Activation of Endogenous Protein Kinase C by Halothane in Synaptosomes

Adult male Sprague-Dawley rats were obtunded with 80% CO²/20% Oxygen²(vol/vol) and decapitated. The cerebral cortices were removed, rapidly chilled, and homogenized in 10 volumes of ice-cold sucrose solution (0.32 M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 micro gram/ml leupeptin). A crude synaptosome fraction (Phosphorus²) was prepared as described. [27]The Phosphorus²was resuspended in oxygenated (95% Oxygen²/5% CO², (vol/vol)) incubation buffer (140 mM NaCl, 5 mM KCl, 5 mM NaHCO³, 1 mM MgCl², 10 mM D-glucose, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4 with NaOH). A lysed synaptosome membrane fraction was prepared by subjecting the Phosphorus²to hypotonic lysis. The Phosphorus²was resuspended in 10 volumes of ice-cold lysis buffer (10 mM HEPES, pH 7.4 with NaOH, 1 mM EDTA, 1 mM dithiothreitol, 1 micro gram/ml leupeptin, 200 micro Meter phenylmethylsulfonyl fluoride, 0.01%(vol/vol) isopropanol), homogenized by ten strokes in a glass/Teflon homogenizer, and incubated on ice for 30 min. The homogenate was centrifuged at 200,000 x g for 20 min. The pellet was resuspended in 5 ml of lysis buffer by brief sonication and homogenization with a glass/Teflon homogenizer on ice and centrifuged at 200,000 x g for 20 min. The washed lysed synaptosomal membranes were resuspended in lysis buffer to a protein concentration of 2 mg/ml (determined by the method of Bradford [28]with bovine serum albumin as standard).

Phosphorylation reactions were carried out at 37 degrees C in a reaction volume of 100 micro liter containing (final concentrations) 50 mM HEPES, pH 7.0 with NaOH, 1 mM ethyleneglycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), 10 mM MgCl², 1 mg/ml lysed synaptosome membranes plus further additives as indicated. Reactions were carried out in 8.8-ml glass vials sealed with Teflon/silicone septa, through which liquid halothane (1–2 micro liter, either pure or diluted with dimethylsulfoxide, injected onto the side of the vial to vaporize) and ATP were injected directly using Hamilton microsyringes. Vapor phase halothane concentrations, which equilibrate within 5 min, [29]were determined in identical samples by gas chromatography. [30]This technique allowed reproducible and stable halothane concentrations using a small reaction volume. After a 5 min preincubation, reactions were initiated by the addition of [gamma-sup 32 Phosphorus]ATP to a final concentration of 20 micro Meter (500–1,000 cpm/pmol), and terminated after 60 s by the addition of 20 micro liter of sodium dodecyl sulfate-stop solution [31]or of 10 micro liter of immunoprecipitation stop solution (10%(wt/vol) sodium dodecyl sulfate, 10 mM EDTA, 10 mM EGTA, 100 mM sodium pyrophosphate, pH 7.4), followed by heating in a 100 degrees Celsius water bath for 2 min. Samples were either analyzed directly by sodium dodecyl sulfate/polyacrylamide gel electrophoresis using 9% acrylamide and autoradiography as described previously [31]or subjected to immunoprecipitation before sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography (see below).

Crude synaptosomes (Phosphorus²fraction) in oxygenated incubation buffer were prelabelled at 37 degrees C for 45 min with 1.5 mCi/ml of [sup 32 Phosphorus]orthophosphate in the presence of 1 mM CaCl², centrifuged at 1,000 x g for 5 min, and resuspended to a protein concentration of 2 mg/ml in oxygenated incubation buffer containing 1 mM CaCl². [32]This preparation contains radioactive ATP generated by intact nerve terminals that contain metabolically active mitochondria, which precludes significant labeling of extrasynaptosomal and nonneuronal proteins. A 45-min prelabeling period is adequate to allow isotopic equilibration between [sup 32 Phosphorus]orthophosphate and ATP such that subsequent increases in protein phosphorylation are due to increases in the degree of labeling rather than exchange of radiolabeled for unlabeled phosphate. [33]Phosphorylation reactions were initiated by the addition of 50 micro liter of the synaptosome suspension to sealed glass vials (see above) containing 3 times the indicated concentrations of okadaic acid, KCl, or ionomycin in a volume of 25 micro liter of incubation buffer (final volume 75 micro liter). Reactions were terminated by the addition of 10 micro liter of immunoprecipitation-stop solution, followed by heating in a 100 degrees C water bath for 2 min.

