Action Of Halothane Research Articles

Volatile Anesthetic Depression of Ca2+ Entry Into and Glutamate Release from Cultured Cerebellar Granule Neurons

Background: Volatile anesthetics (VAs) are known to have actions on a variety of ligand- and voltage gated ion channels, and thereby inhibit neuronal function. VA effects mediated by actions on voltage-gated Ca channels (VGCCs) were determined by studying their effects on the depolarization-induced rise in intracellular Ca2+ transients and the consequent glutamate release in cultured neonatal rat cerebellar granule neurons. Methods: Using a glutamate dehydrogenase-coupled assay for glutamate release, and fura-2 to measure intracellular [Ca2+] ([Ca2+]i), neurons at 37℃ were depolarized by a rapid increase in [K+]o from 5 to 55 mM. Actions of halothane, isoflurane, enflurane, and sevoflurane were compared with effects of altered [Mg2+]o, and by specific blockade of L-, P/Q- and/or N-type VGCC by nicardipine, ω-agatoxin IVA, and ω-conotoxin-GVIA, respectively. Whole-cell patch-clamp studies in these same neurons of VGCC Ba2+ currents were also performed at 22℃. Results: Clinical VA concentrations dose-dependently depressed both peak [Ca2+]i and glutamate release by 35-70%. With N- and/or L-type VGCC blockade, VAs caused a further marked decrease in [Ca2+]i transients. VAs depressed whole cell patch-clamped Ba2+ currents in these granule cell neurons by 35-40%. Conclusions: VAs depress Ca2+ entry by inhibiting a variety of VGCCs, and thereby reduce neuronal glutamate release. This action may contribute to the mechanism of anesthesia as well as provide protection during ischemic insults that cause neuronal injury. Citation: Carl Lynch III, Ning Miao, Kaoru Nagao, Joseph J. Pancrazio. Volatile anesthetic depression of Ca2+ entry into and glutamate release from cultured cerebellar granule neurons. J Anesth Perioper Med 2017; 4: 45-59. doi:10.24015/JAPM.2017.0016This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

ЭКСПЕРИМЕНТАЛЬНОЕ МОДЕЛИРОВАНИЕ ПОСЛЕОПЕРАЦИОННЫХ КОГНИТИВНЫХ РАССТРОЙСТВ У КРЫС

Goal: to develop the design of the experiment for evaluation of cognitive functions and behavior of rats after some aggressive impact imitating anesthesia and surgery in the clinical practice. Methods: Male rats of Wistar stock (n = 40) were used to evaluate long-term behavioral effects of the abdomen surgery and short-term (15-20 min.) and long-term (6 hours) actions of halothane. Simple (2x2) factorial plan was used for the experiment (4 blocks, balanced as per the number of animals from 4 experimental groups, n=2 for each group in the block). For anesthesia induction 4 volumes of % halothane in the air flow (2 l/min) were used, for anesthetic support it was 1.5 volume of % halothane. During surgery the rat was placed on the heated operating table and the mask was used for anesthesia. Laparotomy was performed; empty intestine was identified and irritated for 10 seconds by massage movement of the forefinger and thumb. After that the loop was placed into the abdomen and the wound was sutured layer by layer. Then for three weeks the following behavioral tests were performed in compliance with the protocol: motor activity (Actometer) and exploratory activity in new environment (Open field), social behavior (pair interaction test), novel object recognition, extrapolational deliverance rate, forced swimming, sexual behavior. Ovariectomized female (n = 16) were used as standard opponents for social behavior tests, and hormone-stimulated female rats (n = 26) were used for sexual behavior tests. The results showed that long-term action of halothane vapor caused behavioral changes in rats: it reduces exploratory activity, decreases the novel object recognition index, increases immobility during forced swimming and reduces latent period of extrapolational deliverance. Halothane made no significant impact on motor activity (open field) and sexual behavior of male rats. In general the results of performed tests provide evidence of emotional flattening development in rats. Conclusion: It is feasible to use the experiment design and pool of behavioral tests used in this study for experimental evaluation of inhalation anesthetics effects, their late consequences, and also for searching the ways of management of the developing behavioral disorders.

