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.
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.
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.
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 (VAs) alter Ca2+-dependent signaling in neuronal preparations, including brain slices, isolated synaptosomes, 1–3and cultured neural cells. 4–7They have inhibitory effects on excitability and neurotransmitter release, which are linked with a parallel reduction of cytosolic Ca2+([Ca2+]cyt). Because voltage-dependent calcium channels (VDCCs) play a key role in controlling Ca2+entry and in initiating cellular responses to stimulation through an elevation of [Ca2+]cyt, they are postulated to be a major target of VAs. However, intracellular calcium can also be increased without VDCC activation through receptor-mediated pathways. Net cellular responsiveness is dependent on the Ca2+availability controlled by the Ca2+-regulatory system, consisting of VDCCs, plasma membrane Ca2+–adenosine triphosphatase (PMCA), sarcoplasmic–endoplasmic reticular Ca2+–ATPase (SERCA), the Na+–Ca2+exchanger, and mitochondrial Ca2+sequestration. Different studies showed that VAs also have an inhibitory effect on PMCA 8–10and the Na+–Ca2+exchanger, 11,12but a facilitating effect on Ca2+release or leakage from the intracellular stores. 13,14VA inhibition of VDCCs alone would lead to a lower [Ca2+]cytafter stimulation, but inhibition of the Ca2+-removal mechanisms and enhanced release from intracellular stores would lead to a higher [Ca2+]cyt. To answer the question:“How do VAs depress excitable cells” requires the evaluation of the Ca2+homeostatic mechanisms that might be affected by VAs.
In isolated rat synaptosomes, we demonstrated that halothane and isoflurane inhibited K+-evoked [Ca2+]cyttransient. 15Within synaptosomes, which are pinched-off nerve endings, only a small amount of mitochondria can be detected, other organelles, however, are scarce. 16Therefore, the capacity for intracellular Ca2+regulation by synaptosomes is relatively limited compared with intact cells. Also, as resealed vesicles, synaptosomes may potentially have nonspecific leakage pathways. We, therefore, chose neuron-like human SH-SY5Y neuroblastoma cells to study VA effects on Ca2+homeostasis. These excitable cells express many properties of mature sympathetic neurons, including noradrenaline synthesis, L- and N-type VDCCs, muscarinic receptors, and extensive G proteins. 15,16The current study evaluated the Ca2+responses of intact neuron-like cells to VA exposure and depolarization, and to the stimulation of muscarinic receptors. Our results show distinct effects of VAs on K+depolarization and carbachol-mediated [Ca2+]cytresponse in human neuroblastoma SH-SY5Y cells. Experiments using sequential stimulation suggested two possible novel mechanisms to explain these data.
Materials and Methods
All chemicals used for this study were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise indicated.
Undifferentiated human SH-SY5Y neuroblastoma cells (originally provided by Dr. J. Biedler, Sloan-Kettering Institute for Cancer Research, Rye, New York), passages 56-80, were maintained in RPMI 1640 medium (Gibco BRL, Rockville, MD) containing 12% fetal bovine serum (Gibco BRL) and penicillin (100 U/ml) plus streptomycin (100 μg/ml), split and replated weekly or biweekly.
Cells in confluent culture were loaded with 5 μm fura-2 for 30 min at 37°C in the original culture medium. After removal of the dye-containing medium, the cells (still attached) were rinsed twice with fresh Dulbecco phosphate-buffered saline, without CaCl2and MgCl2but with addition of 10 mm glucose (pH 7.4), then gently resuspended in the same saline. After a centrifugation at 300 rpm, cells were resuspended in Dulbecco phosphate-buffered saline with 10 mm glucose addition, aliquoted and stored on ice until use. Ca2+measurements were performed in a closed cuvette (with an effective working volume of 3.9 ml) at 37°C in an SLM 8000 Aminco fluorescence spectrofluorometer (Aminco, Urban, IL) in the same Dulbecco phosphate-buffered saline. The volume of total reaction solution was between 2 and 2.5 ml. To investigate the VA effects on the VDCC-mediated [Ca2+]cytincrease, cells were allowed to equilibrate at 37°C for 5 min, then 1.5 mm CaCl2was added 5 min before the addition of 100 mm KCl. Fluorescence ratios were monitored at 510 nm after excitation at 340 and 380 nm. Maximal and minimal fluorescence ratios were obtained by using 0.01% sodium dodecyl sulphate and 15 mm ethylene diamine tetraacetic acid, and [Ca2+]cytwas then determined from the fluorescence ratios based on the method of Grynkiewicz et al. 17and a Kdof fura for Ca2+of 224 nm. In the VA treatment groups, cells were preincubated with 0.05, 0.15, 0.25, 0.5, and 1.0 mm halothane or isoflurane for 10 min at 37°C before CaCl2addition. 15In another experimental series, cells were challenged with 1 mm carbachol (a muscarinic receptor agonist and Ca2+releaser from the inositol triphosphate (IP3)–sensitive store) instead of KCl to study the VA effects on the receptor-mediated [Ca2+]cytincrease without directly activating VDCCs.
