: functional relevance in contractile tissue
Meyers MB; Zamparelli C; Verzili D; Dicker AP; Blanck TJ; Chiancone E
1995 Jan 9;357(3):230-234, FEBS letters
Sorcin, a 22 kDa calcium binding protein present in abundance in cardiac tissue and in multi-drug resistant cells and previously described as a soluble protein, is now shown to undergo a calcium-dependent translocation process from the cytosol to cellular membranes in both systems. The translocation process takes place also in E. coli BL21 cells that express recombinant sorcin, r-sorcin, and can be exploited in the purification of the protein. Calcium binding to purified r-sorcin occurs at micromolar concentrations of the metal and is accompanied by a conformational change that renders the protein soluble in the non-ionic detergent Triton X-114. This finding suggests that lipids are the target of sorcin on cellular membranes. The possible significance of the calcium-dependent translocation of sorcin in the specialized functions of sorcin-expressing cells is discussed
— id: 45512, year: 1995, vol: 357, page: 230, stat: Journal Article,

The role of L-type voltage-dependent calcium channels in anesthetic depression of contractility
Blanck TJ; Lee DL; Yasukochi S; Hollmann C; Zhang J
1994 ;31(12):207-214, Advances in pharmacology (New York)
— id: 45515, year: 1994, vol: 31, page: 207, stat: Journal Article,

The effects of halothane on voltage-dependent calcium channels in isolated Langendorff-perfused rat heart
Lee DL; Zhang J; Blanck TJ
1994 Nov;81(5):1212-1219, Anesthesiology
BACKGROUND: Halothane has been previously shown in vitro to decrease both the inward calcium current in isolated cells and the density of calcium antagonist binding sites in cardiac sarcolemmal membranes prepared from several species, including humans, presumably contributing to the negative inotropic effects seen with volatile anesthetics. In this study we examined whether halothane produced similar changes in calcium channel antagonist binding characteristics ex vivo in an intact perfused heart by using isradipine, a dihydropyridine calcium channel blocker that binds specifically to the alpha 1 subunit of the L-type voltage-dependent calcium channel. METHODS: The rat hearts were perfused by the Langendorff method in the presence of halothane and unlabeled isradipine. After the hearts were homogenized and prepared into membranes, a radioligand binding assay was performed and binding curves obtained. Data were analyzed by nonlinear regression analysis of a one-site binding equation and were evaluated by a paired t test. RESULTS: Halothane protected or inhibited the binding of unlabeled isradipine to calcium channels in a dose-dependent manner such that as the halothane is removed during the membrane preparation process, previously obscured sites were then available for specific binding of the radioligand. The sites that were protected by halothane had a lower affinity for [3H]-isradipine than controls. CONCLUSIONS: In both isolated membranes and the intact heart, halothane changes the availability of calcium channel antagonist binding sites, indicating a change in conformation of the voltage-dependent calcium channel in the presence of anesthetic. This change may result from a direct effect on the protein or from an indirect effect mediated through the membrane lipid bilayer. It also is demonstrated that halothane 'protected' channels are probably a modified class of channels compared to those in control tissues as exemplified by the much lower affinity that the protected channels have for [3H]-isradipine. We conclude that a major mechanism by which halothane depresses contractility is mediated through the voltage-dependent calcium channel, and this process results from a conformational change in the channel
— id: 23995, year: 1994, vol: 81, page: 1212, stat: Journal Article,

Lonidamine-mediated respiratory changes in rat heart myocytes: a re-examination of the functional response of mitochondrial cytochrome c oxidase
Sarti P; Antonini G; Arancia G; Blanck TJ; Citro G; Meloni A; Molinari A; Malatesta F
1994 Jun 15;47(12):2221-2225, Biochemical pharmacology
Respiratory activity of intact cardiac myocytes isolated from rats treated with lonidamine (LND) has been examined under conditions where cytochrome oxidase turns over at its maximal rate. Compared to myocytes isolated from control rat hearts, those treated with LND displayed a 60% increase in the cytochrome oxidase-dependent rate of respiration; electron microscopy revealed, in agreement with the literature, that the membrane structure of the mitochondrion had become disorganized. The increase in the rate of oxygen consumption was correlated with the (partial) impairment of the membrane ability to maintain the proton electrochemical potential gradient which normally inhibits oxidase activity. Results are discussed with reference to previous reports showing no effect of LND on cytochrome c oxidase activity. The evidence reported better clarifies the contribution of cytochrome oxidase to the demonstrated energetic failure displayed by cells treated with LND
— id: 45514, year: 1994, vol: 47, page: 2221, stat: Journal Article,

Increased activation of L-type voltage-dependent calcium channels is associated with glycine enhancement of N-methyl-D-aspartate-stimulated dopamine release in global cerebral ischemia/reperfusion
Werling LL; Hoehner PJ; Hurt KJ; Fox LG; Blanck TJ; Rosenthal RE; Fiskum G
1994 Jul;63(1):215-221, Journal of neurochemistry
We investigated the relationships among N-methyl-D-aspartate, glycine, L-type voltage-dependent calcium channels, and [3H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [3H]PN200-110 ([3H]isradipine) to L-type voltage-dependent calcium channels, that open as a consequence of N-methyl-D-aspartate-induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage-sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N-methyl-D-aspartate-stimulated [3H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N-methyl-D-aspartate-stimulated [3H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N-methyl-D-aspartate-stimulated release in all treatment groups. The enhancement of N-methyl-D-aspartate-stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30-min reperfused animals. These data suggest that glycine may facilitate opening of the voltage-dependent calcium channels activated by N-methyl-D-aspartate and that this facilitation is blocked by the antagonist nitrendipine
— id: 45513, year: 1994, vol: 63, page: 215, stat: Journal Article,

