Halothane increases smooth muscle protein phosphatase in airway smooth muscle

BACKGROUND: Halothane relaxes airway smooth muscle, in part, by decreasing the force produced for a given intracellular [Ca(2+)] (i.e., Ca(2+) sensitivity) during muscarinic stimulation, an effect produced by a decrease in regulatory myosin light-chain (rMLC) phosphorylation. The authors tested the hypothesis that halothane reduces rMLC phosphorylation during muscarinic stimulation at constant intracellular [Ca(2+)] by increasing smooth muscle protein phosphatase (SMPP) activity, without changing myosin light-chain kinase (MLCK) activity. METHODS: Enzyme activities were assayed in beta-escin permeabilized strips of canine tracheal smooth muscle. Under conditions of constant intracellular [Ca(2+)], the rate of rMLC phosphorylation was measured by Western blotting during inhibition of SMPP with microcystin-LR (to assay MLCK activity) or during inhibition of MLCK by wortmannin and adenosine triphosphate depletion (to assay SMPP activity). The effect of halothane (0.8 mm) on enzyme activities and isometric force during stimulation with 0.6 microm Ca(2+) and 10 microm acetylcholine was determined. RESULTS: Halothane produced a 14 +/- 8% (mean +/- SD) decrease in isometric force by significantly reducing rMLC phosphorylation (from 32 +/- 9% to 28 +/- 9%). Halothane had no significant effect on any parameter of a monoexponential relation fit to the data for the MLCK activity assay. In contrast, halothane significantly decreased the half-time for rMLC dephosphorylation in the SMPP activity assay (from 0.74 +/- 0.28 min to 0.44 +/- 0.10 min), indicating that it increased SMPP activity. CONCLUSIONS: Halothane decreases Ca(2+) sensitivity and rMLC phosphorylation in airway smooth muscle during muscarinic receptor stimulation by increasing SMPP activity, without affecting MLCK, probably by disrupting receptor G-protein signaling pathways that inhibit SMPP.     

Effects of Primary Alcohols on Airway Smooth Muscle

Background: The investigation examined whether primary alcohols could be used as tools to explore the mechanism of anesthetic actions in airway smooth muscle (ASM). The hypothesis was that, like volatile anesthetics, the primary alcohols relax intact ASM by decreasing intracellular Ca2+ concentration ([Ca2+]i) and by inhibiting agonist-induced increases in the force developed for a given [Ca2+]i (Ca2+ sensitivity).

Method: The effects of butanol, hexanol, and octanol on isometric force in canine tracheal smooth muscle were examined. The effects of hexanol on [Ca2+]i (measured with fura-2) and the relationship between force and [Ca2+]i were studied during membrane depolarization provided by KCl and during muscarinic stimulation provided by acetylcholine.

Results: The primary alcohols relaxed ASM contracted by KCl or acetylcholine in a concentration-dependent manner, with potency increasing as chain length increased. The alcohols could completely relax the strips, even during maximal stimulation with 10 [mu]m acetylcholine (median effective concentrations of 28 +/- 12, 1.3 +/- 0.4, and 0.14 +/- 0.05 mm [mean +/- SD] for butanol, hexanol, and octanol, respectively). Hexanol decreased both [Ca2+]i and force in a concentration-dependent manner. Hexanol decreased Ca2+ sensitivity during muscarinic stimulation but had no effect on the force-[Ca2+]i relationship in its absence.     

