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.     

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 microM 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 microM 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. CONCLUSIONS: The authors conclude that halothane reduces sarcoplasmic reticulum Ca2+ content in ASM cells via increased Ca2+ leak through both IP3 receptor and ryanodine receptor channels. Effects on IP3 receptor channels are both direct and indirect via elevation of IP3 levels.     

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.     

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 [Ca2+] (i.e., Ca2+ 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 [Ca2+] 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 [Ca2+], 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 [mu]m Ca2+ and 10 [mu]m 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.     

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.     

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.     

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.     

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 (< or = 30 microm) of ketamine slightly enhanced norepinephrine-induced contraction, whereas higher concentrations (> or = 100 microM) of ketamine inhibited both norepinephrine- and KCl-induced contractions. In the fura-2-loaded strips, ketamine (> or = 100 microM) 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. CONCLUSIONS: The action of ketamine on contractile response to norepinephrine consists of endothelium-dependent vasoconstricting and endothelium-independent vasodilating components. The direct vasorelaxation is largely a result of reduction of[Ca2+]i in vascular smooth muscle cells. The [Ca2+]i-reducing effects are caused by inhibitions of both voltage-gated Ca2+ influx and norepinephrine-induced Ca2+ release from the intracellular stores.     

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.     

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 microm 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 microm GTPgammaS, 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. CONCLUSIONS: The authors noted dual effects of hexanol and halothane. In the presence of muscarinic receptor stimulation, hexanol, like halothane, decreases calcium sensitivity by interfering with heterotrimeric G-protein function. However, in the absence of muscarinic receptor stimulation, hexanol and halothane slightly increase calcium sensitivity by a G-protein-mediated process not sensitive to pertussis toxin. Hexanol may represent a useful experimental tool to study the effect of anesthetics on heterotrimeric G-protein function.     

Anesthetics Inhibit Membrane Receptor Coupling to the Gq/11 Heterotrimeric G Protein in Airway Smooth Muscle

Background: Some anesthetics relax airway smooth muscle in part by inhibiting acetylcholine-induced increases in Ca2+ sensitivity, an effect associated with inhibition of guanosine nucleotide exchange at the [alpha] subunit of the Gq/11 (G[alpha]q/11) heterotrimeric G protein. This study tested the hypothesis that these anesthetic effects are not unique to the muscarinic receptor but are a general property of the heptahelical receptors that increase Ca2+ sensitivity in airway smooth muscle.

Methods: Anesthetic effects on agonist-induced increases in Ca2+ sensitivity were measured in porcine airway smooth muscle strips permeabilized with S. aureus [alpha]-toxin. Anesthetic effects on basal (without agonist stimulation) and agonist-promoted G[alpha]q/11 guanosine nucleotide exchange were determined in crude membranes prepared from porcine airway smooth muscle. The nonhydrolyzable, radioactive form of guanosine 5'-triphosphate was used as the reporter for nucleotide exchange at G[alpha]q/11.

Results: Acetylcholine, endothelin-1, and histamine caused a concentration-dependent increase in Ca2+ sensitivity. Halothane (0.67 +/- 0.07 mm) and hexanol (10 mm) significantly inhibited the increase in Ca2+ sensitivity induced by each agonist. Each agonist also caused a time- and concentration-dependent increase in G[alpha]q/11 nucleotide exchange. Neither anesthetic had an effect on basal G[alpha]q/11 nucleotide exchange, whereas halothane and hexanol significantly inhibited the increase in G[alpha]q/11 nucleotide exchange promoted by each agonist.     

Agonist-induced calcium regulation in freshly isolated renal microvascular smooth muscle cells.

The studies presented here were performed to determine the effect of agonist stimulation on the cytosolic free Ca2+ concentration ([Ca2+]i) in single smooth muscle cells, freshly isolated from afferent arterioles and interlobular arteries averaging between 10 to 40 microns in diameter. Microvessels were obtained from male Sprague-Dawley rats using an iron oxide collection technique followed by collagenase digestion. Freshly isolated microvascular smooth muscle cells (MVSMC) were loaded with fura 2 and studied using fluorescence photometry techniques. The resting [Ca2+]i averaged 67 +/- 3 nM (N = 82 cells). Increasing the extracellular K+ concentration significantly increased [Ca2+]i dose-dependently (P < 0.05). Involvement of extracellular Ca2+ in the response to KCl-induced depolarization was also evaluated. Resting [Ca2+]i increased approximately 132% from 40 +/- 5 nM to 93 +/- 26 nM in response to 90 mM extracellular KCl. This change was abolished in nominally Ca(2+)-free conditions and markedly attenuated by diltiazem. Inhibition of K+ channels with charybdotoxin or tetraethylammonium chloride produced a modest transient increase in [Ca2+]i during the response to 30 mM K+ and had no detectable effect on responses to 90 mM K+. Studies were also performed to establish whether freshly isolated renal MVSMC exhibit appropriate responses to receptor-dependent physiological agonists. Angiotensin II (100 nM) increased cell Ca2+ from 97 +/- 10 nM to 265 +/- 47 nM (N = 12 cells). Similarly, 100 microM ATP increased MVSMC [Ca2+]i from a control level of 71 +/- 14 nM to 251 +/- 47 nM (N = 11 cells). Norepinephrine administration caused [Ca2+]i to increase from 63 +/- 4 nM to 212 +/- 47 nM (N = six cells), and vasopressin increased [Ca2+]i from 86 +/- 10 nM to 352 +/- 79 nM (N = five cells). These data demonstrate that receptor-dependent and -independent vasoconstrictor agonists increase [Ca2+]i in MVSMC, freshly isolated from rat preglomerular vessels. Furthermore, the ability to measure [Ca2+]i in responses to physiological stimuli in these single cells permits investigation of signal transduction mechanisms involved in regulating renal microvascular resistance.     

