Lidocaine increases intracellular sodium concentration through voltage-dependent sodium channels in an identified lymnaea neuron

BACKGROUND: The local anesthetic lidocaine affects neuronal excitability in the central nervous system; however, the mechanisms of such action remain unclear. The intracellular sodium concentration ([Na]i) and sodium currents (INa) are related to membrane potential and excitability. Using an identifiable respiratory pacemaker neuron from Lymnaea stagnalis, the authors sought to determine whether lidocaine changes [Na]i and membrane potential and whether INa is related to these changes. METHODS: Intracellular recording and sodium imaging were used simultaneously to measure membrane potentials and [Na]i, respectively. Measurements for [Na]i were made in normal, high-Na, and Na-free salines, with membrane hyperpolarization, and with tetrodotoxin pretreatment trials. Furthermore, changes of INa were measured by whole cell patch clamp configuration. RESULTS: Lidocaine increased [Na]i in a dose-dependent manner concurrent with a depolarization of the membrane potential. In the presence of high-Na saline, [Na]i increased and the membrane potential was depolarized; the addition of lidocaine further increased [Na]i, and the membrane potential was further depolarized. In Na-free saline or in the presence of tetrodotoxin, lidocaine did not change [Na]i. Similarly, hyperpolarization of the membrane by current injections also prevented the lidocaine-induced increase of [Na]i. In the patch clamp configuration, membrane depolarization by lidocaine led to an inward sodium influx. A persistent reduction in membrane potential, resulting from lidocaine, brings the cell within the window current of INa where sodium channel activation occurs. CONCLUSION: Lidocaine increases intracellular sodium concentration and promotes excitation through voltage-dependent sodium channels by altering membrane potential in the respiratory pacemaker neuron.     

Local Anesthetics Modulate Neuronal Calcium Signaling through Multiple Sites of Action

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.     

ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/- mouse alpha-cells

Patch-clamp recordings and glucagon release measurements were combined to determine the role of plasma membrane ATP-sensitive K+ channels (KATP channels) in the control of glucagon secretion from mouse pancreatic alpha-cells. In wild-type mouse islets, glucose produced a concentration-dependent (half-maximal inhibitory concentration [IC50]=2.5 mmol/l) reduction of glucagon release. Maximum inhibition (approximately 50%) was attained at glucose concentrations >5 mmol/l. The sulfonylureas tolbutamide (100 micromol/l) and glibenclamide (100 nmol/l) inhibited glucagon secretion to the same extent as a maximally inhibitory concentration of glucose. In mice lacking functional KATP channels (SUR1-/-), glucagon secretion in the absence of glucose was lower than that observed in wild-type islets and both glucose (0-20 mmol/l) and the sulfonylureas failed to inhibit glucagon secretion. Membrane potential recordings revealed that alpha-cells generate action potentials in the absence of glucose. Addition of glucose depolarized the alpha-cell by approximately 7 mV and reduced spike height by 30% Application of tolbutamide likewise depolarized the alpha-cell (approximately 17 mV) and reduced action potential amplitude (43%). Whereas insulin secretion increased monotonically with increasing external K+ concentrations (threshold 25 mmol/l), glucagon secretion was paradoxically suppressed at intermediate concentrations (5.6-15 mmol/l), and stimulation was first detectable at >25 mmol/l K+. In alpha-cells isolated from SUR1-/- mice, both tolbutamide and glucose failed to produce membrane depolarization. These effects correlated with the presence of a small (0.13 nS) sulfonylurea-sensitive conductance in wild-type but not in SUR1-/- alpha-cells. Recordings of the free cytoplasmic Ca2+ concentration ([Ca2+]i) revealed that, whereas glucose lowered [Ca2+]i to the same extent as application of tolbutamide, the Na+ channel blocker tetrodotoxin, or the Ca2+ channel blocker Co2+ in wild-type alpha-cells, the sugar was far less effective on [Ca2+]i in SUR1-/- alpha-cells. We conclude that the KATP channel is involved in the control of glucagon secretion by regulating the membrane potential in the alpha-cell in a way reminiscent of that previously documented in insulin-releasing beta-cells. However, because alpha-cells possess a different complement of voltage-gated ion channels involved in action potential generation than the beta-cell, moderate membrane depolarization in alpha-cells is associated with reduced rather than increased electrical activity and secretion.     