Sevoflurane: A Potent General Anesthetic Enhancer of Kv Channel Activation

General anesthetics have been shown to both inhibit and potentiate various families of ion channels. However, the molecular bases of these actions are not understood. Previous work with K-Shaw2 explored structural determinants of inhibition by alkanols and the volatile general anesthetics halothane and isoflurane (Barber et al. Biophys J. 2011, in press). In this study, we report that sevoflurane, an inhaled anesthetic currently used in human general anesthesia, exhibits the singular ability to reversibly potentiate K-Shaw2 currents investigated in Xenopus oocytes under two-electrode voltage-clamp conditions (∼80% potentiation at 1 mM). This result is in sharp contrast to the action of multiple other inhaled (halothane, isoflurane, desflurane, chloroform, and 1-alkanols) and injected (propofol) general anesthetics, which are inhibitory. This potentiation occurs over a clinically relevant concentration range (0.05 - 1 mM) in a dose-dependent manner that suggests two binding sites with distinct apparent affinities: a high-affinity site (K1 = 60 μM) and a low affinity site (K2 = 4 mM). Furthermore, a double mutation involving the S6 segment and the S4-S5 linker (A326V/A417V) abolishes the potentiation by sevoflurane. This effect is especially interesting because this double mutation also abolishes the inhibitory action of halothane, suggesting that these inhaled anesthetics share effector sites but influence gating in opposite ways. Semiquantitative kinetic modeling suggests that preferential binding of sevoflurane to resting and activated closed states shifts the channels into a novel gating mode with an enhanced open probability. Additional screening of related voltage-gated K channels at a various doses of sevoflurane revealed only modest potentiation of Kv1.2 no effect on Kv1.3, Kv2.1, Kv3.4 and Kv4.2. This study provides a novel framework for further investigations of the structural basis of general anesthetic action.

Functional consequences of mutations in the Drosophila histamine receptor HCLB

The gene hclB encodes a histamine-gated chloride channel subunit in Drosophila melanogaster. Mutations in hclB lead to defects in the visual system and altered sensitivity to the action of ivermectin. To investigate whether this member of the Cys-loop receptors is common across the Insecta, we analysed the genomes of seven other insect species (Diptera, Hymenoptera, Coleoptera) and revealed orthologues of hclB in all of them. Sequence comparisons showed high identity levels between the orthologues, indicating similar constraints and conserved function between the species. Two D. melanogaster mutants, hclBT1 (P293S) and hclBT2 (W111*, a null mutation) were tested for the lapse into, and recovery from, paralysis induced by high temperature or the anaesthetic action of halothane. At 41°C, the hclBT2 flies lapsed into coma faster than wild-type or the hclBT1 flies, while both mutants recovered more slowly. A substantially impaired recovery rate was also observed in hclBT1 after anaesthesia with halothane. Enhanced synaptic signalling at low-intensity light stimuli was registered on electroretinograms recorded from the two mutant strains. Our results suggest that HCLB may play an essential and conserved role in insect neurophysiology.

Reciprocal modulation of I h and I TASK in thalamocortical relay neurons by halothane

By combining electrophysiological, immunohistochemical, and computer modeling techniques, we examined the effects of halothane on the standing outward current (I (SO)) and the hyperpolarization-activated current (I (h)) in rat thalamocortical relay (TC) neurons of the dorsal lateral geniculate nucleus (dLGN). Hyperpolarizing voltage steps elicited an instantaneous current component (I (i)) followed by a slower time-dependent current that represented I (h). Halothane reduced I (h) by shifting the voltage dependency of activation toward more negative potentials and by reducing the maximal conductance. Moreover, halothane augmented I (i) and I (SO). During the blockade of I (h) through Cs+, the current-voltage relationship of the halothane-sensitive current closely resembled the properties of a current through members of the TWIK-related acid-sensitive K+ (TASK) channel family (I (TASK)). Computer simulations in a single-compartment TC neuron model demonstrated that the modulation of I (h) and I (TASK) is sufficient to explain the halothane-induced hyperpolarization of the membrane potential observed in current clamp recordings. Immunohistochemical staining revealed protein expression of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel proteins HCN1, HCN2, and HCN4. Together with the dual effect of halothane on I (h) properties, these results suggest that I (h) in TC neurons critically depends on HCN1/HCN2 heterodimers. It is concluded that the reciprocal modulation of I (h) and I (TASK) is an important mechanism of halothane action in the thalamus.

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.

