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Meeting Abstracts  |   October 2004
Isoflurane Reduces the Carbachol-evoked Ca2+Influx in Neuronal Cells
Author Affiliations & Notes
  • Alexandra Corrales, B.S.
    *
  • Fang Xu, Ph.D.
  • Zayra V. 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, New York, New York.
Article Information
Meeting Abstracts   |   October 2004
Isoflurane Reduces the Carbachol-evoked Ca2+Influx in Neuronal Cells
Anesthesiology 10 2004, Vol.101, 895-901. doi:
Anesthesiology 10 2004, Vol.101, 895-901. doi:
THE role of muscarinic receptors in analgesia and anesthesia is controversial. In the brainstem muscarinic receptors modulate the level of consciousness,1 and in cortical regions2,3 and striatum4 they affect memory and learning. At the spinal level muscarinic receptors inhibit glutamate release5 and enhance γ-aminobutyric acid release.6 Muscarinic agonists have been reported to enhance antinociceptive effects.7–11 However, there are also reports showing that muscarinic block enhances the analgesic or anesthetic action of various drugs.12,13 Halothane and isoflurane have been reported to depress muscarinic receptor function,14–17 and inhibition of the muscarinic signaling has variable effects on the minimal alveolar anesthetic concentration of inhaled anesthetics.18 Therefore, the role of muscarinic receptors in analgesia and anesthesia seems to be complex and unclear and hence it requires further study.
We have previously reported that isoflurane and halothane reduce the carbachol-evoked [Ca2+]cyttransient and that such an effect is eliminated in the presence of KCl.19 In addition, we have shown that the isoflurane-sensitive component of the carbachol evoked [Ca2+]cyttransient requires full caffeine-sensitive Ca2+stores and that the elimination of the isoflurane-sensitivity of the carbachol response requires Ca2+release through the ryanodine channels.20 In SH-SY5Y cells activation of muscarinic receptors by carbachol stimulates the formation of inositol triphosphate (IP3).21,22 In many cell types, including SH-SY5Y cells, stimulation of IP3 formation causes, via  IP3 receptor activation, the release of Ca2+from intracellular IP3-sensitive stores, as well as the entry of Ca2+via  plasma membrane cation channels, at times referred to as capacitative Ca2+entry or store-operated Ca2+channels.21,23–27 In this study we used the human neuroblastoma SH-SY5Y cell line to investigate the modulation of the muscarinic response by isoflurane in a homogenous population of neuronal cells and to determine whether the isoflurane-sensitive component of the carbachol-evoked [Ca2+]cyttransient involves Ca2+release from intracellular stores or Ca2+entry through the plasma membrane.
Materials and Methods
Cell Culture and Solutions
SH-SY5Y human neuroblastoma cells were cultured in Roswell Park Memorial Institute 1640 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). Experiments were performed on monolayer of cells as previously reported.20 Cells were plated on glass coverslips (25-mm diameter) at a density of 2–4 × 104cells/ml (2 ml cell suspension/35 mm culture dish) and used when they formed a confluent monolayer (∼10–16 days after plating). During experimentation cells were continuously perfused with a HEPES buffer containing (in mM) 140 NaCl, 5 KCl, 5 NaHCO3, 10 HEPES, 1 MgCl2, 1.5 CaCl2, 1 adenosine triphosphate, and 10 glucose (pH 7.4). 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). The exchange of the solution was carried out with a manifold. The solutions containing 1 mm carbachol, with and without 100 nm conotoxin GVIA, 10 μm nitrendipine, or 100 μm La3+, 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 use as previously described.19 
Ca2+Measurements
SH-SY5Y cells were loaded with the fluorescent Ca2+indicator Fura-228 by incubating the cells attached 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. After loading, cells were 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 HEPES buffer at 37°C before being exposed to the various drugs. The HEPES buffer without or containing the drugs was perfused at a speed of 250 μl/ml.
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 connected to a high-speed multiwavelength illuminator (DeltaRAM V; Photon Technology International Inc., Lawrenceville, NJ). The excitation wavelengths for Fura-2 (340 nm and 380 nm) were alternately generated by a monochromator (every 0.02 s). The emitted fluorescence (from the alternated excitation at 340 and 380 nm) from 15 to 20 cells was filtered with the fluorescence barrier filter BA 515 nm, collected with a photomultiplier (PMT01–710; Photon Technology International Inc.), and digitized at 50 Hz.
Data Analysis
Data collection and analysis was carried out using the software Felix (version 1.42a, Photon Technology International), Clampfit (Pclamp 8; Axon Instruments, Foster City, CA), and GraphPad Prism (GraphPad Software, Inc., San Diego, CA). For each treatment (corresponding to data in each figure), experiments were done 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 figures the data represent the delta ratio (Δ ratio) of the emission of Fura-2 at 515 nm generated by excitation at 340 and 380 nm (ratio 340/380).
Statistical Analysis
Comparison between different groups was performed using unpaired two-tailed Student t  test when there was only one treatment (fig. 1) and one-way analysis of variance Newman-Keuls test when there was more than one treatment (figs. 2 and 3) using the GraphPad Prism (GraphPad Software, Inc.) software.
