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© 1999 International Anesthesia Research Society
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Abstract |
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We evaluated the effects of volatile anesthetics on T-type calcium current (ICa,T) present in four different cell types using the whole cell version of the patch clamp technique. In dorsal root ganglion neurons and in two neuroendocrine cells—adrenal glomerulosa cells (AG) and thyroid C-cells—ICa,T was reversibly decreased by volatile anesthetics at clinically relevant concentrations, with isoflurane and enflurane being more potent that halothane. In AG cells, the most sensitive cell type tested, ICa,T was reduced 47% ± 4% (n = 6) by isoflurane (0.7 mM) and 56% ± 2% (n = 5) by enflurane (1.2 mM), but by only 24% ± 1% (n = 5; P < 0.05) by halothane (0.7 mM). Isoflurane caused a significant increase in the rate of deactivation of ICa,T in AG cells. In ventricular myocytes, however, ICa,T was much less sensitive to both isoflurane and halothane. The differential sensitivity of ICa,T in various cell types to the anesthetics may reflect differences in the channels expressed in these tissues or differences in the cellular intermediates involved in anesthetic action. Depression of ICa,T in neuronal cells may contribute to anesthetic action through decreases in cellular excitability.
Implications: Using the patch clamp technique, we showed that T-type calcium channels, which promote cellular excitability, are inhibited by volatile anesthetics in neuronal and neuroendocrine cells, but not in ventricular myocytes. Inhibition of neuronal T-type channels may contribute to the mechanism of action of volatile anesthetics.
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Introduction |
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The low-voltage–activated, rapidly inactivating T-type calcium channel is found throughout the nervous system and plays several important roles in the regulation of neuronal excitability. For example, T-type calcium currents (ICa,T) contribute to rhythmic membrane potential oscillations and synchronized firing patterns observed in some types of central neurons (1,2), as well as afterdeploarizations that elicit burst firing in both central and peripheral neurons (3,4). In addition, dendritic T-type channels may modulate synaptic function by altering the processing of postsynaptic potentials (5,6).
Given the multiple functional roles of T-type channels in the processing and transmission of information in the nervous system, these channels could be a target for central nervous system depressants, such as volatile anesthetics. Indeed, volatile anesthetics depress ICa,T in several cell types (7–12), although the magnitude of the effect varies. However, ICa,T from other cells seems to be quite resistant to the anesthetics (13). One explantation for this apparent discrepancy may be that different T-type channels are expressed in different cells. Although a T-type calcium channel has recently been cloned from rat brain (14), differences in the biophysical and pharmacological properties of ICa,T from different cell types suggest that more than one T-type channel may exist (15–19). In addition, the use of a variety of anesthetics in these studies complicates the issue because T-type channels may not be equally sensitive to all volatile anesthetics (9).
The purpose of the present study was to examine the effect of volatile anesthetics on ICa,T measured in several different cell types all known to express a well characterized ICa,T. We have previously reported (9) anesthetic effects on ICa,T from a clonal neuroendocrine thyroid C-cell line. We have extended our observations to include the effects of these drugs on three other cell types. In adrenal glomerulosa (AG) cells, ICa,T is involved in aldosterone secretion (20), whereas in dorsal root ganglion (DRG) neurons, ICa,T regulates burst firing behavior (3). We also examined the effect of anesthetics on ICa,T in a nonneuronal cell type, cardiac ventricular myocytes (VM).
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Methods |
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Thyroid C cells (TC) (rat medullary thyroid carcinoma cell line, 6–23, clone 6) were obtained from American Type Culture Collection (Rockville, MD) and maintained at 37°C in Dulbecco modified Eagle medium (DMEM) (GIBCO, Gaithersburg, MD) containing 10% equine serum (Hyclone, Logan, UT), 50 U/mL penicillin, and 50 µg/mL streptomycin (Sigma Chemical Co., St. Louis, MO) as previously described (9). Cells were used within 1–4 days after plating on glass coverslips coated with poly-D-lysine (Sigma).
Calf adrenal glomerulosa cells (2–7 days old) were prepared as described previously (20). Briefly, thin layers of zona glomerulosa were dissected from adrenal cortex, digested with collagenase (Worthington, Freehold, NJ) at 37°C for 10 min, and dispersed by mechanical agitation. The cells were filtered through 20-mm nylon mesh (Tetko, Elmsford, NY), centrifuged, purified on a two-step (30%/56%) discontinuous Percoll gradient (Pharmacia, Piscataway NJ), and resuspended in Krebs-Ringer bicarbonate buffer containing (in mM): NaCl 120, NaHCO3 25, KCl 3.5, MgSO4 1.2, NaH2PO4 1.2, CaCl2 1.25, 0.1% dextrose, and 0.2% bovine serum albumin, equilibrated with 95% air/5% CO2 (pH 7.4). Cells were plated on glass coverslips and used within 10 h after isolation.
