Isoflurane Reduction of Carbachol-evoked Cytoplasmic Calcium Transients Is Dependent on Caffeine-sensitive Calcium Stores

Experiments were performed on monolayers of SH-SY5Y human neuroblastoma cells (originally provided by June Biedler, Ph.D., Sloan-Kettering Institute for Cancer Research, Rye, NY at the providing time; Dr. Biedler is currently a Distinguished Resident Scientist, Fordham University, Bronx, NY), passages 60–94. Cells were cultured in RPMI1640 medium with l-glutamine, supplemented with penicillin (50 U/ml), streptomycin (50 μg/ml), and 12% fetal bovine serum at 37°C, in a humidified atmosphere containing 5% CO². All cell culture components were Gibco BRL products purchased from Life Technologies (Rockville, MD). Cells were plated on glass coverslips (25-mm diameter) at a density of approximately 2–4 × 10⁴cells/ml (2 ml/Petri dish) and used when they formed a confluent monolayer (i.e. , between 8–20 days after plating). The bath solution was a HEPES buffer containing (in mm) 140 NaCl, 5 KCl, 5 NaHCO³, 10 HEPES, 1 MgCl², 1.5 CaCl², 1 ATP, and 10 glucose (pH 7.4). All experiments were performed at 37°C, and the temperature was controlled with a Dual Heater controller TC-344A and an inline heater SH-27B (Warner Instruments Inc., Hamden CT). All the solutions and instruments were prewarmed to 37°C before being used. The exchange of the solution was done using a manifold, with its common outlet placed in the entrance of the heater inline. The solutions containing [200 mm] KCl (EM-Science), [10 mm] caffeine, [1 mm] carbachol, or [200 μm] ryanodine were prepared using the HEPES buffer. Saturated isoflurane (Ohmeda Caribe Inc., Guayana, PR) solutions were prepared in HEPES buffer 24 h in advance in gas-tight containers and diluted to the final concentration (1 mm) immediately before used as previously described. 16 

SH-SY5Y cells were loaded with the fluorescent Ca²⁺indicator Fura-2 17by incubating the cells on coverslips in the culture medium containing 5 μm of the acetoxymethyl ester of the dye (Fura-2 AM; Molecular Probes, Eugene, OR) for 30 min under culture conditions. 16After loading, cells were gently washed three times with the HEPES buffer, and the coverslips were placed into the perfusion chamber and perfused (250 μl/min) for 30 min with the buffer at 37°C to allow their equilibration with the experimental environment before being exposed to the various drugs. The bath solution alone and containing the individual or combination of drugs was perfused at 250 μl/min for various periods as indicated in the figure legends.

The perfusion chamber was set on an inverted microscope (DIAPHOT 300; Nikon, Melville, NY) equipped with a ×40 oil-immersion objective (N.A.1.30, Nikon). The microscope was attached to a high-speed multiwavelength illuminator (DeltaRAM V; Photon Technology International, Inc., PTI, Lawrenceville, NJ). The excitation wavelengths for Fura-2 (340 nm and 380 nm) were alternately (every 0.02 s) generated with a monochromator. The emitted fluorescence (from the alternated excitation at 340 and 380 nm) from 15–20 cells was filtered with the fluorescence barrier filter BA 515 nm, collected with a photomultiplier (PMT01–710, Photon Technology International), and digitized at 50 Hz.

Data collection and analysis was carried out with Felix (version 1.42a, Photon Technology International) and Clampfit (Pclamp 8; Axon Instruments, Foster City, CA) software. For each treatment (corresponding to data in each figure), experiments were performed under different conditions on sister cultures (same plating day) and on three to five culture sets (different plating days). The averaged traces shown in the figures were obtained by lining up the peak values for the evoked [Ca²⁺]^cyttransients. Unless otherwise indicated, the areas were obtained over a period of 300 s starting from the onset of the carbachol-evoked [Ca²⁺]^cyttransient on each trace. In the figures, the data represent the ratio (in protocols and steady states) or the Δ ratio (for peak and areas) of the emission of Fura-2 at 515 nm obtained with 340 and 380 nm excitations (ratio 340/380). Control steady-state levels were measured just before the addition of any compound, and the new steady-state level after the addition of a particular compound was measured just before the addition of the subsequent compound. Steady-state levels measurements are expressed as ratio (340/380) values.