Time-resolved optical spectroscopy on intact myocytes
Antonini G; Malatesta F; Sarti P; Blanck TJ; Brunori M
1993 Mar;4(1):41-46, Cardioscience
Myocytes prepared from perfused rat heart were studied spectroscopically using a photodiode array spectrophotometer adapted to a rapid mixing stopped-flow apparatus. The isolated cells were found to be viable for 3 to 4 hours, i.e. over the total time of the experiments. Sodium ascorbate and tetramethyl-para-phenylenediamine were used as exogenous reductants. Cytoplasmic and mitochondrial membranes were found to be freely permeable to tetramethyl-para-phenylenediamine. The use of singular value decomposition proved to be powerful in resolving the spectral contributions of the chromophoric components within the overall absorption spectrum. Spectral resolution was improved by adding carbon monoxide at a concentration that kept myoglobin fully saturated without affecting the activity of cytochrome c oxidase. The redox state of cytochrome c and cytochrome a was observed during the steady-state consumption of oxygen and during the reduction following the exhaustion of oxygen. The redox state of the two chromophores was found to be approximately equal and close to 25-30% oxidized during steady-state respiration; during the final reduction they changed simultaneously. These experiments suggest that in living cells, as in the purified enzyme, the rate limiting step of the turnover of cytochrome oxidase is the internal transfer of electrons from cytochrome a to cytochrome a3
— id: 45517, year: 1993, vol: 4, page: 41, stat: Journal Article,

2,3,7,8-tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts
Canga L; Paroli L; Blanck TJ; Silver RB; Rifkind AB
1993 Dec;44(6):1142-1151, Molecular pharmacology
Binding by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor leads to transcriptional activation of several genes and a toxicity syndrome that includes tumor promotion, wasting, hormonal and immune system dysfunction, and death. Recent findings indicate that TCDD may also affect cardiac function. Here, we used the chick embryo, a TCDD-sensitive species, to further characterize the effects of TCDD on ventricular muscle contraction and on cardiac myocyte [Ca2+]i assessed with fura 2. The results show that TCDD causes an evolving sequence of contractile defects, independent of changes in diet, first impairing cAMP-modulated contraction (after 48 hr) and later (by seven days) decreasing responses to [Ca2+]o. Phenobarbital, even at high doses, failed to affect the inotropic response to isoproterenol, supporting the specificity of the ventricular contractile effects of TCDD. TCDD treatment also depressed inotropic responses to theophylline and forskolin, indicating that it has a post-beta-adrenergic receptor effect on cAMP action. In contrast to its depression of responses to beta-adrenergic stimuli and to [Ca2+]o, TCDD did not affect initial tensions of ventricular muscle stimulated at 1 Hz or the force-frequency response up to 1 Hz, indicating that TCDD-treated ventricles can respond normally at slow rates of stimulation. TCDD treatment depressed lusitropic (relaxation) responses to isoproterenol and to increasing [Ca2+]o indicating that it impairs the ability of the sarcoplasmic reticulum to sequester Ca2+. Fura 2-based measurements showed that [Ca2+]i was nearly doubled after TCDD treatment. The increase in [Ca2+]i is consistent with the decrease in the contractile response to [Ca2+]o, amelioration of the response to isoproterenol by subphysiologic concentrations of [Ca2+]o, and intermittent lack of response to electrical stimulation in high K+ observed in ventricles from TCDD-treated embryos. TCDD treatment also depressed the initial increase in [Ca2+]i by isoproterenol, consistent with the decreased contractile response to isoproterenol. The findings show that TCDD causes well defined, progressive impairment of avian ventricular responses to inotropic stimuli, providing new evidence that the heart is a target of TCDD action and that TCDD disturbs intracellular calcium processing
— id: 45516, year: 1993, vol: 44, page: 1142, stat: Journal Article,

Halothane does not alter Ca2+ affinity of troponin C
Blanck TJ; Chiancone E; Salviati G; Heitmiller ES; Verzili D; Luciani G; Colotti G
1992 Jan;76(1):100-105, Anesthesiology
Troponin C has been suggested as a possible target for the negative inotropic action of volatile anesthetics. This study has examined the effect of halothane on the structure and response of isolated cardiac troponin C to Ca2+ and the response of skinned soleus and cardiac muscle fibers to Ca2+. The high-affinity Ca(2+)-binding sites of cardiac troponin C were assessed by measurement of the change in intrinsic tyrosine fluorescence and ultraviolet circular dichroism in response to Ca2+ in the presence and absence of halothane. Halothane (0.9 mM, 1.4%) did not alter the 45% enhancement in intrinsic tyrosine fluorescence that occurs with saturation of the high-affinity sites or change the Ca2+ concentration at which half-maximal enhancement occurred. The molar ellipticity in the far ultraviolet region, a measure of the secondary structure, increased to a similar extent with addition of 10(-6) M Ca2+ in the absence and presence of 1.0 mM (1.6%) halothane. The binding rate of the sulfhydryl reagent, 5,5'-dithiobis (2-nitrobenzoic acid), to troponin C in response to Ca2+ titration was used as a measure of the integrity of the low-affinity Ca(2+)-binding site in troponin C in the presence and absence of 1.0 mM (1.6%) halothane. The rate of reaction was stimulated twofold, and the half maximal effect was observed at pCa 4.8 +/- 0.2 in both control and halothane-treated samples. Halothane (5 mM; 7.8%) did not change the pCa/tension response of skinned soleus fibers; the data were fit to the Hill equation and yielded dissociation constants of 6.2 x 10(-7) M for control and halothane-treated specimens.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 45522, year: 1992, vol: 76, page: 100, stat: Journal Article,