Intracellular sodium increase induced by external calcium removal in human sperm

In human sperm, removal of external calcium produces a fast Na(+)-dependent depolarization that is presumably due to sodium permeation through calcium channels. Calcium restoration produces a ouabain-sensitive hyperpolarization that brings the membrane potential to values frequently more negative than resting. In this work, we show evidence indicating that external calcium removal induces an increase in the intracellular sodium ([Na(+)](i)) and that this phenomenon is related to the Na(+)-dependent depolarization. Calcium restoration blocked the [Na(+)](i) increase and then produced a slow decrease that was inhibited by ouabain. The [Na(+)](i) increase was inhibited by nanomolar-micromolar calcium or by millimolar magnesium, which has been previously shown to inhibit the Na(+)-dependent depolarization. This evidence supports the hypothesis that, in zero-calcium medium, a calcium channel that would contribute to resting intracellular calcium levels allows sodium permeation, producing depolarization and a significant [Na(+)](i) increase. Sodium loading would stimulate the Na(+),K(+)-ATPase, the activity of which contributes to the sperm hyperpolarization observed upon calcium restoration.     

Differential contribution of sodium channel subtypes to action potential generation in unmyelinated human C-type nerve fibers

BACKGROUND: Multiple voltage-dependent sodium channels (Na(v)) contribute to action potentials and excitability of primary nociceptive neurons. The aim of the current study was to characterize subtypes of Na(v) that contribute to action potential generation in peripheral unmyelinated human C-type nerve fibers. METHODS: Registration of C-fiber compound action potentials and determination of membrane threshold was performed by a computerized threshold tracking program. Nerve fibers were stimulated with a 1-ms current pulse either alone or after a small ramp current lasting 300 ms. RESULTS: Compound C-fiber action potentials elicited by supramaximal 1-ms current pulses were rather resistant to application of tetrodotoxin (30-90 nM). However, the same concentrations of tetrodotoxin strongly reduced the peak height and elevated membrane threshold of action potentials evoked at the end of a 300-ms current ramp. A similar effect was observed during application of lidocaine and mexiletine (50 microM each). CONCLUSIONS: These data indicate that more than one type of Na(v) contributes to the generation of action potentials in unmyelinated human C-type nerve fibers. The peak height of an action potential produced by a short electrical impulse is dependent on the activation of tetrodotoxin-resistant ion channels. In contrast, membrane threshold and action potential peak height at the end of a slow membrane depolarization are regulated by a subtype of Na(v) with high sensitivity to low concentrations of tetrodotoxin, lidocaine, and mexiletine. The electrophysiologic and pharmacologic characteristics may indicate the functional activity of the Na(v) 1.7 subtype of voltage-dependent sodium channels.     

Modulation of Cardiac Sodium Current by alpha sub 1 -stimulation and Volatile Anesthetics

Background: alpha1 -adrenoceptor stimulation is known to produce electrophysiologic changes in cardiac tissues, which may involve modulations of the fast inward Na sup + current (INa). A direct prodysrhythmic alpha1 -mediated interaction between catecholamines and halothane has been demonstrated, supporting the hypothesis that generation of halothane-epinephrine dysrhythmias may involve slowed conduction, leading to reentry. In this study, we examined the effects of a selective alpha1 -adrenergic receptor agonist, methoxamine, on cardiac INa in the absence and presence of equianesthetic concentrations of halothane and isoflurane in single ventricular myocytes from adult guinea pig hearts.

Methods: INa was recorded using the standard whole-cell configuration of the patch-clamp technique. Voltage clamp protocols initiated from two different holding potentials (VH) were applied to examine state-dependent effects of methoxamine in the presence of anesthetics. Steady state activation and inactivation and recovery from inactivation were characterized using standard protocols.