The effects of ethanol on CA(2+) sensitivity in airway smooth muscle

Halothane and other volatile anesthetics relax air-way smooth muscle (ASM) in part by decreasing the amount of force produced for a given intracellular Ca(2+) concentration (the Ca(2+) sensitivity) during muscarinic receptor stimulation. To determine whether this is a unique property of the volatile anesthetics, we tested the hypothesis that ethanol, another compound with anesthetic properties, also inhibits calcium sensitization induced by muscarinic stimulation of ASM. A beta-escin permeabilized canine tracheal smooth muscle preparation was used. Ethanol was applied to permeabilized muscles stimulated with calcium in either the absence or presence of acetylcholine. In intact ASM, ethanol produced incomplete relaxation (approximately 40%) at concentrations up to 300 mM. Ethanol significantly increased Ca(2+) sensitivity both in the presence and the absence of muscarinic receptor stimulation. Although ethanol did not affect regulatory myosin light chain (rMLC) phosphorylation during stimulation with Ca(2+) alone, it decreased rMLC phosphorylation by Ca(2+) during muscarinic receptor stimulation. Ethanol, like volatile anesthetics, inhibits increases in rMLC phosphorylation produced by muscarinic receptor stimulation at constant [Ca(2+)](i). However, despite this inhibition, the net effect of ethanol is to increase Ca(2+) sensitivity (defined as the force maintained for a given [Ca(2+)](i)) by a mechanism that is independent of changes in rMLC phosphorylation. IMPLICATIONS: In permeabilized airway smooth muscle, ethanol, like volatile anesthetics, inhibits increases in regulatory protein phosphorylation caused by stimulation of the muscle when intracellular calcium concentration is constant. However, unlike volatile anesthetics, ethanol causes a net increase in force through a process not dependent on protein phosphorylation, an action favoring bronchoconstriction.     

Effects of Intravenous Anesthetics on Ca2+ Sensitivity in Canine Tracheal Smooth Muscle

Background: Halothane and other volatile anesthetics relax airway smooth muscle in part by decreasing the amount of force produced for a particular intracellular calcium concentration (the Ca2+ sensitivity) during muscarinic receptor stimulation. In this study, ketamine, propofol, and midazolam were evaluated to determine whether the inhibitory effect of volatile anesthetics on this signal transduction pathway is a general property of other types of anesthetic drugs.

Methods: A [beta]-escin permeabilized canine tracheal smooth muscle preparation was used. Ketamine, propofol, and midazolam, in concentrations producing near-maximal relaxation in intact airway smooth muscle (200 [mu]M, 270 [mu]M, and 100 [mu]M, respectively), were applied to permeabilized muscles stimulated with calcium in either the absence or the presence of muscarinic receptor stimulation provided by acetylcholine. The effect of halothane also was evaluated.

Results: Confirming previous studies, halothane (0.75 mM) decreased calcium sensitivity during muscarinic receptor stimulation. None of the intravenous anesthetics studied affected Ca2+ sensitivity, either in the absence or the presence of muscarinic receptor stimulation.     

Effects of Halothane on Sarcoplasmic Reticulum Calcium Release Channels in Porcine Airway Smooth Muscle Cells

Background: Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ "leak" through both inositol trisphosphate (IP3) and ryanodine receptor channels.

Methods: Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy.

Results: Halothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 [mu]m Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45 +/- 13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65 +/- 13% and 68 +/- 22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 [mu]m ryanodine blunted the [Ca2+]i response to halothane by 32 +/- 13% and 39 +/- 21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane.     

Dual Effects of Hexanol and Halothane on the Regulation of Calcium Sensitivity in Airway Smooth Muscle

Background: Contraction of airway smooth muscle is regulated by receptor-coupled mechanisms that control the force developed for a given cytosolic calcium concentration (i.e., calcium sensitivity). Halothane antagonizes acetylcholine-induced increases in calcium sensitivity by inhibiting GTP-binding (G)-protein pathways. The authors tested the hypothesis that hexanol, like halothane, inhibits agonist-induced increases in calcium sensitivity in airway smooth muscle by inhibiting G-protein pathways.

Methods: Calcium sensitivity was assessed using [alpha]-toxin-permeabilized canine tracheal smooth muscle. In selected experiments, regulatory myosin light chain phosphorylation was also determined by Western blotting in the presence and absence of 10 mm hexanol and/or 100 [mu]m acetylcholine.

Results: Hexanol (10 mm) and halothane (0.76 mm) attenuated acetylcholine-induced calcium sensitization by decreasing regulatory myosin light chain phosphorylation during receptor stimulation. Hexanol also inhibited increases in calcium sensitivity due to direct stimulation of heterotrimeric G-proteins with tetrafluoroaluminate but not with 3 [mu]m GTP[gamma]S, consistent with prior results obtained with halothane. In contrast, in the absence of receptor stimulation, both compounds produced a small increase in calcium sensitivity by a G-protein-mediated increase in regulatory myosin light chain phosphorylation that was not affected by pertussis toxin treatment.     