To investigate the contribution of different calcium stores and regulatory mechanisms on evoked [Ca2+]cytincreases, sequential stimulation was applied: the cells, in the presence and absence of VA, were challenged by either 100 mm KCl followed by 1 mm carbachol, or 1 mm carbachol followed by 100 mm KCl, and the [Ca2+]cytchanges on stimulant additions were monitored as described herein previously. In addition, effects of caffeine (a releaser of intracellular Ca2+) and of thapsigargin (a specific inhibitor of endoplasmic reticular Ca2+pumps, commonly used for depleting Ca2+stores) on the [Ca2+]cytchanges evoked by sequential stimulation were also studied in the absence of VA.
Volatile Anesthetic Application and Quantification
Dulbecco phosphate-buffered saline saturated with halothane or isoflurane (> 8 h under stirring in closed glass vials) was used. The VA content in these saturated solutions was determined by using a gas chromatograph (GC-17A; Shimadzu Corp., Kyoto, Japan) as described previously. 15The saturated halothane and isoflurane concentration in Dulbecco phosphate-buffered saline was 18.03 ± 0.16 mm and 14.07 ± 0.21 mm, respectively. The actual working concentrations of VAs in the liquid phase in the measuring cuvette were also confirmed by gas chromatographic measurements.
Comparison between control and VA-treated groups or among different groups was performed using Dunnett or Student-Newman-Keuls tests, respectively, using SIGMASTAT software (Jandel Scientific Corp., San Rafael, CA). Significance was considered to be P < 0.05.
Volatile Anesthetics Enhanced K+-evoked [Ca2+]cytTransient
Enhancement Decreased with Increasing Volatile Anesthetic Concentration
The basal [Ca2+]cytmeasured was 86 ± 24 nm (mean ± SD, n = 180) in SH-SY5Y cells incubated in nominally Ca2+-free buffer. Upon adding 1.5 mm CaCl2, the [Ca2+]cytreached a higher steady state level of 153 ± 30 nm (mean ± SD, n = 60). In the presence of 1.5 mm CaCl2, cells responded to depolarization induced by adding 100 mm KCl, with a rapid increase in [Ca2+]cytfollowed by a decay to a plateau level (fig. 1A). This response is triggered by an influx of external Ca2+through VDCCs opened by membrane depolarization and cannot be induced in the absence of extracellular Ca2+(data not shown). Accordingly, pretreatment with 10 μm nitrendipine (a specific inhibitor of L-type VDCCs) or 100 nm ω-conotoxin GVIa (CgTx, a specific inhibitor of N-type VDCCs) inhibited K+-evoked [Ca2+]cytincrease by 34 and 70%, respectively. Coapplication of 10 μm nitrendipine and 100 nm CgTx almost abolished the K+-evoked response (96% inhibition), indicating that L- and N-Type Ca2+channels are the predominant VDCCs in SH-SY5Y cells (fig. 1B), which is consistent with the findings of others. 18,19
In SH-SY5Y cells, halothane and isoflurane (0.05–1 mm in reaction solutions, 0.2–4 and 0.15–3 minimum alveolar concentration [MAC] for halothane and isoflurane, respectively) both enhanced the magnitude of the K+-evoked [Ca2+]cytpeak in a biphasic manner. The magnitude of the enhancement was greatest at relatively low VA concentrations, became smaller with the increasing VA concentration, and eventually became insignificant at the highest VA concentration (1 mm) used (fig. 2). Moreover, the potency of halothane and isoflurane was comparable.
