Involvement of Ca2+ Sensitization in Ropivacaine-induced Contraction of Rat Aortic Smooth Muscle

Background: The mechanisms of amino-amide local anesthetic agent-induced vasoconstriction remain unclear. The current study was designed to examine the roles of the protein kinase C (PKC), Rho kinase, and p44/42 mitogen-activated protein kinase (p44/42 MAPK) signaling pathways in calcium (Ca2+)-sensitization mechanisms in ropivacaine-induced vascular contraction.

Methods: Endothelium-denuded rat aortic rings, segments, and strips were prepared. The cumulative dose-response relations of contraction and intracellular Ca2+ concentration to ropivacaine were tested, using isometric force transducers and a fluorometer, respectively. The dose-dependent ropivacaine-induced phosphorylation of PKC and p44/42 MAPK and the membrane translocation of Rho kinase were also detected using Western blotting.

Results: Ropivacaine induced a dose-dependent biphasic contractile response and an increase in intracellular Ca2+ concentration of rat aortic rings, increasing at concentrations of 3 x 10-5 m to 3 x 10-4 m and decreasing from 10-3 m to 3 x 10-3 m, with a greater tension/intracellular Ca2+ concentration ratio than that induced with potassium chloride. The contraction was attenuated in a dose-dependent manner, by the PKC inhibitors bisindolylmaleimide I and calphostin C, the Rho-kinase inhibitor Y 27632, and the p44/42 MAPK inhibitor PD 098059. Ropivacaine also induced an increase in phosphorylation of PKC and p44/42 MAPK, and membrane translocation of Rho kinase in accordance with the contractile responses, which were also significantly inhibited by bisindolylmaleimide I and calphostin C, Y 27632, and PD 098059, correspondingly.     

Isoflurane Reduces the Carbachol-evoked Ca2+ Influx in Neuronal Cells

Background: The authors previously reported that the isoflurane-caused reduction of the carbachol-evoked cytoplasmic Ca2+ transient increase ([Ca2+]cyt) was eliminated by K+ or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca2+]cyt transient involved Ca2+ influx through the plasma membrane.

Methods: Perfused attached human neuroblastoma SH-SY5Y cells were exposed to carbachol (1 mm, 2 min) in the absence and presence of isoflurane (1 mm) and in the absence and presence of extracellular Ca2+ (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La3+ (100 [mu]m), of the L-type Ca2+ channel blocker nitrendipine (10 [mu]m), and of the N-type Ca2+ channel blocker [omega]-conotoxin GVIA (0.1 [mu]m) on isoflurane modulation of the carbachol-evoked [Ca2+]cyt transient. [Ca2+]cyt was detected with fura-2 and experiments were carried out at 37[degrees]C.

Results: Isoflurane reduced the peak and area of the carbachol-evoked [Ca2+]cyt transient in the presence but not in the absence of extracellular Ca2+. La3+ had a similar effect as the removal of extracellular Ca2+. [omega]-Conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response.     

Inhibitory Effects of Diazepam and Midazolam on Ca2+ and K+ Channels in Canine Tracheal Smooth Muscle Cells

Background: Benzodiazepines have a direct bronchodilator action in airway smooth muscle, but the mechanisms by which these agents produce muscle relaxation are not fully understood. The current study was performed to identify the effects of the benzodiazepines diazepam and midazolam on Ca2+ and K+ channels in canine tracheal smooth muscle cells.

Methods: Whole-cell patch-clamp recording techniques were used to evaluate the effects of the benzodiazepines diazepam (10-8 to 10-3 M) and midazolam (10-8 to 10-3 M) on inward Ca2+ and outward K (+) channel currents in dispersed canine tracheal smooth muscle cells. The effects of the antagonists flumazenil (10-5 M) and PK11195 (10-5 M) on these channels were also studied.

Results: Each benzodiazepine tested significantly inhibited Ca2+ currents in a dose-dependent manner, with 10-6 M diazepam and 10-5 M midazolam each causing approximately 50% depression of peak voltage-dependent Ca2+ currents. Both benzodiazepines promoted the inactivated state of the channel at more-negative potentials. The Ca2+ -activated and voltage-dependent K+ currents were inhibited by diazepam and midazolam (> 10-5 M and > 10-4 M, respectively). Flumazenil and PK11195 had no effect on these channel currents or on the inhibitory effects of the benzodiazepines.     