Halothane Attenuates Calcium Sensitization in Airway Smooth Muscle by Inhibiting G-proteins

Background: Halothane directly relaxes airway smooth muscle partly by decreasing the Ca2+ sensitivity. In smooth muscle, receptor stimulation is thought to increase Ca2+ sensitivity via a cascade of heterotrimeric and small monomeric guanine nucleotide-binding proteins (G-proteins). Whether this model is applicable in the airway and where halothane acts in this pathway were investigated.

Methods: A [small beta, Greek]-escin-permeabilized canine tracheal smooth muscle preparation was used. Exoenzyme C3 of Clostridium botulinum, which inactivates Rho monomeric G-proteins, was used to evaluate the involvement of this protein in the Ca2+ sensitization pathway. The effects of halothane on different stimulants acting at different levels of signal transduction were compared: acetylcholine on the muscarinic receptor, aluminum fluoride (AIF4-) on heterotrimeric G-proteins, and guanosine 5'-O-(3-thiotriphosphate) (GTP sub [small gamma, Greek] S) on all G-proteins.

Results: Exoenzyme C3 equally attenuated acetylcholine- and AIF4 (-) -induced Ca2+ sensitization, suggesting that these pathways are both mediated by Rho. Halothane applied before stimulation equally attenuated acetylcholine- and AIF4- -induced Ca2+ sensitization. However, when added after Ca2+ sensitization was established, the effect of halothane was greater during Ca2+ sensitization induced by acetylcholine compared with AIF4-, which, along with the previous result, suggests that halothane may interfere with dissociation of heterotrimeric G-proteins. Halothane applied during GTP sub [small gamma, Greek] S-induced Ca2+ sensitization had no significant effect on force, suggesting that halothane has no effect downstream from monomeric G-proteins.     

Halothane Attenuation of Calcium Sensitivity in Airway Smooth Muscle: Mechanisms of Action during Muscarinic Receptor Stimulation

Background: In airway smooth muscle, muscarinic receptor stimulation is thought to increase calcium (Ca2+) sensitivity via a guanosine 5'-triphosphate (GTP)-binding protein/protein kinase C (PKC)-mediated mechanism. This study tested the hypothesis that halothane reduces Ca2+ sensitivity during muscarinic receptor stimulation by inhibiting these second messenger pathways.

Methods: A beta-escin permeabilized canine tracheal smooth muscle preparation was used in which the cytosolic Ca2+ concentration ([Ca sup 2+]i) is controlled and the GTP-binding protein/PKC pathways remain intact and can be activated. The muscarinic receptor was activated with acetylcholine plus GTP; the GTP-binding proteins were directly activated with a nonhydrolyzable form of GTP, guanosine 5'-O-(3-thiotriphosphate; GTP gamma S); and PKC was directly activated with the PKC agonist phorbol 12,13-dibutyrate (PDBu).

Results: Free Ca2+ caused a concentration-dependent increase in force. Acetylcholine plus GTP significantly decreased the median effective concentration for free Ca2+ from 0.52 +/- 0.06 micro Meter to 0.21 +/- 0.02 micro Meter, demonstrating an increase in Ca2+ sensitivity. Halothane (0.99 +/- 0.04 mM, equivalent to approximately 4 minimum alveolar concentration in dogs) significantly attenuated this increase in Ca2+ sensitivity induced by acetylcholine plus GTP, increasing the median effective concentration for free Ca2+ from 0.21 +/- 0.02 micro Meter to 0.31 +/- 0.03 micro Meter. However, halothane did not affect the increases in Ca2+ sensitivity induced by GTP gamma S or PDBu.     

Role of Intracellular Ca2+ Stores in the Inhibitory Effect of Halothane on Airway Smooth Muscle Contraction

Background: Halothane directly inhibits contraction of airway smooth muscle, mainly by decreasing the intracellular concentration of free Ca2+ ([Ca2+]i). The role of intracellular Ca2+ stores, sarcoplasmic reticulum, is still unclear. We investigated the role of sarcoplasmic reticulum in the inhibitory effect of halothane on contraction of airway smooth muscle by measuring [Ca2+]i and intracellular concentration of inositol 1,4,5-triphosphate ([IP3]i), a second messenger for release of Ca2+ from sarcoplasmic reticulum.

