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Following injury, primary sensory neurons undergo changes that drive central sensitization and contribute to the maintenance of persistent hypersensitivity. NR2B expression in the dorsal root ganglia (DRG) has not been previously examined in neuropathic pain models. Here, we investigated if changes in NR2B expression within the DRG are associated with hypersensitivities that result from peripheral nerve injuries. This was done by comparing the NR2B expression in the DRG derived from two modalities of the spared nerve injury (SNI) model, since each variant produces different neuropathic pain phenotypes. Using the electronic von Frey to stimulate the spared and non-spared regions of the hindpaws, we demonstrated that sural-SNI animals develop sustained neuropathic pain in both regions while the tibial-SNI animals recover. NR2B expression was measured at Day 23 and Day 86 post-injury. At Day 23 and 86 post-injury, sural-SNI animals display strong hypersensitivity, whereas tibial-SNI animals display 50 and 100% recovery from post-injury-induced hypersensitivity, respectively. In tibial-SNI at Day 86, but not at Day 23 the perinuclear region of the neuronal somata displayed an increase in NR2B protein. This retention of NR2B protein within the perinuclear region, which will render them non-functional, correlates with the recovery observed in tibial-SNI. In sural-SNI at Day 86, DRG displayed an increase in NR2B mRNA which correlates with the development of sustained hypersensitivity in this model. The increase in NR2B mRNA was not associated with an increase in NR2B protein within the neuronal somata. The latter may result from a decrease in kinesin Kif17, since Kif17 mediates NR2B transport to the soma's plasma membrane. In both SNIs, microglia/macrophages showed a transient increase in NR2B protein detected at Day 23 but not at Day 86, which correlates with the initial post-injury induced hypersensitivity in both SNIs. In tibial-SNI at Day 86, but not at Day 23, satellite glia cells (SGCs) displayed an increase in NR2B protein. This study is the first to characterize of cell-specific changes in NR2B expression within the DRG following peripheral nerve injury. We discuss how the observed NR2B changes in DRG can contribute to the different neuropathic pain phenotypes displayed by each SNI variant.

Peripheral nerve injury alters the expression of hundreds of proteins in dorsal root ganglia (DRG). Targeting some of these proteins has led to successful treatments for acute pain, but not for sustained post-operative neuropathic pain. The latter may require targeting multiple proteins. Since a single microRNA (miR) can affect the expression of multiple proteins, here, we describe an approach to identify chronic neuropathic pain-relevant miRs. We used two variants of the spared nerve injury (SNI): Sural-SNI and Tibial-SNI and found distinct pain phenotypes between the two. Both models induced strong mechanical allodynia, but only Sural-SNI rats maintained strong mechanical and cold allodynia, as previously reported. In contrast, we found that Tibial-SNI rats recovered from mechanical allodynia and never developed cold allodynia. Since both models involve nerve injury, we increased the probability of identifying differentially regulated miRs that correlated with the quality and magnitude of neuropathic pain and decreased the probability of detecting miRs that are solely involved in neuronal regeneration. We found seven such miRs in L3-L5 DRG. The expression of these miRs increased in Tibial-SNI. These miRs displayed a lower level of expression in Sural-SNI, with four having levels lower than those in sham animals. Bioinformatic analysis of how these miRs could affect the expression of some ion channels supports the view that, following a peripheral nerve injury, the increase of the seven miRs may contribute to the recovery from neuropathic pain while the decrease of four of them may contribute to the development of chronic neuropathic pain. The approach used resulted in the identification of a small number of potentially neuropathic pain relevant miRs. Additional studies are required to investigate whether manipulating the expression of the identified miRs in primary sensory neurons can prevent or ameliorate chronic neuropathic pain following peripheral nerve injuries.

Background: Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons., Methods: DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine., Results: The mean KCl-induced calcium transient was greater in P7 neurons (P < 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (P < 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages., Conclusions: Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.

Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2). We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2) over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2) or the phosphoinositide 3-kinase (PI3K)/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase Cγ (PLCγ) pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCγ signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs.