Halothane, enflurane, and isoflurane stimulate calcium leakage from rabbit sarcoplasmic reticulum
Blanck TJ; Peterson CV; Baroody B; Tegazzin V; Lou J
1992 May;76(5):813-821, Anesthesiology
The sarcoplasmic reticulum (SR) controls uptake and release of Ca2+ in muscle. Little information is available regarding the effect of volatile anesthetics on Ca2+ release from SR isolated from normal skeletal muscle, even though an abnormality of Ca2+ handling is implicated in malignant hyperthermia. In this study we used a Ca2+ electrode to monitor continuously the release of Ca2+ from SR and the effect of volatile anesthetics on this process. We found that halothane, enflurane, and isoflurane at 0.6, 0.7, and 0.8 vol%, respectively, each increased the velocity of Ca2+ leakage by at least 150% when compared to control. Ruthenium red, a blocker of the SR Ca(2+)-release channel, was shown to have no effect on the velocity of Ca2+ leakage. Halothane and isoflurane both shortened the time at which Ca2+ leakage began (T) in a dose-dependent fashion. Halothane at 4.8 vol% decreased T from 293 +/- 21 s to 149 +/- 20 s. Isoflurane (4.8 vol%) decreased T to 203 +/- 16 s, and enflurane at 5 vol% had little effect, decreasing T to 259 +/- 19 s. We noted a marked stimulation in the ATPase activity of the SR by all three volatile anesthetics. Halothane at 0.63 vol%, isoflurane at 0.42 vol%, and enflurane at 0.62 vol% each increased ATPase activity by at least 300%. We conclude that the stimulation of the velocity of Ca2+ leakage by the volatile anesthetics is related to the more rapid depletion of ATP, but that the shortening of the onset of Ca2+ leakage is a independent phenomenon with a markedly different dose dependence.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 45519, year: 1992, vol: 76, page: 813, stat: Journal Article,

Depression of calcium channel blocker binding to rat brain membranes by halothane
Drenger B; Heitmiller ES; Quigg M; Blanck TJ
1992 May;74(5):758-761, Anesthesia & analgesia
The present study evaluates the action of volatile anesthetics on the voltage-dependent Ca2+ channels in isolated rat brain membranes, measured as changes in binding of the Ca2+ channel blocker [3H]isradipine to these membranes. Equilibrium binding studies with increasing concentrations of [3H]isradipine (0.01-1 nM) in the presence of halothane (1.9%), isoflurane (2.3%), and enflurane (4.8%) at 25 degrees C were performed. Only halothane produced a significant depression in the specific binding of isradipine to the brain membranes at 0.5 and 1.0 nM [3H]isradipine (P = 0.028 and 0.018, respectively). Isoflurane and enflurane had such inconsistent effects that the data were inconclusive. Halothane produced a significant dose-dependent inhibition of binding, the maximum inhibition being 44% (P less than 0.005). Nonlinear regression analysis fit of the binding data indicates halothane produced a 48% decrease (P less than 0.05) in the maximal number of binding sites (Bmax) with no effect on the dissociation constant (Kd). As voltage-dependent Ca2+ channels are important in mediating neurotransmission, the marked decrease in channel number (Bmax) associated with halothane exposure suggests that this phenomenon might be related to the mechanism of general anesthesia
— id: 45521, year: 1992, vol: 74, page: 758, stat: Journal Article,

Alteration of voltage-dependent calcium channels in canine brain during global ischemia and reperfusion
Hoehner PJ; Blanck TJ; Roy R; Rosenthal RE; Fiskum G
1992 May;12(3):418-424, Journal of cerebral blood flow & metabolism
Elevated intracellular calcium (iCa2+) plays an important role in the pathophysiology of ischemic brain damage. The mechanisms by which iCa2+ increases are uncertain. Recent evidence implicates the voltage-dependent calcium channel (VDCC) as a likely site for the alteration in Ca2+ homeostasis during ischemia. The purpose of this study was to determine whether VDCCs are altered by global ischemia and reperfusion in a canine cardiac arrest, resuscitation model. We employed the radioligand, [3H]PN200-110, to quantitate the equilibrium binding characteristics of the VDCCs in the cerebral cortex. Twenty-five adult beagles were separated into four experimental groups: (a) nonischemic controls, (b) those undergoing 10-min ventricular fibrillation and apnea, (c) those undergoing 10-min ventricular fibrillation and apnea followed by spontaneous circulation and controlled respiration for 2 and (d) 24 h. Brain cortex samples were taken prior to killing of the animal, frozen immediately in liquid nitrogen, and crude synaptosomal membranes isolated by differential centrifugation/filtration. After 10 min of ischemia the maximal binding (Bmax) of [3H]PN200-110 increased to greater than 250% of control values (control Bmax 11.16 +/- 0.98; ischemic 28.35 +/- 2.78 fmol/mg protein; p less than 0.05). Bmax returned to near control values after 2 h of reperfusion but remained significantly greater than the control at 24 h. Although the affinity constant (Kd) (control = 0.12 +/- 0.03 nM) appeared to increase with ischemia and normalize with reperfusion, the changes were not statistically significant. We conclude that the binding of [3H]PN200-110 to L-type VDCCs is increased after 10 min of global ischemia/anoxia produced by ventricular fibrillation and apnea in the dog.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 45520, year: 1992, vol: 12, page: 418, stat: Journal Article,

