Introduction

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Using biochemical, electrophysiological, and molecular biological techniques, a number of membrane signaling proteins have been shown to be affected by volatile anesthetics (VAs). Among membrane proteins, ligand-gated ion channels such as -aminobutyric acid_A (Lin et al., 1992), nicotinic (Scheller et al., 1997), and N-methyl-D-aspartate (Yamamura et al., 1990) receptors; G protein-linked receptors such as muscarinic receptors (Durieux, 1995); protein kinase C (PKC; Hemmings and Adamo, 1994; Slater et al., 1997); and voltage-gated ion channels (Franks and Lieb, 1994) are all the possible candidates. Ca²⁺ current through high-voltage-gated calcium channels (HVGCCs) has been shown to be inhibited by VAs in several preparations such as myocardial tissue (Lynch et al., 1981; Terrar and Victory, 1988b; Bosnjak et al., 1991; Pancrazio, 1996), neurons (Study, 1994), and secretory cells (Pancrazio et al., 1993; McDowell et al., 1996). Recently, it has been shown that the VAs halothane (HAL) and isoflurane (ISO) have a common inhibitory effect on the voltage-dependent opening of P/Q, L, N, and R types of HVGCCs expressed in isolation in Xenopus laevis oocytes (Kamatchi et al., 1999). In contrast, consensus regarding VA effects on PKC activity has remained elusive. For example, the activity of PKC was stimulated by HAL in synaptosomes and brain cytosol (Tsuchiya et al., 1988; Hemmings and Adamo, 1996), and VA activation of PKC has been suggested to inhibit m1 muscarinic receptors (Minami et al., 1997). On the other hand, VAs and alcohol were shown to inhibit PKC and to prevent its translocation to the membrane (Slater et al., 1997). In view of such conflicting findings regarding the action of PKC, we elected to examine how VAs would influence PKC modulation of HVGCCs.

Isolated expression of HVGCCs in X. laevis oocytes revealed that PKC activation potentiated 1E and 1B channel currents, whereas 1A and 1C currents were unaltered (Stea et al., 1995a). Immunocytochemical studies have shown that 1E subunit is widely distributed in the brain and potentially modulated by phosphorylation with PKC in addition to other kinases (Yokoyama et al., 1995). Therefore, 1E- type channels were used as a model to study the effect of VAs on PKC-enhanced opening of the HVGCCs by expressing them in X. laevis oocytes. The oocyte model provides a convenient assay system for investigating the modulation of cloned ion channels because these cells endogenously express constituents of several second-messenger pathways, including protein kinase A and PKC. In this investigation, PKC was activated directly by the application of phorbol-12-myristate-13-acetate (PMA) or 1,2-dioctanoyl-sn-glycerol (DOG) or indirectly with acetyl--methylcholine (MCh), an m1 muscarinic receptor agonist. The influence of VAs on 1E channels was studied before, during, or after the application of PKC activators. In addition, the effects of VAs were examined after PKC was depleted from the oocytes.

	Materials and Methods

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Oocyte Harvesting and Microinjection. Mature female X. laevis frogs were obtained from Xenopus I (Ann Arbor, MI), housed in an established frog colony, and fed regular frog brittle twice weekly. For the removal of oocytes, a frog was anesthetized in 500 ml of 0.2% 3-aminobenzoic acid ethyl ester (Sigma Chemical Co., St. Louis, MO) in water until unresponsive to a painful stimulus. The anesthetized frog was placed supine on ice, and an incision of ~1.5 cm long was made through both the skin and muscle layers of one lower abdominal quadrant. A section of the ovary was exteriorized, and a lobule of oocytes (~200) was removed. The wound was closed in two layers, and the animal was allowed to recover from anesthesia, kept in a separate tank overnight, and returned to the colony the next day. The oocytes were washed twice in Ca²⁺-free solution (82.5 mM NaCl, 2 mM KCl, 1.8 mM MgCl₂, 5 mM HEPES, pH 7.5), followed by collagenase (type 1A; Sigma Chemical Co.) treatment (1 mg/ml in Ca²⁺-free solution). The oocytes were agitated in this solution for a period of 2 to 3 h at room temperature to remove the follicular cell layer. Defolliculation was confirmed by microscopic examination. The oocytes were washed twice in Ca²⁺-free solution and transferred to modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.41 mM CaCl₂, 0.30 mM CaNO₃, 0.82 mM MgSO₄, 15 mM HEPES, pH 7.4) containing 10 µg/ml gentamycin sulfate. They were allowed to recover for 3 to 10 h at 16°C before cDNA injection. Nuclear (germinal vesicle) injection (Nanoject; Drummond Scientific Co., Broomall, PA) was performed using 9.2 nl of a 1:1:1 mix (molar ratio; not exceeding a total of 3 ng of cDNA) of rat brain 1E, 1B, and 2/ cDNA subunits subcloned in the mammalian expression vector pMT2 (Stea et al., 1995a). For the coexpression of muscarinic m1 receptor with the Ca²⁺ channel, 1 ng of rat m1 receptor cDNA subcloned in pcDNA 3.1 (InVitrogen, Carlsbad, CA) was included with the above mix. The oocytes were returned to Barth's solution and incubated at 16°C for 6 to 8 days before current recording.