Effects of Isoflurane, Sevoflurane, and Halothane on Myofilament Ca2+ Sensitivity and Sarcoplasmic Reticulum Ca2+ Release in Rat Ventricular Myocytes

Background: The aim of this study was to describe and compare the effects of isoflurane, sevoflurane, and halothane at selected concentrations (i.e., concentrations that led to equivalent depression of the electrically evoked Ca2+ transient) on myofilament Ca2+ sensitivity, sarcoplasmic reticulum (SR) Ca2+ content, and the fraction of SR Ca2+ released during electrical stimulation (fractional release) in rat ventricular myocytes.

Methods: Single rat ventricular myocytes loaded with fura-2 were electrically stimulated at 1 Hz, and the Ca2+ transients and contractions were recorded optically. Cells were exposed to each anesthetic for 1 min. Changes in myofilament Ca2+ sensitivity were assessed by comparing the changes in the Ca2+ transient and contraction during exposure to anesthetic and low Ca2+. SR Ca2+ content was assessed by exposure to 20 mm caffeine.

Results: Isoflurane and halothane caused a depression of myofilament Ca2+ sensitivity, unlike sevoflurane, which had no effect on myofilament Ca2+ sensitivity. All three anesthetics decreased the electrically stimulated Ca2+ transient. SR Ca2+ content was reduced by both isoflurane and halothane but was unchanged by sevoflurane. Fractional release was reduced by both isoflurane and sevoflurane, but was unchanged by halothane.     

Effects of Volatile Anesthetics on Store-operated Ca2+ Influx in Airway Smooth Muscle

Background: In airway smooth muscle (ASM), volatile anesthetics deplete sarcoplasmic reticulum (SR) Ca2+ stores by increasing Ca2+ "leak." Accordingly, SR replenishment becomes dependent on Ca2+ influx. Depletion of SR Ca2+ stores triggers Ca2+ influx via specific plasma membrane channels, store-operated Ca2+ channels (SOCC). We hypothesized that anesthetics inhibit SOCC triggered by increased SR Ca2+ "leak," preventing SR replenishment and enhancing ASM relaxation.

Methods: In porcine ASM cells, SR Ca2+ was depleted by cyclopiazonic acid or caffeine in 0 extracellular Ca2+, nifedipine and KCl (preventing Ca2+ influx through L-type and SOCC channels). Extracellular Ca2+ was rapidly introduced to selectively activate SOCC. After SOCC activation, SR was replenished and the protocol repeated in the presence of 1 or 2 minimum alveolar concentration halothane, isoflurane, or sevoflurane. In other cells, characteristics of SOCC and interactions between acetylcholine (Ach) and volatile anesthetics were examined.

Results: Cyclopiazonic acid produced slow SR leak, whereas the caffeine response was transient in ASM cells. Reintroduction of extracellular Ca2+ rapidly increased [Ca2+]i. This influx was insensitive to nifedipine, SKF-96365, and KBR-7943, inhibited by Ni2+ and blockade of inositol 1,4,5-triphosphate-induced SR Ca2+ release, and enhanced by ACh. Preexposure to 1 or 2 minimum alveolar concentration halothane completely inhibited Ca2+ influx when extracellular Ca2+ was reintroduced, whereas isoflurane and sevoflurane produced less inhibition. Only halothane and isoflurane inhibited ACh-induced augmentation of Ca2+ influx.     

Isoflurane Neuroprotection in Rat Hippocampal Slices Decreases with Aging: Changes in Intracellular Ca2+ Regulation and N-methyl-d-aspartate Receptor-mediated Ca2+ Influx

Background: Most in vitro neuroprotection studies with isoflurane have involved cells obtained during the embryonic or early postnatal period. However, in mature rodents, isoflurane neuroprotection does not persist. The authors determined whether neuroprotection of hippocampal slices with isoflurane decreases with aging and is due to decreased intracellular Ca2+ regulation and survival protein phosphorylation.

