Effects of enflurane on the voltage-gated membrane currents of bovine adrenal chromaffin cells

Neuroscience Letters, 146 (1992) 147-151

147

© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 09057

Effects of enflurane on the voltage-gated membrane currents of bovine adrenal chromaffin cells Joseph J. Pancrazio a'b, W y u n K o n Park c and Carl Lynch

III a

Departments of~Anesthesiology and bBiomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (USA) and CDepartment of Anesthesiology, Yonsei Medical College, Seoul (South Korea) (Received 17 June 1992; Revised version received 3 August 1992; Accepted 9 August 1992)

Key words: Adrenal; Anesthetic; Chromaffin; Enflurane; Ion channel; Patch clamp The effects of the volatile anesthetic enflurane on voltage-gated ionic currents of bovine adrenal chromaffin cells were studied using the patch clamp technique. Bath application of 3.5% (1.7 mM) enflurane decreased the outward Ca:+-dependent K ÷ current (Irtca)) 'hump' by 88_+6% (mean_+S.E.M., n=5 cells) and the peak inward Ca 2÷ current by 60_+3% (n=5), whereas the Ca2+-independent K ÷ current fell by only 34_+3% (n=5) and peak inward Na + current was unchanged. Exposure of excised patch 'BK' Ca2+-dependent K ÷ channels to 3.5% enflurane revealed that the anesthetic directly suppressed the channel probability of opening by 68_+10% (n=4) with no effect on open state conductance. The differential sensitivity of depolarizing and hyperpolarizing current pathways may contribute to the biphasic response, excitation and depression, observed in certain neuronal systems in response to this inhalational anesthetic.

General anesthetics are known to inhibit as well as enhance electrical excitability in neuronal tissue. Of the inhalational agents currently employed, only enflurane has been associated with seizure activity at clinically relevant concentrations [19, 21]. In vitro spontaneous bursting has been reported in isolated neurons after administration of low to moderate dosages of enflurane [6, 14, 15] with depression occurring at higher concentrations [15]. It is unclear, however, the extent which anestheticinduced alterations of excitatory synaptic transmission [14, 16], GABA-mediated pathways [18], or intrinsic bioelectrical properties [5, 6] may account for the excitatory effects of enflurane. Utilizing the patch clamp method, we now report the actions of enflurane on voltage-gated ionic currents of chromaffin cells. Bovine adrenal chromaffin cells, which are considered useful models of neuronal electrophysiology, were generously supplied by Dr. Y.I. Kim. Cells were isolated according to a previously reported method [8] with modifications [4] and affixed to poly-L-lysine-coated glass coverslips. The external bathing solution contained (in mM): NaCI 140, KC1 5, CaC12 2, MgC12 1, HEPES 10, pH 7.4 with NaOH. In order to observe whole-cell outCorrespondence: J.J. Pancrazio, Department of Anesthesiology, Box 238, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.

ward K ÷ currents, the internal pipette solution contained: KC1 100, KOH 42, HEPES 5, EGTA 10, CaC12 1, Mg-ATP (equine salt) 5, pH 7.4 with KOH. In order to isolate whole-cell inward Ca 2÷ and Na ÷ currents, the pipette solution was adjusted to: CsC1 120, tetraethylammonium (TEA) chloride 20, HEPES 10, EGTA-CsOH 11, pH 7.4 with TEA-OH. Unitary K ÷ channel activity was observed by exposing both sides of a membrane patch to a solution yielding an approximate free Ca 2+ concentration of ~900 nM according to Stockbridge [25]: KC1 100, KOH 42, CaC12 9.32, EGTA 10, HEPES 10, pH 7.4 with KOH. All solutions were filtered through a 0.2 gm filter before use. Whole-cell and inside-out patch clamp recordings were made at room temperature (2224°C) as described by Hamill et al. [9]. Measurements were performed using the Axopatch 200 amplifier with series resistance compensation typically set at 75% or greater during whole-cell recordings. Patch electrodes were fabricated from borosilicate glass model 1B150F-4 (World Precision Instruments) and the tips were heatpolished using a Narishige microforge. Data acquisition was accomplished using an Axon Instruments pCLAMP system with a 386-compatible microcomputer. For whole-cell recordings, the holding potential was set to -80 mV and to correct for leakage and capacitive currents, conventional - P / n correction was employed [1]. By this method, the currents evoked by 4