Electrophysiological Recording. Macroscopic currents were recorded using the two-electrode voltage-clamp technique with an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). The amplifier was linked to an interface and an IBM-PC compatible computer equipped with pClamp software (version 5.6; Axon Instruments) for data acquisition. Leak currents were subtracted using the P/4 procedure. Microelectrodes were filled with 3 M CsCl; typical resistances were 0.5 to 2.5 M. KCl-Agar bridges were used as ground electrodes to minimize any junction potential attributable to changes in ionic composition of the bath solution. The oocytes were placed in a recording chamber (500-µl volume) and superfused at a rate of 5 ml/min with recording solution containing 40 mM Ba(OH)₂, 50 mM NaOH, 2 mM KOH, 5 mM HEPES, and 0.4 mM niflumic acid, neutralized to pH 7.4 with methanesulfonic acid. Ba²⁺ was used as the channel-permeant ion, and niflumic acid was included in the recording solution to block intrinsic Ca²⁺-activated Cl channels. I_Ba was elicited for a duration of 850 ms by depolarizing the oocytes to 0 mV from a holding potential of 80 mV. Appropriate current-voltage relations for these currents were confirmed before drug application.

Equilibration with VAs. Output of HAL or ISO from calibrated anesthetic-specific vaporizers was bubbled through a reservoir filled with 30 to 40 ml of superfusion solution. Air at a flow rate of 500 ml/min was used as the carrier gas, and a minimum of 7 to 10 min of bubbling was allowed for equilibration with VA. The superfusion of the oocyte with the continuously bubbled solution was maintained throughout the recording of I_Ba. In the case of combined treatment with VA and other drugs, the VA-equilibrated superfusion solution in addition contained the final concentration of the respective drug. Lack of significant loss of VA from the recording chamber was verified by analysis of triplicate aqueous samples from the chamber that equilibrated with air (1:4, air/solution) in a gas chromatograph (Aerograph 940; Varian Analytical Instruments, Walnut Creek, CA) calibrated with standards for HAL or ISO. Results were converted to concentrations in liquid using aqueous/gaseous partition coefficients at 25°C (Firestone et al., 1986) and averaged to obtain the values stated in the report.

Treatment Schedules. In all oocytes exhibiting significant I_Ba, measurements were obtained under control conditions after one or two sequential drug applications and after drug washout. Control I_Ba was recorded at least 5 min after the oocyte was impaled and I_Ba had stabilized. In general, the recording solution was exchanged for 30 s (~5 chamber volumes), and I_Ba was recorded 2 min after beginning the exchange. To minimize the loss of ISO or HAL to the atmosphere, solutions containing VA were superfused continuously. Two to four minutes after washout of drug, I_Ba was recorded. Five basic schedules of drug application, mentioned briefly later, were used to examine the actions/interactions of PKC activators and VAs. Details of various treatment schedules are included in the correspondingly numbered figure legends, and the tables provide details of current amplitudes: schedule 1, control response with PKC activators (Fig. 1 and Table 1); schedule 2, administration of PKC activators first, followed by the addition of VA (Fig. 2 and Table 2); schedule 3, administration of VA first, followed by the addition of the activators of PKC (Fig. 3 and Table 3); schedule 4, simultaneous administration of PKC activators and VA together (Fig. 4 and Table 4); and schedule 5, effect of VA in PKC-depleted oocytes (Fig. 5 and Table 5).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Effects of PKC activators in oocytes expressing m1 receptors and 1E1B2/-subunits (schedule 1). Oocytes were held at 80 mV and depolarized to 0 mV. The oocytes were allowed to equilibrate in the recording solution for at least 5 min before the control IBa was recorded. After the control measurements, the oocytes of this group were perfused with any one of the following drugs: PMA, 4-PDDC, DOG (not shown), and MCh (1 or 10 µM) for a period of 30 s. IBa was recorded after 2 min from the beginning of perfusion. However, in the case of PMA or DOG, the oocytes were depolarized to the test potential approximately at an interval of 30 s from the second minute of their perfusion to obtain the maximum potentiation of IBa. This is based on our preliminary studies that the effect of PMA reached its maximum (potentiation) in ~3 min in 90% of the oocytes. The response was considered to have reached saturation if the current was approximately equal to its previous response or within ±10%. The trace shown here for PMA was obtained from an oocyte in which the IBa reached its maximum in the third minute. Refer to Table 1 for the averaged values and statistical significance.