Comparison between different groups was performed using the unpaired or paired two-tailed t  test with Sigma software (Jandel Scientific Corp., San Rafael, CA).

Atropine (1 μm), a muscarinic blocker, completely (>99%) blocked the main peak of the carbachol-evoked [Ca²⁺]^cyttransient (fig. 1, 90 sec ). Carbachol activated a small nonmuscarinic increase in [Ca²⁺]^cytthat was activated later than the main muscarinic-evoked [Ca²⁺]^cytincrease (fig. 1A) and it represented 7.4% of the total area of the carbachol-evoked [Ca²⁺]^cyttransient (fig. 1B, Total ). In the following sections, the area measurements for the carbachol-evoked [Ca²⁺]^cyttransients were performed over the first 300 s after the onset of the carbachol-evoked [Ca²⁺]^cyttransient; during this period, the nonmuscarinic component represents less than 2% of the area (fig. 1B, 1st 300 sec ).

Figure 2shows the averaged response for the carbachol-evoked [Ca²⁺]^cyttransient before and after the exposure to 1 mm isoflurane. Isoflurane significantly reduced the peak and the area of the carbachol-evoked [Ca²⁺]^cyttransient (fig. 2A and B). Continuous stimulation with 200 mm KCl reduced the carbachol-evoked [Ca²⁺]^cytresponse (fig. 2D,vs.  A). However, if the cells were continuously stimulated with 200 mm KCl, 1 mm isoflurane did not produce additional changes in the peak height or the area of the carbachol-evoked [Ca²⁺]^cytresponse (fig. 2D and E). Isoflurane produced a slight increase in the steady-state level of [Ca²⁺]^cytthat only became significant when the paired data were considered (fig. 2C). However, continuous KCl stimulation produced a large increase in the [Ca²⁺]^cytsteady-state level, and this increase was attenuated by isoflurane (fig. 2F). Isoflurane also reduced the peak and area values of the KCl-evoked [Ca²⁺]^cyttransient (fig. 3). We previously reported that isoflurane had a biphasic concentration-dependent effect on the KCl-evoked [Ca²⁺]^cyttransient, increasing the transient at low concentrations and decreasing it at higher concentrations. 16In our previous study, 1 mm isoflurane produced no significant change compared with the control value, 16but in this study it decreased the KCl-evoked [Ca²⁺]^cyttransient, suggesting that a sensitivity increase in the isoflurane effect mediates the reduction of the KCl-evoked [Ca²⁺]^cyttransient. This difference may have resulted from the different conditions under which the cells were grown (high-density confluent multilayer 16,vs.  monolayer). However, in both studies it is clear that continuos treatment with KCl eliminated the inhibition by both halothane 16and isoflurane (present study) of the carbachol-evoked [Ca²⁺]^cytresponse.

We then investigated whether a direct depletion of the caffeine-sensitive Ca²⁺stores could also eliminate the isoflurane-mediated reduction of the carbachol-evoked [Ca²⁺]^cyttransient. We previously showed that the carbachol-evoked [Ca²⁺]^cyttransient involves Ca²⁺release mostly from the inositol tri-phosphate-sensitive Ca²⁺store but also from caffeine-sensitive stores. 16In this study, we found that pretreatment with 10 mm caffeine reduced the carbachol-evoked [Ca²⁺]^cytresponse (fig. 4A,vs.  fig. 2A, controls ). In the presence of caffeine, isoflurane did not produce an additional reduction of the carbachol-evoked [Ca²⁺]^cytresponse (fig. 4), suggesting that this action of isoflurane requires a full caffeine-sensitive Ca²⁺store. The steady-state level of [Ca²⁺]^cytwas not affected by isoflurane or caffeine when added individually, and it only displayed a slightly significant increase when they were applied together (fig. 4C).