MARCKS (myristoylated alanine-rich C-kinase substrate) or synapsin 1 were immunoprecipitated from phosphorylated lysed synaptosomal membranes or intact synaptosomes using a previously described method. [34]Samples were incubated with 10 micro liter of rabbit antiserum to either rat brain MARCKS (G73) or bovine synapsin 1 (G486) at 4 degrees C for 1 h, followed by immunoprecipitation with formalin-fixed S. aureus cells (Pansorbin, Calbiochem, La Jolla, CA). After sodium dodecyl sulfate/polyacrylamide gel electrophoresis, phosphorylation was quantified by scanning and analysis with a PhophorImager autoradiography system (Molecular Dynamics, Sunnyvale, CA).

Thymol-free halothane was from Halocarbon Products (North Augusta, SC). The PKC inhibitor peptide (PKC¹⁹-36), [35]Calcium²+/calmodulin-dependent protein kinase II inhibitor peptide ([Ala²⁸⁶]CaMKII²⁸¹-302), and cyclic AMP-dependent protein kinase inhibitor peptide (PKI⁴-24)[36]were synthesized by The Rockefeller University Protein Sequencing Facility. [gamma-sup 32 Phosphorus]ATP and [sup 32 Phosphorus]orthophosphate were from Dupont-New England Nuclear (Boston, MA). Nonidet P-40 and sodium dodecyl sulfate were from Pierce Chemical Co. (Rockford, IL). Okadaic acid was from LC Service Corp. (Woburn, MA). Bovine serum albumin, ionomycin, and 8-bromo-cyclic AMP were from Sigma (St. Louis, MO).

Curve-fitting was performed using the Hill equation (SigmaPlot version 2.0, Jandel). Statistical significance was assessed by the indicated tests using the PHARM/PCS Pharmacologic Calculation System (version 4.2, Springer, New York, NY). These studies were approved by the Cornell University Medical College Institutional Animal Care and Use Committee.

The effects of halothane were examined on basal and Calcium²+-dependent phosphorylation of endogenous substrate proteins in a calmodulin-depleted membrane fraction prepared from lysed rat cortical synaptosomes, a subcellular fraction that is enriched in intact nerve terminals. In the presence of the Calcium²+ chelator EGTA without added Calcium²+, minimal phosphorylation of endogenous proteins was observed in a 60-s assay at 37 degrees C as analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography of the dried gels. The addition of 2.4 vol% halothane had minimal effects on the low basal phosphorylation activity (data not shown). The addition of Calcium²+ resulted in marked Calcium²+-dependent phosphorylation of a number of proteins (Figure 1). The most prominent phosphoproteins observed had apparent M^r(relative molecular weight) values of 83,000, 47,000, and 46,000, although numerous other less prominent phosphoproteins were observed. Halothane stimulated the phosphorylation of these three prominent phosphoproteins, as well as the phosphorylation of a number of other proteins with higher M^rvalues (Figure 1, lanes 3–4). The phosphorylation of at least one protein, with an apparent M^r[approximately equal] 40,000, was inhibited by the addition of halothane.