Fig. 1. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient in the presence, but not in the absence, of extracellular Ca2+. The application of isoflurane was started 10 min before the 2 min carbachol stimulation. (  A–D  ) Cells exposed to 1 mm carbachol (CAB) in the absence or presence of 1 mm isoflurane and in the presence of physiologic concentration of extracellular Ca2+(1.5 mm). The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (  E–H  ) Cells exposed to 1 mm carbachol in the absence or presence of 1 mm isoflurane after reducing the concentration of extracellular Ca2+to 150 μm. The averaged carbachol-evoked [Ca2+]cyttransient (  E  ), and the corresponding measurements: peaks (  F  ), areas (  G  ), and widths at 50% peak height (  H  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380* ms), and Width at 50% peak (ms). The horizontal bars in A and E indicate a 2-min period. The results were expressed as mean ± SEM, except for panels A and E, where they were expressed only as means. Asterisks indicate a statistically significant difference  (*P  < 0.001, unpaired two-tailed Student  t  tests) between control and isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and isoflurane in the presence of either high (1.5 mm) or low (150 μm) extracellular Ca2+(data not shown). The data for parts A–C are taken from an earlier study and are shown here for comparison purposes. The (n) indicate the number of experiments for each condition  .
Fig. 1. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient in the presence, but not in the absence, of extracellular Ca2+. The application of isoflurane was started 10 min before the 2 min carbachol stimulation. (  A–D  ) Cells exposed to 1 mm carbachol (CAB) in the absence or presence of 1 mm isoflurane and in the presence of physiologic concentration of extracellular Ca2+(1.5 mm). The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (  E–H  ) Cells exposed to 1 mm carbachol in the absence or presence of 1 mm isoflurane after reducing the concentration of extracellular Ca2+to 150 μm. The averaged carbachol-evoked [Ca2+]cyttransient (  E  ), and the corresponding measurements: peaks (  F  ), areas (  G  ), and widths at 50% peak height (  H  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380* ms), and Width at 50% peak (ms). The horizontal bars in A and E indicate a 2-min period. The results were expressed as mean ± SEM, except for panels A and E, where they were expressed only as means. Asterisks indicate a statistically significant difference 
	(*P  < 0.001, unpaired two-tailed Student  t  tests) between control and isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and isoflurane in the presence of either high (1.5 mm) or low (150 μm) extracellular Ca2+(data not shown). The data for parts A–C are taken from an earlier study and are shown here for comparison purposes. The (n) indicate the number of experiments for each condition 
	.
Fig. 1. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient in the presence, but not in the absence, of extracellular Ca2+. The application of isoflurane was started 10 min before the 2 min carbachol stimulation. (  A–D  ) Cells exposed to 1 mm carbachol (CAB) in the absence or presence of 1 mm isoflurane and in the presence of physiologic concentration of extracellular Ca2+(1.5 mm). The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (  E–H  ) Cells exposed to 1 mm carbachol in the absence or presence of 1 mm isoflurane after reducing the concentration of extracellular Ca2+to 150 μm. The averaged carbachol-evoked [Ca2+]cyttransient (  E  ), and the corresponding measurements: peaks (  F  ), areas (  G  ), and widths at 50% peak height (  H  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380* ms), and Width at 50% peak (ms). The horizontal bars in A and E indicate a 2-min period. The results were expressed as mean ± SEM, except for panels A and E, where they were expressed only as means. Asterisks indicate a statistically significant difference  (*P  < 0.001, unpaired two-tailed Student  t  tests) between control and isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and isoflurane in the presence of either high (1.5 mm) or low (150 μm) extracellular Ca2+(data not shown). The data for parts A–C are taken from an earlier study and are shown here for comparison purposes. The (n) indicate the number of experiments for each condition  .
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Fig. 2. La3+reduces the carbachol-evoked [Ca2+]cyttransient and eliminates the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of La3+was started 10 min before the carbachol (CAB) stimulation and the application of 1 mm isoflurane (Iso) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of La3+, or La3+plus isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380 * ms), and Width at 50% peak (ms). The horizontal bar in A indicates a 2-min period. The results were expressed as mean ± SEM, except for panel A where they were expressed only as means. Statistically significant differences with control are indicated as follows:  *P  < 0.001,  **P  < 0.01,  #P  < 0.05 (Newman-Keuls test). No statistical difference was found between the groups treated with La3+and with La3+plus isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown)  .
Fig. 2. La3+reduces the carbachol-evoked [Ca2+]cyttransient and eliminates the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of La3+was started 10 min before the carbachol (CAB) stimulation and the application of 1 mm isoflurane (Iso) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of La3+, or La3+plus isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380 * ms), and Width at 50% peak (ms). The horizontal bar in A indicates a 2-min period. The results were expressed as mean ± SEM, except for panel A where they were expressed only as means. Statistically significant differences with control are indicated as follows: 
	*P  < 0.001, 
	**P  < 0.01, 
	#P  < 0.05 (Newman-Keuls test). No statistical difference was found between the groups treated with La3+and with La3+plus isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown) 
	.
Fig. 2. La3+reduces the carbachol-evoked [Ca2+]cyttransient and eliminates the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of La3+was started 10 min before the carbachol (CAB) stimulation and the application of 1 mm isoflurane (Iso) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of La3+, or La3+plus isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380 * ms), and Width at 50% peak (ms). The horizontal bar in A indicates a 2-min period. The results were expressed as mean ± SEM, except for panel A where they were expressed only as means. Statistically significant differences with control are indicated as follows:  *P  < 0.001,  **P  < 0.01,  #P  < 0.05 (Newman-Keuls test). No statistical difference was found between the groups treated with La3+and with La3+plus isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown)  .