Carbachol-evoked [Ca2+]cytTransient Was Decreased by Volatile Anesthetic
In SH-SY5Y cells, carbachol has been shown to increase [Ca2+]cytby increasing IP3production through activation of muscarinic receptors, resulting in Ca2+release from IP3-sensitive stores. 20,21In the presence of 1.5 mm CaCl2, 1 mm carbachol generated a change in [Ca2+]cytin the SH-SY5Y cells similar to that observed with addition of 100 mm KCl (fig. 1A). The carbachol-evoked response can also be induced in nominally Ca2+-free buffer (data not shown), indicating an independence from external Ca2+, which is consistent with the carbachol effect mediated through Ca2+release from IP3-sensitive store. In contrast to K+-evoked [Ca2+]cyttransient, the carbachol-evoked peak response was decreased by halothane (0.05–1 mm, 0.2–4 MAC) in a dose-dependent manner (fig. 3), indicating that the Ca2+loading into or release from the intracellular stores or removal of [Ca2+]cytwas affected by halothane pretreatment.
Sequential Stimulation Indicates a Dynamic Interaction between Distinctive Intracellular Ca2+Stores
The existence of two distinct intracellular Ca2+stores (i.e. , caffeine- and IP 3-sensitive stores) is known. 22,23Our experiments using K+and carbachol to induce [Ca2+]cyttransients indicated a possible interaction of VA with the intracellular Ca2+stores. To investigate whether distinctive Ca2+stores were involved in the VA effects on evoked [Ca2+]cytchanges and what the contributions were of the different stores, experiments were performed with two different sequential stimulation patterns to evoke [Ca2+]cytchanges: either 100 mm KCl followed by 1 mm carbachol or 1 mm carbachol followed by 100 mm KCl, in the presence and absence of various pharmacologic agents. Cells responded to the stimulation sequence KCl (100 mm) → carbachol (1 mm), with two [Ca2+]cytpeaks (fig. 4A). In the presence of 10 mm caffeine, a stimulator of ryanodine receptors, both peaks were largely decreased (fig. 4B), consistent with caffeine-induced Ca2+depletion of caffeine-sensitive stores. Depletion took place without the occurrence of observable changes in baseline [Ca2+]cytlevels before stimulation (before caffeine: 105 ± 5 nm; after caffeine: 102 ± 5 nm; mean SEM; n = 28). In the presence of 1 μm thapsigargin, an inhibitor of the endoplasmic reticular Ca2+pump (commonly used for depleting the IP3-sensitive Ca2+stores), however, only the carbachol peak was abolished (fig. 4C). A coapplication of 10 mm caffeine plus 1 μm thapsigargin (fig. 4D) showed effects on both peaks: the K+peak was largely reduced and the carbachol peak abolished, suggesting that each individual agent has its specific target. Although pretreatment with 10 mm caffeine plus 1 mm carbachol eliminated the K+peak (fig. 4E), pretreatment with 1 μm thapsigargin plus 1 mm carbachol only reduced the K+peak (fig. 4F) to the comparable extent, as with pretreatment solely with 1 mm carbachol (fig. 5). Statistically significant differences among drug treatments are summarized in figures 4G and 4H. Cells also responded to the stimulation sequence carbachol → KCl with two [Ca2+]cytpeaks (fig. 5A). However, the K+peak after the carbachol peak was much smaller than the K+peak elicited alone (fig. 4G,vs. fig. 5, the control columns). Caffeine (10 mm) treatment significantly reduced the carbachol peak, and almost abolished the following K+induced peak (fig. 5). The reduction in the carbachol peak (50%) was less than when the carbachol peak followed the K+peak (fig. 4H,vs. fig. 5, the control columns). thapsigargin (1 m) application significantly decreased the carbachol peak, but not the following K+peak (fig. 5). After a coapplication of 10 mm caffeine and 1 m thapsigargin, both peaks were eliminated (fig. 5). Statistical analyses of data obtained from these series of experiments are shown in figure 5.