Methods: [Ca2+]i was monitored by measuring the 500-nm light emission ratio (F340/F380) of a Ca2+ indicator fura-2 with isometric tension of canine tracheal smooth muscle strip. During Ca2+-free conditions, carbachol (10-5 M) was introduced with pretreatment of halothane (0-3%). During Ca2+-free conditions, 20 mM caffeine, a Ca (2+-induced) Ca2+ release channel opener, was introduced with or without halothane. We measured [IP3]i during exposure to carbachol and halothane by radioimmunoassay technique.

Results: Pretreatment with halothane significantly diminished carbachol-induced increases in [Ca2+]i by 77% and muscle tension by 83% in a dose-dependent manner. Simultaneous administration of halothane significantly enhanced caffeine-induced transient increases in [Ca2+] (i) and muscle tension in a dose-dependent manner, by 97% and 69%, respectively. Pretreatment with halothane abolished these responses. Rapid increase in [IP3]i produced by carbachol was significantly inhibited by 32% by halothane in a dose-dependent manner.     

Mechanisms of Direct Inhibitory Action of Ketamine on Vascular Smooth Muscle in Mesenteric Resistance Arteries

Background: Ketamine was previously suggested to relax vascular smooth muscle by reducing the intracellular Ca2+ concentration ([Ca2+]i). However, no direct evidence is available to indicate that ketamine reduces the [Ca2+]i in vascular smooth muscle of systemic resistance arteries.

Methods: Endothelium-intact or -denuded smooth muscle strips were prepared from rat small mesenteric arteries. Isometric force and [Ca2+]i were measured simultaneously in the fura-2-loaded, endothelium-denuded strips. In some experiments, only isometric force was measured in either the endothelium-intact or [beta]-escin-treated, endothelium-denuded strips.

Results: In the endothelium-intact strips, lower concentrations (<= 30 [mu]m) of ketamine slightly enhanced norepine-phrine-induced contraction, whereas higher concentrations (>= 100 [mu]m) of ketamine inhibited both norepinephrine- and KCl-induced contractions. In the fura-2-loaded strips, ketamine (>= 100 [mu]m) inhibited the increases in both [Ca2+]i and force induced by either norepinephrine or KCl. Ketamine also inhibited the norepinephrine-induced increase in [Ca2+]i after treatment with ryanodine. In the absence of extracellular Ca2+, ketamine notably inhibited the norepinephrine-induced increase in [Ca2+]i, whereas it only minimally inhibited caffeine-induced increase in [Ca2+]i. Ketamine had little influence on the [Ca2+]i-force relation during force development to stepwise increment of extracellular Ca2+ concentration during either KCl depolarization or norepinephrine stimulation. Ketamine did not affect Ca2+-activated contractions in the [beta]-escin membrane-permeabilized strips.     

Comparison of Volatile Anesthetic Actions on Intracellular Calcium Stores of Vascular Smooth Muscle: Investigation in Isolated Systemic Resistance Arteries

Background: Volatile anesthetic actions on intracellular Ca2+ stores (i.e., sarcoplasmic reticulum [SR]) of vascular smooth muscle have not been fully elucidated.

Methods: Using isometric force recording method and fura-2 fluorometry, the actions of four volatile anesthetics on SR were studied in isolated endothelium-denuded rat mesenteric arteries.

Results: Halothane (>= 3%) and enflurane (>= 3%), but not isoflurane and sevoflurane, increased the intracellular Ca2+ concentration ([Ca2+]i) in Ca2+-free solution. These Ca2+-releasing actions were eliminated by procaine. When each anesthetic was applied during Ca2+ loading, halothane (>= 3%) and enflurane (5%), but not isoflurane and sevoflurane, decreased the amount of Ca2+ in the SR. However, if halothane or enflurane was applied with procaine during Ca2+ loading, both anesthetics increased the amount of Ca2+ in the SR. The caffeine-induced increase in [Ca2+]i was enhanced in the presence of halothane (>= 1%), enflurane (>= 1%), and isoflurane (>= 3%) but was attenuated in the presence of sevoflurane (>= 3%). The norepinephrine-induced increase in [Ca2+]i was enhanced only in the presence of sevoflurane (>= 3%). Not all of these anesthetic effects on the [Ca2+]i were parallel with the simultaneously observed anesthetic effects on the force.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Effects of primary alcohols on airway smooth muscle

