Isoflurane reduction of carbachol-evoked cytoplasmic calcium transients is dependent on caffeine-sensitive calcium stores

BACKGROUND: Many muscarinic functions are relevant to anesthesia, and alterations in muscarinic activity affect the anesthetic/analgesic potency of various drugs. Volatile anesthetics have been shown to depress muscarinic receptor function, and inhibition of the muscarinic signaling pathway alters the minimal alveolar anesthetic concentration of inhaled anesthetics. The purpose of this investigation was to determine in a neuronal cell which source of Ca2+ underlying the carbachol-evoked transient increase in cytoplasmic Ca2+ was reduced by isoflurane. METHODS: Experiments were performed at 37 degrees C on continuously perfused monolayers of human neuroblastoma SH-SY5Y cells using Fura-2 as the cytoplasmic Ca2+ indicator. Carbachol (1 mm) was applied to evoke a transient increase in cytoplasmic Ca2+. RESULTS: Isoflurane (1 mm) reduces the carbachol-evoked transient increase in cytoplasmic Ca2+, and this isoflurane action is eliminated when the cells are continuously stimulated with 200 mm KCl or pretreated with 10 mm caffeine or 200 microm ryanodine. CONCLUSIONS: Isoflurane reduction of the carbachol-evoked transient increase in cytoplasmic Ca2+ requires full caffeine-sensitive Ca2+ stores and Ca2+ release from the caffeine-sensitive stores through the ryanodine-sensitive Ca2+ release channels. The results indicate that isoflurane interferes with a muscarinic Ca2+ signaling through a mechanism downstream from the muscarinic receptors.     

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.     

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 reduces the carbachol-evoked Ca2+ influx in neuronal cells

BACKGROUND: The authors previously reported that the isoflurane-caused reduction of the carbachol-evoked cytoplasmic Ca transient increase ([Ca]cyt) was eliminated by K or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca]cyt transient involved Ca 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 Ca (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La (100 microm), of the L-type Ca channel blocker nitrendipine (10 microm), and of the N-type Ca channel blocker omega-conotoxin GVIA (0.1 microm) on isoflurane modulation of the carbachol-evoked [Ca]cyt transient. [Ca]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 [Ca]cyt transient in the presence but not in the absence of extracellular Ca. La had a similar effect as the removal of extracellular Ca. Omega-conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response. CONCLUSIONS: The current data are consistent with previous observations in that the carbachol-evoked [Ca]cyt transient involves both Ca release from intracellular Ca stores and Ca entry through the plasma membrane. It was found that isoflurane attenuates the carbachol-evoked Ca entry. The isoflurane sensitive Ca entry involves a cationic channel different from the L- or N- type voltage-dependent Ca channels. These results indicate that isoflurane attenuates the carbachol-evoked [Ca]cyt transient at a site at the plasma membrane that is distal to the muscarinic receptor.     

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.     

Volatile Anesthetics Affect Calcium Mobilization in Bovine Endothelial Cells

Background: The site where volatile anesthetics inhibit endothelium-dependent, nitric oxide-mediated vasodilation is unclear. To determine whether anesthetics could limit endothelium-dependent nitric oxide production by inhibiting receptor-mediated increases in cytosolic Calcium2+, experiments were performed to see if the inhalational anesthetics halothane, isoflurane, and enflurane affect intracellular Calcium2+ ([Ca2+]i) transients induced by the agonists bradykinin and adenosine triphosphate in cultured bovine aortic endothelial cells.

Methods: Bovine aortic endothelial cells, which had been loaded with the fluorescent Calcium2+ indicator Fura-2, were added to medium preequilibrated with volatile anesthetic (1.25% and 2.5% for isoflurane, 1.755 and 3.5% for enflurane, and 0.75% and 1.5% for halothane). In Calcium2+ -containing medium, intracellular Calcium sup 2+ transients were elicited in response to bradykinin (10 nM and 1 micro Meter) or adenosine triphosphate (1 micro Meter and 100 micro Meter).

Results: Both bradykinin and adenosine triphosphate triggered a rapid rise to peak [Ca2+]i followed by a gradual decline to a plateau above the resting level. Although basal [Ca2+]i was unaltered by the anesthetics, both halothane and enflurane, in a dose-dependent manner, depressed the peak and plateau of the [Ca2+] sub i transient elicited by 10 nM bradykinin, whereas isoflurane had no effect. When [Ca2+]i transients were elicited by 1 micro Meter bradykinin, halothane (1% and 5%) did not alter peak and plateau levels. Halothane and enflurane also decreased [Ca2+]i transients evoked by 1 micro Meter and 100 micro Meter adenosine triphosphate, whereas isoflurane also had no effect in this setting.     

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.     

Volatile Anesthetics Increase Intracellular Calcium in Cerebrocortical and Hippocampal Neurons

Background: An increase in intracellular calcium concentration ([Ca2+]i) in neurons has been proposed as an important effect of volatile anesthetics, because they alter signaling pathways that influence neurotransmission. However, the existing data for anesthetic-induced increases in [Ca2+]i conflict.