Intracellular free calcium accumulation in ferret vascular smooth muscle during crystalloid and blood cardioplegic infusions.

OBJECTIVE: The effects of magnesium- and potassium-based crystalloid and blood-containing cardioplegic solutions on coronary smooth muscle intracellular free calcium ([Ca2+]i) accumulation and microvascular contractile function were examined. METHODS: Isolated ferret hearts were subjected to hyperkalemic (25 mmol/L K+) blood cardioplegic infusion, hypermagnesemic (25 mmol/L Mg2+, K+-free) crystalloid cardioplegic infusion, or hyperkalemic crystalloid cardioplegic infusion for 1 hour. Coronary arterioles were isolated, cannulated, and loaded with fura 2. Reactivity and [Ca2+]i were assessed with videomicroscopy. [Ca2+]i was measured at baseline and after application of 50 mmol/L KCl. In addition, [Ca2+]i and vascular contraction were measured during exposure to Mg2+ and K+ cardioplegic solution at both 4 degrees C and 37 degrees C. RESULTS: From a baseline [Ca2+]i of 177 +/- 52 nmol/L, K+ cardioplegic infusion (302 +/- 80 nmol/L potassium) markedly increased [Ca2+]i, whereas blood cardioplegic infusion (214 +/- 53 nmol/L) and Mg2+ cardioplegic infusion (180 +/- 42 nmol/L) did not alter [Ca2+]i. Although a difference between groups in percentage contraction after application of 50 mmol/L KCl was not observed, [Ca2+]i increased significantly more in vessels in the control group (764 +/- 327 nmol/L) and the K+ crystalloid cardioplegic infusion group (698 +/- 215 nmol/L) than in vessels in the blood cardioplegic infusion group (402 +/- 45 nmol/L) and the Mg2+ cardioplegic infusion group (389 +/- 80 nmol/L). Mg2+ cardioplegic solution induced no microvascular contraction at either 4 degrees C or 37 degrees C, nor was an increase in [Ca2+]i observed. K+ cardioplegic solution induced microvascular contraction at 37 degrees C but not at 4 degrees C; it increased [Ca2+]i at both 4 degrees C and 37 degrees C. CONCLUSION: An Mg2+-based cardioplegic solution, or appropriate Mg2+ or blood supplementation of a K+ crystalloid cardioplegic solution, may decrease the accumulation of [Ca2+]i in the vascular smooth muscle during ischemic arrest.     

Anesthetics inhibit membrane receptor coupling to the Gq/11 heterotrimeric G protein in airway smooth muscle

BACKGROUND: Some anesthetics relax airway smooth muscle in part by inhibiting acetylcholine-induced increases in Ca2+ sensitivity, an effect associated with inhibition of guanosine nucleotide exchange at the alpha subunit of the Gq/11 (Galphaq/11) heterotrimeric G protein. This study tested the hypothesis that these anesthetic effects are not unique to the muscarinic receptor but are a general property of the heptahelical receptors that increase Ca2+ sensitivity in airway smooth muscle. METHODS: Anesthetic effects on agonist-induced increases in Ca2+ sensitivity were measured in porcine airway smooth muscle strips permeabilized with S. aureus alpha-toxin. Anesthetic effects on basal (without agonist stimulation) and agonist-promoted Galphaq/11 guanosine nucleotide exchange were determined in crude membranes prepared from porcine airway smooth muscle. The nonhydrolyzable, radioactive form of guanosine 5'-triphosphate was used as the reporter for nucleotide exchange at Galphaq/11. RESULTS: Acetylcholine, endothelin-1, and histamine caused a concentration-dependent increase in Ca2+ sensitivity. Halothane (0.67 +/- 0.07 mM) and hexanol (10 mM) significantly inhibited the increase in Ca2+ sensitivity induced by each agonist. Each agonist also caused a time- and concentration-dependent increase in Galphaq/11 nucleotide exchange. Neither anesthetic had an effect on basal Galphaq/11 nucleotide exchange, whereas halothane and hexanol significantly inhibited the increase in Galphaq/11 nucleotide exchange promoted by each agonist. CONCLUSION: These data suggest that inhibition of agonist-promoted guanosine nucleotide exchange at Galphaq/11 by some anesthetics may be a general property of heptahelical receptors involved cellular processes mediated by Galphaq/11, including muscarinic, endothelin-1, and histamine receptor activation of Ca2+ sensitivity.     