Spectral analysis of cytochromes in rat heart myocytes: transient and steady-state photodiode array spectrophotometry measurements
Sarti P; Antonini G; Malatesta F; D'Itri E; Brunori M; Blanck TJ
1992 Nov 15;299(1):8-14, Archives of biochemistry & biophysics. ABB
Myocytes prepared from rat heart have been studied by optical spectroscopy using a photodiode array spectrophotometer adapted to a stopped flow apparatus (PASF). The isolated cells were viable for 3-4 h (i.e., over the total time of the experiments), as tested employing morphological parameters of cell damage, reactivity toward trypan blue, and the ability to use succinate in the absence and presence of digitonin. Respiration was activated by addition of sodium ascorbate and tetramethyl-para-phenylenediamine (TMPD) as exogenous reductants, in order to single out the contributions of cytochrome c and cytochrome c oxidase among the complexes of the mitochondrial respiratory chain. TMPD was shown to be freely permeable across cytoplasmic and mitochondrial membranes, with a measured KD = 0.9 mM. The use of singular value decomposition analysis coupled to PASF acquisition proved very powerful in resolving statically and kinetically, in the millisecond time region, the spectral contributions of the cytochromes. Spectral analysis was improved by adding carbon monoxide at concentrations which did not affect cytochrome c oxidase activity, but kept myoglobin fully saturated (and thus uninfluential to absorbance changes)
— id: 45518, year: 1992, vol: 299, page: 8, stat: Journal Article,

Evidence for a halothane-induced reduction in maximal calcium-activated force in mammalian myocardium
Berman MR; Casella ES; Blanck TJ
1991 ;301(1):191-197, Advances in experimental medicine & biology
— id: 45526, year: 1991, vol: 301, page: 191, stat: Journal Article,

Mechanisms of anesthetic action in skeletal, cardiac, and smooth muscle
Blanck, Thomas JJ; Wheeler, David Martyn
New York : Plenum Press, 1991,
— id: 2125, year: 1991, vol: , page: , stat: ,

Hemodynamic effects and onset time of increasing doses of vecuronium in patients undergoing myocardial revascularization
Chen BB; Nyhan DP; Blanck TJ
1991 Dec;5(6):569-573, Journal of cardiothoracic & vascular anesthesia
Study objectives were (1) to compare the hemodynamic effects of increasing doses of vecuronium, given as a bolus during induction of anesthesia using high-dose fentanyl, in patients undergoing myocardial revascularization; and (2) to determine whether increasing the dose of vecuronium would decrease the onset time to maximal depression of twitch response. Forty patients scheduled for elective coronary artery bypass surgery were randomly assigned to four equal groups to receive either 0.1, 0.2, 0.3, or 0.4 mg/kg of vecuronium. Hemodynamic measurements and neuromuscular blockade were recorded at five time points: A, awake state; B, anesthetized state after the administration of fentanyl, 10 micrograms/kg; C, 2 minutes after vecuronium bolus; D, 5 minutes after vecuronium bolus; and E, after intubation. Increasing the dose of vecuronium from 0.1 to 0.2 mg/kg decreased the onset time from 3.8 +/- 0.3 minutes to 1.8 +/- 0.2 minutes (P less than 0.05). However, higher doses of vecuronium (0.3 or 0.4 mg/kg) did not result in further decreases in onset time. There were no significant differences in any hemodynamic parameter measured among the four groups in the anesthetized baseline state. Compared with the anesthetized state, the administration of vecuronium resulted in few alterations in hemodynamics within the groups studied. There were no changes in any hemodynamic parameter at 2 and 5 minutes following administration of 0.4 mg/kg of vecuronium. There were also no dose-related changes in any hemodynamic parameter. Thus, high doses of vecuronium of up to 0.4 mg/kg may be administered to patients with coronary artery disease with few hemodynamic changes.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 45524, year: 1991, vol: 5, page: 569, stat: Journal Article,

Volatile anesthetics depress calcium channel blocker binding to bovine cardiac sarcolemma
Drenger B; Quigg M; Blanck TJ
1991 Jan;74(1):155-165, Anesthesiology
Volatile anesthetics produce their negative inotropic effect on the heart mainly by interference with calcium homeostasis in the myocardial cell. In order to elucidate the mechanism of the depression, we have evaluated the effect of the volatile anesthetics on the binding of the calcium channel blocker [3H]nitrendipine to purified bovine cardiac sarcolemma. The radioligand binding studies were carried out at 25 degrees C, with increasing concentrations of [3H]nitrendipine (0.01-1 nM), in the presence or absence of unlabeled nitrendipine to determine specific binding, and with or without 1.9% halothane, 2.3% isoflurane, and 4.8% enflurane. Separately, [3H]nitrendipine was measured in the presence of increasing doses of halothane (0.78, 1.33, 1.90, and 2.57%). Kinetic studies of association and dissociation rate were performed with 1.90% halothane and 1 nM [3H]nitrendipine at different time intervals. All three volatile anesthetics produced depression of [3H]nitrendipine binding to the isolated cardiac sarcolemma. Only halothane produced a significant depression in binding, ranging between 59 and 66% (P less than 0.05), depending on the concentration of [3H]nitrendipine used. Isoflurane produced 29-38% depression, and enflurane produced 5-22% depression. Halothane also produced a significant (P less than 0.01) dose-dependent decrease in [3H]nitrendipine-specific binding. The kinetic binding experiments demonstrated that the time course for halothane's effect on association and dissociation of [3H]nitrendipine was 5 min for the half-maximum effect; the maximal reduction in binding capacity was at 15-30 min (P less than 0.05). Scatchard analysis revealed that all three volatile anesthetics produced reduction in the maximal number of binding sites; however, they varied in their effect on binding affinity. Only halothane produced a homogenous increase in the dissociation constant, signifying reduced affinity of the Ca2+ blocker to the channel. We suggest that the volatile anesthetics produce conformational changes in these channels consistent with their ability to depress channel-mediated Ca2+ influx into myocytes
— id: 45525, year: 1991, vol: 74, page: 155, stat: Journal Article,