×
Fig. 3. Nitrendipine a L-type Ca2+channel blocker reduces the carbachol-evoked [Ca2+]cyttransient but does not eliminate the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of the calcium channel blocker was started 10 min before the carbachol (CAB) stimulation; and the application of isoflurane (1 mm) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. (  A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of ω-conotoxin GVIA (CTX, 100 nm). (  B  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of nitrendipine (NTP, 10 μm); and of NTP plus 1 mm isoflurane (Iso). The corresponding measurements: peaks (  C  ), areas (  D  ), and widths at 50% peak height (  E  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of either ω-conotoxin GVIA, NTP, and NTP plus isoflurane (NTP+Iso). Units: Peaks (Δ ratio 340/380); Areas (ratio 340/380 * ms) and Width at 50% peak (ms). The horizontal bars in A and B indicate a 2-min period. The results were expressed as mean in panels A and B and as mean ± SEM in panels C, D, and E. Statistically significant differences from control are indicated as follows:  *P  < 0.001,  **P  < 0.01; and statistically significant differences from the NTP group are indicated as follows:  #P  < 0.05 (Newman-Keuls test). The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown)  .
Fig. 3. Nitrendipine a L-type Ca2+channel blocker reduces the carbachol-evoked [Ca2+]cyttransient but does not eliminate the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of the calcium channel blocker was started 10 min before the carbachol (CAB) stimulation; and the application of isoflurane (1 mm) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. (  A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of ω-conotoxin GVIA (CTX, 100 nm). (  B  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of nitrendipine (NTP, 10 μm); and of NTP plus 1 mm isoflurane (Iso). The corresponding measurements: peaks (  C  ), areas (  D  ), and widths at 50% peak height (  E  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of either ω-conotoxin GVIA, NTP, and NTP plus isoflurane (NTP+Iso). Units: Peaks (Δ ratio 340/380); Areas (ratio 340/380 * ms) and Width at 50% peak (ms). The horizontal bars in A and B indicate a 2-min period. The results were expressed as mean in panels A and B and as mean ± SEM in panels C, D, and E. Statistically significant differences from control are indicated as follows: 
	*P  < 0.001, 
	**P  < 0.01; and statistically significant differences from the NTP group are indicated as follows: 
	#P  < 0.05 (Newman-Keuls test). The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown) 
	.
Fig. 3. Nitrendipine a L-type Ca2+channel blocker reduces the carbachol-evoked [Ca2+]cyttransient but does not eliminate the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of the calcium channel blocker was started 10 min before the carbachol (CAB) stimulation; and the application of isoflurane (1 mm) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. (  A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of ω-conotoxin GVIA (CTX, 100 nm). (  B  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of nitrendipine (NTP, 10 μm); and of NTP plus 1 mm isoflurane (Iso). The corresponding measurements: peaks (  C  ), areas (  D  ), and widths at 50% peak height (  E  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of either ω-conotoxin GVIA, NTP, and NTP plus isoflurane (NTP+Iso). Units: Peaks (Δ ratio 340/380); Areas (ratio 340/380 * ms) and Width at 50% peak (ms). The horizontal bars in A and B indicate a 2-min period. The results were expressed as mean in panels A and B and as mean ± SEM in panels C, D, and E. Statistically significant differences from control are indicated as follows:  *P  < 0.001,  **P  < 0.01; and statistically significant differences from the NTP group are indicated as follows:  #P  < 0.05 (Newman-Keuls test). The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown)  .
×
Results
Isoflurane Sensitivity of the Carbachol-evoked [Ca2+]cytTransient is Dependent on Extracellular Ca2+
We have reported that isoflurane (1 mm) reduced the carbachol-evoked [Ca2+]cyttransient (fig. 1A-C).20 The isoflurane effect includes a reduction in the peak and area under the peak but not in the width at 50% peak height of the carbachol-evoked [Ca2+]cyttransient (fig. 1A, B, C, D). When the concentration of extracellular Ca2+was reduced from 1.5 mm to 150 μm, carbachol still evoked a [Ca2+]cyttransient but its magnitude was lower and its decay was speeded up, as indicated by the reduction in the width at 50% peak height (fig. 1E, F, G, H). These results are consistent with previous observations in SH-SY5Y cells and indicate that the carbachol-evoked [Ca2+]cyt-transient results from Ca2+release from intracellular Ca2+stores and Ca2+entry through the plasma membrane.21,24,27 Interestingly, in the presence of low extracellular Ca2+, the carbachol-evoked [Ca2+]cyttransient became insensitive to isoflurane (fig. 1E–Hversus  1A–D).
Isoflurane Sensitivity of the Carbachol-evoked [Ca2+]cytTransient is Eliminated by Exposing the Cells to La3+, a Nonselective Cationic Channel Blocker
Removal of extracellular Ca2+, even for short periods, may induce partial depletion of intracellular Ca2+stores. Hence, the elimination of the isoflurane-sensitive component of the carbachol-evoked cytoplasmic Ca2+response may still involve reduction of Ca2+release from intracellular store rather than elimination of Ca2+entry through the plasma membrane. To distinguish between these possibilities we blocked Ca2+entry through the plasma membrane by using the nonselective cationic channel blocker La3+. La3+has been shown to block various voltage-dependent Ca2+channels,29,30 as well as other cationic channels known as capacitative Ca2+channels.26 La3+alone did not significantly affect the peak but decreased the area and reduced the width at 50% peak height of the carbachol-evoked [Ca 2+]cyttransient (fig. 2). These results suggest that Ca2+entry through the plasma membrane mostly contributes to the decay phase of the carbachol-evoked [Ca2+]cyttransient, whereas the decrease in the carbachol-evoked [Ca2+]cytpeak in low extracellular Ca2+may reflect partial Ca2+depletion from intracellular Ca2+stores. In the presence of La3+, isoflurane did not produce an additional change in the carbachol-evoked [Ca2+]cyttransient (fig. 2). The effect of La3+was stronger than the effect of isoflurane (fig. 2Aversus  fig. 1A), and the main difference was that La3+, but not isoflurane, strongly decreased the width of the carbachol-evoked [Ca2+]cyttransient (fig. 1Dversus  fig. 2D). The latter suggests that the isoflurane effect appears to be mostly at the plasma membrane, probably by blocking a cationic channel.