Methods: Changes in [Ca2+]i were measured using fura-2 fluorescence spectroscopy in rat cortical brain slices at 90, 185, 370, and 705 [micro sign]M isoflurane. To define the causes of an increase in [Ca2+]i, slices were studied in Ca2+-free medium, in the presence of Ca2+-channel blockers, and in the presence of the Ca2+-release inhibitor azumolene. The authors compared the effect of the volatile anesthetic with that of the nonanesthetic compound 1,2-dichlorohexafluorocyclobutane. Single-dose experiments in CA1 neurons in hippocampal slices with halothane (360 [micro sign]M) and in acutely dissociated CA1 neurons with halothane (360 [micro sign]M) and isoflurane (445 [micro sign]M) also were performed.

Results: Isoflurane at 0.5, 1, and 2 minimum alveolar concentrations increased basal [Ca2+]i in cortical slices in a dose-dependent manner (P < 0.05). This increase was not altered by Ca2+-channel blockers or Ca2+-free medium but was reduced 85% by azumolene. The nonanesthetic 1,2-dichlorohexafluorocyclobutane did not increase [Ca2+] (i). In dissociated CA1 neurons, isoflurane reversibly increased basal [Ca (2+)]i by 15 nM (P < 0.05). Halothane increased [Ca2+]i in dissociated CA1 neurons and CA1 neurons in hippocampal slices by approximately 30 nM (P < 0.05).     

Local Anesthetics Modulate Neuronal Calcium Signaling through Multiple Sites of Action

Background: Local anesthetics (LAs) are known to inhibit voltage-dependent Na+ channels, as well as K+ and Ca2+ channels, but with lower potency. Since cellular excitability and responsiveness are largely determined by intracellular Ca2+ availability, sites along the Ca2+ signaling pathways may be targets of LAs. This study was aimed to investigate the LA effects on depolarization and receptor-mediated intracellular Ca2+ changes and to examine the role of Na+ and K+ channels in such functional responses.

Methods: Effects of bupivacaine, ropivacaine, mepivacaine, and lidocaine (0.1-2.3 mm) on evoked [Ca2+]i transients were investigated in neuronal SH-SY5Y cell suspensions using Fura-2 as the intracellular Ca2+ indicator. Potassium chloride (KCl, 100 mm) and carbachol (1 mm) were individually or sequentially applied to evoke increases in intracellular Ca2+. Coapplication of LA and Na+/K+ channel blockers was used to evaluate the role of Na+ and K+ channels in the LA effect on the evoked [Ca2+]i transients.

Results: All four LAs concentration-dependently inhibited both KCl- and carbachol-evoked [Ca2+]i transients with the potency order bupivacaine > ropivacaine > lidocaine >= mepivacaine. The carbachol-evoked [Ca2+]i transients were more sensitive to LAs without than with a KCl prestimulation, whereas the LA-effect on the KCl-evoked [Ca2+]i transients was not uniformly affected by a carbachol prestimulation. Na+ channel blockade did not alter the evoked [Ca2+]i transients with or without a LA. In the absence of LA, K+ channel blockade increased the KCl-, but decreased the carbachol-evoked [Ca2+]i transients. A coapplication of LA and K+ channel blocker resulted in larger inhibition of both KCl- and carbachol-evoked [Ca2+]i transients than by LA alone.     

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 Volatile Anesthetics on N-methyl-d-aspartate Excitotoxicity in Primary Rat Neuronal-Glial Cultures

Background: Volatile anesthetics are known to ameliorate experimental ischemic brain injury. A possible mechanism is inhibition of excitotoxic cascades induced by excessive glutamatergic stimulation. This study examined interactions between volatile anesthetics and excitotoxic stress.

Methods: Primary cortical neuronal-glial cultures were exposed to N-methyl-d-aspartate (NMDA) or glutamate and isoflurane (0.1-3.3 mm), sevoflurane (0.1-2.9 mm), halothane (0.1-2.9 mm), or 10 [mu]m (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801). Lactate dehydrogenase release was measured 24 h later. In other cultures, effects of volatile anesthetics on Ca++ uptake and mitochondrial membrane potential were determined in the presence or absence of NMDA (0-200 [mu]m).

Results: Volatile anesthetics reduced excitotoxin induced lactate dehydrogenase release by up to 52% in a dose-dependent manner. At higher concentrations, this protection was reversed. When corrected for olive oil solubility, the three anesthetics offered equivalent protection. MK-801 provided near-complete protection. Ca++ uptake was proportionally reduced with increasing concentrations of anesthetic but did not account for reversal of protection at higher anesthetic concentrations. Given equivalent NMDA-induced Ca++ loads, cells treated with volatile anesthetic had greater lactate dehydrogenase release than those left untreated. At protective concentrations, volatile anesthetics partially inhibited NMDA-induced mitochondrial membrane depolarization. At higher concentrations, volatile anesthetics alone were sufficient to induce mitochondrial depolarization.     