View this table: [in this window] [in a new window]   TABLE 1 Effects of PKC activators in oocytes expressing m1 receptors and 1E1B2/-subunits (schedule 1) The oocytes were held at 80 mV and depolarized to 0 mV for a period of 850 ms to activate IBa as described in the text. The peak and late currents indicate the maximum amplitude and the current at 830 ms, respectively. The oocytes were perfused with PMA for a period of 30 s, and IBa was recorded between min 2 and 4 thereafter by depolarization as mentioned in schedule 1 (see legend for Fig. 1 for details). The current reached its maximum amplitude at 3 min after the perfusion of PMA in ~90% of the oocytes. MCh at 1 or 10 µM was perfused for 30 s, and the current was recorded 2 min after the beginning of perfusion as mentioned in schedule 1. 4-PDDC was perfused for a period of 30 s, and the current was recorded after 2 min. Refer to Fig. 1 for the prototype current tracings for the values shown here. Values are given as mean ± S.E.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effects of subsequent treatment with VAs on the response to PKC activators (in the ongoing presence of PMA/DOG) in oocytes expressing 1E1B2/-subunits (schedule 2). This schedule was basically the continuation of schedule 1 but with additional examination of the effect of VA after the presumed phosphorylation of the 1E channels. The oocytes were allowed to equilibrate in the recording solution for at least 5 min before the control IBa was recorded. After obtaining maximum potentiation with PMA or DOG, oocytes were perfused with a solution containing VA (in presence of PMA or DOG). The IBa was recorded after 2 min from the beginning of the perfusion of the VA. This was followed by wash protocol. See Table 2 for the statistical significance of the result shown here.

View this table: [in this window] [in a new window]   TABLE 2 Effects of subsequent treatment with VAs on the response to PKC activators (in the ongoing presence of PMA/DOG) in oocytes expressing 1E1B2/-subunits (schedule 2) The oocytes were held at 80 mV and depolarized to 0 mV. The control measurements were taken at least 5 min after the oocytes were impaled. The treatment schedule is as shown in schedule 2. Briefly, after obtaining maximum potentiation with PMA or DOG as mentioned in the legends to Fig. 2, the oocytes were perfused with a solution containing VA (in the presence of PMA or DOG). The IBa was recorded after 2 min from the beginning of perfusion with VA. This was followed by the wash protocol. Refer to Fig. 2 for the prototype current tracings for the values shown here. Values are given as mean ± S.E.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effects of prior treatment with VAs on the response to PKC activators in oocytes expressing m1 receptors and 1E1B2/-subunits (schedule 3). This protocol was the reverse of schedule 2, that is, the PKC activators were tested after the administration of VAs. After the control measurements, the oocytes were perfused with either VA, and the IBa was recorded 2 min from the beginning of the perfusion. This was followed immediately with the perfusion of a solution of PMA or DOG or MCh (10 µM) that was equilibrated with the respective VA. The IBa was recorded 2 min from the beginning of the perfusion of this combination. This was followed by wash protocol. See Table 3 for averaged results and significance.