Halothane inhibits binding of calcium channel blockers to cardiac sarcolemma
Drenger B; Runge SR; Hoehner P; Quigg M; Blanck TJ
1991 ;301(1):109-114, Advances in experimental medicine & biology
— id: 45527, year: 1991, vol: 301, page: 109, stat: Journal Article,

Halothane depresses D600 binding to bovine heart sarcolemma
Hoehner PJ; Quigg MC; Blanck TJ
1991 Dec;75(6):1019-1024, Anesthesiology
Volatile anesthetics exert their negative inotropic effects by interfering with Ca2+ homeostasis in the myocardial cell. The mechanism of this dose-dependent action is uncertain. 3H-D600 (3H-Gallopamil), a Ca(2+)-channel antagonist, binds to the voltage-dependent Ca2+ channels (VDCC) in a specific, saturable, and reversible manner. We used this ligand to study the effect of halothane on the binding characteristics of the VDCC in purified bovine heart sarcolemma. Cardiac sarcolemmal vesicles were isolated from fresh bovine heart by differential centrifugation and filtration. 3H-D600 equilibrium binding assays were performed in the presence or absence of 1.0 mM unlabeled D600 to determine total and nonspecific binding in room air and at 0.7, 1.3, and 2.5% (vol/vol) halothane. Halothane produced a significant dose-dependent and reversible depression of 3H-D600 specific binding in bovine heart sarcolemma. Depression was completely reversed when halothane had evaporated from the samples prior to filtration. Halothane 1.3% (vol/vol) produced a 40% reduction in the maximum binding capacity. The dissociation constant was not affected by any concentration of halothane. One mechanism by which the volatile anesthetics may induce negative inotropism is through the reduction of functional VDCCs in the heart, leading to reduction of Ca2+ entry. The results of this study support this hypothesis
— id: 45523, year: 1991, vol: 75, page: 1019, stat: Journal Article,

Volatile anesthetic effects on left ventricular relaxation in swine
Humphrey LS; Stinson DC; Humphrey MJ; Finney RS; Zeller PA; Judd MR; Blanck TJ
1990 Oct;73(4):731-738, Anesthesiology
The effects of halothane (0.5, 1.0, and 1.5%; n = 10), enflurane (1.0, 2.0, and 3.0%; n = 8), and isoflurane (0.75, 1.5, and 2.25%; n = 8) on isovolumic relaxation were studied in open-chest swine. The time constant for isovolumic left ventricular pressure decline, T, was determined at each anesthetic concentration at the intrinsic heart rate and during atrial pacing to 150 beats per min. The effect of increased left ventricular afterload on T was investigated by partial occlusion of the thoracic aorta to raise the left ventricular systolic pressure to baseline in the presence of volatile anesthetics, and 20% above baseline in the absence of volatile anesthetics. Heart rate and left ventricular systolic pressure decreased substantially with all three anesthetics, whereas left ventricular end-diastolic pressure increased (by 3-4 mmHg). Relaxation time constants increased with all three anesthetics at the intrinsic heart rate; when the heart rate was controlled by pacing, T increased in the halothane and enflurane, but not in the isoflurane, experiments. T was significantly prolonged (by 30-100%) by partial aortic occlusion in the presence of anesthetic, but not in the control measurements. T did not change significantly in the isoflurane experiments when atrial pacing was employed with partial aortic occlusion. The volatile anesthetics, particularly halothane, seem to impair the relaxation process of the left ventricle; further investigation of the mechanisms of this interference, such as anesthetic effects on intracellular calcium movement and total left ventricular load, is warranted
— id: 45528, year: 1990, vol: 73, page: 731, stat: Journal Article,

Depression of myocardial force and stiffness without change in crossbridge kinetics: effects of volatile anesthetics reproduced by nifedipine
Chung OY; Blanck TJ; Berman MR
1989 Sep;71(3):444-448, Anesthesiology
The authors examined the effects of nifedipine, a sarcolemmal slow Ca2+ channel blocker, on dynamic stiffness and force of rabbit right ventricular trabeculum and papillary muscle in Ba2+ contracture, in an attempt to reproduce the effects of halothane, enflurane, and isoflurane on a similar preparation as reported by Shibata et al. Once barium contracture force was established, muscle length was perturbed with small amplitude sinusoidal oscillations in the frequency range of 0.1-100 Hz. Nifedipine 1 microM was then added to the superfusate and dynamic stiffness was again measured. Additional barium was used to determine restoration of contracture force to and beyond control levels. Nifedipine produced a significant decrease in contracture force and high-frequency stiffness with no effect on the frequency (fmin) at which stiffness amplitude exhibited a minimum (P less than 0.005). Contracture force and stiffness could be restored by adding additional barium to the nifedipine-treated muscles. These results are similar to those reported by Shibata et al. using volatile anesthetics. Since nifedipine, which acts specifically at the sarcolemmal slow Ca2+ channel, affects contracture force and dynamic stiffness in this preparation in a manner similar to the volatile anesthetics, the authors suggest that the anesthetics studied by Shibata et al. may well exert a significant component of their negative inotropic activity via their action on the sarcolemmal slow Ca2+ channel
— id: 45529, year: 1989, vol: 71, page: 444, stat: Journal Article,