Neonatal (7–15 day old) rats were killed in accordance with approved animal care protocols of the University of Virginia. Individual dorsal root ganglia were carefully removed from the cervical, thoracic, and lumbar regions of the spinal column using a dissecting microscope and placed in 0.25% trypsin solution (Sigma) containing 2 mg/mL collagenase (Sigma) at 37°C for 30 min. The ganglia were then gently triturated using flame polished Pasteur pipettes. Cells were plated on glass coverslips coated with poly-L-lysine (Sigma) and maintained in an incubator at 37°C in DMEM containing 10% fetal bovine serum (Hyclone), 50 U/mL penicillin, and 50 µg/mL streptomycin for up to 3 days. Healthy appearing, spherical cells were studied as soon as 2 h after isolation and usually not more than 2 days after isolation because the cells tended to grow processes in which membrane potential could not be adequately controlled. ICa,T was usually expressed by medium- to large-sized cells approximately 20–30 µm in diameter.
English shorthair guinea pigs were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) in accordance with approved animal care protocols of the University of Virginia. The heart was quickly excised and perfused using a Langendorff perfusion system, first for 5 min with a Ca2+-free Tyrode's solution containing (in mM): NaCl 118, KCl 5, MgSO4 1.2, NaHCO3 26, glucose 11, ethylenediaminetetraacetic acid 0.1, and pyruvic acid 5, then with Tyrode's supplemented with 25 µM CaCl2, 0.5 mg/mL albumin, and 0.5 mg/mL collagenase type II for 20 min or until the heart appeared flaccid. The ventricles were then removed, minced, and incubated for 30 min in an enzyme solution bubbled gently with 95% O25% CO2. The resulting slurry was filtered through 200-µm nylon mesh and centrifuged at 80g for 2 min. Cells were washed twice in Krebs-Henseleit buffer with 0.2 mM CaCl2, resuspended in buffer with 1 mM CaCl2, and stored in an incubator at 37°C for up to 8 h. Only rod-shaped cells with apparent striations that remained quiescent in solution containing 2 mM CaCl2 were studied.
Patch pipettes were made using a two-stage pipette puller (Narishige USA, Greenvale, NY) and were heat-polished on a microforge (ALA Scientific Instruments, Westbury, NY). Whole cell calcium currents were recorded from individual cells using commercial patch clamp amplifiers (Model 8900; Dagan Corporation, Minneapolis, MN; and Axopatch 200 and 200A; Axon Instruments, Foster City, CA). Data acquisition was performed by using pClamp version 5.5.1 (Axon Instruments) with a microcomputer. Currents were digitized, and leak and capacitative currents were subtracted from current records by adding the currents obtained from four hyperpolarizing pulses each equal to one-fourth the magnitude of the depolarizing test pulse. Current records were analyzed offline using a program developed in the laboratory (21), and exponential fits to current traces were calculated using Clampfit version 6 (Axon Instruments).
For each cell type studied, the external and internal recording solutions were designed to facilitate the measurement of calcium current and minimized the contributions of other voltage-dependent currents (Tables 1 and 2). Tetrodotoxin (TTX; Sigma; 2 mg/mL) was always included in the extracellular solution in experiments on VM to eliminate currents carried by Na+ channels. The absence of extracellular Na+ eliminated voltage-activated Na+ currents in DRG, AG, and TC cells; therefore, most studies in these cells were performed without TTX. Volatile anesthetics were introduced into the external solutions by vigorously bubbling them with filtered air passed through drug specific anesthetic vaporizers. Control solutions were bubbled with filtered air alone. Vaporizers were set to deliver the desired anesthetic doses (in vol %), which were chosen to represent multiples of the minimum alveolar anesthetic concentration (MAC) for each drug. Concentrations of anesthetics in solution were determined by using gas chromatography, as previously described (22). Isoflurane and enflurane were obtained from Ohmeda (Liberty Corner, NJ), and halothane was obtained from Halcarbon Laboratories (River Edge, NJ).
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Calcium currents were recorded at room temperature before and after application of anesthetic, and after washout of anesthetic (recovery) for each cell. In VM, DRG, and TC cells, ICa,T was recorded during depolarizing tests potential from a holding potential of -80 mV. Peak inward currents were compared at test potentials at which ICa,T was maximal. In AG cells, ICa,T tail currents were elicited by 15-ms depolarizing steps from a holding potential of -90 mV, followed by a repolarizing step to -50 mV. The amplitude of the tail current measured 1 ms after repolarization from a test potential of 20 mV was taken as the peak ICa,T in AG cells. Anesthetic-induced decreases in peak ICa,T for all cell types are reported as percent changes from an average of the control and recovery values to eliminate time-dependent changes in the current.