148

hyperpolarizing subpulses of amplitude P/4 were added to the current elicited from a depolarizing command voltage from the holding potential of magnitude P. The magnitude of the whole-cell Ca2+-dependent K + current (IK~Ca)) was represented by the outward current 'hump' apparent from the current-voltage (I-V) curve [17], whereas the amplitude of the current at +90 mV was used to approximate the CaZ+-independent K ÷ current (IK) (Fig. 1A). IK(Ca)was quantified by drawing a tangent between the inflection points on each side of the hump and measuring the distance from the peak outward current and a point along the tangent at the corresponding voltage. Enflurane was equilibrated in the bathing solution by bubbling filtered air through a calibrated vaporizer for at least 20 min. Anesthetic levels, which were set with the vaporizer in terms of volume % and were determined with a gas chromatograph sampled directly from the recording chamber, are listed in Table I. Control, anesthetic treatment, and recovery phases were performed on each cell tested. Data are represented as mean+S.E.M. and the number of cells tested (n). Statistical significance of a drug effect was determined using Student's t-test. Values of P<0.05 were considered to be significant. As previously shown by Marty and Neher [17], the I-V curve from a typical bovine adrenal chromaffin cell exhibited a characteristic shape in the presence of external Ca 2+ (Fig. 1A). Application of Ca2+-free external solution (Ca 2÷ replaced with 0.5 m M EGTA) ablated the Ca2+-dependent hump (n=6). Typically, the outward current evoked by voltages more positive than +60 mV was larger in the absence of external Ca 2+ due to the additional flux of K* through Ca 2+ channels which are ordinarily K+ impermeant [17]. Administration of 3.5% (1.7 mM) enflurane produced a dramatic reduction in outward current as shown in Fig. lB. In contrast, peak inward Na ÷ current (INa) was insensitive to the volatile

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ANESTHETIC CONCENTRATIONS (PERCENT IN AIR AND M M IN SOLUTION) A N D PERCENT CONTROL 1K~c~l, IK A N D Ica A F T E R APPLICATION OF E N F L U R A N C E Concentrations in solution reported as mean_+standard deviation (n=45 trials), whereas percent control current given as mean _+ S.E.M. and the number of cells examined in parentheses. Anesthetic concentration

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Membrane Potential (mY) Fig. I. Voltage-activated membrane currents from bovine adrenal chromaffin cells. A: outward current vs. voltage (I-V) relation for a cell in normal external Ca 2÷ (2 mM) and after treatment with Ca2+-free solution (external Ca 2÷ replaced with 0.5 m M EGTA). The Ca2+-dependent K ÷ current (IK~c~))'hump' is abolished in the absence of Ca 2÷ entry. As shown, 1K(Ca~was quantified by drawing a tangent between the inflection points on each side of the hump and measuring the distance from the peak outward current and a point along the tangent at the corresponding voltage. The Ca2+-independent K ÷ current (IK) was estimated as the current at +90 mV beyond the hump. B: effect of the inhalational anesthetic enflurane on voltage-gated membrane currents. Current traces shown were elicited by depolarizing pulses 80 ms in duration from -30 to +30 mV in 20 mV increments. Records are from the same cell before and during exposure to 3.5% (1.7 mM) enflurane (denoted by e). C: I-V relations for the cell identified in B. Enflurane primarily depresses the Ca2*-dependent K + current (1K(c~) hump.