The roles of specific protein kinases and/or protein phosphatases in mediating the effects of halothane on Calcium²+-dependent phosphorylation were investigated further by the use of selective inhibitors. In the presence of [Ala²⁸⁶]CaMKII²⁸¹-302, a selective inhibitor of Calcium²+/calmodulin-dependent protein kinase II (CaMKII) based on an intramolecular autoinhibitory peptide domain [35](Figure 1, lanes 1–4), the phosphorylation pattern observed was not significantly different from that observed in the absence of inhibitor, as expected in the absence of added calmodulin (data not shown). However, we routinely included this inhibitor in all assays of Calcium²+-dependent phosphorylation to eliminate the possible involvement of CaMKII in the observed effects of halothane. In the presence of PKC¹⁹-36, a selective inhibitor of PKC based on an intramolecular autoinhibitory peptide domain [35](Figure 1, lanes 5–8), the phosphorylation of the three prominent phosphoproteins (M^r= 83,000, 47,000, and 46,000) was significantly inhibited in the absence or presence of halothane. The inhibitor did not completely inhibit PKC, because some phosphorylation of these proteins was evident in its presence (Figure 1, lanes 5–6), and halothane was able to stimulate the residual PKC activity (Figure 1, lanes 7–8). In contrast, the phosphorylation of numerous other proteins was not affected by this inhibitor, including that of a number of proteins that were observed in the presence of [Ala²⁸⁶]CaMKII²⁸¹-302 and in the presence of both inhibitors (data not shown). Okadaic acid [37]is a polyether toxin produced by marine dinoflagellates that is a potent inhibitor of serine/threonine-specific protein phosphatase-1 and -2A, and at higher concentrations, of protein phosphatase-2B, which together constitute most of the phosphatase activity in synaptosomes. [38]Calcium²+-dependent phosphorylation of most phosphoproteins was enhanced in the presence of 2 micro Meter okadaic acid, which should completely inhibit protein phosphatase-1, -2A, and -2B and has been shown to inhibit the majority of phosphatase activity in synaptosomes. [38]Further stimulation of the okadaic acid-enhanced phosphorylation of many of these proteins was observed with the addition of halothane (Figure 1, lanes 9–12), while the phosphorylation of the M sub r [approximately equal] 40,000 protein was still inhibited.

The effect of halothane on phosphorylation catalyzed by PKC was investigated further by analyzing the phosphorylation of the specific endogenous PKC substrate MARCKS, an in vivo substrate for PKC that appears to regulate membrane-cytoskeleton interactions. [39]In rat brain, MARCKS is a major PKC substrate with an M^rof 83,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis that is concentrated in synaptosomal membranes. [40]The phosphorylation of MARCKS was analyzed in lysed rat synaptosomal membranes by use of immunoprecipitation with a specific antiserum to MARCKS [40](a representative experiment is shown in Figure 2; pooled data are given in Table 1). Halothane (2.4 vol%) stimulated basal Calcium²+-dependent phosphorylation of MARCKS in lysed synaptic membranes 2.1-fold; this effect was inhibited by PKC¹⁹-36, which also inhibited basal MARCKS phosphorylation in the absence of halothane because of basal endogenous PKC activity.

A dose-response analysis of the effect of halothane on the phosphorylation of the M^r= 83,000 phosphoprotein, identified as MARCKS by immunoprecipitation, was carried out in lysed synaptic membranes (Figure 3). A steep monophasic increase in phosphorylation was observed between 1.6 and 2.4 vol% halothane, which reached a plateau above 2.4 vol% halothane. Curve-fitting of the data to the Hill equation indicated an EC sub 50 of 1.8 vol% halothane with a Hill coefficient of 7.2. The low basal phosphorylation of MARCKS in this experiment compared to the data in Table 1, which varied between different preparations, resulted in a 5.3-fold increase in phosphorylation produced by 2.4 vol% halothane.

The phosphorylation of MARCKS was studied in intact synaptosomes by metabolic prelabelling of endogenous ATP pools with [sup 32 Phosphorus]orthophosphate followed by immunoprecipitation of MARCKS. [32,40]Analysis of proteins phosphorylated in prelabelled synaptosomes under basal conditions revealed intense labeling of multiple proteins; the phosphorylation pattern detected by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis was too complex for meaningful analysis (data not shown). Immunoprecipitation of MARCKS from prelabelled synaptosomes was used as a measure of endogenous PKC activity and showed that MARCKS was phosphorylated under basal conditions. Exposure to 2.4 vol% halothane for 60 s stimulated the basal phosphorylation of MARCKS 1.4-fold (a representative experiment is shown in Figure 4; pooled data are given in Table 1). MARCKS phosphorylation was potentiated by halothane in the absence or presence of okadaic acid, which produced only a modest increase in basal MARCKS phosphorylation.