Methods: Hippocampal slices from 5-day-old, 1-month-old, and 19- to 23-month-old rats were deprived of oxygen and glucose for 5-30 min in media bubbled with 1% isoflurane. Cell death was assessed in the CA1, CA3, and dentate regions, and intracellular Ca2+ concentration was measured in CA1 neurons. N-methyl-d-aspartate receptor (NMDAR)-dependent Ca2+ influx was measured and the phosphorylation of NMDARs, and the survival proteins Akt and mitogen-activated protein kinase p42/44 were quantified.

Results: Twenty minutes of oxygen and glucose deprivation killed approximately 40-60% of neurons in CA3 and dentate in all age groups. Isoflurane, 1%, reduced death of CA1, CA3, and dentate neurons in slices from 5-day-old rats but not those from 23-month-old rats. In 5-day slices, isoflurane attenuated NMDAR-mediated Ca2+ influx, whereas in aging slices, Ca2+ influx was increased protein kinase C. In aging slices, isoflurane did not increase the phosphorylation of Akt and p42/44.     

Sevoflurane Inhibits Angiotensin II-induced, Protein Kinase C-mediated but Not Ca2+-elicited Contraction of Rat Aortic Smooth Muscle

Background: Whether volatile anesthetics attenuate angiotensin II-mediated vascular tone has not been determined. The current study was designed to investigate the effects of sevoflurane on the angiotensin II-stimulated, Ca2+- and protein kinase C (PKC)-mediated contraction of rat aortic smooth muscle.

Methods: The dose-dependent effects of sevoflurane on angiotensin II (10-7 m)-induced contraction, the increase in intracellular Ca2+ concentration, and PKC phosphorylation of rat aortic smooth muscle were measured using an isometric force transducer, a fluorometer, and Western blotting, respectively.

Results: Angiotensin II induced a transient increase in intracellular Ca2+ concentration, phosphorylation of Ca2+-dependent PKC (cPKC)-[alpha], and consequently, a transient contraction of rat aortic smooth muscle. Phosphorylation of the Ca2+-independent PKC-[epsilon] was not detected. The angiotensin II-induced contraction was almost completely abolished by removing extracellular Ca2+ and was significantly inhibited by the selective cPKC inhibitor Go 6976 (10-5 m) but was not inhibited by the nonselective PKC inhibitor Ro 31-8425 (10-5 m). Sevoflurane dose-dependently inhibited the angiotensin II-induced contraction, with reductions of 14.2 +/- 5.2% (P > 0.05), 26.7 +/- 8.9% (P < 0.05), and 38.5 +/- 12.8% (P < 0.01) (n = 10) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively. The angiotensin II-elicited increase in intracellular Ca2+ concentration was not significantly influenced by 3.4, 5.1, or 8.5% sevoflurane. However, cPKC-[alpha] phosphorylation induced by angiotensin II was inhibited dose dependently by 1.7, 3.4, and 5.1% sevoflurane, with depressions of 20.5 +/- 14.2% (P > 0.05), 37.0 +/- 17.8% (P < 0.05), and 62.5 +/- 12.2% (P < 0.01) (n = 4), respectively.     

Droperidol Inhibits Intracellular Ca2+, Myofilament Ca2+ Sensitivity, and Contraction in Rat Ventricular Myocytes

Background: Droperidol has recently been associated with cardiac arrhythmias and sudden cardiac death. Changes in action potential duration seem to be the cause of the arrhythmic behavior, which can lead to alterations in intracellular free Ca2+ concentration ([Ca2+]i). Because [Ca2+]i and myofilament Ca2+ sensitivity are key regulators of myocardial contractility, the authors' objective was to identify whether droperidol alters [Ca2+]i or myofilament Ca2+ sensitivity in rat ventricular myocytes and to identify the cellular mechanisms responsible for these effects.

Methods: Freshly isolated rat ventricular myocytes were obtained from adult rat hearts. Myocyte shortening, [Ca2+]i, nitric oxide production, intracellular pH, and action potentials were monitored in cardiomyocytes exposed to droperidol. Langendorff perfused hearts were used to assess overall cardiac function.