A study of the effect of hemorrhage on the cardiorespiratory actions of halothane, isoflurane and sevoflurane in the dog

ObjectiveTo compare the cardiorespiratory changes induced by equipotent concentrations of halothane (HAL), isoflurane (ISO) and sevoflurane (SEVO) before and after hemorrhage. Study designProspective, randomized clinical trial. AnimalsTwenty-four healthy adult dogs weighing 15.4 ± 3.4 kg (mean ± SD). MethodsAnimals were randomly allocated to one of three groups (n = 8 per group). In each group, anesthesia was maintained with 1.5 minimum alveolar concentration of HAL (1.3%), ISO (1.9%) and SEVO (3.5%) in oxygen. Controlled ventilation was performed to maintain eucapnia. Cardiorespiratory variables were evaluated at baseline (between 60 and 90 minutes after induction), immediately after and 30 minutes after the withdrawal of 32 mL kg−1 of blood (40% of the estimated blood volume) over a 30-minute period. ResultsDuring baseline conditions, ISO and SEVO resulted in higher cardiac index (CI) than HAL. Heart rates were higher with SEVO at baseline, while mean arterial pressure (MAP) and mean pulmonary arterial pressure did not differ between groups. Although heart rate values were higher for ISO and SEVO after hemorrhage, only ISO resulted in a higher CI when compared with HAL. In ISO-anesthetized dogs, MAP was higher immediately after hemorrhage, and this was related to better maintenance of CI and to an increase in systemic vascular resistance index from baseline. ConclusionsAlthough the hemodynamic responses of ISO and SEVO are similar in normovolaemic dogs, ISO results in better maintenance of circulatory function during the early period following a massive blood loss. Clinical relevanceInhaled anesthetics should be used judiciously in animals presented with blood loss. However, if an inhalational agent is to be used under these circumstances, ISO may provide better hemodynamic stability than SEVO or HAL.

Antagonistic Actions of Halothane and Sevoflurane on Spontaneous Ca2+Release in Rat Ventricular Myocytes

Halothane has been reported to sensitize Ca(2+) release from the sarcoplasmic reticulum (SR), which is thought to contribute to its initial positive inotropic effect. However, little is known about whether isoflurane or sevoflurane affect the SR Ca(2+) release process, which may contribute to the inotropic profile of these anesthetics. Mild Ca(2+) overload was induced in isolated rat ventricular myocytes by increase of extracellular Ca(2+) to 2 mM. The resultant Ca(2+) transients due to spontaneous Ca(2+) release from the SR were detected optically (fura-2). Cells were exposed to 0.6 mM anesthetic for a period of 4 min, and the frequency and amplitude of spontaneous Ca(2+) transients were measured. Halothane caused a temporary threefold increase in frequency and decreased the amplitude (to 54% of control) of spontaneous Ca(2+) transients. Removal of halothane inhibited spontaneous Ca release before it returned to control. In contrast, sevoflurane initially inhibited frequency of Ca(2+) release (to 10% of control), whereas its removal induced a burst of spontaneous Ca(2+) release. Isoflurane had no significant effect on either frequency or amplitude of spontaneous Ca(2+) release on application or removal. Sevoflurane was able to ameliorate the effects of halothane on the frequency and amplitude of spontaneous Ca(2+) release both on application and wash-off. Application of halothane and removal of sevoflurane sensitize the SR Ca(2+) release process (and vice versa on removal). Sevoflurane reversed the effects of halothane, suggesting they may act at the same subcellular target on the SR.

Vasoconstricting and vasodilating actions of halothane are unrelated to Ser19 MLC phosphorylation

Contraction or relaxation in smooth muscle is primarily dependent on Ser19 phosphorylation/dephosphorylation of the 20 kDa regulatory myosin light chain (MLC). In isolated mesenteric resistance blood vessels, halothane has a transient vasoconstricting action related to Ca2+release from ryanodine-sensitive intracellular Ca2+ stores, and a long-lasting vasodilating action. Whether these vasoconstricting and vasodilating actions of halothane depend on Ser19 phosphorylation/dephosphorylation of MLC has not been fully elucidated. Methods Vessels are cannulated and held at a constant transmural pressure (40mmHg) and image analysis is used to measure vessel diameter. Ser19 phosphorylation of MLC is quantified using the Western blotting technique and an Odyssey Infrared Imaging System. Results In physiologic saline solution (PSS), halothane (4.5 vol%) produced transient contraction to 51% of maximal and an increase in Ser 19 phosphorylation of MLC. Following a 30 min. incubation in PSS containing 0Ca2+ + 2mM EGTA, both halothane contraction and Ser19 phosphorylation were reduced. After incubation for 10 min in the kinase inhibitor wortmannin (10-6M), halothane contraction was reduced but Ser19 phosphorylation was not affected. After a 15 min incubation with the phosphatase inhibitor calyculin A (3 X 10-8M), halothane contraction was 52% of maximal with an increase in Ser19 phosphorylation, but a small contraction persisted in calyculin while MLC phosphorylation decreased below baseline. With halothane in PSS alone, the vessel diameter returned to baseline after approx. 20 sec, however the Ser19 phosphorylation decreased only a small amount. Conclusion The vasoconstricting and vasodilating actions of halothane appear to be poorly correlated with changes in MLC Ser19 phosphorylation levels.

