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Meeting Abstracts  |   March 1996
Activation of Endogenous Protein Kinase C by Halothane in Synaptosomes
Author Notes
  • (Hemmings, Jr.) Associate Professor of Anesthesiology and Pharmacology; Director of Research in the Department of Anesthesiology.
  • (Adamo) Research Specialist in Anesthesiology.
  • Received from Cornell University Medical College, New York, New York. Submitted for publication May 11, 1995. Accepted for publication November 21, 1995. Supported in part by a Foundation for Anesthesia Education and Research/BOC Anesthesiology Young Investigator Award and a Cornell Scholar Award in Biomedical Science. MARCKS antibody G73 was a gift of A. C. Nairn, and bovine synapsin 1 antibody G486 was a gift of A. J. Czernik, both of The Rockefeller University.
  • Address correspondence to Dr. Hemmings: Department of Anesthesiology, Cornell University Medical College, Box 50, LC-203, 525 East 68th Street, New York, New York 10021. Address electronic mail to: hchemmi@med.cornell.edu.
Article Information
Meeting Abstracts   |   March 1996
Activation of Endogenous Protein Kinase C by Halothane in Synaptosomes
Anesthesiology 3 1996, Vol.84, 652-662. doi:
Anesthesiology 3 1996, Vol.84, 652-662. doi:
GENERAL anesthetics alter synaptic transmission in the central nervous system through presynaptic and post-synaptic mechanisms. The effects of general anesthetics on presynaptic mechanisms and intracellular neuronal signaling pathways are not well characterized compared to their effects on postsynaptic mechanisms. [1] Protein kinase C (PKC), an important family of intracellular protein kinases that regulate neuronal function, [2,3] has been implicated as a potential target for general anesthetic action in numerous studies. [4–17] PKC exists as a number of structurally related isoforms distinguished by their regulatory domains and cofactor dependence. [18] The Calcium2+-dependent or conventional isoforms of PKC are components of the phospholipase C/inositol triphosphate/diacylglycerol signal transduction pathway. They are serine/threonine-specific protein kinases that are activated by the lipid second messenger 1,2-diacyl-sn-glycerol, by phospholipids, such as phosphatidylserine, and by Calcium2+ through specific interactions with the regulatory domain of PKC. [18,19] Physiologic activation of conventional PKC isoforms (alpha, beta 1, beta 2, and gamma, which are abundant in mammalian brain [19]) occurs when diacylglycerol is generated in response to activation of cell surface receptors coupled to phospholipase C. Binding of diacylglycerol increases the affinity of PKC for its cofactors Calcium2+ and phosphatidylserine, facilitates PKC translocation and binding to cell membranes, and increases PKC catalytic activity. [20] Phosphorylation of specific proteins by activated PKC is the effector mechanism for regulation of a number of neuronal processes, many of which are also sensitive to general anesthetics, such as the release of neurotransmitters, [21] ion channel function, [22] and neurotransmitter receptor desensitization. [23] .
Previous studies of general anesthetic effects on PKC activity have yielded contradictory results. For example, halothane has been found to inhibit [14] or stimulate [15] purified brain PKC in vitro and to stimulate PKC activity in brain cytosol [7] and in intact neurosecretory cells. [10] These differences may be explained in part by variations in the conditions used to analyze PKC activity in vitro, [15] because the properties of PKC are highly dependent on the lipid preparations and protein substrates used in the assay, [24] and in some instances, by differences in the particular PKC isoforms present. We previously showed that clinically effective concentrations of halothane and propofol stimulate PKC activation when assayed with a physiologically relevant lipid bilayer preparation [15] by increasing the affinity of PKC for Calcium2+, phosphatidylserine, and diacylglycerol. [25] However, the pharmacologic significance of in vitro studies of anesthetic effects on PKC activation is limited by the use of artificial lipid preparations and substrates.