Multiple Effects of Volatile Anesthetic on Ca2+Homeostasis in Undifferentiated Human SH-SY5Y Neuroblastoma Cells
Using two different sequential stimulation patterns, possible differential effects of volatile anesthetic on the voltage-sensitive Ca2+entry and on the contribution of intracellular Ca2+to the evoked [Ca2+]cyttransient were investigated. The cells were stimulated either by 100 mm KCl followed by 1 mm carbachol (figs. 6A and B) or by 1 mm carbachol followed by 100 mm KCl (figs. 7A and B). In the stimulation sequence KCl → carbachol, the KCl-induced peak [Ca2+]cytresponse was enhanced by halothane and isoflurane (0.05–1 mm;figs. 2 and 6C). The resultant carbachol-evoked peak response was not statistically significantly affected by halothane (fig. 6D) and isoflurane (not shown).
In the stimulation sequence carbachol → KCl, the K+-induced peak resulting from carbachol stimulation in the controls (no halothane) was significantly lower than the K+peak without carbachol prestimulation (comparing fig. 7Dwith fig. 6C). However, the carbachol-evoked peak response in [Ca2+]cytwas dose-dependently reduced by halothane toward the level found in the carbachol peak resulting from KCl prestimulation (comparing fig. 7Cwith fig. 6D). The K+-induced peak resulting from carbachol stimulation was significantly increased by halothane in a similar manner as observed in halothane-treated cells stimulated by KCl without carbachol prestimulation (comparing fig. 7Dwith fig. 6C). In this case, however, the K+peak was increased toward the level found in the nontreated cells stimulated solely with KCl.
Halothane and isoflurane both increased the baseline level of [Ca2+]cytbefore stimulation with KCl or carbachol (table 1). However, the halothane and isoflurane induced increase in baseline [Ca2+]cytbefore stimulation did not correlate with the concentration dependence of VA action on the K+-evoked [Ca2+]cytpeak (figs. 2 and 6C) or with the concentration dependence of VA action on the carbachol-evoked [Ca2+]cytpeak, suggesting that more than one mechanism is involved in the halothane and isoflurane modulation of Ca2+homeostasis.
The current study shows that halothane and isoflurane at clinically relevant concentrations both affected the K+- and carbachol-evoked [Ca2+]cyttransients in undifferentiated human SH-SY5Y neuroblastoma cells. The direction (increase or decrease) and magnitude of the VA action on the stimulated Ca2+responses was found to be dependent on the type of stimulation (KCl or carbachol) and on the Ca2+content within the intracellular Ca2+stores. Halothane and isoflurane enhanced the K+-evoked [Ca2+]cyttransient whether intracellular Ca2+stores were full or partially depleted. Halothane reduced the carbachol-evoked [Ca2+]cyttransient when the intracellular Ca2+stores were full, but had no effect on the carbachol-evoked [Ca2+]cyttransient when a K+stimulation had partially depleted the caffeine-sensitive intracellular Ca2+stores. Previously reported effects of VA on various Ca2+homeostatic mechanisms, including PMCA, Na+–Ca2+exchanger, Ca2+-activated Ca2+release, and VDCCs, are not sufficient to explain all of our observations. Therefore, two other mechanisms of modification of Ca2+homeostasis are considered: one that necessitates a volatile anesthetic–sensitive unidirectional Ca2+-translocation pathway between the caffeine-sensitive and the IP3-sensitive Ca2+stores and one that necessitates the existence of volatile anesthetic–sensitive plasma membrane capacitative Ca2+entry channels.