BACKGROUND: The investigation examined whether primary alcohols could be used as tools to explore the mechanism of anesthetic actions in airway smooth muscle (ASM). The hypothesis was that, like volatile anesthetics, the primary alcohols relax intact ASM by decreasing intracellular Ca2+ concentration ([Ca2+]i) and by inhibiting agonist-induced increases in the force developed for a given [Ca2+]i (Ca2+ sensitivity). METHOD: The effects of butanol, hexanol, and octanol on isometric force in canine tracheal smooth muscle were examined. The effects of hexanol on [Ca2+]i (measured with fura-2) and the relationship between force and [Ca2+]i were studied during membrane depolarization provided by KCl and during muscarinic stimulation provided by acetylcholine. RESULTS: The primary alcohols relaxed ASM contracted by KCl or acetylcholine in a concentration-dependent manner, with potency increasing as chain length increased. The alcohols could completely relax the strips, even during maximal stimulation with 10 microM acetylcholine (median effective concentrations of 28 +/- 12, 1.3 +/- 0.4, and 0.14 +/- 0.05 mM [mean +/- SD] for butanol, hexanol, and octanol, respectively). Hexanol decreased both [Ca2+]i and force in a concentration-dependent manner. Hexanol decreased Ca2+ sensitivity during muscarinic stimulation but had no effect on the force-[Ca2+]i relationship in its absence. CONCLUSIONS: Primary alcohols produce reversible, complete relaxation of ASM, with potency increasing as chain length increases, by decreasing [Ca2+]i and inhibiting increases in Ca2+ sensitivity produced by muscarinic receptor stimulation. These actions mimic those of volatile anesthetics on ASM, a circumstance suggesting that the primary alcohols may be useful tools for further exploring mechanisms of anesthetic effects on ASM.     

The Action of Sevoflurane on Vascular Smooth Muscle of Isolated Mesenteric Resistance Arteries (Part 2): Mechanisms of Endothelium-independent Vasorelaxation

Background: The precise mechanisms behind the direct inhibitory action of sevoflurane on vascular smooth muscle have not been fully elucidated.

Methods: Endothelium-denuded smooth muscle strips were prepared from rat small mesenteric arteries. Isometric force and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded strips. In another series of experiments, only isometric force was measured in the [beta]-escin-membrane-permeabilized strips.

Results: Sevoflurane (3-5%) inhibited the increases in both the [Ca2+]i and the force induced by either norepinephrine (0.5-10 [mu]m) or 40 mm K+. Sevoflurane still inhibited the increase in [Ca2+]i induced by norepinephrine after depletion of intracellular Ca2+ stores with ionomycin, although it little influenced the increase in [Ca2+]i induced by norepinephrine after treatment with verapamil. In the fura-2-loaded membrane-intact muscle, sevoflurane caused a rightward shift of Ca2+-force relation during force development to stepwise increment of extracellular Ca2+ concentration during 40-mm K+ depolarization in either the presence or the absence of norepinephrine. In contrast, sevoflurane did not influence Ca2+-activated contraction in the [beta]-escin-permeabilized muscle, in which [alpha]-adrenergic receptor coupling was not retained.     

Calcium Concentration-dependent Mechanisms through which Ketamine Relaxes Canine Airway Smooth Muscle

Background: Ketamine is a potent bronchodilator that, in clinically used concentrations, relaxes airway smooth muscle in part by a direct effect. This study explored the role of calcium concentration (Ca2+) in this relaxation.

Methods: Canine trachea smooth muscle strips were loaded with the fluorescent probe fura-2 and mounted in a spectrophotometric system to measure force and intracellular calcium concentration ([Ca2+]i) simultaneously. Calcium influx was estimated using a manganese quenching technique. Cyclic nucleotides in the airway smooth muscle strips were measured by radioimmunoassay.

Results: In smooth muscle strips stimulated with submaximal (0.1 micro Meter) and maximal (10 micro Meter) concentrations of acetylcholine, ketamine caused a concentration-dependent decrease in force and [Ca2+]i. The sensitivity of the force response to ketamine significantly decreased as the intensity of muscarinic receptor stimulation increased; the median effective concentration for relaxation induced by ketamine was 59 micro Meter and 850 micro Meter for tissue contracted by 0.1 micro Meter or 10 micro Meter acetylcholine, respectively (P < 0.05). In contrast, the sensitivity of the [Ca2+] sub i response did not depend on the intensity of muscarinic receptor stimulation. Ketamine at 1 mM significantly inhibited calcium influx. Ketamine did not significantly increase cyclic nucleotide concentrations.     