Multiple actions of halothane on contractile response to noradrenaline in isolated mesenteric resistance arteries

Halothane, a volatile anaesthetic, produces systemic hypotension and significantly alters organ blood flow. Isometric force was recorded in isolated rat small mesenteric arteries to investigate its action on contractile response to noradrenaline, the sympathetic neurotransmitter. Halothane (1-5%) enhanced contractile response to noradrenaline in the endothelium-intact arteries, but had little influence in the endothelium-denuded arteries. However, halothane consistently inhibited the noradrenaline response in the endothelium-denuded arteries pretreated with ryanodine (10 microM). The enhancement of the contractile response to noradrenaline in the endothelium-intact arteries was unaffected by treatment with N(G)-nitro L-arginine, tetraethylammonium, apamin, charybdotoxin, indomethacin, diclofenac, nordihydroguaiaretic acid, BQ-123, BQ-788, losartan, ketanserin, or superoxide dismutase. Halothane prolonged vasorelaxation after washout of noradrenaline in the endothelium-denuded arteries. Both ryanodine and vanadate (0.1-0.3 mM), a putative inhibitor of the plasma membrane Ca2+-ATPase, also prolonged the vasorelaxation. Halothane still prolonged the vasorelaxation in the ryanodine-treated arteries, but not in the vanadate-treated arteries. Halothane decreased the pD2 value for the pCa-force relation in the beta-escin-permeabilised, endothelium-denuded arteries. Halothane appears to influence contractile response to noradrenaline through multiple actions including endothelium-dependent enhancing, endothelium-independent enhancing, and endothelium-independent inhibitory actions. Nitric oxide, endothelium-derived hyperpolarising factor, cyclooxygenase products, lipoxygenase products, endothelin-1, angiotensin-II, serotonin, and superoxide anions are not involved in the endothelium-dependent enhancement. The endothelium-independent enhancement is presumably due to its ability to stimulate Ca2+ release from the ryanodine-sensitive intracellular stores, while the endothelium-independent inhibition is due, at least in part, to depressed Ca2+-activation of contractile proteins. Halothane may inhibit the plasma membrane Ca2+-ATPase of vascular smooth muscle cells.

Electrical synapses between Bergmann glial cells and Purkinje neurones in rat cerebellar slices

In the present study, we directly demonstrated electrical coupling between Bergmann glial cells (BG) and Purkinje neurones (PN) in acutely isolated cerebellar slices, prepared from 15 to 30 days old Sprague–Dawley rats. Electrical coupling between these two cells was identified by dual whole-cell voltage clamp, which allowed direct recording of junctional current. Whole-cell recordings from PN-PN, PN-BG and BG-BG pairs were made using Nomarski optics and infrared visualisation, which allowed precise morphological identification of cells. Junctional currents were recorded by applying hyper/and depolarising voltage sequences ranging from −120 to +40 mV (voltage step 10 mV) to one of the cells in the pair, while ion currents were measured from both cells. As has been shown before, junctional currents were frequently observed in BG-BG pairs: we found electrical coupling in 27 out of 34 pairs analysed. When the similar protocol was applied to the PN-BG pairs, junctional currents were found in 61 out of 87 pairs analysed. The electrical coupling was bi-directional as similar junctional currents were observed in PN when voltage step protocol was applied to BG. No electrical coupling was observed in PN-PN pairs ( n = 21). To correlate the appearance of these currents with gap junctions we treated slices with octanol (200 μM) or halothane (500 μM)—known inhibitors of gap junction conductance. Both agents applied for 5 min resulted in a complete inhibition of junctional currents in PN-BG pair. The washout (15 min) led to a complete recovery of junctional currents after treatment with octanol; the action of halothane was irreversible. Finally, we found that filling the BG by Alexa Fluor 488 results in staining of adjacent PN (in 11 out of 23 pairs tested). We conclude therefore that cerebellar neurones and glial cells are directly connected via gap junctions.