The effect of halothane, enflurane, and isoflurane on the dynamic stiffness of rabbit papillary muscle
Shibata T; Blanck TJ; Sagawa K; Hunter W
1989 Mar;70(3):496-502, Anesthesiology
The authors examined the effect of halothane, enflurane, and isoflurane on the dynamic stiffness of rabbit papillary muscles in Ba2+ contracture. Ca2+ was replaced by Ba2+ in order to constantly activate myofibrils. The dynamic stiffness of the contractured muscle was examined by exposing the muscle to sinusoidal length perturbations at frequencies of 0.05-30 Hz under two concentrations of anesthetic, approximately 0.5, and 1.5-2 mM, and at two Ba2+ concentrations, 0.5 and 1.5-2 mM. The anesthetics had no effect on the frequency (fmin) at which minimum stiffness occurred, but markedly decreased the stiffness modulus at high frequencies (Khi). The decrease in Khi was significant for all anesthetics at the P less than 0.05 level. Increasing the Ba2+ concentration from 0.5 to 1.5-2 mM in the presence of 0.5 mM of anesthetic resulted in a return of Khi to control levels. The authors conclude that halothane, enflurane, and isoflurane did not alter actin-myosin ATPase kinetics, because fmin was unchanged, but decreased the number of crossbridge interactions, because Khi was significantly decreased by all three anesthetics
— id: 45530, year: 1989, vol: 70, page: 496, stat: Journal Article,

Halothane decreases calcium channel antagonist binding to cardiac membranes
Blanck TJ; Runge S; Stevenson RL
1988 Nov;67(11):1032-1035, Anesthesia & analgesia
The effect of halothane concentration on the binding of the calcium antagonist, [3H] nitrendipine (3HNTP), to rat and rabbit heart membranes was examined in vitro because it has been hypothesized that one mechanism by which halothane depresses cardiac contractility is by interfering with Ca2+ channel function. Membranes were incubated for 90 minutes in a closed system with 3HNTP and increasing concentrations of halothane. The amount of 3HNTP bound to membranes was quantified by radioligand binding technique and liquid scintillation counting. It was found in both the rat and rabbit cardiac membranes that halothane (0.4-2.0%) caused a dose-dependent decrease in specific 3HNTP binding (P less than 0.0001). The decrease in 3HNTP binding caused by halothane was also found to be reversible. These results indicate that halothane interferes with one property of the Ca2+ channel and suggest that this may be one possible mechanism for the negative inotropic action of halothane
— id: 45531, year: 1988, vol: 67, page: 1032, stat: Journal Article,

Thiopental does not alter Ca2+ uptake by cardiac sarcoplasmic reticulum
Blanck TJ; Stevenson RL
1988 Apr;67(4):346-348, Anesthesia & analgesia
The effect of thiopental on Ca2+ uptake by cardiac sarcoplasmic reticulum (SR) isolated from the rabbit was examined to clarify the role of the sarcoplasmic reticulum in the negative inotropic action of thiopental. Thiopental, from 0 to 378 microM, did not alter the rate of Ca2+ uptake by the SR. We also compared the ATP dependence of Ca2+ uptake in the presence and absence of 284 microM thiopental. The Km for ATP and the Vmax of Ca+ uptake were unaffected by thiopental. It is concluded that thiopental does not alter Ca2+ uptake by the SR and that the negative inotropic effects of thiopental occur at other sites in the myocardial cell
— id: 45532, year: 1988, vol: 67, page: 346, stat: Journal Article,

The effect of volatile anesthetics on the pH dependence of calcium uptake by cardiac sarcoplasmic reticulum
Casella ES; Suite ND; Fisher YI; Blanck TJ
1987 Sep;67(3):386-390, Anesthesiology
The effect of volatile anesthetics (VA) on the pH dependence of calcium uptake by cardiac sarcoplasmic reticulum (SR) was studied. SR was incubated at 37 degrees C with 45CaCl2 in the control state (no anesthetic) and in the presence of each of the VA from pH 6.6-7.6. The VA used were: halothane, 1.3%; enflurane, 1.8%; and isoflurane, 1.2%. In the control state, the initial rate of calcium uptake, measured after a 2-min incubation, was maximal at pH 6.8 (mean +/- SEM: 665 +/- 37 nmoles/mg) and markedly inhibited at pH 7.6 (107 +/- 9 nmoles/mg). In the presence of the VA, the calcium uptake rate was mildly depressed (7-32%) at pH 6.6-6.8, unchanged at pH 7.0, and greatly enhanced (52-78%) at pH 7.2-7.6, when compared to control. The maximal uptake of calcium by the SR at a calcium concentration of 10(-6)M, measured by a 20-min incubation, had a similar pH dependence in the control state, with a decline first evident at pH 7.2 and a 50% drop in the maximal uptake of calcium from pH 7.0-7.6. The presence of the VA was associated with a uniform depression of the maximal uptake of calcium by the SR at all pH levels measured. In view of these findings, it appears that pH does affect SR function in the presence of VA. This alteration of the pH effect by VA may be a factor responsible for discrepancies in results previously reported by investigators studying the effects of VA on the uptake of calcium by the SR
— id: 45534, year: 1987, vol: 67, page: 386, stat: Journal Article,