Volatile Anesthetics Enhanced the K+-evoked [Ca2+]cytTransient
The observed augmentation by halothane and isoflurane of the K+-evoked [Ca2+]cyttransient was surprising because we previously showed that halothane and isoflurane decreased the K+-evoked [Ca2+]cyttransient in rat brain synaptosomes, 15and the VDCC activity in SH-SY5Y cells. 4However, an increase in resting [Ca2+]cytby VAs was reported by several authors using rat brain slices and dissociated neurons, 24,25and enhancing and inhibitory 26effects by VAs on NMDA-evoked [Ca2+]cytresponses have been observed in cultured neural cells and in rat brain slices. In the current study, membrane depolarization by high K+induces Ca2+entry through VDCCs and this in turn induces Ca2+release from intracellular Ca2+stores. The augmentation of the K+-stimulated [Ca2+]cytby halothane and isoflurane (fig. 2) is not related to a potentiating effect of Ca2+entry through the VDCCs because VAs have been shown to decrease L-type 5and N-type (our recent observation) Ca2+currents in human SH-SY5Y neuroblastoma cells. The observed enhancement in the K+-stimulated [Ca2+]cyttransient by halothane and isoflurane is most likely related to an alteration in Ca2+regulation at sites different than VDCCs. The overall VA effect on the K+-induced peak represents the sum of two opposing effects: (1) inhibition of VDCCs, leading to a decrease in the [Ca2+]cyt, and (2) inhibition of intracellular Ca2+-removal mechanisms (PMCA, SERCA, or Na+–Ca2+exchanger) and enhancement of Ca2+release from intracellular Ca2+stores (ryanodine or IP3receptors), leading to an increase in [Ca2+]cyt. At lower VA levels, VDCCs are only partially inhibited so that the VA effects leading to an increase in the K+-evoked [Ca2+]cyttransient will be predominant. At high VA levels, however, increased inhibition of VDCCs and decreased Ca2+entry would result in a decrease of the amount of intracellular Ca2+released. This interpretation would be consistent with the observation that the VA-caused increase in the K+-evoked [Ca2+]cyttransient is manifested at the lowest VA, whereas its magnitude decreases as the VA increases.
Little is known about VA effects on Ca2+–ATPases or Na+–Ca2+exchangers of SH-SY5Y cells. Inhibitory effects by VAs have been observed on PMCA activities in several cultured neural cell lines 9,10,25and in isolated rat cortical synaptosomes. 8Inhibitory VA effects on Na+–Ca2+exchange activity have also been shown in cultured neural cells 11and in neonatal myocardium, 12but not in isolated synaptic plasma membranes. 27The effect of the VA on SERCA depends on the SERCA isoform. 28,29Volatile anesthetics inhibit SERCA 2a in cardiac sarcoplasmic reticulum, whereas they activate SERCA 1 in skeletal muscle sarcoplasmic reticulum. 29The predominant isoform in the brain is SERCA 2b 30and the VA effect on the neuronal SERCA has not been defined. Volatile anesthetics can also cause Ca2+release, and decrease Ca2+uptake into sarcoplasmic reticulum by activating sarcoplasmic reticulum Ca2+-release channels. 14,31In cardiac and smooth muscle, halothane, but not isoflurane, enhances Ca2+release through the ryanodine-sensitive Ca2+release channel. 32,33We found that halothane and isoflurane had similar potencies for the K+-evoked [Ca2+]cyttransient, suggesting that the neuronal ryanodine-sensitive Ca2+release channels might be affected equally by these VAs.
Volatile Anesthetics Reduced Carbachol-evoked [Ca2+]cytTransient
Unlike the K+-evoked [Ca2+]cyttransient primarily mediated by VDCCs through membrane depolarization, the carbachol-stimulated [Ca2+]cyttransient does not necessitate external Ca2+, is mediated through an activation of muscarinic receptors, and involves Ca2+release mainly from the IP3-sensitive stores. In contrast to the K+-evoked [Ca2+]cyttransient, the receptor-mediated peak [Ca2+]cyttransient was inhibited by halothane (fig. 3). This is consistent with previous observations in different muscle preparations in which halothane treatment caused a net loss of Ca2+from IP3-sensitive stores, 31,34,35which would in turn lead to a reduction in Ca2+availability during carbachol stimulation. In SH-SY5Y cells, muscarinic agonists increase [Ca2+]cytand IP3level in a concentration-dependent manner. 36Halothane has been shown to decrease the carbachol-induced [Ca2+]cytincrease and reduce IP3formation in an airway smooth-muscle preparation. 37Several other studies have shown that VAs depress IP3-mediated [Ca2+]cytincrease in response to stimuli such as vasopressin 38and thyrotropin-releasing hormone. These observations would indicate that inhibition of carbachol-evoked [Ca2+]cyttransient by VA in SH-SY5Y cells might be attributed to reduction in IP3production. In addition, VAs may also decrease the carbachol-evoked [Ca2+]cyttransient by reducing the activity of muscarinic receptors. 39,40In SH-SY5Y cells, however, halothane has been reported to enhance IP3formation. 41However, these authors also reported halothane inhibition of K+-evoked [Ca2+]cytand a lack of effect of halothane on carbachol-evoked [Ca2+]cyt, results that are dramatically different than ours, which we attribute to either a different stage of differentiation of SH-SY5Y cells or a difference in experimental conditions, such as the period of time the cells are exposed to halothane. 7Experiments in more comparable experimental conditions need to be undertaken to explain these differences.