Results: Droperidol (0.03-1 [mu]m) caused concentration-dependent decreases in peak [Ca2+]i and shortening. Droperidol inhibited 35 mm KCl-induced increase in [Ca2+]i, with little direct effect on sarcoplasmic reticulum Ca2+ stores. Droperidol had no effect on action potential duration but caused a rightward shift in the concentration-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. Droperidol decreased pHi and increased nitric oxide production. Droperidol exerted a negative inotropic effect in Langendorff perfused hearts.     

Effects of Halothane and Isoflurane on the Intracellular Ca2+ Transient in Ferret Cardiac Muscle

Background: Halothane and isoflurane depress myocardial contractility by decreasing transsarcolemmal Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum. Decreases in Ca2+ sensitivity of the contractile proteins have been shown in skinned cardiac fibers, but the relative importance of this effect in intact living myocardium is unknown. The aims of this study were to assess whether halothane and isoflurane decrease myofibrillar Ca2+ sensitivity in intact, living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+ sensitivity versus changes in myoplasmic Ca2+ availability caused by these anesthetics.

Methods: The effects of halothane and isoflurane (0-1.5 times the minimum alveolar concentration (MAC) in three equal increments) on isometric and isotonic variables of contractility and on the intracellular calcium transient were assessed in isolated ferret right ventricular papillary muscle microinjected with the Ca2+-regulated photoprotein aequorin. The intracellular calcium transient was analyzed in the context of a multicompartment model of intracellular Ca2+ buffers in mammalian ventricular myocardium.

Results: Halothane and isoflurane decreased contractility, time-to-peak force, time to half-isometric relaxation, and intracellular Ca2+ transient in a reversible, concentration-dependent manner. Halothane, but not isoflurane, slowed the increase and the decrease of the intracellular Ca2+ transient. Increasing extracellular Ca2+ in the presence of anesthetic to produce peak force equal to control values increased intracellular Ca2+ to values higher than control values.     

Mg2+ Dependence of Halothane-induced Ca2+ Release from the Sarcoplasmic Reticulum in Skeletal Muscle from Humans Susceptible to Malignant Hyperthermia

Background: Recent work suggests that impaired Mg2+ regulation of the ryanodine receptor is a common feature of both pig and human malignant hyperthermia. Therefore, the influence of [Mg2+] on halothane-induced Ca2+ release from the sarcoplasmic reticulum was studied in malignant hyperthermia-susceptible (MHS) or -nonsusceptible (MHN) muscle.

Methods: Vastus medialis fibers were mechanically skinned and perfused with solutions containing physiologic (1 mm) or reduced concentrations of free [Mg2+]. Sarcoplasmic reticulum Ca2+ release was detected using fura-2 or fluo-3.

Results: In MHN fibers, 1 mm halothane consistently did not induce sarcoplasmic reticulum Ca2+ release in the presence of 1 mm Mg2+. It was necessary to increase the halothane concentration to 20 mm or greater before Ca2+ release occurred. However, when [Mg2+] was reduced below 1 mm, halothane became an increasingly effective stimulus for Ca2+ release; e.g., at 0.4 mm Mg2+, 58% of MHN fibers responded to halothane. In MHS fibers, 1 mm halothane induced Ca2+ release in 57% of MHS fibers at 1 mm Mg2+. Reducing [Mg2+] increased the proportion of MHS fibers that responded to 1 mm halothane. Further experiments revealed differences in the characteristics of halothane-induced Ca2+ release in MHS and MHN fibers: In MHN fibers, at 1 mm Mg2+, halothane induced a diffuse increase in [Ca2+], which began at the periphery of the fiber and spread slowly inward. In MHS fibers, halothane induced a localized Ca2+ release, which then propagated along the fiber. However, propagated Ca2+ release was observed in MHN fibers when halothane was applied at an Mg2+ concentration of 0.4 mm or less.     

