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Pain Medicine  |   October 2003
Isoflurane Reduction of Carbachol-evoked Cytoplasmic Calcium Transients Is Dependent on Caffeine-sensitive Calcium Stores
Author Affiliations & Notes
  • Alexandra Corrales, B.S.
    *
  • Fang Xu, Ph.D.
  • Zayra Garavito-Aguilar, B.S.
    *
  • Thomas J. J. Blanck, M.D., Ph.D.
  • Esperanza Recio-Pinto, Ph.D.
    §
  • *Research Assistant, †Assistant Professor of Anesthesiology, ‡Professor of Anesthesiology, §Associate Professor of Anesthesiology, Anesthesiology Department, New York University School of Medicine.
  • Received from the Anesthesiology Department, New York University School of Medicine, New York, New York.
Article Information
Pain Medicine
Pain Medicine   |   October 2003
Isoflurane Reduction of Carbachol-evoked Cytoplasmic Calcium Transients Is Dependent on Caffeine-sensitive Calcium Stores
Anesthesiology 10 2003, Vol.99, 882-888. doi:
Anesthesiology 10 2003, Vol.99, 882-888. doi:
MANY muscarinic functions are relevant to anesthesia and alterations in muscarinic activity affect the anesthetic/analgesic potency of various drugs. Brain stem muscarinic signaling modulates the level of consciousness, 1 whereas cortical muscarinic signaling affects memory and learning. 2,3 Spinal muscarinic receptors inhibit glutamate release 4 and enhance γ-aminobutyric acid release. 5 Muscarinic agonists have been reported to enhance antinociceptive effects, and this enhancement is blocked by pretreatment with M1, M2, or M3 muscarinic receptor antagonists or with antisense inhibition of M1 receptors. 6–8 However, there are also reports indicating that muscarinic block enhances the analgesic/anesthetic action of various drugs, and that some of these drugs induce muscarinic block. 9,10 Halothane and isoflurane have been shown to depress muscarinic receptor function, 11–14 and inhibition of the muscarinic signaling pathway has been shown to have variable effects on the minimal alveolar anesthetic concentration of inhaled anesthetics. 15 Therefore, the role of muscarinic receptors in analgesia/anesthesia seems to be complex and can either augment or attenuate the anesthetic potency of the volatile anesthetics.
Muscarinic activation leads to a transient elevation of cytoplasmic Ca2+([Ca2+]cyt) through Ca2+release from intracellular stores. We investigated the action of volatile anesthetics on the [Ca2+]cyttransients evoked by a muscarinic agonist, carbachol, in human SH-SY5Y cells. We previously reported that halothane reduced the carbachol-evoked [Ca2+]cyttransient in a concentration-dependent manner, and that sustained stimulation with KCl eliminated this effect of halothane. 16 The Ca2+contributing to the carbachol-evoked [Ca2+]cyttransient seems to derive from both inositol tri-phosphate-sensitive and caffeine-sensitive Ca2+stores. 16 The purpose of this investigation was to determine which source of Ca2+underlying the carbachol-evoked [Ca2+]cyttransient was reduced by isoflurane.
Materials and Methods
Cell Culture and Solutions
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 
Ca2+Measurements
SH-SY5Y cells were loaded with the fluorescent Ca2+indicator Fura-2 17 by 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. 16 After 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 Analysis
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.
Statistical Analysis
Comparison between different groups was performed using the unpaired or paired two-tailed t  test with Sigma software (Jandel Scientific Corp., San Rafael, CA).
Results
Atropine Blocked the Carbachol-evoked [Ca2+]cytTransient
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  ).
Fig. 1. Atropine blocks the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 μm atropine. (A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of atropine. Number sign  indicates the location of the carbachol-evoked [Ca2+]cytpeak in the presence of atropine. The inset  shows the steady-state levels of [Ca2+]cyin the absence and presence of atropine. (B  ) The maximal response at the time of the carbachol-evoked [Ca2+]cytpeak in the absence of atropine (90 sec  ) and in the presence of atropine (520 sec  ) (left  ); and the area values during either the entire (Total  ) or the first 300 s (1st 300 sec  ) of the carbachol-evoked [Ca2+]cyttransient (right  ). Asterisk  indicates a statistically significant difference (P  < 0.0001, paired t  test) between control and atropine conditions.
Fig. 1. Atropine blocks the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 μm atropine. (A 
	) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of atropine. Number sign 
	indicates the location of the carbachol-evoked [Ca2+]cytpeak in the presence of atropine. The inset 
	shows the steady-state levels of [Ca2+]cyin the absence and presence of atropine. (B 
	) The maximal response at the time of the carbachol-evoked [Ca2+]cytpeak in the absence of atropine (90 sec 
	) and in the presence of atropine (520 sec 
	) (left 
	); and the area values during either the entire (Total 
	) or the first 300 s (1st 300 sec 
	) of the carbachol-evoked [Ca2+]cyttransient (right 
	). Asterisk 
	indicates a statistically significant difference (P 
	< 0.0001, paired t 
	test) between control and atropine conditions.