Results: Methoxamine decreased INa in a concentration- and voltage-dependent manner, being more potent at the depolarized VH. Halothane and isoflurane interacted synergistically with methoxamine to suppress INa near the physiologic cardiac resting potential of -80 mV. The effect of methoxamine with anesthetics appeared to be additive when using a VH of -110 mV, a potential where no Na sup + channels are in the inactivated state. Methoxamine in the absence and presence of anesthetics significantly shifted the half maximal inactivation voltage in the hyperpolarizing direction but had no effect on steady-state activation.     

Suppression of energy requirement by lidocaine in the ischemic mouse brain

Effects of lidocaine on parameters of membrane functional integrity were investigated in the mouse brain. Changes in the direct-current potential shift in the cerebral cortex provoked by decapitation ischemia were compared in animals given lidocaine (0.05, 0.25, or 1.0 micromol, intracerebroventricular) or saline 15 minutes before ischemia. The brain content of adenosine 5'-triphosphate (ATP) was measured in animals subjected to 0, 0.5, 1, and 2 minutes of decapitation ischemia, and the effect of preischemic administration of lidocaine (0.25 micromol, intracerebroventricular) was evaluated. Na+, K+-ATPase, and Ca2+-ATPase activity was evaluated in brains pretreated with lidocaine (0.25 micromol, intracerebroventricular) or saline 15 minutes before decapitation. Changes in the intracellular Ca concentration ([Ca2+]i) were evaluated in hippocampal slices and the effects of lidocaine (50, 100, or 400 microM) were assessed in the hippocampal CA1 field and dentate gyrus at pH 7.4 and pH 6.8 every 60s for a duration of 50 min. The preischemic administration of lidocaine (1.0 and 0.25 micromol) delayed the onset of anoxic depolarization to 49 seconds and 44 seconds, respectively, as compared with that in the saline group at 27 seconds. Lidocaine maintained ATP levels higher than those in corresponding saline groups, values being 165% after 1 minute of ischemia and 212% after 2 minutes, respectively. Lidocaine did not affect Na+, K+-ATPase, and Ca2+-ATPase activity. Lidocaine did not affect changes in the [Ca2+]i in either area at either pH. The findings may suggest that lidocaine maintains the energy level by delaying depolarization in neurons, which may contribute to removal of cytosolic Ca2+ in ischemic states.     

Ketamine Inhibits Sodium Currents in Identified Cardiac Parasympathetic Neurons in Nucleus Ambiguus

Background: Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated the possibility that ketamine increases heart rate by inhibiting the central cardiac parasympathetic mechanisms.

Methods: We used a novel in vitro approach to study the effect of ketamine on the identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording, and ketamine effects on isolated potassium (K+) and sodium (Na+) currents were studied.

Results: Cardiac nucleus ambiguus neurons (n = 14) were inherently silent, but depolarization evoked sustained action potential trains with little delay or adaptation. Ketamine (10 [mu]m) reduced this response but had no effect on the voltage threshold for action potentials (n = 14;P > 0.05). The current-voltage relations for the transient K+ current and the delayed rectified K+ current (n = 5) were unaltered by ketamine (10 [mu]m-1 mm). Ketamine depressed the total Na+ current dose-dependently (10 [mu]m-1 mm). In addition, ketamine shifted the Na+ current inactivation curves to more negative potentials, thus suggesting the enhancement of the Na+ channel inactivation (P < 0.05; n = 7). In the presence of Cd2+, ketamine (10 [mu]m) continued to inhibit voltage-gated Na+ currents, which recovered completely within 10 min.     

The role of membrane depolarization in norepinephrine-induced contractions of the rabbit mesenteric resistance artery