To test whether the isoflurane-mediated reduction of the carbachol-evoked [Ca²⁺]^cytrequired Ca²⁺release from caffeine-sensitive store through ryanodine-sensitive Ca²⁺release channels, we blocked ryanodine-sensitive Ca²⁺release channels by exposing the cells to 200 μm ryanodine. Because activation of Ca²⁺release channels is required to obtain maximal channel block by ryanodine, 18,19the following protocol was developed. Cells were pretreated for 5 min with ryanodine, exposed to a short KCl pulse (to activate ryanodine-sensitive Ca²⁺release channels), and after 18 min (time required for Ca²⁺refilling of intracellular Ca²⁺stores) exposed to carbachol. The level of channel block was determined by measuring the ryanodine action on two consecutive KCl-evoked [Ca²⁺]^cyttransients (fig. 5). When cells were exposed to ryanodine, the initial response was an increase in the [Ca²⁺]^cyt(fig. 5, arrow ), which has been reported to reflect activation of ryanodine-sensitive Ca²⁺channels. 19,20Ryanodine showed only a small blocking effect on the first KCl-evoked [Ca²⁺]^cytresponse (fig. 5C, 1st ), even when cells were exposed to ryanodine for 48 h (data not shown); however, ryanodine strongly blocked the second KCl-evoked [Ca²⁺]^cytresponse (fig. 5C, 2nd ). In contrast to the continuous exposure to KCl (fig. 2D), the KCl pulse did not eliminate the isoflurane-mediated reduction of the carbachol-evoked [Ca²⁺]^cyttransient (fig. 6); although the isoflurane-mediated area reduction was significant, the peak reduction did not reach a significant level (fig. 6A and B). Ryanodine pretreatment did not significantly decrease the carbachol-evoked [Ca²⁺]^cytresponse (peak or area) (fig. 6A and B, control vs.  fig. 6D and E, control ), in contrast to continuous exposure to KCl (fig. 2B, control vs.  E, control ) or caffeine treatment (fig. 4B, control vs.  fig. 2B, control ). However, ryanodine pretreatment still eliminated the isoflurane-mediated reduction of the carbachol-evoked [Ca²⁺]^cyttransient (fig. 6D and E), suggesting that the isoflurane-mediated reduction of the carbachol-evoked [Ca²⁺]^cyttransient requires Ca²⁺release from the caffeine-sensitive Ca²⁺stores through ryanodine-sensitive Ca²⁺-release channels. The steady-state level of [Ca²⁺]^cytwas slightly increased after the KCl pulse (fig. 6C) or after ryanodine pretreatment (fig. 6F); under both of these conditions, the addition of isoflurane did not produce additional changes in the steady-state level of [Ca²⁺]^cyt(fig. 6C and F).

The purpose of this study was to determine the intracellular source of the portion of the carbachol-evoked [Ca²⁺]^cyttransient that was reduced by volatile anesthetics. Our previous work suggested that this portion of Ca²⁺was derived from the caffeine-sensitive Ca²⁺store. 16We have demonstrated in this study that the portion of the carbachol-evoked [Ca²⁺]^cyttransient that is reduced by halothane is also reduced by isoflurane, that it arises from the caffeine-sensitive Ca²⁺store, and that it is released from the caffeine-sensitive store through ryanodine-sensitive Ca²⁺release channels.

The role of muscarinic receptors in analgesia/anesthesia is complex because, as discussed previously, both activation and blockade of muscarinic receptors have been shown to be analgesic. Our results indicate that, at the concentrations used, isoflurane blocks only part of the carbachol-evoked [Ca²⁺]^cytresponse, apparently at site that is distal to the muscarinic receptor. This isoflurane-sensitive component may be contributing to the anesthetic potency of isoflurane. The previously reported variable effects of muscarinic blockers on the minimum alveolar concentration of inhaled anesthetics could reflect differences in the magnitude of this isoflurane-sensitive component in various brain/spinal regions, where the muscarinic blockers were applied.