Fig. 1. Atropine blocks the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 μm atropine. (A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of atropine. Number sign  indicates the location of the carbachol-evoked [Ca2+]cytpeak in the presence of atropine. The inset  shows the steady-state levels of [Ca2+]cyin the absence and presence of atropine. (B  ) The maximal response at the time of the carbachol-evoked [Ca2+]cytpeak in the absence of atropine (90 sec  ) and in the presence of atropine (520 sec  ) (left  ); and the area values during either the entire (Total  ) or the first 300 s (1st 300 sec  ) of the carbachol-evoked [Ca2+]cyttransient (right  ). Asterisk  indicates a statistically significant difference (P  < 0.0001, paired t  test) between control and atropine conditions.
×
Isoflurane Reduced the Carbachol-evoked [Ca2+]cytTransient and Continuous Stimulation with KCl Eliminated the Isoflurane-mediated Reduction of the Carbachol-evoked [Ca2+]cytTransient
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. 2Dvs.  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. 16 In our previous study, 1 mm isoflurane produced no significant change compared with the control value, 16 but 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 16 and isoflurane (present study) of the carbachol-evoked [Ca2+]cytresponse.
Fig. 2. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient, and this reduction is eliminated by continuous exposure to KCl. (A, B  ) Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation. (A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. (B  ) The mean peak and area values of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C  ) The corresponding [Ca2+]cytsteady-state levels (left  ) and the paired [Ca2+]cytsteady-state levels (control and isoflurane measurements performed on the same cells) obtained from data shown in figures 2 and 4, in the absence and presence of isoflurane. (D, E  ) Cells were treated as described for panels A  and B  but in the presence of 200 mm KCl. The application of KCl was started 5 min before the carbachol stimulation. (D  ) The averaged carbachol-evoked [Ca2+]cyttransients in the presence of KCl and in the absence and presence of isoflurane. (E  ) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the presence of KCl and in the absence and presence of isoflurane. (F  ) The corresponding [Ca2+]cytsteady-state levels. Asterisks  indicate a statistically significant difference (**P  < 0.001, unpaired t  test; *P  < 0.005, paired t  test) between control and a given group.
Fig. 2. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient, and this reduction is eliminated by continuous exposure to KCl. (A, B 
	) Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation. (A 
	) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. (B 
	) The mean peak and area values of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C 
	) The corresponding [Ca2+]cytsteady-state levels (left 
	) and the paired [Ca2+]cytsteady-state levels (control and isoflurane measurements performed on the same cells) obtained from data shown in figures 2 and 4, in the absence and presence of isoflurane. (D, E 
	) Cells were treated as described for panels A 
	and B 
	but in the presence of 200 mm KCl. The application of KCl was started 5 min before the carbachol stimulation. (D 
	) The averaged carbachol-evoked [Ca2+]cyttransients in the presence of KCl and in the absence and presence of isoflurane. (E 
	) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the presence of KCl and in the absence and presence of isoflurane. (F 
	) The corresponding [Ca2+]cytsteady-state levels. Asterisks 
	indicate a statistically significant difference (**P 
	< 0.001, unpaired t 
	test; *P 
	< 0.005, paired t 
	test) between control and a given group.
Fig. 2. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient, and this reduction is eliminated by continuous exposure to KCl. (A, B  ) Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation. (A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. (B  ) The mean peak and area values of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C  ) The corresponding [Ca2+]cytsteady-state levels (left  ) and the paired [Ca2+]cytsteady-state levels (control and isoflurane measurements performed on the same cells) obtained from data shown in figures 2 and 4, in the absence and presence of isoflurane. (D, E  ) Cells were treated as described for panels A  and B  but in the presence of 200 mm KCl. The application of KCl was started 5 min before the carbachol stimulation. (D  ) The averaged carbachol-evoked [Ca2+]cyttransients in the presence of KCl and in the absence and presence of isoflurane. (E  ) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the presence of KCl and in the absence and presence of isoflurane. (F  ) The corresponding [Ca2+]cytsteady-state levels. Asterisks  indicate a statistically significant difference (**P  < 0.001, unpaired t  test; *P  < 0.005, paired t  test) between control and a given group.
×
Fig. 3. Isoflurane reduces the KCl-evoked [Ca2+]cyttransient. The conditions are the same as described in figure 1B. (A  ) The averaged KCl-evoked [Ca2+]cyttransients in the absence and presence of 1 mm isoflurane (ISO). (B  ) The mean peak and area values for the KCl-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. The corresponding steady-state levels of [Ca2+]cytare shown in figure 2F. Asterisks  indicate a statistically significant difference (P  < 0.001, unpaired t  test) between control and isoflurane conditions.
Fig. 3. Isoflurane reduces the KCl-evoked [Ca2+]cyttransient. The conditions are the same as described in figure 1B. (A 
	) The averaged KCl-evoked [Ca2+]cyttransients in the absence and presence of 1 mm isoflurane (ISO). (B 
	) The mean peak and area values for the KCl-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. The corresponding steady-state levels of [Ca2+]cytare shown in figure 2F. Asterisks 
	indicate a statistically significant difference (P 
	< 0.001, unpaired t 
	test) between control and isoflurane conditions.