Anesthetic Preconditioning Enhances Ca2+ Handling and Mechanical and Metabolic Function Elicited by Na+-Ca2+ Exchange Inhibition in Isolated Hearts

Background: Anesthetic preconditioning (APC) is well known to protect against myocardial ischemia-reperfusion injury. Studies also show the benefit of Na+-Ca2+ exchange inhibition on ischemia-reperfusion injury. The authors tested whether APC plus Na+-Ca2+ exchange inhibitors given just on reperfusion affords additive protection in intact hearts.

Methods: Cytosolic [Ca2+] was measured by fluorescence at the left ventricular wall of guinea pig isolated hearts using indo-1 dye. Sarcoplasmic reticular Ca2+-cycling proteins, i.e., Ca2+ release channel (ryanodine receptor [RyR2]), sarcoplasmic reticular Ca2+-pump adenosine triphosphatase (SERCA2a), and phospholamban were measured by Western blots. Hearts were assigned to seven groups (n = 8 each): (1) time control; (2) ischemia; (3, 4) 10 [mu]m Na+-Ca2+ exchange inhibitor KB-R7943 (KBR) or 1 [mu]m SEA0400 (SEA), given during the first 10 min of reperfusion; (5) APC initiated by sevoflurane (2.2%, 0.41 +/- 0.03 mm) given for 15 min and washed out for 15 min before ischemia-reperfusion; (6, 7) APC plus KBR or SEA.

Results: The authors found that APC reduced the increase in systolic [Ca2+], whereas KBR and SEA both reduced the increase in diastolic [Ca2+] on reperfusion. Each intervention improved recovery of left ventricular function. Moreover, APC plus KBR or SEA afforded better functional recovery than APC, KBR, or SEA alone (P < 0.05). Ischemia-reperfusion-induced degradation of major sarcoplasmic reticular Ca2+-cycling proteins was attenuated by APC, but not by KBR or SEA.     

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.     

Isoflurane Activates PKC and Ca2+-Calmodulin-dependent Protein Kinase II via MAP Kinase Signaling in Cultured Vascular Smooth Muscle Cells

Background: Protein kinase C (PKC) and Ca2+-calmodulin-dependent protein kinase II (CaMKII) have been implicated in isoflurane-increased force in skinned femoral arterial strips. The extracellular signal-regulated kinases (ERK1/2) of mitogen-activated protein kinase have been shown to be target effectors of PKC and CaMKII. This study examined the role of the ERK1/2 signaling pathway in isoflurane activation of PKC and CaMKII using cultured vascular smooth muscle cells.

Methods: Vascular smooth muscle cells were prepared by cell migration from isolated rabbit femoral arterial segments. Growth of passage of vascular smooth muscle cells (80-90% confluence, passage 5-10) was arrested for 48 h before experiments, during which time phorbol 1,3-diaceylester treatment was used to down-regulate PKC. Cells were treated for 30 min with one of the inhibitors of mitogen-activated protein kinase kinase (PD98059), PKC (Go6976 and bisindolylmaleimide), or CaMKII (KN-93 and KN-62) at 10 [mu]m. After administration of isoflurane, vascular smooth muscle cells were frozen rapidly, homogenized, and centrifuged. The homogenates were used for identification of phosphorylated ERK1/2 or for further centrifugation to separate the membrane from the cytosol for identification of PKC isoforms ([alpha] and ) by Western blotting.

Results: Isoflurane increased ERK1/2 phosphorylation in a dose-dependent manner and reached a plateau at 10 min. PD98059 or down-regulated PKC blocked the increase of phosphorylated ERK1/2 levels by isoflurane, and bisindolylmaleimide, KN-93, or KN-62, but not by Go6976 reduced levels of phosphorylated ERK1/2. The membrane fraction of PKC but not of PKC[alpha] was increased by isoflurane.     

Propofol Increases Myofilament Ca2+ Sensitivity and Intracellular pH via Activation of Na+-H+ Exchange in Rat Ventricular Myocytes

Background: The objectives were to determine the extent and mechanism of action by which propofol increases myofilament Ca2+ sensitivity and intracellular pH (pHi) in ventricular myocytes.