149

agent (data not shown). In Fig. 1C, the I-V plots of outward current before, during and after exposure to enflurane show that the anesthetic diminishes IK(C,) as well as the outward current (IK) evoked by depolarized potentials beyond the hump. Enflurane at 3.5% decreased I~:(c,) by 88_+6% (n=5) whereas IK, elicited by a command potential to +90 mV, fell by 34 _+3% (n=5). To determine whether or not the observed decrease in Ir(ca) could be attributed to a reduction in Ca 2÷entry, the effect of enflurane on inward Ca 2÷ current (/Ca) was evaluated. Since the high-voltage activated Ic, typically inactivates much more slowly than IN,, IC, was readily distinguished from IN, for quantitative analysis. Peak Ic,, evoked by potentials from -10 to +10 mV, ranged from -50 to -220 pA. As illustrated in Fig. 2A, the inhalational anesthetic substantially reduced/Ca and increased the rate of inactivation. Overall, peak Ic, fell by 60+3% (n=5) with exposure to 3.5% enflurane. The rate of inactivation, measured 100-350 ms from the onset of the voltage pulse to ensure adequate separation from IN,, was enhanced by the inhalational anesthetic. This inactivation phase was adequately fit by a single exponential decay function for inward currents evoked by potentials ranging from -20 to +20 mV. In response to a command voltage to +10 mV, the time constant of inactivation, rh, fell from 2.87+0.60 s (n=10) to 0.61 _+0.11 s with exposure to 3.5% enflurane. Fig. 2B summarizes the dose-dependent reductions in rh by enflurane. Typical I-V plots shown in Fig. 2C suggest that the anesthetic had little or no effect on the voltagedependence of activation. This observation was confirmed quantitatively by scaling the current magnitude at each potential to the constant field equation and fitting the resulting values to a Boltzmann function. No shift in voltage dependence was observed. With the exception of INa, the actions of enflurane on each of the currents examined were dose-dependent. Table I summarizes the reductions achieved by different anesthetic concentrations o n / K ( C a ) , / C a and I K. C o m p a r a t i v e l y , IK(Ca) appeared most sensitive to the volatile anesthetic. Since enflurane reduces Ic, rh, the time integral of/Ca, which is proportional to C a 2+ entry, is further diminished possibly resulting in the greater decrease in IK(Ca). The exact quantitative relationship between Ca z+ entry and Ca2+-dependent K ÷ channel activation is unclear, therefore we performed inside-out patch clamp recordings to determine if the anesthetic can directly affect Ca2+-dependent K + channel conductance or activity. Marty and Neher [17] have previously shown that bovine adrenal chromaffin cells express large conductance 'BK' Ca2+-dependent channels which underlie the macroscopically observed IK(Ca)"Fig. 3 depicts data segments from a typical patch containing 3 BK channels evoked by a transmembrane voltage of +30 mV before and during

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Membrane Potential (mY) Fig. 2. The effect of enflurane on voltage-gated Ca 2÷ current (Ic,) of adrenal chromaffin cells. Cells were dialyzed with a solution replacing K ÷ with Cs ÷ and TEA to suppress outward K + currents. The initial inward transient current represents rapid Na ÷ current which appeared unaffected by the anesthetic. Enflurane treatment is denoted by o. A: 3.5% (1.7 mM) enflurane reversibly diminishes/ca evoked by a command pulse to 0 mV from a holding potential of - 8 0 mV. B: dosedependent reductions in the time constant of inactivation, r,, o f / c a evoked by different potentials. All anesthetic-treated rh values are significantly (P<0.01) different from control, rh values between concentrations are significantly different (P<0.05) except for currents elicited by a test pulse to +20 mV. C: the I-V plots for control, enflurane treatment, and recovery show that anesthetic-induced reduction in /Ca is voltage-independent.

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Fig. 3. Effect of 3.5% enflurane on an excised inside-out membrane patch containing 3 BK channels in the presence of-900 nM free Ca2. on both sides of the membrane patch. The membrane patch was voltageclamped to +30 mV. Channel openings (outward current) are shown as upward deflections. A BK channel subconductance state (indicated by o), which was approximatelyone-third the magnitude of the more prevalent larger open channel conductance state, was rarely observed. These traces are representativeconsecutivedata segments from records 90 s in duration. In the presence of the anesthetic, Po decreases with no effect on open channel amplitude; Po decreased from 0.286 to 0.023 with enflurane treatment.