The Sensitivity of G Protein-activated K+ Channels toward Halothane Is Essentially Determined by the C Terminus

G protein-activated K(+) channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. When coexpressed with the m(2) acetylcholine (ACh) receptor in Xenopus oocytes, agonist-activated GIRK1(F137S)- and GIRK2-mediated currents are inhibited by halothane, whereas in the absence of ACh, high concentrations of halothane induce GIRK1(F137S)-mediated currents. To elucidate the molecular mechanism of halothane action on GIRK currents of different subunit compositions, we constructed deletion mutants of GIRK1(F137S) (GIRK1(Delta363*)) and GIRK2 (GIRK2(Delta356)) lacking the C-terminal ends, as well as chimeric GIRK channels. Mutated GIRK channels showed normal currents when activated by ACh but exhibited different pharmacological properties toward halothane. GIRK2(Delta356) showed no sensitivity against the inhibitory action of halothane but was activated by halothane in the absence of an agonist. GIRK1(Delta363*) was activated by halothane more efficiently. Currents mediated by chimeric channels were inhibited by anesthetic concentrations that were at least 30-fold lower than those necessary to decrease GIRK2 wild type currents. Glutathione S-transferase pulldown experiments did not show displacement of bound Gbetagamma by halothane, indicating that halothane does not interfere with Gbetagamma binding. Single channel experiments revealed an influence of halothane on the gating of the channels: The agonist-induced currents of GIRK1 and GIRK2, carried mainly by brief openings, were inhibited, whereas higher concentrations of the anesthetic promoted long openings of GIRK1 channels. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of current modulation.

Inhibitory effects of halothane on the thermogenic pathway in brown adipocytes: localization to adenylyl cyclase and mitochondrial fatty acid oxidation

Volatile anesthetics such as halothane efficiently inhibit nonshivering thermogenesis as well as the cellular manifestation of that phenomenon: norepinephrine-induced respiration in brown adipocytes. To identify the molecular site(s) of action of such anesthetics, we have examined the effect of halothane on the sequential intracellular steps from the interaction of norepinephrine with isolated brown adipocytes to the stimulation of mitochondrial respiration (=thermogenesis). We did not identify an inhibition at the level of the adrenergic receptors, but a first site of inhibition was identified as the generation of cAMP by adenylyl cyclase; this led to inhibition of norepinephrine-induced expression of the uncoupling protein-1 (UCP1) gene and reduced norepinephrine-induced lipolysis as secondary effects. Although an inhibition of lipolysis in itself would inhibit thermogenesis, circumvention of this inhibition revealed that a second, postlipolytic, site of inhibition existed: halothane also inhibited the stimulatory effect of exogenous fatty acids on cellular respiration. This inhibition was independent of the presence of UCP1 in the mitochondria of the cells and was thus not directly on the thermogenic uncoupling mechanism. Since not only fatty acid oxidation but also pyruvate oxidation were inhibited by halothane in isolated mitochondria, whereas glycerol-3-phosphate oxidation was not, the second site of action of halothane, evident when cyclase/lipolytic inhibition was circumvented, was located to the respiratory chain, complex I. The results thus explain the inhibition of nonshivering thermogenesis by identifying two sites of action of halothane in brown adipocytes. In addition, the results may open for new formulations of the molecular background to anesthesia.

Effect of halothane on the release of [Ca2+]i in dorsal root ganglion neurons.

We investigated the effect of the volatile anaesthetic halothane on [Ca2+]i of dorsal root ganglion neurons. Halothane was able to increase [Ca2+]i in those neurons in a dose-dependent manner and independent of extracellular calcium. However, halothane action was inhibited by BAPTA-AM, suggesting the involvement of intracellular calcium stores. Dantrolene, an inhibitor of ryanodine-sensitive calcium stores had no effect while 2-APB, an inhibitor of IP3-sensitive calcium store reduced by 78% the halothane-evoked increase on [Ca2+]i. These data suggests that halothane increased [Ca2+]i of ganglion neurons through calcium release from IP3-sensitive calcium store. One possible consequence of the halothane action is to alter presynaptic activity and signaling pathways that influence neurotransmission.

Ca2+- and myosin phosphorylation-independent relaxation by halothane in K+-depolarized rat mesenteric arteries.