Fig. 3. Isoflurane reduces the KCl-evoked [Ca2+]cyttransient. The conditions are the same as described in figure 1B. (A  ) The averaged KCl-evoked [Ca2+]cyttransients in the absence and presence of 1 mm isoflurane (ISO). (B  ) The mean peak and area values for the KCl-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. The corresponding steady-state levels of [Ca2+]cytare shown in figure 2F. Asterisks  indicate a statistically significant difference (P  < 0.001, unpaired t  test) between control and isoflurane conditions.
×
Isoflurane-mediated Reduction of the Carbachol-evoked [Ca2+]cytTransient Required Full Caffeine-sensitive Ca2+Stores
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. 16 In this study, we found that pretreatment with 10 mm caffeine reduced the carbachol-evoked [Ca2+]cytresponse (fig. 4Avs.  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).
Fig. 4. Caffeine treatment eliminates isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the presence of 10 mm caffeine (CAF) and in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation, and the application of caffeine was started 5 min before the carbachol stimulation. (A  ) The averaged carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (B  ) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C  ) The corresponding steady-state levels of [Ca2+]cyt. Asterisk  indicates a statistically significant difference (P  < 0.05, unpaired t  test) between control and a given group.
Fig. 4. Caffeine treatment eliminates isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the presence of 10 mm caffeine (CAF) and in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation, and the application of caffeine was started 5 min before the carbachol stimulation. (A 
	) The averaged carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (B 
	) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C 
	) The corresponding steady-state levels of [Ca2+]cyt. Asterisk 
	indicates a statistically significant difference (P 
	< 0.05, unpaired t 
	test) between control and a given group.
Fig. 4. Caffeine treatment eliminates isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the presence of 10 mm caffeine (CAF) and in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation, and the application of caffeine was started 5 min before the carbachol stimulation. (A  ) The averaged carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (B  ) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C  ) The corresponding steady-state levels of [Ca2+]cyt. Asterisk  indicates a statistically significant difference (P  < 0.05, unpaired t  test) between control and a given group.
×
Isoflurane-mediated Reduction of the Carbachol-evoked [Ca2+]cytTransient Required Ca2+Release from the Caffeine-sensitive Store through the Ryanodine-sensitive Ca2+Release Channels
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, 18,19 the 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. 19,20 Ryanodine 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).
Fig. 5. Ryanodine decreases the KCl-evoked [Ca2+]cyttransient. Cells were stimulated two consecutive times with 200 mm KCl in the absence (A  ) and presence (B  ) of 200 μm ryanodine (Ry). The application of ryanodine was started 5 min before the first KCl stimulation, and KCl was applied for 2 min. The time interval between the first and second KCl pulse was 18 min. When cells were exposed to ryanodine, the initial response was an increase in the [Ca2+]cyt(indicated with an arrow  ). (C  ) The mean peak values for the first and the second KCl-evoked [Ca2+]cyttransients in the absence (open circles  ) and presence (filled circles  ) of ryanodine. Asterisk  and number sign  indicate a statistically significant difference (P  < 0.02 and P  < 0.05, respectively; unpaired t  test) between control and ryanodine groups.
Fig. 5. Ryanodine decreases the KCl-evoked [Ca2+]cyttransient. Cells were stimulated two consecutive times with 200 mm KCl in the absence (A 
	) and presence (B 
	) of 200 μm ryanodine (Ry). The application of ryanodine was started 5 min before the first KCl stimulation, and KCl was applied for 2 min. The time interval between the first and second KCl pulse was 18 min. When cells were exposed to ryanodine, the initial response was an increase in the [Ca2+]cyt(indicated with an arrow 
	). (C 
	) The mean peak values for the first and the second KCl-evoked [Ca2+]cyttransients in the absence (open circles 
	) and presence (filled circles 
	) of ryanodine. Asterisk 
	and number sign 
	indicate a statistically significant difference (P 
	< 0.02 and P 
	< 0.05, respectively; unpaired t 
	test) between control and ryanodine groups.
Fig. 5. Ryanodine decreases the KCl-evoked [Ca2+]cyttransient. Cells were stimulated two consecutive times with 200 mm KCl in the absence (A  ) and presence (B  ) of 200 μm ryanodine (Ry). The application of ryanodine was started 5 min before the first KCl stimulation, and KCl was applied for 2 min. The time interval between the first and second KCl pulse was 18 min. When cells were exposed to ryanodine, the initial response was an increase in the [Ca2+]cyt(indicated with an arrow  ). (C  ) The mean peak values for the first and the second KCl-evoked [Ca2+]cyttransients in the absence (open circles  ) and presence (filled circles  ) of ryanodine. Asterisk  and number sign  indicate a statistically significant difference (P  < 0.02 and P  < 0.05, respectively; unpaired t  test) between control and ryanodine groups.