The phosphorylation of synapsin 1, a synaptic vesicle-associated protein involved in regulating synaptic vesicle-cytoskeleton interactions, [41]was studied in intact synaptosomes prelabelled with [sup 32 Phosphorus]orthophosphate [42]by immunoprecipitation of synapsin 1. Immunoprecipitation of synapsin 1 (M^r= 80,000/75,000)[43]from prelabelled synaptosomes showed that synapsin 1 was phosphorylated under basal conditions (a representative experiment is shown in Figure 5; pooled data are given in Table 2). Halothane (2.4 vol%) did not significantly affect basal synapsin 1 phosphorylation or synapsin 1 phosphorylation stimulated by KCl-evoked depolarization, which induces voltage-dependent Calcium²+ channel opening, Calcium²+ influx, and activation of Calcium²+/calmodulin-dependent synapsin 1 kinases. [42]To bypass voltage-dependent Calcium²+ channel involvement, ionomycin, a Calcium²+ ionophore, was used to allow direct Calcium sup 2+ entry. Halothane stimulated ionomycin-induced synapsin 1 phosphorylation 1.8-fold. Synapsin 1 phosphorylation induced by KCl or ionomycin occurred primarily on the 35 kDa proteolytic fragment, which contains the Calcium²+-dependent phosphorylation sites 2 and 3, as indicated by phosphopeptide analysis after proteolytic digestion [42](data not shown).

The effects of halothane were examined on basal and cAMP-dependent phosphorylation of endogenous substrate proteins in a membrane fraction prepared from lysed rat cortical synaptosomes. In the presence of EGTA and a peptide inhibitor (PKI⁴-24) of cAMP-dependent protein kinase (PKA), [36]halothane had minimal effects on the low basal phosphorylation of endogenous proteins. The most striking effect of halothane under these conditions was a consistent reduction in the phosphorylation of a protein of M^r[approximately equal] 40,000 (Figure 1). Addition of 8-bromo-cAMP, a nonhydrolyzable cAMP analog, stimulated the phosphorylation of four major phosphoprotein bands known as the synapsins [41](a representative experiment is shown in Figure 6), as well as of a number of other minor phosphoproteins. The addition of 2.4 vol% halothane appeared to enhance the phosphorylation of most of the cAMP-stimulated phosphoproteins; this effect was not statistically significant when quantified, however (data not shown). Okadaic acid also produced a slight potentiation in the phosphorylation of many of the cAMP-stimulated phosphoproteins; in the presence of okadaic acid, halothane again had no significant effects on the cAMP-stimulated phosphoproteins. Okadaic acid also led to the appearance of a phosphoprotein of M^r= 48,000 that was not observed in its absence; the phosphorylation of this protein was not affected by halothane.

Protein kinase C is a family of a signal transducing enzymes, several isoforms of which are highly concentrated in the nervous system and have been implicated in the modulation of synaptic transmission. [21–23]The results of this study demonstrate that halothane, at clinically relevant concentrations, stimulates the activity of endogenous PKC in a rat brain subcellular fraction enriched in nerve terminals (synaptosomes) and in membranes prepared from this fraction. Evidence for activation of PKC by halothane includes halothane-stimulated phosphorylation of MARCKS, which is a specific substrate for PKC, and inhibition of this effect by a selective PKC inhibitor. Previous studies of anesthetic effects on PKC activity, which have employed purified PKC, artificial lipid preparations, and artificial substrate proteins in vitro, have yielded conflicting results. The current study overcomes many of the limitations inherent in the study of purified PKC in vitro by analyzing anesthetic effects on the phosphorylation of endogenous brain PKC substrates by native PKC in situ in the presence of endogenous membranes.