Effects of halothane on myocardial high-energy phosphate metabolism and intracellular pH utilizing 31P NMR spectroscopy
Murray PA; Blanck TJ; Rogers MC; Jacobus WE
1987 Nov;67(5):649-653, Anesthesiology
Utilizing 31phosphorus nuclear magnetic resonance (NMR) spectroscopy, the authors tested the two hypotheses that the negative inotropic action of halothane is the result of: 1) myocardial intracellular acidosis, and 2) a decrease in myocardial high-energy phosphates. In isolated, paced, Langendorff-perfused rabbit hearts, halothane (1.5 vol %) dissolved in the coronary perfusate produced a 48 +/- 2% decrease (P less than 0.01) in left ventricular developed pressure. In contrast, halothane administration had no significant effect on myocardial intracellular pH (7.18 +/- 0.04 at control vs 7.21 +/- 0.02 during halothane). Halothane exposure decreased (P less than 0.01) the forward rate constant of the creatine kinase reaction by 32 +/- 6%, as measured using saturation transfer NMR, suggesting a decline in the rate of high-energy phosphate metabolism. This was further indicated by a concomitant decrease (P less than 0.05) in myocardial oxygen consumption (20 +/- 5%). During the halothane-induced reduction in left ventricular developed pressure, only small decreases in the myocardial steady state concentrations of phosphocreatine (7 +/- 1%; P less than 0.01) and beta ATP (12 +/- 4%; P less than 0.05), and an increase in Pi (18 +/- 6%; P less than 0.05) were observed. However, similar changes in steady-state high-energy phosphate metabolites were also measured in time-control hearts not exposed to halothane. These results indicate that the negative inotropic action of halothane is not mediated by myocardial intracellular acidosis. Moreover, these findings do not support the concept that the negative inotropic action of halothane is the result of a reduction in myocardial high-energy phosphates
— id: 45533, year: 1987, vol: 67, page: 649, stat: Journal Article,

Intraoperative use of verapamil for nitroglycerin--refractory myocardial ischemia
Humphrey LS; Blanck TJ
1985 Jan;64(1):68-71, Anesthesia & analgesia
— id: 45535, year: 1985, vol: 64, page: 68, stat: Journal Article,

Low molecular weight proteins in human malignant hyperthermic muscle
Blanck TJ; Fisher YI; Thompson M; Muldoon S
1984 Nov;61(5):589-592, Anesthesiology
Malignant hyperthermia (MH) is a pharmacogenetic disorder provoked by volatile anesthetics and depolarizing muscle relaxants. The preoperative diagnosis of MH is difficult because it requires a large muscle biopsy and a laboratory dedicated to such diagnostic studies. The authors performed electrophoresis of six muscles taken from MH patients or their relatives to determine whether the protein composition is different from normal muscle. MH muscle was found to contain large amounts of two low molecular weight proteins (15,000 daltons and 13,500 daltons) that are not present in normal muscle. Although it has not been determined that these differences are specific for MH, this finding eventually might be of assistance in diagnosing MH
— id: 45537, year: 1984, vol: 61, page: 589, stat: Journal Article,

Immediate improvement of dysfunctional myocardial segments after coronary revascularization: detection by intraoperative transesophageal echocardiography
Topol EJ; Weiss JL; Guzman PA; Dorsey-Lima S; Blanck TJ; Humphrey LS; Baumgartner WA; Flaherty JT; Reitz BA
1984 Dec;4(6):1123-1134, Journal of the American College of Cardiology
To ascertain the immediate effects of coronary artery bypass grafting on regional myocardial function, intraoperative transesophageal two-dimensional echocardiograms were obtained in 20 patients using a 3.5 MHz phased array transducer at the tip of a flexible gastroscope. Cross-sectional images of the left ventricle were obtained at multiple levels before skin incision and were repeated serially before and immediately after cardiopulmonary bypass. Using a computer-aided contouring system, percent systolic wall thickening was determined for eight anatomic segments in each patient at similar loading conditions (four each at mitral and papillary muscle levels). Of the 152 segments analyzed, systolic wall thickening improved from a prerevascularization mean value (+/- SEM) of 42.7 +/- 2.9% to a postrevascularization mean value of 51.6 +/- 2.6% (p less than 0.001). Thickening improved most in those segments with the worst preoperative function (p less than 0.001). Chest wall echocardiograms obtained 8.4 +/- 2.3 days after operation showed no deterioration or further improvement in segmental motion compared with transesophageal echocardiograms obtained after revascularization. Thus: regional myocardial function frequently improves immediately after bypass grafting, with increases in regional thickening being most marked in those segments demonstrating the most severe preoperative dysfunction, and this improvement appears to be sustained; and in some patients, chronic subclinical ischemic dysfunction is present which can be improved by revascularization
— id: 45536, year: 1984, vol: 4, page: 1123, stat: Journal Article,

Malignant hyperthermia
Blanck TJ; Gruener RP
1983 Aug 1;32(15):2287-2289, Biochemical pharmacology
— id: 45538, year: 1983, vol: 32, page: 2287, stat: Journal Article,