Changes in membrane potential during norepinephrine-induced contractions in the rabbit mesenteric resistance artery (3rd or 4th branch) were investigated using microelectrodes. Norepinephrine at concentrations greater than 10(-6) M depolarized the membrane and induced contractions dose-dependently. Maximum effects were produced by 10(-4) M norepinephrine. Depolarization was maintained at almost steady level during 15 min application of norepinephrine. During the same period, contractions continued with slight decay. Oscillatory contractions were observed at more than 3 X 10(-6) M norepinephrine, and occasionally persisted throughout the application of norepinephrine. Treatments with Ca2+-rich 1 mM EGTA solution, 10(-5) M diltiazem, 3 X 10(-6) M D600 and 1 mM La3+ did not significantly affect the amount of depolarization induced by 10(-4) M norepinephrine; however, contractions were greatly inhibited by these treatments. Replacement of Na+ by choline markedly reduced depolarization while contractions were not affected. In Ca2+ -free Na+-free solution, no depolarization was induced, while contractions were still produced by norepinephrine, indicating that Cl- was not essential for membrane depolarization. These results suggest that contractions of the rabbit mesenteric resistance artery to norepinephrine are mainly due to the enhanced influx of extracellular Ca2+ which is not dependent on potential-sensitive mechanisms. Depolarization is thought to be due to the increase in the membrane permeability to Na+ and Cl- which is coincidentally produced by norepinephrine. The membrane potential oscillations were dependent on Ca entry but could not be shown to be the result of fluctuations in Ca current.     

Role of muscle in regulating extracellular [K+

There is a positive association between diets rich in potassium, control of blood pressure, and prevention of stroke. Extracellular [K+] is regulated closely to maintain normal membrane excitability by the concerted regulatory responses of muscle and kidney. Although kidney is responsible for ultimately matching K+ output to K+ intake each day, muscle contains more than 90% of the body's K+ and can buffer changes in extracellular fluid [K+] by either acutely taking up extracellular fluid K+ or releasing intracellular fluid K+ from muscle. It long has been assumed that the changes in muscle K+ transport are a function of sodium pump (Na,K-adenosine triphosphatase [Na, K-ATPasel]) abundance, especially that of the alpha2 isoform, which predominates in skeletal muscle. To test the physiologic significance of changes in muscle Na,K-ATPase expression, we developed the K+ clamp, which measures insulin-stimulated cellular K+ uptake in vivo in the conscious rat. By using the K+ clamp we discovered that significant insulin resistance to cell K+ uptake occurs as follows: (1) early in K+ deprivation before a decrease in muscle sodium pump pool size, and (2) during glucocorticoid treatment, which increases muscle Na,K-ATPase alpha2 levels greater than 50%. We also discovered that adaptation of renal and extrarenal K+ handling to altered K+ balance often occurs without changes in plasma [K+], supporting a feedforward mechanism involving K+ sensing in the splanchnic bed and adjustment of K+ handling. These findings establish the advantage of combining molecular analyses of Na,K-ATPase expression and activity with systems analyses of cellular K+ uptake and excretion in vivo to reveal regulatory mechanisms operating to control K+ homeostasis.     

Differential Contribution of Sodium Channel Subtypes to Action Potential Generation in Unmyelinated Human C-type Nerve Fibers

Background: Multiple voltage-dependent sodium channels (Nav) contribute to action potentials and excitability of primary nociceptive neurons. The aim of the current study was to characterize subtypes of Nav that contribute to action potential generation in peripheral unmyelinated human C-type nerve fibers.

Methods: Registration of C-fiber compound action potentials and determination of membrane threshold was performed by a computerized threshold tracking program. Nerve fibers were stimulated with a 1-ms current pulse either alone or after a small ramp current lasting 300 ms.

Results: Compound C-fiber action potentials elicited by supramaximal 1-ms current pulses were rather resistant to application of tetrodotoxin (30-90 nm). However, the same concentrations of tetrodotoxin strongly reduced the peak height and elevated membrane threshold of action potentials evoked at the end of a 300-ms current ramp. A similar effect was observed during application of lidocaine and mexiletine (50 [mu]m each).     