×
Fig. 6. Ryanodine treatment eliminates the isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min after a transient (2 min) stimulation with 200 mm KCl, in the absence (A–C  ) or presence (D–F  ) of 200 μm ryanodine (Ry) and in the absence or presence of 1 mm isoflurane (ISO), respectively. The application of the KCl stimulation was done 18 min before the carbachol stimulation. Ryanodine application was started 5 min before the KCl stimulation, and the isoflurane application was started 10 min before the carbachol stimulation. The averaged carbachol-evoked [Ca2+]cyttransients (A  ), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (B  ), following the KCl pulse are shown in the absence and presence of isoflurane. In the presence of ryanodine, the averaged carbachol-evoked [Ca2+]cyttransients (D  ), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (E  ), following the KCl pulse are shown in the absence and presence of ISO. (C, F  ) The corresponding [Ca2+]cytsteady-state levels. Symbols indicate a statistically significant difference between control and a given group (*P  < 0.005, @P  < 0.02 unpaired t  test; #P  < 0.005, **P  < 0.0001, paired t  test).
Fig. 6. Ryanodine treatment eliminates the isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min after a transient (2 min) stimulation with 200 mm KCl, in the absence (A–C 
	) or presence (D–F 
	) of 200 μm ryanodine (Ry) and in the absence or presence of 1 mm isoflurane (ISO), respectively. The application of the KCl stimulation was done 18 min before the carbachol stimulation. Ryanodine application was started 5 min before the KCl stimulation, and the isoflurane application was started 10 min before the carbachol stimulation. The averaged carbachol-evoked [Ca2+]cyttransients (A 
	), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (B 
	), following the KCl pulse are shown in the absence and presence of isoflurane. In the presence of ryanodine, the averaged carbachol-evoked [Ca2+]cyttransients (D 
	), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (E 
	), following the KCl pulse are shown in the absence and presence of ISO. (C, F 
	) The corresponding [Ca2+]cytsteady-state levels. Symbols indicate a statistically significant difference between control and a given group (*P 
	< 0.005, @P 
	< 0.02 unpaired t 
	test; #P 
	< 0.005, **P 
	< 0.0001, paired t 
	test).
Fig. 6. Ryanodine treatment eliminates the isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min after a transient (2 min) stimulation with 200 mm KCl, in the absence (A–C  ) or presence (D–F  ) of 200 μm ryanodine (Ry) and in the absence or presence of 1 mm isoflurane (ISO), respectively. The application of the KCl stimulation was done 18 min before the carbachol stimulation. Ryanodine application was started 5 min before the KCl stimulation, and the isoflurane application was started 10 min before the carbachol stimulation. The averaged carbachol-evoked [Ca2+]cyttransients (A  ), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (B  ), following the KCl pulse are shown in the absence and presence of isoflurane. In the presence of ryanodine, the averaged carbachol-evoked [Ca2+]cyttransients (D  ), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (E  ), following the KCl pulse are shown in the absence and presence of ISO. (C, F  ) The corresponding [Ca2+]cytsteady-state levels. Symbols indicate a statistically significant difference between control and a given group (*P  < 0.005, @P  < 0.02 unpaired t  test; #P  < 0.005, **P  < 0.0001, paired t  test).
×
Discussion
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. 16 We 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, 19,21 reduces 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, 22 and perhaps isoflurane, 16 actually 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), 23,24 or 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. 25 At 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, 26 and in smooth muscle cells halothane decreases the carbachol-evoked [Ca2+]cytincrease and reduces inositol tri-phosphate production. 27 Therefore, 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.