Isoflurane Sensitivity of the Carbachol-evoked [Ca2+]cytTransient is not Eliminated by either ω-Conotoxin GVIA, an N-type Ca2+Channel Blocker, or by Nitrendipine, an L-type Ca2+Channel Blocker
Carbachol, through activation of muscarinic receptors, has been shown to affect voltage-dependent Ca2+channels31,32 and to allow Ca2+entry through other nonselective cationic channels.33–35 In SH-SY5Y cells, the predominant voltage-dependent Ca2+channels are L-type and N-type.36,37 We tested whether these voltage-dependent channels contributed to the carbachol-evoked [Ca2+]cyttransient and, if so, whether they were the isoflurane targets underlying the isoflurane reduction in the carbachol response. It was found that the N-type Ca2+channel blocker ω-conotoxin GVIA at a supramaximal concentration (100 nm) did not affect the carbachol-evoked [Ca2+]cyttransient (fig. 3A), whereas the L-type Ca2+channel blocker nitrendipine at a supramaximal concentration (10 μm) reduced the carbachol-evoked [Ca2+]cyttransient (fig. 3B). This indicated that under these conditions, exposure to carbachol increases Ca2+entry through L-type, but not N-type, Ca2+channels. Surprisingly, in the presence of nitrendipine, isoflurane further reduced the peak and area of the carbachol-evoked [Ca2+]cyttransient (fig. 3C, D) without affecting the width at 50% peak (fig. 3E). Therefore, the isoflurane effect on the carbachol-evoked [Ca2+]cyttransient is not attributable to the isoflurane effects on the L-type or N- type Ca2+channels but to an isoflurane effect on a La3+-sensitive plasma membrane cationic channel.
Discussion
As previously reported,21,24,27 it was found that in the human neuroblastoma cell line SH-SY5Y cells, the carbachol-evoked [Ca2+]cyttransient involves both Ca2+release from intracellular Ca2+stores and Ca2+entry through the plasma membrane. Moreover, we found that the blocking effect of isoflurane on the carbachol-evoked [Ca2+]cyttransient appears to be mediated by blocking the carbachol-evoked Ca2+entry through the plasma membrane. This isoflurane sensitive Ca2+entry involves a cationic channel that is different from the L-type or N-type voltage-dependent Ca2+channels. These results together with our previous observations19,20 indicate that at the concentrations used, isoflurane blocks only part of the carbachol-evoked [Ca2+]cytresponse, apparently at a site at the plasma membrane that is distal to the muscarinic receptor.
In SH-SY5Y cells the carbachol-evoked [Ca2+]cyttransient is blocked by atropine20,38 and is resistant to the N-type channel blocker (ω-conotoxin).38 Previously, it was found that the carbachol-evoked [Ca2+]cytincrease was also resistant to a maximal effective concentration (1 μm) of the L-type channel blocker dihydropyridine +PN 200–110.38 However, in this study we found that the L-type channel blocker, nitrendipine (10 μm) reduced the carbachol-evoked [Ca2+]cyttransient without eliminating its sensitivity to isoflurane. This was surprising because volatile anesthetics are known to reduce the magnitude of L-type Ca2+channel currents.39,40 If L-type channels are contributing to the carbachol-evoked [Ca2+]cyttransient, blocking them should reduce the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient, which did not occur. One possible explanation is that at the high concentration of nitrendipine used in this study, nitrendipine may be reducing the carbachol-evoked [Ca2+]cyttransient by interfering with the G-protein-linked muscarinic receptors rather than by blocking L-type Ca2+currents.41–43 
Because La3+, but not N-type or L-type Ca2+channel blockers, eliminated the isoflurane action on the carbachol-evoked [Ca2+]cyttransient, isoflurane may be reducing the carbachol-evoked [Ca2+]cyttransient by blocking a cationic channel at the plasma membrane. As La3+, but not isoflurane, reduced the width at 50% peak, it indicates that SH-SY5Y cells may express several nonvoltage dependent cationic channels that mediate Ca2+influx upon muscarinic activation and that isoflurane acts only in a subgroup of these channels.