Early effects of parathyroid hormone on membrane potential of rat osteoblasts in culture: role of cAMP and Ca2+

Microelectrodes were used to investigate the possible involvement of cAMP and Ca2+ ions in the parathyroid hormone's, bPTH(1-34), effect on the membrane potential of rat osteoblasts in primary culture. Parathyroid hormone (10(-7) M) depolarized cell membrane by 25.0 +/- 6.1 mV (mean +/- standard deviation, SD; n = 17). Blocking Ca2+ influx with the Ca channel blocker cobalt revealed two phases in the hormone effect: a rapid and slight membrane hyperpolarization followed by sustained depolarization. In addition, cobalt significantly (p less than 0.01) decreased the magnitude of the PTH depolarizing action. The addition of dibutyryl-cAMP (10(-3) M) to the perfusion solution also resulted in a biphasic effect. At a lower concentration (10(-4) M), dibutyryl-cAMP produced only membrane hyperpolarization, suggesting a cAMP dose dependence of the opposite membrane potential changes. Forskolin (10(-5) M) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (10(-4) M) mimicked the depolarizing effect of PTH. IBMX at a low concentration (5 x 10(-6) M) potentiated the depolarizing effect of PTH. Increases in [Ca2+]i using Ca2+ ionophore A23187 and intracellular injection of CaCl2 or inositol trisphosphate decreased the PTH depolarizing action, whereas intracellular injection of EGTA enhanced this effect. These results indicate that PTH evokes a biphasic change in rat osteoblast membrane potential that seems to be mediated by an increase in cAMP and modulated by intracellular calcium.     

Lidocaine Suppresses the Anoxic Depolarization and Reduces the Increase in the Intracellular Ca sup 2+ Concentration in Gerbil Hippocampal Neurons

Background: The movement of ions, particularly Ca2+, across the plasma membrane of neurons is regarded as an initial element of the development of ischemic neuronal damage. Because the mechanism by which lidocaine protects neurons against ischemia is unclear, the effects of lidocaine on the ischemia-induced membrane depolarization, histologic outcome, and the change in the intracellular Ca2+ concentration in the gerbil hippocampus were studied.

Methods: The changes in the direct-current potential shift in the hippocampal CA1 area produced by transient forebrain ischemia for 4 min were compared in animals given lidocaine (0.8 micro mol administered intracerebroventricularly) 10 min before ischemia and those given saline. The histologic outcome was evaluated 7 days after ischemia by assessing delayed neuronal death in hippocampal CA1 pyramidal cells in these animals. In a second study, hypoxia-induced intracellular Ca2+ increases were evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effects of lidocaine (10, 50, and 100 micro Meter) on the Ca2+ accumulation were examined. In addition, the effect of lidocaine (100 micro Meter) drug perfusion with a Ca2+ -free ischemia-like medium was investigated.

Results: The preischemic administration of lidocaine delayed the onset of the ischemia-induced membrane depolarization (anoxic depolarization) and reduced its maximal amplitude. The histologic outcome was improved by the preischemic treatment with lidocaine. The in vitro hypoxia-induced increase in the intracellular concentration of Ca2+ was suppressed by the perfusion with lidocaine-containing mediums (50 and 100 micro Meter), regarding the initiation and the extent of the increase. The hypoxia-induced intracellular Ca2+ elevation in the Ca2+ -free condition was similar to that in the Ca2+ -containing condition. Perfusion with lidocaine (100 micro Meter) inhibited this elevation in the Ca2+ -free condition.     

The Local Anesthetic n-Butyl-p-Aminobenzoate Selectively Affects Inactivation of Fast Sodium Currents in Cultured Rat Sensory Neurons

Background: Aqueous suspensions of the local anesthetic n-butyl-p-aminobenzoate (BAB), epidurally applied in terminal cancer patients, resulted in a sensory blockade, lasting up to several months. To investigate the mechanism of action on the cellular level, the effect of 100 micro Meter BAB on Sodium sup + action potentials and on Sodium sup + currents in dorsal root ganglion neurons from neonatal rats was studied.

Methods: Small neurons grown in cell culture were selected for patch-clamp measurements. Both Sodium sup + action potentials, evoked by current pulses of increasing amplitude (current clamp) and Sodium sup + currents, activated at different membrane potentials (voltage clamp), were investigated in the absence and presence of 100 micro Meter BAB. The local anesthetic was applied by external perfusion for 2 or 10 min.

Results: In the presence of 100 micro Meter BAB, either the firing threshold was raised or the action potential was abolished. The maximal peak conductances, underlying the fast sodium current INa,F and the slow sodium current INa,S, were not changed. However, the inactivation of INa,F Was increased by BAB. The sigmoid inactivation curve shifted 12 mV toward hyperpolarizing membrane voltages, whereas no changes were found for the inactivation of the slow Sodium sup + current. Only at short exposure times of 2 min, the effects of BAB could be reversed during a 10-min wash-out.     