References
Brann MR, Ellis J, Jorgensen H, Hill-Eubanks D, Jones SV: Muscarinic acetylcholine receptor subtypes: Localization and structure/function. Prog Brain Res 1993; 98: 121–7Brann, MR Ellis, J Jorgensen, H Hill-Eubanks, D Jones, SV
Fibiger HC, Damsma G, Day JC: Behavioral pharmacology and biochemistry of central cholinergic neurotransmission. Adv Exp Med Biol 1991; 295: 399–414Fibiger, HC Damsma, G Day, JC
Durieux ME: Muscarinic signaling in the central nervous system. Recent developments and anesthetic implications. A nesthesiology 1996; 84: 173–89Durieux, ME
Li DP, Chen SR, Pan YZ, Levey AI, Pan HL: Role of presynaptic muscarinic and GABA(B) receptors in spinal glutamate release and cholinergic analgesia in rats. J Physiol 2002; 543: 807–18Li, DP Chen, SR Pan, YZ Levey, AI Pan, HL
Baba H, Kohno T, Okamoto M, Goldstein PA, Shimoji K, Yoshimura M: Muscarinic facilitation of GABA release in substantia gelatinosa of the rat spinal dorsal horn. J Physiol 1998; 508: 83–93Baba, H Kohno, T Okamoto, M Goldstein, PA Shimoji, K Yoshimura, M
Naguib M, Yaksh TL: Characterization of muscarinic receptor subtypes that mediate antinociception in the rat spinal cord. Anesth Analg 1997; 85: 847–53Naguib, M Yaksh, TL
Bartolini A, Ghelardini C, Fantetti L, Malcangio M, Malmberg-Aiello P, Giotti A: Role of muscarinic receptor subtypes in central antinociception. Br J Pharmacol 1992; 105: 77–82Bartolini, A Ghelardini, C Fantetti, L Malcangio, M Malmberg-Aiello, P Giotti, A
Ghelardini C, Galeotti N, Bartolini A: Loss of muscarinic antinociception by antisense inhibition of M(1) receptors. Br J Pharmacol 2000; 129: 1633–40Ghelardini, C Galeotti, N Bartolini, A
Shiraishi M, Minami K, Uezono Y, Yanagihara N, Shigematsu A: Inhibition by tramadol of muscarinic receptor-induced responses in cultured adrenal medullary cells and in Xenopus laevis oocytes expressing cloned M1 receptors. J Pharmacol Exp Ther 2001; 299: 255–60Shiraishi, M Minami, K Uezono, Y Yanagihara, N Shigematsu, A
Durieux ME: Inhibition by ketamine of muscarinic acetylcholine receptor function. Anesth Analg 1995; 81: 57–62Durieux, ME
Minami K, Yanagihara N, Toyohira Y, Tsutsui M, Shigematsu A, Wada A, Izumi F: Isoflurane inhibits nicotinic acetylcholine receptor-mediated 22Na+ influx and muscarinic receptor-evoked cyclic GMP production in cultured bovine adrenal medullary cells. Naunyn Schmiedebergs Arch Pharmacol 1994; 349: 223–9Minami, K Yanagihara, N Toyohira, Y Tsutsui, M Shigematsu, A Wada, A Izumi, F
Durieux ME: Halothane inhibits signaling through m1 muscarinic receptors expressed in Xenopus oocytes. A nesthesiology 1995; 82: 174–82Durieux, ME
Minami K, Minami M, Harris RA: Inhibition of 5-hydroxytryptamine type 2A receptor-induced currents by n  -alcohols and anesthetics. J Pharmacol Exp Ther 1997; 281: 1136–43Minami, K Minami, M Harris, RA
Do SH, Kamatchi GL, Durieux ME: The effects of isoflurane on native and chimeric muscarinic acetylcholine receptors: The role of protein kinase C. Anesth Analg 2001; 93: 375–81Do, SH Kamatchi, GL Durieux, ME
Ishizawa Y: Selective blockade of muscarinic receptor subtypes in the brain stem reticular formation in rats: Effects on anesthetic requirements. Brain Res 2000; 873: 124–6Ishizawa, Y
Xu F, Zhang J, Recio-Pinto E, Blanck TJ: Halothane and isoflurane augment depolarization-induced cytosolic Ca2+transients and attenuate carbachol-stimulated Ca2+transients. A nesthesiology 2000; 92: 1746–56Xu, F Zhang, J Recio-Pinto, E Blanck, TJ
Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–50Grynkiewicz, G Poenie, M Tsien, RY
Tanna B, Welch W, Ruest L, Sutko JL, Williams AJ: Interactions of a reversible ryanoid (21-amino-9alpha-hydroxy-ryanodine) with single sheep cardiac ryanodine receptor channels. J Gen Physiol 1998; 112: 55–69Tanna, B Welch, W Ruest, L Sutko, JL Williams, AJ
Fill M, Copello JA: Ryanodine receptor calcium release channels. Physiol Rev 2002; 82: 893–922Fill, M Copello, JA
Fessenden JD, Wang Y, Moore RA, Chen SR, Allen PD, Pessah IN: Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line. Biophys J 2000; 79: 2509–25Fessenden, JD Wang, Y Moore, RA Chen, SR Allen, PD Pessah, IN
Zimanyi I, Buck E, Abramson JJ, Mack MM, Pessah IN: Ryanodine induces persistent inactivation of the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum. Mol Pharmacol 1992; 42: 1049–57Zimanyi, I Buck, E Abramson, JJ Mack, MM Pessah, IN
Pancrazio JJ, Lynch C III: Differential anesthetic-induced opening of calcium-dependent large conductance channels in isolated ventricular myocytes. Pflugers Arch 1994; 429: 134–6Pancrazio, JJ Lynch, C
Peterson BZ, DeMaria CD, Adelman JP, Yue DT: Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type calcium channels. Neuron 1999; 22: 549–58Peterson, BZ DeMaria, CD Adelman, JP Yue, DT
Zuhlke RD, Pitt GS, Deisseroth K, Tsien RW, Reuter H: Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 1999; 399: 159–62Zuhlke, RD Pitt, GS Deisseroth, K Tsien, RW Reuter, H
Karon BS, Autry JM, Shi Y, Garnett CE, Inesi G, Jones LR, Kutchai H, Thomas DD: Different anesthetic sensitivities of skeletal and cardiac isoforms of the Ca-ATPase. Biochemistry 1999; 38: 9301–7Karon, BS Autry, JM Shi, Y Garnett, CE Inesi, G Jones, LR Kutchai, H Thomas, DD