Methods: Freshly isolated adult rat ventricular myocytes were used for the study. Cardiac myofibrils were extracted for assessment of myofibrillar actomyosin adenosine triphosphatase (ATPase) activity. Myocyte shortening (video edge detection) and pHi (2',7'-bis-(2-carboxyethyl)-5(6')-carboxyfluorescein, 500/440 ratio) were monitored simultaneously in individual cells field-stimulated (0.3 Hz) and superfused with HEPES-buffered solution (pH 7.4, 30[degrees]C).

Results: Propofol (100 [mu]m) reduced the Ca2+ concentration required for activation of myofibrillar actomyosin ATPase from pCa 5.7 +/- 0.01 to 6.6 +/- 0.01. Increasing pHi (7.05 +/- 0.03 to 7.39 +/- 0.04) with NH4Cl increased myocyte shortening by 35 +/- 12%. Washout of NH4Cl decreased pHi to 6.82 +/- 0.03 and decreased myocyte shortening to 52 +/- 10% of control. Propofol caused a dose-dependent increase in pHi but reduced myocyte shortening. The propofol-induced increase in pHi was attenuated, whereas the decrease in myocyte shortening was enhanced after pretreatment with ethylisopropyl amiloride, a Na+-H+ exchange inhibitor, or bisindolylmaleimide I, a protein kinase C inhibitor. Propofol also attenuated the NH4Cl-induced intracellular acidosis, increased the rate of recovery from acidosis, and attenuated the associated decrease in myocyte shortening. Propofol caused a leftward shift in the extracellular Ca2+-shortening relation, and this effect was attenuated by ethylisopropyl amiloride.     

Anesthetic-induced Alteration of Ca2+ Homeostasis in Neural Cells: A Temperature-sensitive Process That Is Enhanced by Blockade of Plasma Membrane Ca2+-ATPase Isoforms

Background: Many inhalation anesthetics at clinically relevant concentrations inhibit plasma membrane Ca2+-adenosine triphosphatase (PMCA) ion pumping in brain synaptic membranes and in cultured cells of neural origin. In this study, the authors investigated the effect of inhalation anesthetics on cytosolic calcium homeostasis in cortical neurons maintained at physiologic and room temperatures and on cortical neurons and pheochromocytoma cells with antisense blockade of specific PMCA isoforms.

Methods: Using Ca2+-specific confocal microfluorimetry, the anesthetic effects on Ca2+ dynamics were examined in mouse embryonic cortical neurons in association with ligand-stimulated Ca2+ influx. Studies were done at 21 [degree sign]C and 37 [degree sign]C. Mouse embryonic cortical neurons with oligodeoxyribonucleotide blockade of PMCA2 expression and transfected rat pheochromocytoma cells with blocked expression of PMCA1 were also examined.

Results: Baseline and poststimulation peak cytosolic calcium concentrations ([Ca2+]i) were increased, and Ca2+ clearance was delayed in cells exposed at 37 [degree sign]C, but not at 21 [degree sign]C, to concentrations 相似文献   

Thiopental Alters Contraction, Intracellular Ca2+, and pH in Rat Ventricular Myocytes

Background: Myocardial contractility is regulated by intracellular concentration of free Ca2+ ([Ca2+]i) and myofilament Ca2+ sensitivity. The objective of this study was to elucidate the direct effects of thiopental on cardiac excitation-contraction coupling using individual, field-stimulated ventricular myocytes.

Methods: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i (340/380 ratio) and myocyte shortening (video-edge detection) were monitored simultaneously in individual cells field-stimulated at 0.3 Hz. Amplitude and timing of myocyte shortening and [Ca2+]i were compared before and after addition of thiopental. Intracellular pH was measured with the pH indicator, BCECF (500/440 ratio). Real-time uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. One hundred thirty-two cells were studied.

Results: Field stimulation increased [Ca2+]i from 85 +/- 10 nM to 355 +/- 22 nM (mean +/- SEM). Myocytes shortened by 10% of resting cell length (127 +/- 5 [micro sign]m). Times to peak [Ca2+]i and shortening were 139 +/- 6 and 173 +/- 7 msec, respectively. Times to 50% recovery for [Ca2+]i and shortening were 296 +/- 6 and 290 +/- 6 ms, respectively. Addition of thiopental (30-1,000 [micro sign]M) resulted in dose-dependent decreases in peak [Ca2+]i and myocyte shortening. Thiopental altered time to peak and time to 50% recovery for [Ca2+]i and myocyte shortening and inhibited the rate of uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles. Thiopental did not, however, alter the amount of Ca2+ released in response to caffeine in sarcoplasmic reticulum vesicles or intact cells. Thiopental (100 [micro sign]M) increased intracellular pH and caused an upward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. These effects were abolished by ethylisopropyl amiloride, an inhibitor of Na+ -H+ exchange.     