The experiments reported here examined the effects of halothane on protein phosphorylation in synaptosomes, a nerve terminal preparation that has been subjected to extensive biochemical and pharmacologic analysis. [26]Detailed studies in intact synaptosomes [27,32,43]have identified six major substrates for depolarization-induced, Calcium sup 2+-dependent protein phosphorylation. These substrates include the synapsins (synapsin Ia, Ib, IIa, and IIb), a family of neuron-specific synaptic vesicle-associated proteins that are substrates for CaMKII and PKA, [41]and two substrates for PKC, MARCKS [44]and GAP-43 (also known as B-50 or neuromodulin). [45]Our initial studies examined the effects of halothane on protein phosphorylation in a calmodulin-depleted synaptic membrane fraction in which MARCKS can be phosphorylated by endogenous PKC in a native lipid environment. [46]In the presence of Calcium²+ and [gamma-sup 32 Phosphorus]ATP, three prominent phosphoproteins were observed with M^rvalues of 83,000, 47,000, and 46,000. The protein kinase responsible for the phosphorylation of these proteins was identified as PKC, because phosphorylation occurred in the presence of Calcium²+ and the absence of calmodulin, and it was inhibited by a peptide inhibitor selective for PKC but not by one selective for CaMKII (the major Calcium²+/calmodulin-dependent protein kinase in brain [47]). Based on these observations, the M^r= 83,000 protein is identified as MARCKS and the M^r= 46,000 protein as GAP-43; the identity of the M^r= 83,000 protein as MARCKS was confirmed by immunoprecipitation. The M^r= 47,000 protein is probably MacMARCKS, a MARCKS-related PKC substrate that undergoes depolarization-induced phosphorylation in synaptosomes. [48]Two of these proteins probably correspond to the cytosolic proteins of M^r= 80,000 and 47,000 phosphorylated by purified PKC in response to halothane in a high-speed supernatant fraction prepared from rat cerebral cortex. [7].

Halothane stimulated MARCKS phosphorylation with a calculated EC⁵⁰of 1.8 vol%, which is 1.5–2 times the EC⁵⁰(MAC) of halothane in vivo (0.88–1.24 vol% in rats). This is somewhat lower then the EC⁵⁰of 2.2 vol%(at 30 degrees C) for activation of purified brain PKC in the presence of an artificial lipid vesicle preparation. [15]The observation that okadaic acid potentiates but does not prevent the halothane-stimulated phosphorylation of MARCKS, GAP-43, and the M^r= 47,000 protein indicates that halothane exerts its effect by activation of PKC rather than by inhibition of okadaic acid-sensitive protein phosphatases, which have been shown previously to dephosphorylate both MARCKS [49]and GAP-43. [50]The halothane-induced reduction in the phosphorylation of the unidentified M sub r [approximately equal] 40,000 protein appears to be due to inhibition of an unidentified protein kinase with high basal activity rather than to activation of protein phosphatase-1, -2A, or -2B, because the effect persisted in the presence of okadaic acid.

Basal phosphorylation of MARCKS in the presence of extracellular Calcium²+ was detected in intact synaptosomes, as has been observed previously. [32,44,46]Halothane caused a 1.4-fold increase in the basal phosphorylation of MARCKS in 60 s. The magnitude of this effect is comparable to that reported for treatment of synaptosomes with 40 mM KCl (1.9-fold) or 100 micro mM beta-phorbol 12,13-dibutyrate (1.8-fold). [32]Stimulation of MARCKS phosphorylation by halothane in intact synaptosomes was not due to inhibition of okadaic acid-sensitive protein phosphatases, because the effect was also observed in the presence of okadaic acid. The minimal effect of okadaic acid on basal MARCKS phosphorylation indicates that basal MARCKS phosphatase activity in synaptosomes is low, as also observed in Swiss 3T3 cells. [49]Halothane-stimulated phosphorylation of MARCKS in these preparations is therefore due to stimulation of PKC activity rather than to inhibition of protein phosphatase activity.

We have used phosphorylation of synapsin 1 to assess anesthetic effects on endogenous Calcium²+/calmodulin- and cyclic AMP-dependent phosphorylation in synaptosomes. Synapsin 1 is phosphorylated in intact synaptosomes under basal conditions. [32,42]Phosphorylation of synapsin 1 is stimulated by agents that elevate intracellular Calcium²+, such as KCl, 4-aminopyridine, veratridine, A23187, or ionomycin [27,32,51,52]through activation of Calcium²+/calmodulin-dependent protein kinase I (which phosphorylates site 1) and CaMKII (which phosphorylates sites 2 and 3). Site 1 is also phosphorylated by increases in cyclic AMP, [42,52]through activation of PKA. [41]Manipulations that cause Calcium sup +-dependent phosphorylation of synapsin 1 result in increased release of neurotransmitter by disrupting the ternary complex of synapsin 1 bound to actin and synaptic vesicles. [41]A variety of physiologic and pharmacologic manipulations have been shown to affect phosphorylation of synapsin 1 through changes in intracellular Calcium²+ or cyclic AMP. [53].