Martin AK, Nahorski SR, Willars GB: Complex relationship between Ins(1, 4, 5)P3 accumulation and Ca2+-signalling in a human neuroblastoma reveled by cellular differentiation. Br J Pharmacol 1999; 126: 1559–66Martin, AK Nahorski, SR Willars, GB
Yamakage M, Kohro S, Matsuzaki T, Tsuchida H, Namiki A: Role of intracellular Ca2+ stores in the inhibitory effect of halothane on airway smooth muscle contraction. A nesthesiology 1998; 89: 165–73Yamakage, M Kohro, S Matsuzaki, T Tsuchida, H Namiki, A
Fig. 1. Atropine blocks the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 μm atropine. (A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of atropine. Number sign  indicates the location of the carbachol-evoked [Ca2+]cytpeak in the presence of atropine. The inset  shows the steady-state levels of [Ca2+]cyin the absence and presence of atropine. (B  ) The maximal response at the time of the carbachol-evoked [Ca2+]cytpeak in the absence of atropine (90 sec  ) and in the presence of atropine (520 sec  ) (left  ); and the area values during either the entire (Total  ) or the first 300 s (1st 300 sec  ) of the carbachol-evoked [Ca2+]cyttransient (right  ). Asterisk  indicates a statistically significant difference (P  < 0.0001, paired t  test) between control and atropine conditions.
Fig. 1. Atropine blocks the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 μm atropine. (A 
	) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of atropine. Number sign 
	indicates the location of the carbachol-evoked [Ca2+]cytpeak in the presence of atropine. The inset 
	shows the steady-state levels of [Ca2+]cyin the absence and presence of atropine. (B 
	) The maximal response at the time of the carbachol-evoked [Ca2+]cytpeak in the absence of atropine (90 sec 
	) and in the presence of atropine (520 sec 
	) (left 
	); and the area values during either the entire (Total 
	) or the first 300 s (1st 300 sec 
	) of the carbachol-evoked [Ca2+]cyttransient (right 
	). Asterisk 
	indicates a statistically significant difference (P 
	< 0.0001, paired t 
	test) between control and atropine conditions.
Fig. 1. Atropine blocks the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 μm atropine. (A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of atropine. Number sign  indicates the location of the carbachol-evoked [Ca2+]cytpeak in the presence of atropine. The inset  shows the steady-state levels of [Ca2+]cyin the absence and presence of atropine. (B  ) The maximal response at the time of the carbachol-evoked [Ca2+]cytpeak in the absence of atropine (90 sec  ) and in the presence of atropine (520 sec  ) (left  ); and the area values during either the entire (Total  ) or the first 300 s (1st 300 sec  ) of the carbachol-evoked [Ca2+]cyttransient (right  ). Asterisk  indicates a statistically significant difference (P  < 0.0001, paired t  test) between control and atropine conditions.
×
Fig. 2. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient, and this reduction is eliminated by continuous exposure to KCl. (A, B  ) Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation. (A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. (B  ) The mean peak and area values of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C  ) The corresponding [Ca2+]cytsteady-state levels (left  ) and the paired [Ca2+]cytsteady-state levels (control and isoflurane measurements performed on the same cells) obtained from data shown in figures 2 and 4, in the absence and presence of isoflurane. (D, E  ) Cells were treated as described for panels A  and B  but in the presence of 200 mm KCl. The application of KCl was started 5 min before the carbachol stimulation. (D  ) The averaged carbachol-evoked [Ca2+]cyttransients in the presence of KCl and in the absence and presence of isoflurane. (E  ) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the presence of KCl and in the absence and presence of isoflurane. (F  ) The corresponding [Ca2+]cytsteady-state levels. Asterisks  indicate a statistically significant difference (**P  < 0.001, unpaired t  test; *P  < 0.005, paired t  test) between control and a given group.
Fig. 2. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient, and this reduction is eliminated by continuous exposure to KCl. (A, B 
	) Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation. (A 
	) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. (B 
	) The mean peak and area values of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C 
	) The corresponding [Ca2+]cytsteady-state levels (left 
	) and the paired [Ca2+]cytsteady-state levels (control and isoflurane measurements performed on the same cells) obtained from data shown in figures 2 and 4, in the absence and presence of isoflurane. (D, E 
	) Cells were treated as described for panels A 
	and B 
	but in the presence of 200 mm KCl. The application of KCl was started 5 min before the carbachol stimulation. (D 
	) The averaged carbachol-evoked [Ca2+]cyttransients in the presence of KCl and in the absence and presence of isoflurane. (E 
	) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the presence of KCl and in the absence and presence of isoflurane. (F 
	) The corresponding [Ca2+]cytsteady-state levels. Asterisks 
	indicate a statistically significant difference (**P 
	< 0.001, unpaired t 
	test; *P 
	< 0.005, paired t 
	test) between control and a given group.