There are at least two possible candidates for the isoflurane-sensitive plasma-membrane cationic channel, an inositol IP3-activated IP3-channel and a capacitative Ca2+channel. There is evidence suggesting the presence of plasma membrane-IP3 receptors in mammalian neurons. IP3-activated inward Ba2+currents have been recorded in excised inside-out patches of primary cultured Purkinje cells44 and in olfactory neurons of rat.45 However, halothane has been shown to increase, rather than decrease, Ca2+currents through IP3 receptors.46 Moreover, it has been argued that there are no IP3 receptors on the plasma membrane but a group of IP3 receptors located very close to the plasma membrane that on activation in turn activate cationic channels on the plasma membrane.47,48 Capacitative Ca2+influx is mediated by channels that are opened in response to depletion of intracellular Ca2+stores.49 There appear to be various types of capacitative Ca2+channels.49,50 Opening of capacitative channels after activation of G-protein-linked receptors, such as muscarinic receptors, involves receptor-mediated activation of phospholipase C and Ca2+release by IP3.49 Isoflurane has been shown to inhibit the histamine-induced Ca2+influx in primary cultures of human endothelial cells.51 In rat glioma C6 cells, volatile anesthetics appear to have different inhibitory effects on capacitative Ca2+influx such that strong inhibition is observed with halothane but not with enflurane.52 It is then possible that isoflurane is inhibiting muscarinic-activated capacitative Ca2+influx in the SH-SY5Y cells.
We would like to postulate that this isoflurane-sensitive cationic channel contributes either to the anesthetic potency or to the side effects of isoflurane. The previously reported variable effects of muscarinic blockers (as with other G-protein linked receptors) on the minimal alveolar anesthetic concentration of inhaled anesthetics might in part reflect differences in magnitude of the muscarinic-mediated modulation of various cationic channels. Although the muscarinic-mediated activation of the isoflurane-sensitive cationic channel might reduce the isoflurane potency, the muscarinic-mediated inhibition of voltage-dependent channels31,32 might increase the isoflurane potency. The net effect of a muscarinic agent on the isoflurane potency for reducing the muscarinic-evoked increases in [Ca2+]cytwould then depend on the contribution of each of the cationic channels in the different brain and spinal regions where the muscarinic agents are applied.
We previously reported that the isoflurane-action on the carbachol-evoked [Ca2+]cyttransient required that the caffeine-sensitive Ca2+stores were not depleted (by either KCl or caffeine pretreatment) and that ryanodine-sensitive Ca2+release channels were open.19,20 One possible explanation is that there is an open conformation of ryanodine-sensitive Ca2+release channels that interacts with the muscarinic-activated cationic channels and prevents their opening. Another explanation is that because of distinct spatial distribution, Ca2+release through the ryanodine-sensitive channels blocks the isoflurane-sensitive cationic channels, whereas Ca2+release through IP3-sensitive channels opens the isoflurane-sensitive cationic channels.
In summary, we postulate that in SH-SY5Y cells there is an isoflurane-sensitive cationic channel at the plasma membrane that is activated by carbachol and inhibited by La3+, isoflurane, and, possibly, through an interaction with the ryanodine-sensitive Ca2+release channel or by a Ca2+release through these channels (fig. 4). As discussed above, a possible candidate is a isoflurane-sensitive capacitative Ca2+channel. This potential target of isoflurane may serve as a site at which isoflurane may affect at least some of the actions of most of the G-protein linked receptors. The magnitude of the isoflurane effect on a given G-protein linked receptor would then be determined in part by the ability of the receptor to activate these isoflurane-sensitive capacitative Ca2+channels.
Fig. 4. Postulated mechanism for the isoflurane-sensitivity of the carbachol-evoked [Ca2+]cyttransient. (  A  ) We postulate that in SH-SY5Y cells there is an isoflurane-sensitive cationic channel at the plasma membrane (CAPC) that is activated (+) through activation of the muscarinic receptors (M). On its activation, CAPC allows Ca2+entry through the plasma membrane. (  B  ) Ca2+influx through the CAPC is inhibited (-) by isoflurane (ISO), La3+and possibly by either an interaction with the ryanodine-sensitive Ca2+release channel (RYR) or by a Ca2+release through RYR channels located at the caffeine-sensitive Ca2+stores (CAF-stores). Under control conditions, the carbachol-evoked [Ca2+]cyttransient results from Ca2+release from the IP3-sensitive Ca2+stores (IP3-stores) and from at least some of the caffeine-sensitive Ca2+stores,  19 and finally from the Ca2+influx through a putative isoflurane-sensitive plasma membrane cationic channel (CAPC). The carbachol-evoked [Ca2+]cyttransient is reduced by isoflurane before (  C  ) but not after treatment with either KCl or La3+(  D  ). Exposure to KCl depolarizes the plasma membrane leading to opening of voltage-dependent Ca2+channels (VDCC), which in turn leads to opening of RYR channels by a process called Ca2+-induced Ca2+release.  IP3  = inositol triphosphate;  IP3R  = IP3-sensitive Ca2+channels  .
Fig. 4. Postulated mechanism for the isoflurane-sensitivity of the carbachol-evoked [Ca2+]cyttransient. (  A  ) We postulate that in SH-SY5Y cells there is an isoflurane-sensitive cationic channel at the plasma membrane (CAPC) that is activated (+) through activation of the muscarinic receptors (M). On its activation, CAPC allows Ca2+entry through the plasma membrane. (  B  ) Ca2+influx through the CAPC is inhibited (-) by isoflurane (ISO), La3+and possibly by either an interaction with the ryanodine-sensitive Ca2+release channel (RYR) or by a Ca2+release through RYR channels located at the caffeine-sensitive Ca2+stores (CAF-stores). Under control conditions, the carbachol-evoked [Ca2+]cyttransient results from Ca2+release from the IP3-sensitive Ca2+stores (IP3-stores) and from at least some of the caffeine-sensitive Ca2+stores,  19and finally from the Ca2+influx through a putative isoflurane-sensitive plasma membrane cationic channel (CAPC). The carbachol-evoked [Ca2+]cyttransient is reduced by isoflurane before (  C  ) but not after treatment with either KCl or La3+(  D  ). Exposure to KCl depolarizes the plasma membrane leading to opening of voltage-dependent Ca2+channels (VDCC), which in turn leads to opening of RYR channels by a process called Ca2+-induced Ca2+release.  IP3  = inositol triphosphate;  IP3R  = IP3-sensitive Ca2+channels 
	.