View this table: [in this window] [in a new window]   TABLE 3 Effects of previous treatment with VAs on the response to PKC activators in oocytes expressing m1 receptors and 1E1B2/-subunits (schedule 3) The oocytes were held at 80 mV and depolarized to 0 mV. The oocytes were allowed to equilibrate in the recording solution for at least 5 min before the control IBa was recorded. This was followed by the application of drugs as mentioned in treatment schedule 3 in the legend for Fig. 3. Briefly, the oocytes were perfused with VA first, and the IBa was recorded 2 min from the beginning of its perfusion. This was followed immediately with the perfusion of a solution of PMA or DOG or MCh equilibrated with VA. The IBa was recorded 2 min from the beginning of the second perfusion. Refer to Fig. 3 for the prototype current tracings for the values shown here. Values are given as mean ± S.E.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of simultaneous treatment with VAs and PKC activators in oocytes expressing m1 receptors and 1E1B2/-subunits (schedule 4). After the control measurements, oocytes were perfused continuously with a superfusion solution containing PMA or MCh (1 or 10 µM), which was equilibrated with the respective VA. The IBa was recorded 2 min from the beginning of perfusion of this combination. See Table 4 for averaged results and statistical significance.

View this table: [in this window] [in a new window]   TABLE 4 Effects of simultaneous treatment with VAs and PKC activators in oocytes expressing m1 receptors and 1E1B2/-subunits (schedule 4) The oocytes were held at 80 mV and depolarized to 0 mV. These experiments were conducted according to schedule 4 (see the legend for Fig. 4). Briefly, after the control measurement, one of the combinations shown here was perfused continuously for a period of 2 min, and the IBa was recorded. The perfusion solution was equilibrated with VAs and in the presence of the other agents as mentioned in the text. Refer to Fig. 4 for the prototype current tracings for the values shown here. Values are given as mean ± S.E.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of PMA and VAs in PKC-depleted oocytes expressing 1E1B2/-subunits (schedule 5). The oocytes used in this group were incubated in Barth's solution containing PMA (500 nM) for ~4 h at 16°C before experimentation. After the control measurements, the oocyte was perfused with PMA or continuously with one of the VAs. The IBa was recorded 2 min from the beginning of perfusion. See Table 5 for averaged results and statistical significance.

View this table: [in this window] [in a new window]   TABLE 5 Effect of PMA and VAs in PKC-depleted oocytes expressing 1E1B2/-subunits (schedule 5) The oocytes used in this group were incubated in Barth's solution containing PMA (500 nM) for ~4 h at 16°C. Next, the oocyte was transferred to the recording chamber and clamped as mentioned in the text. The treatment schedule was as in schedule 5, as in the legend for Fig. 5. The control measurements were taken at least 5 min after the oocytes were impaled. After this, they were perfused with either fresh PMA or VA, and the IBa was recorded in 2 min as mentioned in schedule 5. Refer to Fig. 5 for the prototype current tracings for the values shown here. Values are given as mean ± S.E.

Chemicals. HAL and ISO were purchased from Halocarbon Laboratories (River Edge, NJ) and Ohmeda PPD Inc. (Liberty Corner, NJ), respectively. PMA, 4-phorbol-12,13-didecanoate (4-PDDC; Research Biochemicals, Inc., Natick, MA), and DOG (Calbiochem, San Diego, CA) were dissolved in DMSO (0.05%). MCh (Sigma Chemical Co.) was dissolved in distilled water. All these agents except VAs were prepared as concentrated stock solutions and stored frozen at 20°C. They were diluted to their final concentration in recording solution on the day of the experiment before use. Niflumic acid (Sigma Chemical Co.) was added to the recording solution, which was stirred overnight for it to dissolve.

Data Analysis. The peak represented the maximum amplitude of the inward current. The current amplitude at 830 ms was arbitrarily defined as the late current, which was used as a measure of relative degree of channel inactivation. The data are shown as mean ± S.E., unless otherwise indicated. The data were analyzed using either the PCS program (Pancrazio, 1993) or Clampfit, version 6.0.2 (Axon Instruments). Statistical significance was determined using either Dunnett's t test or paired t test, and P < .05 was considered significant.

	Results

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Approximately 80% of the oocytes injected with Ca²⁺-channel subunits expressed the inward I_Ba on depolarization. The average peak and late I_Ba under control conditions were 1784 ± 179 nA (range, 462 to 6138 nA) and 265 ± 31 nA (range, 70 to 1096 nA), respectively, with the peak observed between 10 and 10 mV and at 62 ± 0.5 ms (n = 52). Typically, the current became outward between 50 and 60 mV. The basic properties of this inward current are similar to those described when cloned neuronal 1E channels from other species were expressed in this system (Stea et al., 1995b).