In contrast to the adult brain, the adult spinal cord is a non-neurogenic environment. Understanding how to manipulate the spinal cord environment to promote the formation of new neurons is an attractive therapeutic strategy for spinal cord injury and disease. The cannabinoid 1 receptor (CB1R) has been implicated as a modulator of neural progenitor cell proliferation and fate specification in the brain; however, no evidence exists for modulation of adult spinal cord progenitor cells. Using adult rat spinal cord primary cultures, we demonstrated that CB1R antagonism with AM251 significantly decreased the number of Nestin(+) cells, and increased the number of βIII tubulin(+) and DCX(+) cells, indicative of neuronal differentiation. AM251's effect was blocked by co-application of the CB1R agonists, WIN 55, 212-2, or ACEA. Consistent with our hypothesis, cultures, and spinal cord slices derived from CB1R knock-out (CB1-/-) mice had significantly higher levels of DCX(+) cells compared to those derived from wild type (CB1+/+) mice, indicative of enhanced neuronal differentiation in CB1-/- spinal cords. Moreover, AM251 promoted neuronal differentiation in CB1+/+, but not in CB1-/- cultures. Since CB1R modulates synaptic transmission, and synaptic transmission has been shown to influence progenitor cell fate, we evaluated whether AM251-induced neuronal differentiation was affected by chronic inactivity. Either the presence of the voltage-dependent sodium channel blocker tetrodotoxin (TTX), or the removal of mature neurons, inhibited the AM251-induced increase in DCX(+) cells. In summary, antagonism or absence of CB1R promotes neuronal differentiation in adult spinal cords, and this action appears to require TTX-sensitive neuronal activity. Our data suggest that the previously detected elevated levels of endocannabinoids in the injured adult spinal cord could contribute to the non-neurogenic environment and CB1R antagonists could potentially be used to enhance replacement of damaged neurons.

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.

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.

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.

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.

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.

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.

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.

Background: Local anesthetics (LAs) are known to inhibit voltage-dependent Na+ channels, as well as K+ and Ca2+ channels, but with lower potency. Since cellular excitability and responsiveness are largely determined by intracellular Ca2+ availability, sites along the Ca2+ signaling pathways may be targets of LAs. This study was aimed to investigate the LA effects on depolarization and receptor-mediated intracellular Ca2+ changes and to examine the role of Na+ and K+ channels in such functional responses., Methods: Effects of bupivacaine, ropivacaine, mepivacaine, and lidocaine (0.1-2.3 mm) on evoked [Ca2+](i) transients were investigated in neuronal SH-SY5Y cell suspensions using Fura-2 as the intracellular Ca2+ indicator. Potassium chloride (KCl, 100 mm) and carbachol (1 mm) were individually or sequentially applied to evoke increases in intracellular Ca2+. Coapplication of LA and Na+/K+ channel blockers was used to evaluate the role of Na+ and K+ channels in the LA effect on the evoked [Ca2+](i) transients., Results: All four LAs concentration-dependently inhibited both KCl- and carbachol-evoked [Ca2+](i) transients with the potency order bupivacaine > ropivacaine > lidocaine >/= mepivacaine. The carbachol-evoked [Ca2+](i) transients were more sensitive to LAs without than with a KCl prestimulation, whereas the LA-effect on the KCl-evoked [Ca2+](i) transients was not uniformly affected by a carbachol prestimulation. Na+ channel blockade did not alter the evoked [Ca2+](i) transients with or without a LA. In the absence of LA, K+ channel blockade increased the KCl-, but decreased the carbachol-evoked [Ca2+](i) transients. A coapplication of LA and K+ channel blocker resulted in larger inhibition of both KCl- and carbachol-evoked [Ca2+](i) transients than by LA alone., Conclusions: Different and overlapping sites of action of LAs are involved in inhibiting the KCl- and carbachol-evoked [Ca2+](i) transients, including voltage-dependent Ca2+ channels, a site associated with the caffeine-sensitive Ca2+ store and a possible site associated with the IP(3)-sensitive Ca2+ store, and a site in the muscarinic pathway. K+ channels, but not Na+ channels, seem to modulate the evoked [Ca2+](i) transients, as well as the LA-effects on such responses.

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.

Unlabelled: 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.

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.

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.

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.

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.

Unlabelled: 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.

Unlabelled: 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.

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.

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.

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.

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.

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.

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.

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)

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)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.