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.     

Effects of Sevoflurane on the Intracellular Ca2+ Transient in Ferret Cardiac Muscle

Background: Sevoflurane depresses myocardial contractility by decreasing transsarcolemmal Ca2+ influx. In skinned muscle fibers, sevoflurane affects actin-myosin cross-bridge cycling, which might contribute to the negative inotropic effect. It is uncertain to what extent decreases in Ca2+ sensitivity of the contractile proteins play a role in the negative inotropic effect of sevoflurane in intact cardiac muscle tissue. The aim of this study was to assess whether sevoflurane decreases myofibrillar Ca2+ sensitivity in intact living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+ sensitivity versus changes in myoplasmic Ca2+ availability by sevoflurane.

Methods: The effects of sevoflurane 0-4.05% vol/vol (0-1.5 minimum alveolar concentration [MAC]) on isometric and isotonic variables of contractility and on the intracellular calcium transient were assessed in isolated ferret right ventricular papillary muscles microinjected with the Ca2+-regulated photoprotein aequorin. The intracellular calcium transient was analyzed in the context of a multicompartment model of intracellular Ca2+ buffers in mammalian ventricular myocardium.

Results: Sevoflurane decreased contractility, time to peak force, time to half isometric relaxation, and the [Ca2+]i transient in a reversible, concentration-dependent manner. Increasing [Ca2+]o in the presence of sevoflurane to produce peak force equal to control increased intracellular Ca2+ transient higher than control.     

Store-operated Ca2+ Influx in Airway Smooth Muscle: Interactions between Volatile Anesthetic and Cyclic Nucleotide Effects

Background: Volatile anesthetics produce bronchodilation in part by depleting sarcoplasmic reticulum Ca2+ stores in airway smooth muscle (ASM). Other bronchodilatory drugs are known to act via cyclic nucleotides (cyclic adenosine 3',5'-cyclic monophosphate, cyclic guanosine 3',5'-cyclic monophosphate). Intracellular Ca2+ regulation in ASM involves plasma membrane Ca2+ influx, including that triggered by sarcoplasmic reticulum Ca2+ depletion (store-operated Ca2+ entry [SOCE]). The authors hypothesized that anesthetics and bronchodilatory agents interact in inhibiting SOCE, thus enhancing ASM relaxation.

Methods: In enzymatically dissociated porcine ASM cells imaged using fluorescence microscopy, sarcoplasmic reticulum Ca2+ was depleted by 1 [mu]m cyclopiazonic acid in 0 extracellular Ca2+, nifedipine, and potassium chloride (preventing Ca2+ influx through L-type channels and SOCE). Extracellular Ca2+ was rapidly reintroduced to selectively activate SOCE in the presence or absence of 1 minimum alveolar concentration (MAC) halothane, isoflurane, or sevoflurane. Anesthetic interference with SOCE regulation by cyclic nucleotides was examined by activating SOCE in the presence of (1) 1 [mu]m acetylcholine, (2) 100 [mu]m dibutryl cyclic adenosine 3',5'-cyclic monophosphate, or (3) 100 [mu]m 8-bromo-cyclic guanosine 3',5'-cyclic monophosphate.

Results: SOCE was enhanced by acetylcholine, whereas volatile anesthetics and both cyclic nucleotides partially inhibited Ca2+ influx. Preexposure to 1 or 2 MAC anesthetic (halothane > isoflurane > sevoflurane) inhibited SOCE. Only halothane and isoflurane inhibited acetylcholine-induced augmentation of Ca2+ influx, and significantly potentiated cyclic nucleotide inhibition such that no influx was observed in the presence of anesthetics and cyclic nucleotides.