exposure to enflurane. Under control conditions, large current deflections averaging 7.1+0.2 pA (n=4) with a probability of channel opening (Po) of 0.114+0.072 (n=4) are clearly evident. Exposure of the membrane patch to a Ca 2+ concentration of~100 nM significantly reduces Po (data not shown) indicating that the large conductance channel openings shown in Fig. 3 are indeed Ca2+-de pendent. We occasionally observed Ca2+-dependent smaller openings which may constitute a subconductance state as described in adrenocortical cells [23]. These events were rare and therefore did not significantly affect the analysis. Anesthetic treatment significantly depressed Po by 68+10% (n=4) with no alteration of the open channel amplitude. These data indicate that while anestheticinduced reductions in Ic, may well contribute to the observed decrease in IK(Ca), enflurane can directly depress BK channel activity. Most of the prior voltage clamp studies of inhalational anesthetics have focussed on the effect of halothane. A recent study on G H 3 clonal pituitary cells [10] showed that INa is relatively unaffected by halothane at the corresponding equipotent dosages utilized in this study of enflurane. The PC12 p h e o c h r o m o c y t o m a cell line, which has properties similar to adrenal chromaffin cells, exhibits depolarization-induced cytoplasmic Ca 2+ rises which appear to be more sensitive to blockade (IC50:0.78 mM) by enflurane than in chromaffin cells [11]. Although clinically relevant concentrations of halothane (1-2%) exert virtually no effect on chromaffin cell voltage-gated C a 2+ entry [20, 25], we observed a substantial reduction of Ic, with enflurane. Consistent with our findings, GOthert and Wendt [7] reported that high KCl-induced catechol-

amine release from chromaffin cells was unaffected by -<14 m M halothane, whereas 1.0 m M enflurane diminishes release by 20%, suggesting that voltage-gated Ca 2+ entry was more sensitive to enflurane than halothane. In guinea pig papillary muscle, Lynch [13] has previously shown that ventricular slow action potentials, employed as an assay for/ca, are reduced by enflurane over a clinically useful concentration range. Likewise, whole-cell voltage clamp measurements from isolated ventricular myocytes revealed depression of Ic, and an increase in the rate of inactivation with enflurane [2] similar to our observations. Alternatively, it is possible that enflurane may unmask a transient Ca 2+ current subtype in chromaffin cells by preferentially blocking a steady-state component [3]. Further experiments utilizing selective Ca 2÷ channel antagonists in combination with enflurane may reveal Ca 2+ channel selectivity of the anesthetic. The present results indicate that enflurane can directly depress CaZ+-dependent K ÷ channel activity independent of Ca 2+ entry. Consistent with the present findings, a past biochemical study utilizing 86Rb + showed that flux through Ca2+-activated K ÷ channels of rat glioma C6 cells is diminished by enflurane with a similar dose-dependence [24]. In isolated hippocampal neurons, the fast and slow afterhyperpolarization phases, which are mediated by Ca2+-dependent K + channels [12], are readily decreased by low to moderate levels of inhalational anesthetics resulting in an increase in excitability, whereas higher concentrations decrease excitability [6]. The relevance of the present findings to clinical anesthesia and seizure activity must be considered with caution since enflurane is likely to affect ligand-gated channels as well [14, 16, 18]. Interestingly, Stevens et al. [21] have shown that enflurane-induced convulsant activity in cats is biphasic. The frequency of seizures increases rapidly from 2 to 4% enflurane with a decrease in occurrence at higher concentrations [21]. Perhaps the differential sensitivity of hyperpolarizing currents, IK(ca) and IK, and depolarizing currents, lc,, and IN,, to enflurane may contribute to this biphasic response. This study was supported by N I H Research G r a n t GM31144-08 to C.L. and a National Research Service Award to J.J.E l Armstrong, C.M. and Bezanilla, F., Charge movement associated with the opening and closing of the activation gates of Na channels, J. Physiol., 63 (1974) 533--552. 2 Bosnjak, Z.J., Supan, F.D. and Rusch, N.J., The effects of halothane, enflurane, and isoflurane on calcium current in isolated canine ventricular cells, Anesthesiology,74 (I 991) 340-345. 3 Bossu,J.-L., De Waard, M. and Feltz, A., Inactivation characteristics reveal two calcium currents in adult bovine chromaffin cells, J. Physiol., 437 (1991) 603 620.