Direct inhibitory mechanisms of halothane on canine tracheal smooth muscle contraction.

Halothane directly relaxes airway smooth muscle. To determine the direct inhibitory mechanisms of halothane on canine tracheal smooth muscle contraction, the effects of this anesthetic on the levels of several intracellular second messengers were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), Ca2+/phospholipid-dependent protein kinase (PKC) translocation, and intracellular cyclic adenosine monophosphate concentration ([cAMP]i). When carbachol (1 microM) was used to increase [Ca2+]i to the same concentration as that induced by high-K+ (72.7 mM), the carbachol-induced contraction was more than twice as great, indicating that carbachol enhances the sensitivity of contractile elements to Ca2+ or activates a Ca(2+)-independent mechanism. Similarly, 12-deoxyphorbol 13-isobutylate, a potent PKC activator, markedly potentiated high-K(+)-induced muscle contraction without an increase of [Ca2+]i. The addition of halothane (0.33, 0.75, 1.15, and 1.47 mM) decreased [Ca2+]i and the muscle tension induced by carbachol. However, the decrease of muscle tension was more marked than that of [Ca2+]i at the higher concentrations. Although [Ca2+]i in the presence of verapamil and carbachol was not affected by halothane, the anesthetic markedly decreased muscle force by decreasing the "Ca2+ sensitization" or the Ca(2+)-independent enhancement of tension observed with carbachol. Halothane (0.75 and 1.47 mM) significantly released the membrane-associated PKC to cytosol, which decreased PKC activity. [cAMP]i of the smooth muscle stimulated by carbachol was moderately but significantly increased by halothane. However, when equivalent relaxation was induced with forskolin, which acts via adenylate cyclase activation, a much higher [cAMP]i was observed, which suggests that halothane acts via an additional pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Volatile Anesthetics with and without Verapamil on Intracellular Activity in Vascular Smooth Muscle

Background: Although halothane and isoflurane inhibit receptor agonist-induced smooth muscle contraction by inhibiting Calcium2+ influx via the L-type voltage-dependent Calcium2+ channels, their effects on pharmacomechanical coupling remained to be clarified. The intracellular action of both anesthetics was studied during agonist-induced contractions using the Calcium2+ channel blocker verapamil.

Methods: Isolated spiral strips of rat thoracic aorta with endothelium removed were suspended for isometric tension recordings in physiologic salt solution. Cytosolic concentration of Calcium2+ ([Ca sup 2+]i) was measured concomitantly using fura-2-Calcium2+ fluorescence. Muscle contraction was evoked by the receptor agonists with 30 nM norepinephrine or 10 micro Meter prostaglandin F2 alpha (PGF2 alpha), followed by exposure to halothane, at 0%, 1%, 2%, and 3% or isoflurane, at 2% and 4%. The effects of the anesthetics were compared with those of 0.1-1 micro Meter verapamil (n = 8 for each condition). To clarify the intracellular action of the volatile anesthetics on agonist-induced contractions, this procedure was repeated for the anesthetics only in the presence of 1 micro Meter verapamil (n = 8 for each condition). The effects of both anesthetics were also examined in nonreceptor-mediated contractions evoked with a 1-micro Meter dose of the protein kinase C activator, 12-deoxyphorbol 13-isobutylate, which increases the Calcium2+ sensitivity of the contractile elements (n = 8 for each).

Results: Halothane, isoflurane, and verapamil suppressed norepinephrine- and PGF2 alpha-induced increases in muscle tension and [Ca sup 2+]i in a concentration-dependent manner. The Calcium2+ -tension regression lines suggested that the volatile anesthetics reduced Calcium2+ sensitivity of the contractile elements during PGF2 alpha-induced contraction. Pretreatment of the muscle strip with verapamil revealed that halothane and isoflurane released Calcium2+ during norepinephrine-induced contraction and that [Ca2+]i -tension relationship was modulated during PGF2 alpha-induced contractions. Halothane at 2% and 3% and isoflurane at 4% suppressed 12-deoxyphorbol 13-isobutylate-induced increases in muscle tension, whereas they enhanced increases in [Ca2+]i, indicating that both anesthetics suppressed Calcium2+ sensitivity during 12-deoxyphorbol 13-isobutylate-induced contraction.     