Fig. 4. Postulated mechanism for the isoflurane-sensitivity of the carbachol-evoked [Ca2+]cyttransient. (  A  ) We postulate that in SH-SY5Y cells there is an isoflurane-sensitive cationic channel at the plasma membrane (CAPC) that is activated (+) through activation of the muscarinic receptors (M). On its activation, CAPC allows Ca2+entry through the plasma membrane. (  B  ) Ca2+influx through the CAPC is inhibited (-) by isoflurane (ISO), La3+and possibly by either an interaction with the ryanodine-sensitive Ca2+release channel (RYR) or by a Ca2+release through RYR channels located at the caffeine-sensitive Ca2+stores (CAF-stores). Under control conditions, the carbachol-evoked [Ca2+]cyttransient results from Ca2+release from the IP3-sensitive Ca2+stores (IP3-stores) and from at least some of the caffeine-sensitive Ca2+stores,  19 and finally from the Ca2+influx through a putative isoflurane-sensitive plasma membrane cationic channel (CAPC). The carbachol-evoked [Ca2+]cyttransient is reduced by isoflurane before (  C  ) but not after treatment with either KCl or La3+(  D  ). Exposure to KCl depolarizes the plasma membrane leading to opening of voltage-dependent Ca2+channels (VDCC), which in turn leads to opening of RYR channels by a process called Ca2+-induced Ca2+release.  IP3  = inositol triphosphate;  IP3R  = IP3-sensitive Ca2+channels  .
×
The authors thank June Biedler, Ph.D. for providing the SH-SY5Y human neuroblastoma cells (Sloan-Kettering Institute for Cancer Research, Rye, New York).
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Fig. 1. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient in the presence, but not in the absence, of extracellular Ca2+. The application of isoflurane was started 10 min before the 2 min carbachol stimulation. (  A–D  ) Cells exposed to 1 mm carbachol (CAB) in the absence or presence of 1 mm isoflurane and in the presence of physiologic concentration of extracellular Ca2+(1.5 mm). The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (  E–H  ) Cells exposed to 1 mm carbachol in the absence or presence of 1 mm isoflurane after reducing the concentration of extracellular Ca2+to 150 μm. The averaged carbachol-evoked [Ca2+]cyttransient (  E  ), and the corresponding measurements: peaks (  F  ), areas (  G  ), and widths at 50% peak height (  H  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380* ms), and Width at 50% peak (ms). The horizontal bars in A and E indicate a 2-min period. The results were expressed as mean ± SEM, except for panels A and E, where they were expressed only as means. Asterisks indicate a statistically significant difference  (*P  < 0.001, unpaired two-tailed Student  t  tests) between control and isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and isoflurane in the presence of either high (1.5 mm) or low (150 μm) extracellular Ca2+(data not shown). The data for parts A–C are taken from an earlier study and are shown here for comparison purposes. The (n) indicate the number of experiments for each condition  .
Fig. 1. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient in the presence, but not in the absence, of extracellular Ca2+. The application of isoflurane was started 10 min before the 2 min carbachol stimulation. (  A–D  ) Cells exposed to 1 mm carbachol (CAB) in the absence or presence of 1 mm isoflurane and in the presence of physiologic concentration of extracellular Ca2+(1.5 mm). The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (  E–H  ) Cells exposed to 1 mm carbachol in the absence or presence of 1 mm isoflurane after reducing the concentration of extracellular Ca2+to 150 μm. The averaged carbachol-evoked [Ca2+]cyttransient (  E  ), and the corresponding measurements: peaks (  F  ), areas (  G  ), and widths at 50% peak height (  H  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380* ms), and Width at 50% peak (ms). The horizontal bars in A and E indicate a 2-min period. The results were expressed as mean ± SEM, except for panels A and E, where they were expressed only as means. Asterisks indicate a statistically significant difference 
	(*P  < 0.001, unpaired two-tailed Student  t  tests) between control and isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and isoflurane in the presence of either high (1.5 mm) or low (150 μm) extracellular Ca2+(data not shown). The data for parts A–C are taken from an earlier study and are shown here for comparison purposes. The (n) indicate the number of experiments for each condition 
	.
Fig. 1. Isoflurane reduces the carbachol-evoked [Ca2+]cyttransient in the presence, but not in the absence, of extracellular Ca2+. The application of isoflurane was started 10 min before the 2 min carbachol stimulation. (  A–D  ) Cells exposed to 1 mm carbachol (CAB) in the absence or presence of 1 mm isoflurane and in the presence of physiologic concentration of extracellular Ca2+(1.5 mm). The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. (  E–H  ) Cells exposed to 1 mm carbachol in the absence or presence of 1 mm isoflurane after reducing the concentration of extracellular Ca2+to 150 μm. The averaged carbachol-evoked [Ca2+]cyttransient (  E  ), and the corresponding measurements: peaks (  F  ), areas (  G  ), and widths at 50% peak height (  H  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380* ms), and Width at 50% peak (ms). The horizontal bars in A and E indicate a 2-min period. The results were expressed as mean ± SEM, except for panels A and E, where they were expressed only as means. Asterisks indicate a statistically significant difference  (*P  < 0.001, unpaired two-tailed Student  t  tests) between control and isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and isoflurane in the presence of either high (1.5 mm) or low (150 μm) extracellular Ca2+(data not shown). The data for parts A–C are taken from an earlier study and are shown here for comparison purposes. The (n) indicate the number of experiments for each condition  .