Under basal conditions, synapsin 1 was phosphorylated in intact synaptosomes in the presence of extracellular Calcium²+, as described previously. [27,32]Halothane did not affect basal synapsin 1 phosphorylation, in contrast to its effect on MARCKS phosphorylation. This observation is consistent with the finding that halothane had no effect on cyclic AMP-stimulated phosphoproteins in a synaptic membrane fraction (this study) or on purified rat CaMKII or bovine PKA activity in vitro. [15]An in vivo study has demonstrated no effect of halothane or ether on synapsin 1 phosphorylation in mouse brain. [54]Depolarization with KCl or treatment with ionomycin stimulated synapsin 1 phosphorylation in synaptosomes. Halothane did not affect KCl depolarization-induced synapsin 1 phosphorylation, which is tightly coupled to Calcium²+ influx through voltage-dependent Calcium²+ channels. [51]This observation suggests that the Calcium²+ channels coupled to synapsin 1 phosphorylation in synaptosomes are not highly sensitive to inhibition by volatile anesthetics. A previous study showed that neuronal Calcium²+ channel types are sensitive to volatile anesthetics, [55]although at least one Calcium²+ channel type appears to be relatively insensitive. [56].

The observation that KCl-stimulated synapsin 1 phosphorylation, which is tightly coupled to specific Calcium²+ channel activation, [51,57]was not sensitive to halothane is consistent with the finding that KCl-evoked synaptosomal glutamate release, which is also tightly coupled to specific Calcium²+ channel activation, [51]is not highly sensitive to volatile anesthetics' effects. [58]The precise Calcium²+ channel type(s) coupled to synapsin 1 phosphorylation and amino acid neurotransmitter release in rat cortical synaptosomes have not been firmly identified and studied directly, however. [57]The interpretation of these data must take into account the fact that physiologic depolarization, which occurs over milliseconds, may exhibit different sensitivity to anesthetics compared to pharmacologic depolarization with agents, such as KCl or veratridine, which produce prolonged depolarization. [32,57]Ionomycin induced a small increase in synapsin 1 phosphorylation compared to KCl, which is consistent with the relative unresponsiveness of synapsin 1 phosphorylation and of glutamate release to the "bulk" increase in cytoplasmic Calcium²+ induced by an ionophore compared to the localized Calcium²+ entry due to Calcium sup 2+ channel activation. [51]Halothane potentiated ionomycin-induced synapsin 1 phosphorylation 1.8-fold. This effects appears to be due to a direct interaction between halothane and the ionophore ionomycin, resulting in more Calcium²+ influx, because halothane also potentiates the ionomycin-induced increase in cytoplasmic Calcium²+ and glutamate release in synaptosomes. [58].

The PKC signaling pathway is complex and subject to regulation at multiple levels. For example, PKC is regulated by multiple intracellular messengers, exists in multiple molecular isoforms and undergoes subcellular translocation and down-regulation. Agonist-induced degradation of membrane phospholipids results in a biphasic peak in the formation of the second messenger diacylglycerol, as well as in the formation of other lipid modulators of PKC activity. [59]The existence of 11 isoforms of PKC in mammals, which differ in their tissue distribution, regional distribution in brain, subcellular localization, and sensitivity to various activators, suggests that different isoforms control specific physiologic functions. [19,60]On agonist-induced stimulation, PKC rapidly translocates from the cytosol to membranes, where it undergoes proteolysis-mediated down-regulation in an isoform-specific manner. [61]The role of these important regulatory mechanisms in determining the ultimate effects of anesthetics and other drugs on the PKC signaling pathway in specific tissues and cells is unclear. Previous studies in intact cells have implicated PKC in the effects of general anesthetics on neurotransmitter release from PC12 cells, [10]prostacyclin production in endothelial cells, [16]protein phosphorylation in neuronal growth cones, [13]smooth muscle contraction, [11]hepatic blood flow, [17]and loss of righting reflex in tadpoles. [12]Several of these studies inferred anesthetic inhibition, rather than activation, of PKC activity based on indirect evidence. The results of the current study demonstrate the importance of direct determinations of anesthetic effects on endogenous PKC activity, translocation, and/or down-regulation in the study of anesthetic interactions with the PKC signaling pathway. The possibility that initial activation of PKC by halothane may result in translocation and down-regulation with subsequent attenuation of PKC-dependent effects is currently under investigation.