The experiments with caffeine show that the isoflurane-mediated blocking action on the carbachol-evoked [Ca²⁺]^cyttransient requires that caffeine-sensitive Ca²⁺stores are not depleted. We show that ryanodine at 200 μm, which is known to be a maximal blocking concentration, 19,21reduces the KCl-evoked [Ca²⁺]^cytresponse by 50% without reducing the carbachol-evoked [Ca²⁺]^cytresponse, and eliminates the isoflurane-mediated blocking effect on the carbachol-evoked [Ca²⁺]^cyttransient. This finding indicates that Ca²⁺release through ryanodine channels is required for the isoflurane-mediated blocking action on the carbachol-evoked [Ca²⁺]^cyttransient. There are several possible explanations for the mechanism by which isoflurane and halothane may reduce the carbachol-evoked [Ca²⁺]^cyttransient. One possibility is that isoflurane and halothane actually block the opening of the ryanodine-sensitive Ca²⁺release channel; however, this appears unlikely because halothane, 22and perhaps isoflurane, 16actually enhance the opening of the ryanodine-sensitive Ca²⁺release channel. A second possibility is that isoflurane decreases the level of Ca²⁺in the caffeine-sensitive Ca²⁺stores by enhancing Ca²⁺release through ryanodine-sensitive Ca²⁺channels. However, further experiments are required to determine whether the isoflurane action on the carbachol-evoked [Ca²⁺]^cyttransient results from a direct or indirect action of isoflurane on the ryanodine-sensitive Ca²⁺channels. As an indirect action, Ca²⁺is released from the caffeine -sensitive stores and shuttled to the inositol tri-phosphate-sensitive stores through volatile-anesthetic-sensitive transporters (e.g. , similar to the binding of Ca²⁺, entering through l-type Ca²⁺channels, to calmodulin), 23,24or Ca²⁺could be released from the caffeine-sensitive stores and inhibits a volatile-anesthetic-sensitive channel (e.g. , capacitative channels at the plasma membrane). In addition, Ca²⁺released through the ryanodine-sensitive Ca²⁺channels could be in close vicinity to Ca²⁺transport systems that are activated by volatile anesthetics such as the sarco/endoplasmic reticulum Ca²⁺-pumping adenosine triphosphatase. 25At this time there is no evidence to implicate any of these or other possible mechanisms. In SH-SY5Y cells, muscarinic agonists increase [Ca²⁺]^cytand the level of inositol tri-phosphate, 26and in smooth muscle cells halothane decreases the carbachol-evoked [Ca²⁺]^cytincrease and reduces inositol tri-phosphate production. 27Therefore, isoflurane may inhibit the carbachol-evoked [Ca²⁺]^cyttransient by reducing the inositol tri-phosphate production. However, a decrease in the inositol tri-phosphate production or any direct isoflurane action on muscarinic receptors (even if they take place) would not explain our observations. If the latter actions were responsible for the isoflurane-mediated decrease in the carbachol evoked [Ca²⁺]^cyttransient, the isoflurane effect under the various conditions studied (continuous KCl, ryanodine or caffeine treatment) would still be expected.

In summary, this study shows that isoflurane interferes with muscarinic-evoked [Ca²⁺]^cyttransients through a mechanism downstream from the muscarinic receptors. Isoflurane reduces the carbachol-evoked [Ca²⁺]^cyttransient. The carbachol-evoked [Ca²⁺]^cyttransient is dependent on Ca²⁺derived from both inositol tri-phosphate- and caffeine-sensitive Ca²⁺stores. Reduction of the carbachol-evoked [Ca²⁺]^cyttransient by isoflurane is eliminated either when the caffeine-sensitive Ca²⁺store is depleted or when the release of Ca²⁺from the caffeine-sensitive Ca²⁺store is blocked by ryanodine. The relationship between the modulation of Ca²⁺homeostasis by volatile anesthetics and the mechanisms of anesthesia remains unclear and must be examined in more complex systems.