×
Fig. 2. La3+reduces the carbachol-evoked [Ca2+]cyttransient and eliminates the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of La3+was started 10 min before the carbachol (CAB) stimulation and the application of 1 mm isoflurane (Iso) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of La3+, or La3+plus isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380 * ms), and Width at 50% peak (ms). The horizontal bar in A indicates a 2-min period. The results were expressed as mean ± SEM, except for panel A where they were expressed only as means. Statistically significant differences with control are indicated as follows:  *P  < 0.001,  **P  < 0.01,  #P  < 0.05 (Newman-Keuls test). No statistical difference was found between the groups treated with La3+and with La3+plus isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown)  .
Fig. 2. La3+reduces the carbachol-evoked [Ca2+]cyttransient and eliminates the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of La3+was started 10 min before the carbachol (CAB) stimulation and the application of 1 mm isoflurane (Iso) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of La3+, or La3+plus isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380 * ms), and Width at 50% peak (ms). The horizontal bar in A indicates a 2-min period. The results were expressed as mean ± SEM, except for panel A where they were expressed only as means. Statistically significant differences with control are indicated as follows: 
	*P  < 0.001, 
	**P  < 0.01, 
	#P  < 0.05 (Newman-Keuls test). No statistical difference was found between the groups treated with La3+and with La3+plus isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown) 
	.
Fig. 2. La3+reduces the carbachol-evoked [Ca2+]cyttransient and eliminates the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of La3+was started 10 min before the carbachol (CAB) stimulation and the application of 1 mm isoflurane (Iso) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. The averaged carbachol-evoked [Ca2+]cyttransient (  A  ) and the corresponding measurements: peaks (  B  ), areas (  C  ), and widths at 50% peak height (  D  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of La3+, or La3+plus isoflurane. Units: Peaks (Δ ratio 340/380), Areas (ratio 340/380 * ms), and Width at 50% peak (ms). The horizontal bar in A indicates a 2-min period. The results were expressed as mean ± SEM, except for panel A where they were expressed only as means. Statistically significant differences with control are indicated as follows:  *P  < 0.001,  **P  < 0.01,  #P  < 0.05 (Newman-Keuls test). No statistical difference was found between the groups treated with La3+and with La3+plus isoflurane. The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown)  .
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Fig. 3. Nitrendipine a L-type Ca2+channel blocker reduces the carbachol-evoked [Ca2+]cyttransient but does not eliminate the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of the calcium channel blocker was started 10 min before the carbachol (CAB) stimulation; and the application of isoflurane (1 mm) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. (  A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of ω-conotoxin GVIA (CTX, 100 nm). (  B  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of nitrendipine (NTP, 10 μm); and of NTP plus 1 mm isoflurane (Iso). The corresponding measurements: peaks (  C  ), areas (  D  ), and widths at 50% peak height (  E  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of either ω-conotoxin GVIA, NTP, and NTP plus isoflurane (NTP+Iso). Units: Peaks (Δ ratio 340/380); Areas (ratio 340/380 * ms) and Width at 50% peak (ms). The horizontal bars in A and B indicate a 2-min period. The results were expressed as mean in panels A and B and as mean ± SEM in panels C, D, and E. Statistically significant differences from control are indicated as follows:  *P  < 0.001,  **P  < 0.01; and statistically significant differences from the NTP group are indicated as follows:  #P  < 0.05 (Newman-Keuls test). The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown)  .
Fig. 3. Nitrendipine a L-type Ca2+channel blocker reduces the carbachol-evoked [Ca2+]cyttransient but does not eliminate the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of the calcium channel blocker was started 10 min before the carbachol (CAB) stimulation; and the application of isoflurane (1 mm) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. (  A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of ω-conotoxin GVIA (CTX, 100 nm). (  B  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of nitrendipine (NTP, 10 μm); and of NTP plus 1 mm isoflurane (Iso). The corresponding measurements: peaks (  C  ), areas (  D  ), and widths at 50% peak height (  E  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of either ω-conotoxin GVIA, NTP, and NTP plus isoflurane (NTP+Iso). Units: Peaks (Δ ratio 340/380); Areas (ratio 340/380 * ms) and Width at 50% peak (ms). The horizontal bars in A and B indicate a 2-min period. The results were expressed as mean in panels A and B and as mean ± SEM in panels C, D, and E. Statistically significant differences from control are indicated as follows: 
	*P  < 0.001, 
	**P  < 0.01; and statistically significant differences from the NTP group are indicated as follows: 
	#P  < 0.05 (Newman-Keuls test). The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown) 
	.