Fig. 2. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient, and this reduction is eliminated by continuous exposure to KCl. (A, B  ) Cells were exposed to 1 mm carbachol (CAB) for 2 min in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation. (A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. (B  ) The mean peak and area values of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C  ) The corresponding [Ca2+]cytsteady-state levels (left  ) and the paired [Ca2+]cytsteady-state levels (control and isoflurane measurements performed on the same cells) obtained from data shown in figures 2 and 4, in the absence and presence of isoflurane. (D, E  ) Cells were treated as described for panels A  and B  but in the presence of 200 mm KCl. The application of KCl was started 5 min before the carbachol stimulation. (D  ) The averaged carbachol-evoked [Ca2+]cyttransients in the presence of KCl and in the absence and presence of isoflurane. (E  ) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the presence of KCl and in the absence and presence of isoflurane. (F  ) The corresponding [Ca2+]cytsteady-state levels. Asterisks  indicate a statistically significant difference (**P  < 0.001, unpaired t  test; *P  < 0.005, paired t  test) between control and a given group.
×
Fig. 3. Isoflurane reduces the KCl-evoked [Ca2+]cyttransient. The conditions are the same as described in figure 1B. (A  ) The averaged KCl-evoked [Ca2+]cyttransients in the absence and presence of 1 mm isoflurane (ISO). (B  ) The mean peak and area values for the KCl-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. The corresponding steady-state levels of [Ca2+]cytare shown in figure 2F. Asterisks  indicate a statistically significant difference (P  < 0.001, unpaired t  test) between control and isoflurane conditions.
Fig. 3. Isoflurane reduces the KCl-evoked [Ca2+]cyttransient. The conditions are the same as described in figure 1B. (A 
	) The averaged KCl-evoked [Ca2+]cyttransients in the absence and presence of 1 mm isoflurane (ISO). (B 
	) The mean peak and area values for the KCl-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. The corresponding steady-state levels of [Ca2+]cytare shown in figure 2F. Asterisks 
	indicate a statistically significant difference (P 
	< 0.001, unpaired t 
	test) between control and isoflurane conditions.
Fig. 3. Isoflurane reduces the KCl-evoked [Ca2+]cyttransient. The conditions are the same as described in figure 1B. (A  ) The averaged KCl-evoked [Ca2+]cyttransients in the absence and presence of 1 mm isoflurane (ISO). (B  ) The mean peak and area values for the KCl-evoked [Ca2+]cyttransients in the absence and presence of isoflurane. The corresponding steady-state levels of [Ca2+]cytare shown in figure 2F. Asterisks  indicate a statistically significant difference (P  < 0.001, unpaired t  test) between control and isoflurane conditions.
×
Fig. 4. Caffeine treatment eliminates isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the presence of 10 mm caffeine (CAF) and in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation, and the application of caffeine was started 5 min before the carbachol stimulation. (A  ) The averaged carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (B  ) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C  ) The corresponding steady-state levels of [Ca2+]cyt. Asterisk  indicates a statistically significant difference (P  < 0.05, unpaired t  test) between control and a given group.
Fig. 4. Caffeine treatment eliminates isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the presence of 10 mm caffeine (CAF) and in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation, and the application of caffeine was started 5 min before the carbachol stimulation. (A 
	) The averaged carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (B 
	) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C 
	) The corresponding steady-state levels of [Ca2+]cyt. Asterisk 
	indicates a statistically significant difference (P 
	< 0.05, unpaired t 
	test) between control and a given group.
Fig. 4. Caffeine treatment eliminates isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min in the presence of 10 mm caffeine (CAF) and in the absence or presence of 1 mm isoflurane (ISO). The application of isoflurane was started 10 min before the carbachol stimulation, and the application of caffeine was started 5 min before the carbachol stimulation. (A  ) The averaged carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (B  ) The mean peak and area values for the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (C  ) The corresponding steady-state levels of [Ca2+]cyt. Asterisk  indicates a statistically significant difference (P  < 0.05, unpaired t  test) between control and a given group.
×
Fig. 5. Ryanodine decreases the KCl-evoked [Ca2+]cyttransient. Cells were stimulated two consecutive times with 200 mm KCl in the absence (A  ) and presence (B  ) of 200 μm ryanodine (Ry). The application of ryanodine was started 5 min before the first KCl stimulation, and KCl was applied for 2 min. The time interval between the first and second KCl pulse was 18 min. When cells were exposed to ryanodine, the initial response was an increase in the [Ca2+]cyt(indicated with an arrow  ). (C  ) The mean peak values for the first and the second KCl-evoked [Ca2+]cyttransients in the absence (open circles  ) and presence (filled circles  ) of ryanodine. Asterisk  and number sign  indicate a statistically significant difference (P  < 0.02 and P  < 0.05, respectively; unpaired t  test) between control and ryanodine groups.