Effects of Isoflurane on Intracellular Calcium and Myocardial Crossbridge Kinetics in Tetanized Papillary Muscles

Background: Isoflurane depresses the intracellular Ca2+ transient and force development during a twitch, but its effects on crossbridge cycling rates are difficult to predict because of the transient nature of the twitch. Measurements of the effects of isoflurane on crossbridge cycling kinetics during tetanic contractions, which provide a steady state level of activation in intact cardiac muscle, have not been previously reported.

Methods: Ferret right ventricular papillary muscles were isolated, and superficial cells were microinjected with the bioluminescent photoprotein aequorin to monitor the intracellular Ca2+ concentration. The rate of tension redevelopment (kTR) was measured during steady state isometric activation (tetanic stimulation, frequency 20 Hz, 1 [mu]m ryanodine, temperature = 30[degrees]C) in the absence of isoflurane (2, 6, and 12 mm extracellular [Ca2+]) and in the presence of 0.5, 1.0, and 1.5 minimum alveolar concentration isoflurane (12 mm extracellular [Ca2+]).

Results: Intracellular [Ca2+], isometric force, and kTR all increased when the extracellular [Ca2+] increased. Isoflurane (0.5, 1.0, and 1.5 minimum alveolar concentration) caused intracellular [Ca2+], isometric force, and kTR to decrease in a dose-dependent manner in the presence of 12 mm extracellular [Ca2+]. In the presence of increasing concentrations of isoflurane, the relation between intracellular [Ca2+] and force remained unchanged, whereas the relation between intracellular [Ca2+] and kTR was shifted toward higher [Ca2+].     

Volatile Anesthetics Selectively Inhibit the Calcium sup 2+ -transporting ATPase in Neuronal and Erythrocyte Plasma Membranes

Background: The activity of the plasma membrane Calcium2+ - transporting adenosine triphosphatase (PMCA) is inhibited by volatile anesthetics at clinical concentrations. The goal of the current study was to determine whether the inhibition is selective as compared to other adenosine triphosphatases (ATPases) and another group of general anesthetics, barbiturates. In addition, the authors determined whether the response to anesthetics of the enzymes in neuronal membranes is similar to that in erythrocyte membranes.

Methods: The effects of halothane, isoflurane, and sodium pentobarbital on four different ATPase activities were studied at 37 degrees C in two distinct plasma membrane preparations, human red blood cells and synaptosomal membranes from rat cerebellum.

Results: Inhibition patterns of the PMCA by halothane and isoflurane at anesthetic concentrations were very similar in red blood cells and synaptosomal membranes. The half-maximal inhibition (I50) occurred at 0.25-0.30 mM halothane and 0.30-0.32 mM isoflurane. The PMCA in both membranes was significantly more sensitive to the inhibitory action of volatile anesthetics (I50 = 0.75-1.15 minimum alveolar concentration) than were other ATPases, such as the Sodium sup +, Potassium sup + -ATPase (I50 [nearly equal] 3 minimum alveolar concentration) or Magnesium sup 2+ -ATPase (I50 greater or equal to 5 minimum alveolar concentration). In contrast, sodium pentobarbital inhibited the PMCA in both membranes only at [nearly equal] 100-200-fold above its anesthetic concentrations. The other ATPases were inhibited at similar pentobarbital concentrations (I50 = 11-22 mM).     

G-protein Activation Decreases Isoflurane Inhibition of N-type Ba2+ Currents

Background: G-protein activation mediates inhibition of N-type Ca2+ currents. Volatile anesthetics affect G-protein pathways at various levels, and activation of G-proteins has been shown to increase the volatile anesthetic potency for inhibiting the electrical-induced contraction in ileum. The authors investigated whether isoflurane inhibition of N-type Ba2+ currents was mediated by G-protein activation.

Methods: N-type Ba2+ currents were measured in the human neuronal SH-SY5Y cell line by using the whole cell voltage-clamp method.

Results: Isoflurane was found to have two effects on N-type Ba2+ currents. First, isoflurane reduced the magnitude of N-type Ba2+ currents to a similar extent (IC50 ~ 0.28 mm) in the absence and presence of GDP[beta]S (a nonhydrolyzable GDP analog). Interestingly, GTP[gamma]S (a nonhydrolyzable GTP analog and G-protein activator) in a dose-dependent manner reduced the isoflurane block; 120 [mu]m GTP[gamma]S completely eliminated the block of 0.3 mm isoflurane and reduced the apparent isoflurane potency by ~ 2.4 times (IC50 ~ 0.68 mm). Pretreatment with pertussis toxin or cholera toxin did not eliminate the GTP[gamma]S-induced protection against the isoflurane block. Furthermore, isoflurane reduced the magnitude of voltage-dependent G-protein-mediated inhibition of N-type Ba2+ currents, and this effect was eliminated by pretreatment with pertussis toxin or cholera toxin.     

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.     

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.     

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 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.     

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   

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. CONCLUSIONS: Depressed myofilament Ca2+ sensitivity contributes to the negative inotropic effects of isoflurane and halothane but not sevoflurane. The decrease in the Ca2+ transient is either responsible for or contributory to the negative inotropic effects of all three anesthetics and is either primarily the result of a decrease in fractional release (isoflurane and sevoflurane) or primarily the result of a decrease in SR Ca2+ content (halothane).