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.     

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 microm), increased [Ca2+]i to approximately 50% of the value induced by 60 mm KCl. Under these conditions, propofol (10, 100 microm) 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 microm). 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 G? 6976 also attenuated propofol-induced increases in tension. CONCLUSION: These results suggest that propofol increases myofilament Ca2+ sensitivity in PASM, and this effect involves the PKC signaling pathway.     

Halothane and isoflurane augment depolarization-induced cytosolic CA2+ transients and attenuate carbachol-stimulated CA2+ transients

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

Halothane and Isoflurane Augment Depolarization-induced Cytosolic CA2+ Transients and Attenuate Carbachol-stimulated CA2+ Transients

Background: Neuronal excitability is in part determined by Ca2+ availability that is controlled by regulatory mechanisms of cytosolic Ca2+ ([Ca2+]cyt). Alteration of any of those mechanisms by volatile anesthetics (VAs) may lead to a change in presynaptic transmission and postsynaptic excitability. Using a human neuroblastoma cell line, the effects of halothane and isoflurane on cytosolic Ca2+ concentration ([Ca2+]cyt) in response to K+ and carbachol stimulation were investigated.

Methods: Volatile anesthetic (0.05-1 mm) action on stimulated [Ca2+]cyt transients were monitored in suspensions of SH-SY5Y cells loaded with fura-2. Potassium chloride (KCl; 100 mm) was used to depolarize and activate Ca2+ entry through voltage-dependent calcium channels; 1 mm carbachol was used to activate muscarinic receptor-mediated inositol triphosphate (IP3)-dependent intracellular Ca2+ release. Sequential stimulations, KCl followed by carbachol and vice versa, were used to investigate interactions between intracellular Ca2+ stores.

Results: Halothane and isoflurane in clinically relevant concentrations enhanced the K+-evoked [Ca2+]cyt transient whether intracellular Ca2+ stores were full or partially depleted. In contrast, halothane and isoflurane reduced the carbachol-evoked [Ca2+]cyt transient when the intracellular Ca2+ stores were full but had no effect when the Ca2+ stores were partially depleted by KCl stimulation.     

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.     

Mechanisms of direct inhibitory action of isoflurane on vascular smooth muscle of mesenteric resistance arteries

BACKGROUND: Isoflurane has been shown to directly inhibit vascular reactivity. However, less information is available regarding its underlying mechanisms in systemic resistance arteries. METHODS: Endothelium-denuded smooth muscle strips were prepared from rat mesenteric resistance arteries. Isometric force and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded strips, whereas only the force was measured in the beta-escin membrane-permeabilized strips. RESULTS: Isoflurane (3-5%) inhibited the increases in both [Ca2+]i and force induced by either norepinephrine (0.5 microM) or KCl (40 mM). These inhibitions were similarly observed after depletion of intracellular Ca2+ stores by ryanodine. Regardless of the presence of ryanodine, after washout of isoflurane, its inhibition of the norepinephrine response (both [Ca2+]i and force) was significantly prolonged, whereas that of the KCl response was quickly restored. In the ryanodine-treated strips, the norepinephrine- and KCl-induced increases in [Ca2+]i were both eliminated by nifedipine, a voltage-gated Ca2+ channel blocker, whereas only the former was inhibited by niflumic acid, a Ca2+-activated Cl- channel blocker. Isoflurane caused a rightward shift of the Ca2+-force relation only in the fura-2-loaded strips but not in the beta-escin-permeabilized strips. CONCLUSIONS: In mesenteric resistance arteries, isoflurane depresses vascular smooth muscle reactivity by directly inhibiting both Ca2+ mobilization and myofilament Ca2+ sensitivity. Isoflurane inhibits both norepinephrine- and KCl-induced voltage-gated Ca2+ influx. During stimulation with norepinephrine, isoflurane may prevent activation of Ca2+-activated Cl- channels and thereby inhibit voltage-gated Ca2+ influx in a prolonged manner. The presence of the plasma membrane appears essential for its inhibition of the myofilament Ca2+ sensitivity.