Differences in baseline [Ca2+]cytmight affect the activation of cellular homeostatic Ca2+-control mechanisms. In the current study, in addition to the variation of baseline [Ca2+]cytof approximately 28% (see Results), we also observed variation in the peak heights of stimulated [Ca2+]cyttransients from experiment to experiment. Nevertheless, halothane and isoflurane consistently enhanced the K+-evoked [Ca2+]cyttransient, reduced the carbachol-evoked [Ca2+]cyttransient before KCl application, and only had a significant effect on the carbachol-evoked [Ca2+]cyttransient after KCl application at the highest anesthetic concentration.
Sequential Stimulation Using KCl and Carbachol
The sequential stimulation data suggest that the caffeine-sensitive and IP3-sensitive Ca2+stores are distinct, but each partially contributes to the K+-evoked and carbachol-evoked Ca2+transient (fig. 8A). The inhibition of the carbachol-evoked [Ca2+]cyttransient by halothane and isoflurane can be attributed to the action of these drugs at several sites, but also suggests, but does not prove, that the IP3-mediated Ca2+release is not stimulated by halothane and isoflurane, unlike the caffeine-sensitive Ca2+release. Conversely, the loss of inhibition by halothane and isoflurane of the carbachol-evoked [Ca2+]cyttransient after KCl stimulation suggests the presence of a novel VA action. One possibility is that there is a VA-sensitive, unidirectional Ca2+-translocation pathway between the caffeine-sensitive and the IP3-sensitive Ca2+stores. This unidirectional Ca2+pathway is reduced in a concentration-dependent manner by halothane and isoflurane and is only apparent when the caffeine-sensitive Ca2+store is relatively full. This could be a direct or an indirect unidirectional Ca2+path, such as a vesicle transport of Ca2+(fig. 8A). A second possible explanation for the loss of inhibition by halothane and isoflurane of the carbachol-evoked [Ca2+]cyttransient after KCl stimulation relates to the potential activity of plasma membrane capacitative Ca2+channels. Plasma membrane capacitative Ca2+channels have been shown to be activated during release of Ca2+from IP3-sensitive Ca2+stores, but also to be inactivated by the elevation of cytosolic Ca2+. 42If the capacitative channels are sensitive to VA, one could observe VA concentration-dependent inhibition of the carbachol-evoked [Ca2+]cyt, as we have. Conversely, KCl stimulation raises [Ca2+]cyt, which might lead to inactivation of capacitative Ca2+channels and removal of halothane and isoflurane inhibition of the carbachol-evoked [Ca2+]cyt. Our experiments monitored global intracellular Ca2+concentration; however, an evaluation of the subcellular heterogeneity of Ca2+concentration would probably help to clarify the existence of the proposed novel mechanisms.
Changes in [Ca2+]cytcaused by halothane and isoflurane can contribute to anesthesia in at least two ways. First, increasing Ca2+release from intracellular stores could facilitate the release of inhibitory neurotransmitters, such as has been suggested for the VA enhancement of γ-aminobutyric acid type A receptor–mediated inhibition in rat hippocampal slices. 43Second, increases in [Ca2+]cytare known to activate Ca2+-dependent K+channels, which play an important role in repolarizing membrane potential. 44The increase in [Ca2+]cytwe observed would be expected to prolong the activity of Ca2+-dependent K+channels, leading to prolonged hyperpolarization and a reduction in neuronal excitability.
In summary, the distinct effects of VA on K+- and carbachol-evoked [Ca2+]cyttransients can be attributed to their effects on multiple Ca2+homeostatic mechanisms in a particular cell model. The net change in intracellular Ca2+level at stimulation depends on the contributions made by several regulatory mechanisms. The previously reported VA effects on individual Ca2+homeostatic mechanisms were consistent with most of our results but could not explain the loss of VA sensitivity of the carbachol-evoked [Ca2+]cyttransient with KCl pretreatment. The data, therefore, are consistent with the presence of a novel VA-sensitive interaction between the caffeine- and IP3-sensitive stores or action of halothane and isoflurane on capacitative Ca2+channels.