Control Response with PKC Activators (Schedule 1). The administration of PMA (500 nM) led to the potentiation of both the peak and late I_Ba through 1E channels (Fig. 1 and Table 1). On an average, with 500 nM PMA, the peak I_Ba was increased by ~50% and the late component was increased by ~200% compared with control. The potentiation with PMA was observed within 1 min after its application and was evident for up to 10 min without any significant decline; the effect of PMA after this period was not examined. The maximum PMA-induced potentiation was seen in ~3 min in 90% of the oocytes and occurred in the remaining 10% between 3 and 4 min. As PMA binds to the membrane tightly, washout for a prolonged period resulted in only a very slow recovery toward the control values.

Administration of Activators of PKC First, Followed by VA (Schedule 2). The results obtained in this section were a continuation of schedule 1. HAL (0.59 mM) or ISO (0.70 mM) were additionally applied after PMA-induced potentiation was maximal (~3 min). The VAs inhibited I_Ba markedly to a level below the pre-PMA control values (~75% of pre-PMA peak I_Ba control; Figs. 2 and 6A and Table 2). However, this VA-induced reduction of the PMA-potentiated I_Ba was proportionally similar (peak, 51 ± 5%; late, 47 ± 3%, combined average of PMA plus HAL and PMA plus ISO of Table 2; n = 16) to their inhibition of control (no PMA) I_Ba (peak, 57 ± 9%; late, 41 ± 16%, combined average of HAL and ISO of Table 3; n = 18). Similar results were obtained when DOG was substituted for PMA; the potentiation induced by DOG was reversed by HAL (Fig. 2 and Table 2). Here, again, the fractional inhibition of DOG-potentiated I_Ba was quantitatively the same as that observed with HAL alone (Table 3). The slowing of inactivation seen with PMA persisted despite the VA-induced depression. In summary, HAL or ISO produced ~50% inhibition of I_Ba regardless of whether it was previously enhanced by PKC activation with PMA or DOG.

Administration of VA First, Followed by Activators of PKC (Schedule 3). As noted earlier, the administration of HAL (0.59 mM) or ISO (0.70 mM) produced a significant inhibition (~50%) of peak and late components of I_Ba compared with control. When PMA or DOG was applied after VA (in the ongoing presence of VA), the I_Ba was still inhibited to the same extent without any significant change. The presence of either HAL or ISO abolished the enhancing effect of PKC stimulation by PMA (or DOG). The inhibitory effect of VAs was extended beyond the direct effect on the 1E channels and appeared to prevent the presumed PKC-induced phosphorylation by PMA or DOG. In all of these experiments, the inherent depressant effects of VAs on I_Ba were reversible with washing (Figs. 3, A-C, and 6A and Table 3).

Application of PKC Activators and VA Together (Schedule 4). To determine how long VA application was required to abolish the PMA potentiation, PMA (or MCh) was applied simultaneously with VA. The administration of the combination of HAL and PMA or ISO and PMA led to a significant inhibition of the peak and late components of I_Ba that was reversible with washing (Figs. 4A and 6A and Table 4). Furthermore, the inhibition produced by these combinations was quantitatively identical with the effect of VAs alone (Figs. 3, A and B, and 6A and Table 3) as if no PMA was present.

View larger version (44K): [in this window] [in a new window]   Fig. 6.   Summary of the effects of the various combinations of VAs with PMA (A) and MCh (10 µM) (B). Left and right, peak and late currents, respectively. These figures were plotted using the values shown in Tables 1 to 4. Values (as percentage) based on the IBa measurements that were converted to percentage of their respective control values. The dotted line represents control (100%). Open columns, PMA or MCh; striped columns, presence of VA. MCh response in the absence of anesthetic is included as a control (data from Table 1). The n value is given in parentheses. aP < .001, bP < .01, cP < .05 compared with the respective control (paired t test). NS, not significant compared with control (paired t test). **P < .001, *P < .01 compared with their respective PMA or VA alone values (paired t test).