The influence of changes in extracellular and intracellular sodium concentration on detrusor contractility

OBJECTIVE: To determine the role of Na+-Ca2+ exchange in the regulation of isolated detrusor smooth muscle contractility. MATERIALS AND METHODS: Isolated guinea-pig detrusor strips were used to record isometric tension generated by; (a) electrical-field stimulation to elicit nerve-mediated responses; and (b) adding carbachol or superfusing with a high-K+ solution. The [Na+] gradient between extracellular and intracellular compartments was altered by: (i) reducing superfusate [Na+] in stages from 140.2 to 10.2 mm; (ii) addition of the cardiac glycoside strophanthidin (200 microm). RESULTS Reducing extracellular [Na+] reversibly reduced the magnitude of nerve-mediated contractions but increased the resting tension and magnitude of carbachol-induced contracture. The mean (sd) [Na+] required for a half-maximum effect on attenuating contractions, at 85.9 (6.2) mm, and developing contracture, at 59.1 (14.3) mm, were significantly different. The time constants of changes to nerve-mediated contractions and carbachol contracture were also significantly different, at 147 (5) vs 1207 (386) s, respectively. These differences suggest that separate mechanisms influence nerve-mediated contraction and contracture in low-Na+ solutions. Exposure to the cardiac glycoside strophanthidin produced a similar effect to low-Na+ solutions for carbachol contracture. Low-Na+ solutions had no significant effect on contractures induced by high extracellular [K+]. CONCLUSION Reducing the transmembrane [Na+] difference increases intracellular [Ca2+]. This increase is largely accommodated in intracellular stores, that can be released by exogenous carbachol. The results are consistent with the presence of a functional Na+-Ca2+ exchanger in the surface membrane. The lack of effect of low-Na+ solutions on contractures evoked by membrane depolarization is consistent with this conclusion. The reduction of the nerve-mediated contraction by low-Na+ solution might result from blockade of the nerve action potential.     

Inhibition of Presynaptic Sodium Channels by Halothane

Background: Recent electrophysiologic studies indicate that clinical concentrations of volatile general anesthetic agents inhibit central nervous system sodium (Na sup +) channels. In this study, the biochemical effects of halothane on Na sup + channel function were determined using rat brain synaptosomes (pinched-off nerve terminals) to assess the role of presynaptic Na sup + channels in anesthetic effects.

Methods: Synaptosomes from adult rat cerebral cortex were used to determine the effects of halothane on veratridine-evoked Na sup + channel-dependent Na sup + influx (using22 Na sup +), changes in intrasynaptosomal [Na sup +] (using ion-specific spectrofluorometry), and neurotoxin interactions with specific receptor sites of the Na sup + channel (by radioligand binding). The potential physiologic and functional significance of these effects was determined by measuring the effects of halothane on veratridine-evoked Na sup + channel-dependent glutamate release (using enzyme-coupled spectrofluorometry).

Results: Halothane inhibited veratridine-evoked22 Na sup + influx (IC50 = 1.1 mM) and changes in intrasynaptosomal [Na sup +] (concentration for 50% inhibition [IC50] = 0.97 mM), and it specifically antagonized [sup 3 H]batrachotoxinin-A 20-alpha-benzoate binding to receptor site two of the Na sup + channel (IC50 = 0.53 mM). Scatchard and kinetic analysis revealed an allosteric competitive mechanism for inhibition of toxin binding. Halothane inhibited veratridine-evoked glutamate release from synaptosomes with comparable potency (IC50 = 0.67 mM).     

Differential Effects of Anesthetic and Nonanesthetic Cyclobutanes on Neuronal Voltage-gated Sodium Channels

Background: Despite their key role in the generation and propagation of action potentials in excitable cells, voltage-gated sodium (Na+) channels have been considered to be insensitive to general anesthetics. The authors tested the sensitivity of neuronal Na+ channels to structurally similar anesthetic (1-chloro-1,2,2-trifluorocyclobutane; F3) and nonanesthetic (1,2-dichlorohexafluorocyclobutane; F6) polyhalogenated cyclobutanes by neurochemical and electrophysiologic methods.