The kinetics of channel opening can be estimated from whole cell currents by assessing the rate at which the current activates after a depolarizing step. ICa,T activation was assessed by fitting the current measured in the first 15 ms after a depolarizing pulse to a third order exponential of the form: I = A*(1-exp[-t/
m])3 where m is the time constant for activation. The rate at which channels close after a rapid return from a depolarizing pulse to a hyperpolarized potential (deactivation) was assessed by fitting tail current traces to a single exponential of the form: I = A*exp(-t/d) where d is the time constant for deactivation. The rate at which channel conductance decreases during a sustained depolarization (inactivation) was assessed either by fitting the inactivating phase of the current trace to a single exponential of the form: I = A*exp(-t/h) where h is the time constant for inactivation, or by normalizing currents before and after isoflurane and analyzing them visually. Cells exhibiting relatively large inward currents during 200-ms test pulses were chosen for analysis of the inactivation kinetics of ICa,T. The minimize capacitative artifacts and the contribution of rapidly deactivating (d ~ 160 µs) high-voltage–activated calcium current present in AG cells, data in the first 1 ms after repolarization were not included in tail current analysis. Finally, the voltage dependence of ICa,T activation in AG cells was assessed by plotting the amplitude of the tail current measured 1 ms after repolarization against the test potential and fitting it to a Boltzmann equation of the form: I/Imax = 1/(1+exp[-(V-V')/k]) where V' is the voltage required for half-maximal activation and k is the slope factor.
Statistical significance was determined by single-factor analysis of variance and Scheffé's post hoc test for comparisons among three means. P values <0.05 were considered significant. All values are reported as means ± SE.
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Results |
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ICa,T was measured in all four cell types studied. In each cell type, ICa,T was activated from a holding potential of either -90 or -80 mV and had a threshold voltage for activation of between -60 and -40 mV. The peak inward current was typically maximal at test potentials between -30 to -20 mV, and the current displayed rapid inactivation during a test potential. These characteristics have been previously described for ICa,T measured in these cells (8,9,20), and they enabled us to distinguish ICa,T from the high-threshold calcium currents present in these cells. There were no obvious differences in the activation or inactivation kinetics of macroscopic ICa,T among the four cell types studied.
In DRG neurons and in the TC and AG cells, isoflurane (0.7 mM) and enflurane (1.2 mM) decreased ICa,T to a greater extent than a clinically equipotent concentration of halothane (0.7 mM) (Fig. 1). With all three anesthetics, the effect was rapid (within approximately 1 min after bath application) and readily reversible. Unlike the other cell types studied, VM expressed ICa,T that was relatively resistant to volatile anesthetics (Fig. 2). Even at higher concentrations, isoflurane (1.2 mM, n = 4) and halothane (1.4 mM, n = 5) each depressed ICa,T by only approximately 20% compared with control. Figure 3 summarizes the effects of a single concentration of each of the three volatile anesthetics on ICa,Tin TC, DRG, and AG cells. At clinically equivalent concentrations, halothane was the least potent anesthetic in all three cell types, whereas there was no statistically significant differences between the effects of enflurane and isoflurane.
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) neurons, thyroid C cells (TC; 
), and adrenal glomerulosa (AG; 
) cells are summarized. The bars represent the percent depression of peak current for each cell type at clinically equivalent concentrations of each anesthetic (mean ± SE). Halothane was the least effective in reducing T-type calcium current in all three cell types. * P < 0.05.The effects of the volatile anesthetics were dose-dependent. In AG cells, the decrease in ICa,T with isoflurane was 26% ± 3% (n = 4) at 0.4 mM and 47% ± 4% (n = 6) at 0.7 mM concentrations. With halothane, ICa,T was diminished by 24% ± 1% (n = 5) at 0.7 mM and by 30% ± 4% (n = 3) at 1.1 mM. In TC cells, a dose-response relationship for all three anesthetics has been well documented (9).
We considered the possibility that isoflurane may alter the voltage dependence of ICa,T activation and/or inactivation. Tail current magnitudes were fit to the Boltzmann function, as described in Methods. Under control conditions, V' and k were -14 ± 2 and 8.2 ± 0.3 mV, (n = 6), respectively. After application of isoflurane (0.7 mM), there was a slight hyperpolarizing shift in the activation curve of 3 ± 1 mV, with an insignificant decrease in the slope factor of 0.8 ± 0.6 mV (P > 0.05; n = 6). In TC cells, isoflurane (0.7 mM) does not alter the voltage dependence of ICa,T inactivation (9).