Effect of VA in PKC-Depleted Oocytes (Schedule 5). The down-regulation of PKC was confirmed as the administration of PMA after the prolonged incubation of oocytes with PMA failed to produce any significant potentiation of I_Ba. However, in these oocytes, HAL or ISO still significantly inhibited the I_Ba, which was quantitatively similar to that of the effect in control oocytes. The inhibitory effects of VAs were reversible with washing (Fig. 5 and Table 5).

	Discussion

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As previously reported, the VAs depressed currents through R-type HVGCCs (Kamatchi et al., 1999) encoded by the 1E subunit. This reversible depression is similar to that seen for other expressed HVGCCs such as P/Q-type (1A), N-type (1B), and L-type (1C) channels (Kamatchi et al., 1999), as well as native Ca²⁺ channels in a variety of tissues (Terrar and Victory, 1988a; Bosnjak et al., 1991; Pancrazio et al., 1992; Study, 1994). This depression may contribute to the VA-induced anesthetic state because nonspecific HVGCC blockade by Cd²⁺ as well as L-type Ca²⁺-channel block by verapamil has been found to decrease the anesthetic requirements for ethanol, pentobarbitone, and ketamine in mice (Dolin and Little, 1986; Shen and Sangiah, 1995). Similarly, blockade of P-type channels causes lethargy and stupor, and N-type channel blockade causes antinociception in mice (Bowersox et al., 1996; Llinás et al., 1989).

In the present study, the application of PMA or DOG for 3 min increased the peak and late current through the expressed 1E-type channels of ~150% and ~300%, respectively, as reported previously (Stea et al., 1995a). Several factors, such as an increased Ca²⁺-channel conductance, probability of opening, and channel open time, could be responsible for this potentiation. This increased peak and late I_Ba were reduced to ~50% of their respective PMA/DOG-enhanced values when the oocytes were exposed to VA (in the presence of PMA/DOG), which typically reduced peak and late I_Ba to 75 and 150%, respectively, of their respective control values (Fig. 6A). Because the late current was still greater than pre-PMA control values, the channels still show the kinetic behavior seen after the presumed PKC-mediated phosphorylation even in the presence of VA. When VAs were administered before PMA/DOG, the current was likewise decreased by ~50% as shown in schedule 3 (Figs. 3 and 6A and Table 3), suggesting that VAs inhibited control as well as the PMA/DOG-potentiated channels in a quantitatively similar manner.

The most interesting feature of the study was that PMA or DOG failed to potentiate I_Ba when either agent was perfused after VA. Similarly, PMA or a low concentration of MCh (1 µM) administered simultaneously with VAs also failed to potentiate the I_Ba (Figs. 4 and 6A and Table 4). The inhibition produced by the simultaneous application of these two agents was quantitatively similar to the effect of VA alone and was complete within 2 min. One possibility is the direct action on PKC because anesthetics and alcohols, such as HAL (0.6 mM), enflurane (1 mM), and ethanol (200 mM), were shown to inhibit PKC (Slater et al., 1993). Since binding sites for both anesthetics/alcohols (Slater et al., 1993) and PMA (Ono et al., 1989; for a review, see Quest, 1996) were reported to be present in the regulatory region of PKC, it is tempting to postulate some specific antagonism in this site. Furthermore, it has been shown that HAL (2.4 vol%) reduced the membrane-associated PKC component in rat synaptosomal preparations (Hemmings and Adamo, 1997). If the action of PMA is initiated by its binding to the membrane with subsequent interaction with PKC (Quest, 1996), it is reasonable to relate the VA blockade of the PMA or DOG actions to the inhibition of translocation of PKC. VAs may have reversibly altered the atmosphere/configuration of the membrane bilayer so that PMA would not bind within the membrane in a manner that would allow PKC activation. However, any explanation of this apparent inhibitory action of VA in blocking PMA effects must be contrasted with the fact that the depressant action of VAs was reversed (as it was not significant compared with control) when either VA was followed by a high concentration of MCh (10 µM) (Figs. 3 and 6B and Table 3). Similarly, VA-induced inhibition of I_Ba was absent when a high concentration of MCh was combined with the anesthetic (Figs. 4 and 6B and Table 4). Thus, the effects of high and low concentrations of MCh are qualitatively different. It is well known that m1 muscarinic receptor stimulation activates G proteins (G_q and G₁₁), which in turn activate phospholipase C, resulting in the production of diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP₃). Although DAG (the membrane constituent for which DOG and PMA are substitutes) activates PKC directly, intracellular calcium concentration released from the endoplasmic reticulum by IP₃ acts as a cofactor in the activation of conventional PKC (cPKC) isoforms (for a review, see Caulfield, 1993). Such a second-messenger pathway culminating in the activation of PKC and leading to the potentiation of 1E channels by 1 µM MCh (EC₅₀) has been shown in our previous study in which the inhibitors of any one of the intermediaries such as the G proteins, phospholipase C, IP₃, intracellular calcium concentration, and PKC led to blockade of the effect of MCh (G. L. Kamatchi, S. N. Tiwari, C. Lynch III, and M. E. Durieux, unpublished observations). Hence, the possibility of an alternative pathway such as direct G protein-mediated modulation or the involvement of any non-PKC effector molecule in the action of MCh seems unlikely. In the present study, in addition to 1 µM MCh, we also used 10 µM MCh because our goal was to activate receptor-mediated PKC to the maximum. An indication for this effect was derived from our previous study in which 12.8 µM MCh saturated the potentiation of 1E channels (G. L. Kamatchi, S. N. Tiwari, C. Lynch III, and M. E. Durieux, unpublished observations).