Methods: Synaptosomes (pinched-off nerve terminals) from adult rat cerebral cortex were used to determine the effects of F3 and F6 on 4-aminopyridine- or veratridine-evoked (Na+ channel-dependent) glutamate release (using an enzyme-coupled spectrofluorimetric assay) and increases in intracellular Ca2+ ([Ca2+]i) (using ion-specific spectrofluorimetry). Effects of F3 and F6 on Na+ currents were evaluated directly in rat lumbar dorsal root ganglion neurons by whole-cell patch-clamp recording.

Results: F3 inhibited glutamate release evoked by 4-aminopyridine (inhibitory concentration of 50% [IC50] = 0.77 mM [~ 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [~ 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50= 0.5 mM [~ 0.5 MAC]) in synaptosomes; F6 had no significant effects up to 0.05 mM (approximately twice the predicted MAC). F3 caused reversible membrane potential-independent inhibition of peak Na+ currents (70 +/- 9% block at 0.6 mM [~ 0.6 MAC]), and a hyperpolarizing shift in the voltage-dependence of steady state inactivation in dorsal root ganglion neurons (-21 +/- 9.3 mV at 0.6 mM). F6 inhibited peak Na+ currents to a lesser extent (16 +/- 2% block at 0.018 mM [predicted MAC]) and had minimal effects on steady state inactivation.     

Hypersensitivity of Malignant Hyperthermia-susceptible Swine Skeletal Muscle to Caffeine Is Mediated by High Resting Myoplasmic [Ca2+]

Background: Malignant hyperthermia (MH) is an inherited pharmacogenetic syndrome that is triggered by halogenated anesthetics and/or depolarizing muscle relaxants. MH-susceptible (MHS) skeletal muscle has been shown to be more sensitive to caffeine-induced contracture than muscle from nonsusceptible (MHN) subjects and is the basis for the most commonly used clinical diagnostic test to determine MH susceptibility.

Methods: We studied the effects of caffeine on myoplasmic free calcium concentration ([Ca2+]i) in MHN and MHS swine muscle fibers by means of Ca2+-selective microelectrodes before and after K+-induced partial depolarization.

Results: [Ca2+]i in untreated MHN fibers was 123 +/- 8 nm versus 342 +/- 33 nm in MHS fibers. Caffeine (2 mm) caused an increase in [Ca2+]i in both groups (296 +/- 41 nm MHN vs. 1,159 +/- 235 nm MHS) with no change in resting membrane potential. When either MHN or MHS, muscle fibers were incubated in 10 mm K+ [Ca2+]i transiently increased to 272 +/- 22 nm in MHN and 967 +/- 38 nm in MHS for 6-8 min. Exposure of MHN fibers to 2 mm caffeine while resting [Ca2+]i was elevated induced an increment in [Ca2+]i to 940 +/- 37 nm. After 6-8 min of exposure to 10 mm K+, [Ca2+]i returned to control levels in all fibers, and the effect of 2 mm caffeine on resting [Ca2+]i returned to control, despite continued partial membrane depolarization.     

Erythrocyte sodium transport in normotensive and hypertensive patients on chronic hemodialysis, acute effect of treatment

Patients on chronic hemodialysis were divided into two groups: normotensive patients (group I) and renal hypertensive patients treated with antihypertensives (group II). The sodium and potassium contents in red blood cells ([Na+]i, [K+]i), ouabain-resistant net sodium uptake (ORNa+ uptake, phi Na), the relative ORNa+ uptake (k), the mean cell hemoglobin concentration (MCHC), and acid-base status were examined just before and after dialysis. The results indicate that in treated renal hypertensive patients k is stimulated and causes lower red blood cell sodium content. The reason for this increase remains obscure: the pattern of alterations of the sodium transport components during dialysis is similar in all patients: [Na+]i and phi Na increase significantly during dialysis, and the increases in [Na+]i are closely related to increases in pH and bicarbonate.