Association of post-anaesthetic hyperthermia with abnormal muscle characteristics: a case report
Kripke BJ; Blanck TJ; Sizemore DA; Comunale FL; Christiansen J; Gruener R
1983 May;30(3 Pt 1):290-294, Canadian Anesthetists' Society journal
A previously healthy 18-year-old male, following appendectomy developed post-anaesthetic hyperthermia (42.1 degrees C) with an elevation of serum creatine kinase and activated partial thromboplastin time. Repeated arterial blood gases were normal. Cooling and anti-pyretic medication did not control the fever. In contrast, sodium dantrolene appeared effective in lowering the patient's temperature and normalizing the vital signs, both acutely and over the following three days. Subsequent muscle biopsy revealed a normal contracture response to caffeine alone or in the presence of halothane. However, the muscle had a larger than normal potentiation of evoked twitch tension in the presence of caffeine and halothane. Electrophoresis of the muscle revealed a marked increase of an unidentified low molecular weight protein. The patient's clinical course, and the results of the muscle studies, suggest that an abnormality of skeletal muscle
— id: 45539, year: 1983, vol: 30, page: 290, stat: Journal Article,

Enflurane and isoflurane stimulate calcium transport by cardiac sarcoplasmic reticulum
Blanck TJ; Thompson M
1982 Feb;61(2):142-145, Anesthesia & analgesia
— id: 45540, year: 1982, vol: 61, page: 142, stat: Journal Article,

A simple closed system for performing biochemical experiments at clinical concentrations of volatile anesthetics
Blanck TJ
1981 Jun;60(6):435-436, Anesthesia & analgesia
— id: 45542, year: 1981, vol: 60, page: 435, stat: Journal Article,

Calcium uptake by isolated sarcoplasmic reticulum: examination of halothane inhibition, pH dependence, and Ca2+ dependence of normal and malignant hyperthermic human muscle
Blanck TJ; Gruener R; Suffecool SL; Thompson M
1981 Jul;60(7):492-498, Anesthesia & analgesia
Ca2+ uptake and release in muscle homogenates and fragmented sarcoplasmic reticulum were examined in biopsy specimens from nonsusceptible and malignant hyperthermia (MH) susceptible patients. Ca2+ flux was examined by the filter binding assay technique using 45Ca. It was found that Ca2+ uptake and release were the same in both normal and MH muscle homogenates. Halothane inhibited the uptake of Ca2+ by the sarcoplasmic reticulum. The halothane inhibition of Ca2+ uptake in normal and MH sarcoplasmic reticulum was fitted to a single line with a correlation coefficient (r) of -0.958. The pH and Ca2+ dependence of Ca2+ uptake were the same for both normal and MH sarcoplasmic reticulum. The pK for Ca2+ uptake is approximately 5.9. It is concluded that the Ca2+ uptake function of the muscle from the five patients with MH examined is not abnormal and might not be the locus for the initiation of MH
— id: 45541, year: 1981, vol: 60, page: 492, stat: Journal Article,

Calcium transport by cardiac sarcoplasmic reticulum: modulation of halothane action by substrate concentration and pH
Blanck TJ; Thompson M
1981 Jun;60(6):390-394, Anesthesia & analgesia
The response of calcium transport to halothane by the cardiac sarcoplasmic reticulum (SR) was investigated to determine whether the SR is a site for anesthetic depression of the myocardium. It was observed that halothane could both stimulate (by 800%) and inhibit (by 500%) calcium transport. The varied effects are dependent on adenosine triphosphate (ATP) and calcium and hydrogen ion concentrations. At 2.25% halothane, the Km for ATP is decreased from 2.35 to 0.712 mM and Vmax is decreased from 292 to 149 nmoles/mg/2min. It was found that the steady-state level of calcium in the SR was decreased by 33% by halothane at pH 6.9, whereas halothane had no effect at pH 7.3. It was concluded that the SR is an unlikely site of halothane-induced myocardial depression in the normal heart when substrate concentrations and pH are maintained. In the ischemic heart in which the pH and substrate concentration have decreased, the interaction of halothane with the SR might contribute to a decrease in Ca2+ for contraction
— id: 45543, year: 1981, vol: 60, page: 390, stat: Journal Article,

Measurement of halothane by ultraviolet spectroscopy
Blanck TJ; Thompson M
1980 Jul;59(7):481-483, Anesthesia & analgesia
Halothane absorbs strongly in the ultraviolet region of the spectrum. This property has been employed to measure the concentration of halothane in samples in which the effect of halothane on enzyme kinetics was being studied. Halothane can be completely extracted into heptane, displays a concentration-dependent linear increase in absorbance over a broad concentration range, and has a molar extinction coefficient of 447 M cm-1 at 208 nm. The procedure described for the measurement of halothane will enable other investigators who do not have a gas chromatograph to measure the concentration of halothane
— id: 45544, year: 1980, vol: 59, page: 481, stat: Journal Article,

Beta-adrenergic receptor ligand binding by rabbit lung
Blanck TJ; Gillis CN
1979 Jun 15;28(12):1903-1909, Biochemical pharmacology
— id: 45545, year: 1979, vol: 28, page: 1903, stat: Journal Article,

Malignant hyperthermia
Blanck TJ; Gruener R; Casey WJ; Stern LZ
1979 May;36(5):351-353, Arizona medicine
— id: 45546, year: 1979, vol: 36, page: 351, stat: Journal Article,

The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts
Blanck TJ; Peterkofsky B
1975 Nov;171(1):259-267, Archives of biochemistry & biophysics. ABB
— id: 45547, year: 1975, vol: 171, page: 259, stat: Journal Article,

The stereochemistry of pyridoxal phosphate enzymes. The absolute stereochemistry of cofactor C' 4 protonation in the transamination of holoserine hydroxymethylase by D-alanine
Voet JG; Hindenlang DM; Blanck TJ; Ulevitch RJ; Kallen RG; Dunathan HC
1973 Feb 10;248(3):841-842, Journal of biological chemistry
— id: 45548, year: 1973, vol: 248, page: 841, stat: Journal Article,