Volatile anesthetics inhibit vascular smooth muscle contraction, but the mechanisms responsible are uncertain. In this study, the effects of halothane on Ca2+ signaling and Ca2+ activation of contractile proteins were examined in high K+-depolarized smooth muscle from rat mesenteric resistance arteries. Vessels were cannulated and held at a constant transmural pressure (40 mmHg). Image analysis and microfluorimetry were used to simultaneously measure vessel diameter and smooth muscle intracellular [Ca2+] concentration ([Ca2+]i). Myosin light chain (MLC) phosphorylation was measured using the Western blotting technique. Step increases in extracellular [Ca2+] concentration (0-10 mM) in high K+ (40 mM)-depolarized smooth muscle produced incremental increases in [Ca2+]i, MLC phosphorylation, and contraction. Halothane (0.5-4.5%) inhibited contraction in a concentration-dependent manner, but the decrease in [Ca2+]i was small, and there was a marked shift in the [Ca2+]i-contraction relationship to the right, indicating an important Ca2+ desensitizing effect. Halothane (0.5-4.5%) did not affect MLC phosphorylation or the [Ca2+]-MLC phosphorylation relationship, but the MLC phosphorylation-contraction relationship was also shifted rightward, indicating an "MLC phosphorylation" desensitizing effect. In contrast, control relaxations produced by the Ca2+ channel blocker nifedipine were accompanied by decreases in both [Ca2+]i and MLC phosphorylation, and nifedipine had no affect on the [Ca2+]i-contraction, [Ca2+]i-MLC phosphorylation, and MLC phosphorylation-contraction relationships. In high K+-depolarized vascular smooth muscle, halothane relaxation is largely mediated by a Ca2+ and MLC phosphorylation desensitizing effect. These results suggest that the relaxing action of halothane is independent of the classic Ca2+-induced myosin phosphorylation contraction mechanism.

Facilitatory action of halothane at subanesthetic concentrations on glutamatergic excitatory synaptic transmission in the CA1 area of adult rat hippocampus

Whole-cell recordings were made from pyramidal cells visually identified in the CA1 field of adult rat hippocampal slices, and the effects of subanesthetic concentrations of halothane on excitatory postsynaptic currents mediated by non- N-methyl- d-aspartate subtype glutamate receptors were investigated. Halothane concentrations were measured by gas chromatography. At concentrations of 0.2 mM and 0.6 mM, halothane reversibly decreased the amplitude of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of Schaffer collateral fibers, and the decrease was accompanied by enhanced paired-pulse facilitation, consistent with the previously reported presynaptic site of halothane's inhibitory action. By contrast, at lower concentrations (0.02 mM and 0.05 mM), halothane increased the amplitude of EPSCs without any appreciable changes in paired-pulse facilitation. Moreover, the frequency of miniature EPSCs arising spontaneously in the presence of tetrodotoxin (mEPSCs) was increased by subanesthetic halothane, but the amplitude of the mEPSCs did not change significantly. These observations suggest that at subanesthetic concentrations halothane postsynaptically enhances glutamatergic excitatory synaptic transmission. This may provide a vital clue to elucidation of the neural mechanisms of the nociceptive reflex enhancement and excitatory state that occur at light levels of anesthesia.

G protein-coupled receptors as direct targets of inhaled anesthetics.

The molecular pharmacology of inhalational anesthetics remains poorly understood. Despite accumulating evidence suggesting that neuronal membrane proteins are potential targets of inhaled anesthetics, most currently favored membrane protein targets lack any direct evidence for anesthetic binding. We report herein the location of the binding site for the inhaled anesthetic halothane at the amino acid residue level of resolution in the ligand binding cavity in a prototypical G protein-coupled receptor, bovine rhodopsin. Tryptophan fluorescence quenching and direct photoaffinity labeling with [(14)C]halothane suggested an interhelical location of halothane with a stoichiometry of 1 (halothane/rhodopsin molar ratio). Radiosequence analysis of [(14)C]halothane-labeled rhodopsin revealed that halothane contacts an amino acid residue (Trp265) lining the ligand binding cavity in the transmembrane core of the receptor. The predicted functional consequence, competition between halothane and the ligand retinal, was shown here by spectroscopy and is known to exist in vivo. These data suggest that competition with endogenous ligands may be a general mechanism of the action of halothane at this large family of signaling proteins.