Ca2+- and Myosin Phosphorylation-independent Relaxation by Halothane in K+-depolarized Rat Mesenteric Arteries

Background: 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.

Methods: 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.

Results: 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.     

Propofol Increases Pulmonary Artery Smooth Muscle Myofilament Calcium Sensitivity: Role of Protein Kinase C

Background: Vascular smooth muscle tone is regulated by changes in intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. These cellular mechanisms could serve as targets for anesthetic agents that alter vasomotor tone. This study tested the hypothesis that propofol increases myofilament Ca2+ sensitivity in pulmonary artery smooth muscle (PASM) via the protein kinase C (PKC) signaling pathway.

Methods: Canine PASM strips were denuded of endothelium, loaded with fura-2/AM, and suspended in modified Krebs- Ringer's buffer at 37[degrees]C for simultaneous measurement of isometric tension and [Ca2+]i.

Results: The KCl (30 mm) induced monotonic increases in [Ca2+]i and tension. Verapamil, an L-type Ca2+ channel blocker, attenuated KCl-induced increases in [Ca2+]i and tension to an equal extent. In contrast, propofol attenuated KCl-induced increases in [Ca2+]i to a greater extent than concomitant changes in tension and caused an upward shift in the peak tension-[Ca2+]i relation. Increasing extracellular Ca2+ in the presence of 30 mm KCl resulted in similar increases in [Ca2+]i in control and propofol-pretreated strips, whereas concomitant increases in tension were greater during propofol administration. The Ca2+ ionophore, ionomycin (0.1 [mu]m), increased [Ca2+]i to approximately 50% of the value induced by 60 mm KCl. Under these conditions, propofol (10, 100 [mu]m) caused increases in tension equivalent to 11 +/- 2 and 28 +/- 3% of the increases in tension in response to 60 mm KCl, whereas [Ca2+]i was slightly decreased. Similar effects were observed in response to the PKC activator, phorbol 12-myristate 13-acetate (PMA, 1 [mu]m). Specific inhibition of PKC with bisindolylmaleimide I before ionomycin administration decreased the propofol- and PMA-induced increases in tension and abolished the propofol- and PMA-induced decreases in [Ca2+]i. Selective inhibition of Ca2+-dependent PKC isoforms with Go 6976 also attenuated propofol-induced increases in tension.     

Halothane decreases calcium sensitivity of rat aortic smooth muscle

PURPOSE: To examine the effect of halothane on the cytosolic Ca2+ concentration ([Ca2+]i)-tension relationship of rat aortic smooth muscle. METHODS: Rat aortic rings without endothelia were loaded with the fluorescent Ca2+ indicator, Fura PE3-AM, and then mounted in organ baths. The changes in isometric tension and [Ca2+]i were measured simultaneously. In one series ionomycin (10 nM-3 microM) was added to normal Krebs' solution cumulatively in the absence and presence of halothane (1.5%, 3%). In the other series, CaCl2 (0.3-3 mM) was added to Ca2+-free Krebs' solution including high KCl (50 mM), phenylephrine (100 nM) or prostaglandin F2alpha (PGF2alpha, 1-3 microM) in the absence and presence of halothane (1.5%, 3%). The linear part of [Ca2+]i-tension relationship was analyzed by a linear regression. RESULTS: Halothane, 1.5%, had no effect on the normal [Ca2+]i-tension relationship obtained with the calcium ionophore, ionomycin (10 nM-3 microM), but halothane 3% decreased the slope of the relationship (0.239 +/- 0.037 for control and 0.110 +/- 0.010 for halothane 3%, P < 0.05). Halothane, 1.5% and 3%, did not change the [Ca2+]i-tension relationship obtained with CaCl2 (0.3-3 mM) in the presence of high KCl (50 mM) or phenylephrine (100 nM). In contrast, halothane, 3%, inhibited the intercept of [Ca2+]i-tension relationship obtained with CaCl2 (0.3-3 mM) in the presence of prostaglandin F2alpha (PGF2alpha, 1-3 microM) (45.708 +/- 4.233 for control and 26.997 +/- 2.522 for halothane 3%, P < 0.01). CONCLUSION: Halothane decreases the Ca2+ sensitivity and that in the presence of PGF2.