Fig. 3. Nitrendipine a L-type Ca2+channel blocker reduces the carbachol-evoked [Ca2+]cyttransient but does not eliminate the isoflurane sensitivity of the carbachol-evoked [Ca2+]cyttransient. The application of the calcium channel blocker was started 10 min before the carbachol (CAB) stimulation; and the application of isoflurane (1 mm) 5 min before the carbachol stimulation. The duration of the carbachol stimulation was 2 min. (  A  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of ω-conotoxin GVIA (CTX, 100 nm). (  B  ) The averaged carbachol-evoked [Ca2+]cyttransients in the absence and presence of nitrendipine (NTP, 10 μm); and of NTP plus 1 mm isoflurane (Iso). The corresponding measurements: peaks (  C  ), areas (  D  ), and widths at 50% peak height (  E  ) of the carbachol-evoked [Ca2+]cyttransient in the absence and presence of either ω-conotoxin GVIA, NTP, and NTP plus isoflurane (NTP+Iso). Units: Peaks (Δ ratio 340/380); Areas (ratio 340/380 * ms) and Width at 50% peak (ms). The horizontal bars in A and B indicate a 2-min period. The results were expressed as mean in panels A and B and as mean ± SEM in panels C, D, and E. Statistically significant differences from control are indicated as follows:  *P  < 0.001,  **P  < 0.01; and statistically significant differences from the NTP group are indicated as follows:  #P  < 0.05 (Newman-Keuls test). The baseline values (absolute ratio values before the addition of carbachol) were not statistically different (  P  > 0.05) between control and the treated groups (data not shown)  .
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Fig. 4. Postulated mechanism for the isoflurane-sensitivity of the carbachol-evoked [Ca2+]cyttransient. (  A  ) We postulate that in SH-SY5Y cells there is an isoflurane-sensitive cationic channel at the plasma membrane (CAPC) that is activated (+) through activation of the muscarinic receptors (M). On its activation, CAPC allows Ca2+entry through the plasma membrane. (  B  ) Ca2+influx through the CAPC is inhibited (-) by isoflurane (ISO), La3+and possibly by either an interaction with the ryanodine-sensitive Ca2+release channel (RYR) or by a Ca2+release through RYR channels located at the caffeine-sensitive Ca2+stores (CAF-stores). Under control conditions, the carbachol-evoked [Ca2+]cyttransient results from Ca2+release from the IP3-sensitive Ca2+stores (IP3-stores) and from at least some of the caffeine-sensitive Ca2+stores,  19 and finally from the Ca2+influx through a putative isoflurane-sensitive plasma membrane cationic channel (CAPC). The carbachol-evoked [Ca2+]cyttransient is reduced by isoflurane before (  C  ) but not after treatment with either KCl or La3+(  D  ). Exposure to KCl depolarizes the plasma membrane leading to opening of voltage-dependent Ca2+channels (VDCC), which in turn leads to opening of RYR channels by a process called Ca2+-induced Ca2+release.  IP3  = inositol triphosphate;  IP3R  = IP3-sensitive Ca2+channels  .
Fig. 4. Postulated mechanism for the isoflurane-sensitivity of the carbachol-evoked [Ca2+]cyttransient. (  A  ) We postulate that in SH-SY5Y cells there is an isoflurane-sensitive cationic channel at the plasma membrane (CAPC) that is activated (+) through activation of the muscarinic receptors (M). On its activation, CAPC allows Ca2+entry through the plasma membrane. (  B  ) Ca2+influx through the CAPC is inhibited (-) by isoflurane (ISO), La3+and possibly by either an interaction with the ryanodine-sensitive Ca2+release channel (RYR) or by a Ca2+release through RYR channels located at the caffeine-sensitive Ca2+stores (CAF-stores). Under control conditions, the carbachol-evoked [Ca2+]cyttransient results from Ca2+release from the IP3-sensitive Ca2+stores (IP3-stores) and from at least some of the caffeine-sensitive Ca2+stores,  19and finally from the Ca2+influx through a putative isoflurane-sensitive plasma membrane cationic channel (CAPC). The carbachol-evoked [Ca2+]cyttransient is reduced by isoflurane before (  C  ) but not after treatment with either KCl or La3+(  D  ). Exposure to KCl depolarizes the plasma membrane leading to opening of voltage-dependent Ca2+channels (VDCC), which in turn leads to opening of RYR channels by a process called Ca2+-induced Ca2+release.  IP3  = inositol triphosphate;  IP3R  = IP3-sensitive Ca2+channels 
	.
Fig. 4. Postulated mechanism for the isoflurane-sensitivity of the carbachol-evoked [Ca2+]cyttransient. (  A  ) We postulate that in SH-SY5Y cells there is an isoflurane-sensitive cationic channel at the plasma membrane (CAPC) that is activated (+) through activation of the muscarinic receptors (M). On its activation, CAPC allows Ca2+entry through the plasma membrane. (  B  ) Ca2+influx through the CAPC is inhibited (-) by isoflurane (ISO), La3+and possibly by either an interaction with the ryanodine-sensitive Ca2+release channel (RYR) or by a Ca2+release through RYR channels located at the caffeine-sensitive Ca2+stores (CAF-stores). Under control conditions, the carbachol-evoked [Ca2+]cyttransient results from Ca2+release from the IP3-sensitive Ca2+stores (IP3-stores) and from at least some of the caffeine-sensitive Ca2+stores,  19 and finally from the Ca2+influx through a putative isoflurane-sensitive plasma membrane cationic channel (CAPC). The carbachol-evoked [Ca2+]cyttransient is reduced by isoflurane before (  C  ) but not after treatment with either KCl or La3+(  D  ). Exposure to KCl depolarizes the plasma membrane leading to opening of voltage-dependent Ca2+channels (VDCC), which in turn leads to opening of RYR channels by a process called Ca2+-induced Ca2+release.  IP3  = inositol triphosphate;  IP3R  = IP3-sensitive Ca2+channels  .
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