Inhibitory action of halothane on rat masculine sexual behavior and sperm motility

Adult male rats were exposed to inhale halothane in the following regime: 15 ppm/4 h/5 days/week/9 weeks. Sexual behavior observations and sperm motility test were made before halothane exposure (0 days) and at 15, 30, 45 and 60 days of exposure. Fifteen days after halothane exposure, this anesthetic inhibited the proportion of animals displaying ejaculation. In those animals ejaculating, halothane produced an inhibition of masculine sexual behavior reflected as an increase in the intromission latency, number of mounts and postejaculatory interval. At 30 days after exposure, only an increase in the intromission latency was observed. At 45 and 60 days, the inhibitory effect of halothane on sexual behavior disappeared. Similarly, at 15 and 30 days, but not at 45 or 60 days of halothane exposure, a reduced sperm motility was observed. Such transient effects of halothane suggest the development of tolerance to the inhibitory actions of this anesthetic on sexual behavior and sperm motility. These halothane effects are in line with an inhibition of masculine sexual behavior after stimulation of the GABAergic system.

Halothane differentially decreases 5-hydroxytryptamine-induced contractions in normal and chronic hypoxic rat pulmonary arteries.

The mechanism of action of halothane is not fully understood in pulmonary circulation and especially in chronic hypertension models. As the 5-hydroxytryptamine (5-HT) pulmonary vasoconstrictor response increases in chronic hypoxic rat, halothane could differentially attenuate this vasoconstriction response on normoxic and chronic hypoxic rats. The effect of halothane on 5-HT-induced contractions on pulmonary arteries isolated from normoxic and chronic hypoxic rats was compared. Rings dissected from proximal pulmonary artery without endothelium were attached to a force transducer to record tone and placed in an organ chamber gassed either by air or air + halothane (1-5%). Contractions induced by (10(-4) M) 5-HT were used to test the effect of halothane on rings isolated from normoxic and chronic hypoxic rats. 5-Hydroxytryptamine-mediated contractions were more sensitive to external calcium in normoxic than chronic hypoxic rings. In calcium-free solution, with verapamil or cadmium the amplitude of remaining 5-HT-induced contractions were greater in chronic hypoxic rings. Halothane (1-5%) decreased 5-HT-mediated contractions in normoxic and chronic hypoxic rings. The effect occurred with no change of pD2 for 5-HT and was more pronounced in normoxic rings. The effect of halothane on both rings was abolished in the absence of external calcium or in the presence of verapamil. In the presence of cadmium, 5% halothane had no effect on normoxic rings but still decreased the remaining 5-HT contraction on chronic hypoxic rings. The findings suggested that halothane decreased sarcolemmal calcium entry in pulmonary artery rings by a cadmium-sensitive pathway in normoxic rats and by a cadmium-insensitive pathway in chronic hypoxic rats.

Modulation of neuronal nicotinic acetylcholine receptors by halothane in rat cortical neurons.

Inhalational general anesthetics have recently been shown to inhibit neuronal nicotinic acetylcholine (ACh) receptors (nnAChRs) expressed in Xenopus laevis oocytes and in molluscan neurons. However, drug actions on these systems are not necessarily the same as those seen on native mammalian neurons. Thus, we analyzed the detailed mechanisms of action of halothane on nnAChRs using rat cortical neurons in long-term primary culture. Currents induced by applications of ACh via a U-tube system were recorded by the whole-cell, patch-clamp technique. ACh evoked two types of currents, alpha-bungarotoxin-sensitive, fast desensitizing (alpha 7-type) currents and alpha-bungarotoxin-insensitive, slowly desensitizing (alpha 4 beta 2-type) currents. Halothane suppressed alpha 4 beta 2-type currents more than alpha 7-type currents with IC(50) values of 105 and 552 microM, respectively. Halothane shifted the ACh dose-response curve for the alpha 4 beta 2-type currents in the direction of lower ACh concentrations and slowed its apparent rate of desensitization. The rate of recovery after washout from halothane block was much faster than the rate of recovery from ACh desensitization. Thus, the halothane block was not caused by receptor desensitization. Chlorisondamine, an irreversible open channel blocker for nnAChRs, caused a time-dependent block that was attenuated by halothane. These results could be accounted for by kinetic simulation based on a model in which halothane causes flickering block of open channels, as seen in muscle nAChRs. Halothane block of nnAChRs is deemed to play an important role in anesthesia via a direct action on the receptor and an indirect action to suppress transmitter release.