All types of cPKC isoforms (, I, II, and ), one isoform of novel PKC (e.g., ), and an atypical PKC (e.g., ) have been shown to be present in X. laevis oocytes (Johnson and Capco, 1997). Among these, the -isoform requires DAG but not Ca²⁺ for its activation, whereas the -isoform requires neither DAG nor Ca²⁺ (Quest, 1996). These PKCs may be the target of intracellular second messengers such as arachidonic acid and free fatty acids because they have been shown to activate PKC independent of phosphatidylserine, DAG, and even Ca²⁺ (Khan et al., 1995). Activation of PKC need not occur exclusively by translocation to the plasma membrane because its translocation to the nucleus and association with cytoskeletal elements on activation are now well documented (Quest, 1996). Such a possibility exists, because muscarinic agonists were shown to be involved in the release of arachidonic acid (Axelrod, 1990). Based on this evidence, it is conceivable that the PKC isoform or isoforms involved in the inhibition of PMA and low concentration of MCh by VAs are identical or from the same source, probably cPKC, as it requires DAG (or PMA/DOG) and Ca²⁺. These agents were susceptible to VAs due to the possible disruption of the membrane bilayer by VAs. On the contrary, the effect of a high concentration of MCh was only partially blocked by VAs in that MCh possibly shared both the membrane-dependent and -independent pools of PKC. Such a possibility can only be speculated, because a good model with which to study the changes at the level of the membrane is not available. However, this is consistent with the finding that PKC may not be the target of VAs as shown from the oocytes in which PKC had been down-regulated. In this particular series of experiments, the down-regulation of PKC was evidenced by the absence of potentiation of I_Ba with the exposure to fresh PMA. However, VAs still caused ~50% decrease in I_Ba (Fig. 5 and Table 5). Based on this evidence, it is reasonable to conclude that VAs inhibit these 1E channels independent of the existing status of the channels.

Another simple explanation exists that is more difficult to test. The VAs and PMA, as well as DOG, are highly lipophilic in character. Considering the high concentrations of the VAs relative to the PMA or DOG, it is conceivable that the anesthetics somehow bind to these agents in aqueous solution and prevent them from entering the membrane. If this is the case, any studies in which VAs are combined with PMA must be viewed with caution. However, examinations of PKC activity in the combined presence of anesthetics and PMA have been performed and have demonstrated an effect of PMA in the presence of VAs (Hemmings and Adamo, 1994, 1997; Slater et al., 1997), suggesting this possibility is unlikely.

Although the direct depression of 1E HVGCC by VAs is similar to that reported for a number of Ca²⁺ channels, the striking result observed in this study is the differing effects of VAs on direct and indirect activation of PKC. The results from these experiments suggest that the presumed membrane-based activation of PKC by PMA or DOG was blocked by VAs, whereas the receptor-based PKC activation (with a high concentration of MCh) was only partially blocked. Although direct VA effects on proteins have received considerable attention, the action of VAs on the cell membrane surface or membrane protein-lipid interface have been demonstrated as well (Miller, 1985; Fraser et al., 1990). Action at such a site might not only alter ion channel behavior, possibly explaining the direct effects on I_Ba, but also influence the function of other membrane-activated components, such as PKC.