151 4 Creutz, C.E., Zaks, W.J., Hamman, H.C., Crane, S., Martin, W.H., Gould, K.L., Oddie, K. and Parsons, S.J., Identification of chromaffin granule proteins: relationship of the chromobindins to calelectrin, synhibin, and substrates p35 and p36, J. Biol. Chem., 262 (1987) 1860-1868. 5 Franks, N.P. and Lieb, W.R., Volatile general anesthetics activate a novel neuronal K ÷ current, Nature, 333 (1988) 662-664. 6 Fujiwara, N., Higashi, H., Nishi, S., Shimoji, K., Sugita, S. and Yoshimura, M., Changes in spontaneous firing patterns of rat hippocampal neurones induced by volatile anesthetics, J. Physiol., 402 (1988) 155-175. 7 GOthert, M. and Wendt, J., Inhibition of adrenal medullary catecholamine secretion by enflurane, Anesthesiology, 46 (1977) 404410. 8 Greenberg, A. and Zinder, O., ~- and fl-receptor control of catecholamine secretion from isolated adrenal medulla cells, Cell. Tissue Res., 226 (1982) 655-665. 9 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches, Pfltigers Arch., 391 (1981) 85-100. 10 Herrington, J., Stern, R.C., Evers, A.S. and Lingle, C.J., Halothane inhibits two components of calcium current in clonal (GH3) pituitary cells, J. Neurosci., 11 (1991) 2226-2240. 11 Kress, H.G., Mtiller, J., Eisert, A., Gilge, U., Tas, P.W. and Koschel, K., Effects of the volatile anesthetics on cytoplasmic Ca 2+ signaling and transmitter release in a neural cell line, Anesthesiology, 74 (1991) 309-319. 12 Lancaster, B., Nicoll, R.A. and Perkel, D.J., Calcium activates two types of potassium channels in rat hippocampal neurons in culture, J. Neurosci., 11 (1991) 23 30. 13 Lynch III, C., Vogel, S., Pratila, M.G. and Sperelakis, N., Enflurane depression of myocardial slow action potentials, J. Pharmacol. Exp. Ther., 222 (1982) 405-409.

14 Maclver, M.B. and Kendig, J.J., Enflurane-induced burst discharge of hippocampal CA1 neurones is blocked by NMDA receptor antagonist APV, Br. J. Anaesth., 63 (1989) 296-305. 15 Maclver, M.B. and Roth, S.H., Anesthetics produce differential actions on the discharge activity of a single neuron, Eur. J. Pharmacol., 139 (1987) 43 52. 16 Martin, D.C., Volatile anesthetics and NMDA receptors - enflurane inhibition of glutamate-stimulated 3H-MK-801 binding and reversal by glycine, Neurosci. Lett., 132 (1991) 73-76. 17 Marty, A. and Neher, E., Potassium channels in cultured bovine adrenal chromaffin cells, J. Physiol., 367 (1985) 117-141. 18 Nakahiro, M., Yeh, J.Z., Brunner, E. and Narahashi, T., General anesthetics modulate GABA receptor channel complex in rat dorsal root ganglion neurons, FASEB J., 3 (1989) 1850-1854. 19 Neigh, J.L., Garman, J.K. and Harp, J.R., The electroencephalographic pattern during anesthesia with ethrane, Anesthesiology, 35 (1971) 482-487. 20 Pancrazio, J.J. and Lynch III, C., Suppression of a Ca2÷-dependent K ÷ conductance in adrenal chromaffin cells by halothane, Anesthesiology, 75 (1991) A1038. 21 Stevens, J.E., Fujinaga, M., Oshima, E. and Mori, K., The biphasic pattern of the convulsive property of enflurane in cats, Br. J. Anaesth., 56 (1984) 395-403. 22 Stockbridge, N., EGTA, Comput. Biol. Med., 17 (1987) 299-304. 23 Tabares, L., Lrpez-Barneo, J. and de Miguel, C., Calcium- and voltage-activated potassium channels in adrenocortical cell membranes, Biochim. Biophys. Acta, 814 (1985) 96-102. 24 Tas, P.W., Kress, H.G. and Koschel, K., Volatile anesthetics inhibit the ion flux through Ca2+-activated K* channels of rat glioma C6 cells, Biochim. Biophys. Acta,983 (1989) 264-268. 25 Yashima, N., Wada, A. and Izumi, F., Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells, Anesthesiology, 64 (1986) 466M72.