Fig. 5. Ryanodine decreases the KCl-evoked [Ca2+]cyttransient. Cells were stimulated two consecutive times with 200 mm KCl in the absence (A 
	) and presence (B 
	) of 200 μm ryanodine (Ry). The application of ryanodine was started 5 min before the first KCl stimulation, and KCl was applied for 2 min. The time interval between the first and second KCl pulse was 18 min. When cells were exposed to ryanodine, the initial response was an increase in the [Ca2+]cyt(indicated with an arrow 
	). (C 
	) The mean peak values for the first and the second KCl-evoked [Ca2+]cyttransients in the absence (open circles 
	) and presence (filled circles 
	) of ryanodine. Asterisk 
	and number sign 
	indicate a statistically significant difference (P 
	< 0.02 and P 
	< 0.05, respectively; unpaired t 
	test) between control and ryanodine groups.
Fig. 5. Ryanodine decreases the KCl-evoked [Ca2+]cyttransient. Cells were stimulated two consecutive times with 200 mm KCl in the absence (A  ) and presence (B  ) of 200 μm ryanodine (Ry). The application of ryanodine was started 5 min before the first KCl stimulation, and KCl was applied for 2 min. The time interval between the first and second KCl pulse was 18 min. When cells were exposed to ryanodine, the initial response was an increase in the [Ca2+]cyt(indicated with an arrow  ). (C  ) The mean peak values for the first and the second KCl-evoked [Ca2+]cyttransients in the absence (open circles  ) and presence (filled circles  ) of ryanodine. Asterisk  and number sign  indicate a statistically significant difference (P  < 0.02 and P  < 0.05, respectively; unpaired t  test) between control and ryanodine groups.
×
Fig. 6. Ryanodine treatment eliminates the isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min after a transient (2 min) stimulation with 200 mm KCl, in the absence (A–C  ) or presence (D–F  ) of 200 μm ryanodine (Ry) and in the absence or presence of 1 mm isoflurane (ISO), respectively. The application of the KCl stimulation was done 18 min before the carbachol stimulation. Ryanodine application was started 5 min before the KCl stimulation, and the isoflurane application was started 10 min before the carbachol stimulation. The averaged carbachol-evoked [Ca2+]cyttransients (A  ), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (B  ), following the KCl pulse are shown in the absence and presence of isoflurane. In the presence of ryanodine, the averaged carbachol-evoked [Ca2+]cyttransients (D  ), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (E  ), following the KCl pulse are shown in the absence and presence of ISO. (C, F  ) The corresponding [Ca2+]cytsteady-state levels. Symbols indicate a statistically significant difference between control and a given group (*P  < 0.005, @P  < 0.02 unpaired t  test; #P  < 0.005, **P  < 0.0001, paired t  test).
Fig. 6. Ryanodine treatment eliminates the isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min after a transient (2 min) stimulation with 200 mm KCl, in the absence (A–C 
	) or presence (D–F 
	) of 200 μm ryanodine (Ry) and in the absence or presence of 1 mm isoflurane (ISO), respectively. The application of the KCl stimulation was done 18 min before the carbachol stimulation. Ryanodine application was started 5 min before the KCl stimulation, and the isoflurane application was started 10 min before the carbachol stimulation. The averaged carbachol-evoked [Ca2+]cyttransients (A 
	), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (B 
	), following the KCl pulse are shown in the absence and presence of isoflurane. In the presence of ryanodine, the averaged carbachol-evoked [Ca2+]cyttransients (D 
	), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (E 
	), following the KCl pulse are shown in the absence and presence of ISO. (C, F 
	) The corresponding [Ca2+]cytsteady-state levels. Symbols indicate a statistically significant difference between control and a given group (*P 
	< 0.005, @P 
	< 0.02 unpaired t 
	test; #P 
	< 0.005, **P 
	< 0.0001, paired t 
	test).
Fig. 6. Ryanodine treatment eliminates the isoflurane-mediated reduction of the carbachol-evoked [Ca2+]cyttransient. Cells were exposed to 1 mm carbachol (CAB) for 2 min after a transient (2 min) stimulation with 200 mm KCl, in the absence (A–C  ) or presence (D–F  ) of 200 μm ryanodine (Ry) and in the absence or presence of 1 mm isoflurane (ISO), respectively. The application of the KCl stimulation was done 18 min before the carbachol stimulation. Ryanodine application was started 5 min before the KCl stimulation, and the isoflurane application was started 10 min before the carbachol stimulation. The averaged carbachol-evoked [Ca2+]cyttransients (A  ), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (B  ), following the KCl pulse are shown in the absence and presence of isoflurane. In the presence of ryanodine, the averaged carbachol-evoked [Ca2+]cyttransients (D  ), and the mean peak and area values for the carbachol-evoked [Ca2+]cyttransients (E  ), following the KCl pulse are shown in the absence and presence of ISO. (C, F  ) The corresponding [Ca2+]cytsteady-state levels. Symbols indicate a statistically significant difference between control and a given group (*P  < 0.005, @P  < 0.02 unpaired t  test; #P  < 0.005, **P  < 0.0001, paired t  test).
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