The m was determined in AG cells for test potentials ranging from -15 to 20 mV. Isoflurane (0.7 mM) did not significantly change the time course of activation of ICa,T at any test potential (Fig. 4). At a test potential of 20 mV, for example, m averaged 2.5 ± 0.4 ms in control versus 2.9 ± 0.7 ms after the administration of isoflurane (n = 4). Similarly, isoflurane had no effect on the time course of inactivation of ICa,T (Fig. 4).
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T-type channels are unique among the voltage-activated calcium channels for their slow rate of deactivation. To examine the effects of isoflurane on ICa,T deactivation, tail current traces evoked in AG cells during repolarization to -50 mV were fit to a single exponential, as described in Methods and shown in Figure 4. In the absence of anesthetics, the
d averaged 17 ± 1 ms (n = 6). In the same six cells, the administration of isoflurane (0.7 mM) caused a significant decrease in d to 11 ± 1 ms (P < 0.05), which was reversible with washout of anesthetic. |
Discussion |
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Our results show that ICa,T recorded from neuronal and neuroendocrine cell types is sensitive to inhibition by volatile anesthetics. Qualitatively similar results have been reported for other neuronal cell types (7,8,10,11,19). However, ICa,T recorded from nonneuronal cells, such as VM, is relatively insensitive to these drugs. Portal vein smooth muscle cells, another nonneuronal cell type, also express ICa,T that is insensitive to volatile anesthetics (13). Interestingly, ICa,T recorded from cardiac Purkinje cells seems to be more sensitive to the volatile drugs than ICa,T in VM (12).
The reason for the differences in anesthetic sensitivity of T-type channels from various tissues is not clear, although it has also been described for other drugs (15,16,19). The presence of two ICa,T with different kinetics and pharmacological sensitivities recorded simultaneously in individual cells (17,18) strongly suggests that more than one T-type channel exists. The recent cloning of a T-type channel from rat brain (14) should help to provide information about the structural diversity of T-type channels in different tissues. We found no evidence for more than one ICa,T in any of the cells used in the present study based on the kinetics or voltage dependence of the macroscopic current.
Another explanation for the differences in anesthetic sensitivity among cell types could be diversity in T-type channel regulation in various tissues. ICa,T is modulated by phosphorylation and interaction with G-proteins, for example (23,24). Only certain cells may possess the receptors and/or intracellular mediators required for expression of anesthetic sensitivity.
The aqueous anesthetic concentrations we report were obtained after bubbling the test solutions with anesthetic delivered by drug-specific vaporizers that were set to deliver approximately twice the MAC value (in gas concentration) for each drug measured in humans at 37°C. At lower temperatures, however, the solubility of these gases increases such that the final concentration measured at 22°C is approximately twice what would be expected at 37°C. At these clinically equivalent concentrations, we found that ICa,T in neuronal and neuroendocrine cells is less sensitive to halothane compared with either isoflurane or enflurane. Others have reported halothane to be equipotent with isoflurane (8,12) or have not made comparisons among volatile anesthetics (7,10,11). The recent study by Todorovic and Lingle (19), however, confirms our finding that ICa,T in DRG is more sensitive to isoflurane than to halothane.
In an attempt to elucidate a biophysical mechanism for the depression of ICa,T by isoflurane, we examined the effect of isoflurane on the kinetics of the macroscopic current. We found that isoflurane caused a significant increase in the deactivation rate of ICa,T, with no effects on activation or inactivation rates. Halothane, which had much greater effects on ICa,T in other studies (8,11) than we observed has been reported to have a variety of effects on macroscopic current kinetics. In GH3 cells, for example, halothane indiscriminately increases the rates of activation, inactivation, and deactivation of ICa,T (11), whereas in DRG neurons (8), the major effect of halothane is the decrease the rate of inactivation. None of these changes in macroscopic current kinetics can adequately explain the observed decreases in peak ICa,T. A better understanding of the effects of the volatile anesthetics will best be achieved by using measurements of single T-type channel activity.
T-type channels play several important roles in the regulation of nervous system excitability and information processing. The presence of T-type channels allows peripheral neurons to generate bursts of action potentials, rather than individual spikes, which may affect central processing of sensory signals (3). In several types of central neurons, T-type channels contribute to the generation of membrane potential oscillations that lead to rhythmic firing of action potentials and alter the ability of these neurons to process incoming synaptic information (1,2). In addition, T-type channels on the dendrites of some central neurons can alter dendritic responses to postsynaptic potentials (5,6), amplifying or inhibiting both excitatory and inhibitory potentials based on previous patterns of membrane excitation. Theoretically, volatile anesthetic inhibition of T-type channels could lead to alterations in neuronal information processing and excitability that may contribute to some of the clinical manifestations of general anesthesia.
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Acknowledgments |
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Supported by National Institutes of Health Grants GM31144 to CL and HL36977 to PB, and by NRSA Fellowship GM17718-02 to TM.
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