This study employs the synaptosome preparation to analyze the effects of halothane on endogenous neuronal protein phosphorylation. Synaptosomes are the simplest subcellular preparation that retains the ability to synthesize ATP, maintain ion gradients, and take up and release neurotransmitters. [26] Lysed synaptosomal membranes contain endogenous brain protein kinases and their substrates and are accessible to both membrane permeable and impermeable reagents, which makes them useful for detailed biochemical studies. Intact synaptosomes, which consist of pinched-off nerve terminals, require metabolic prelabelling to study phosphorylation and are only accessible to membrane permeable reagents or agents that act at the plasma membrane. Protein phosphorylation in intact synaptosomes can be activated directly, by the addition of membrane permeable protein kinase activators or of Calcium2+ ionophores (e.g., ionomycin) or indirectly by depolarization with KCl, which activates voltage-dependent Calcium2+ channels and allows extracellular Calcium sup 2+ influx. Analysis of the sensitivity to anesthetics of protein phosphorylation in synaptosomes activated by these different mechanisms provides the opportunity to probe specific site(s) of anesthetic action. We report evidence that halothane activates endogenous PKC in synaptic membranes and intact synaptosomes to phosphorylate specific endogenous substrate proteins without affecting cAMP-dependent or Calcium2+/calmodulin-dependent phosphorylation.
Methods and Materials
Preparation of 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).
Endogenous Phosphorylation of Lysed Synaptosome Membranes
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).
Phosphorylation in Intact Synaptosomes
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.
Immunoprecipitation of MARCKS and Synapsin 1
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).
Materials
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).
Miscellaneous
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.
Results
Effects of Halothane on Calcium sup 2+-dependent Endogenous Protein Phosphorylation in Synaptic Membranes
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 Mr(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 Mrvalues (Figure 1, lanes 3–4). The phosphorylation of at least one protein, with an apparent Mr[approximately equal] 40,000, was inhibited by the addition of halothane.
Figure 1. Effects of halothane on endogenous Calcium2+-dependent protein phosphorylation in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was incubated under phosphorylating conditions with 1.2 mM CaCl2for 60 sec at 37 degrees Celsius with [gamma-sup 32 Phosphorus]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter [Ala286]CaMKII281-302, lanes 5–8 contained 10 micro Meter PKC19-36, and lanes 9–12 contained 2 micro Meter okadaic acid (OA). Halothane significantly stimulated the phosphorylation of proteins of M sub r = 83,000, Mr= 47,000, and Mr= 46,000 (arrows), which was inhibited by PKC19-36 and slightly potentiated by okadaic acid. The Mr= 83,000 phosphoprotein was identified as MARCKS by immunoprecipitation (vide infra). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left. + and - indicate the presence and absence of halothane, respectively, and duplicate lanes of each sample are shown.
Figure 1. Effects of halothane on endogenous Calcium2+-dependent protein phosphorylation in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was incubated under phosphorylating conditions with 1.2 mM CaCl2for 60 sec at 37 degrees Celsius with [gamma-sup 32 Phosphorus]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter [Ala286]CaMKII281-302, lanes 5–8 contained 10 micro Meter PKC19-36, and lanes 9–12 contained 2 micro Meter okadaic acid (OA). Halothane significantly stimulated the phosphorylation of proteins of M sub r = 83,000, Mr= 47,000, and Mr= 46,000 (arrows), which was inhibited by PKC19-36 and slightly potentiated by okadaic acid. The Mr= 83,000 phosphoprotein was identified as MARCKS by immunoprecipitation (vide infra). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left. + and - indicate the presence and absence of halothane, respectively, and duplicate lanes of each sample are shown.
Figure 1. Effects of halothane on endogenous Calcium2+-dependent protein phosphorylation in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was incubated under phosphorylating conditions with 1.2 mM CaCl2for 60 sec at 37 degrees Celsius with [gamma-sup 32 Phosphorus]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter [Ala286]CaMKII281-302, lanes 5–8 contained 10 micro Meter PKC19-36, and lanes 9–12 contained 2 micro Meter okadaic acid (OA). Halothane significantly stimulated the phosphorylation of proteins of M sub r = 83,000, Mr= 47,000, and Mr= 46,000 (arrows), which was inhibited by PKC19-36 and slightly potentiated by okadaic acid. The Mr= 83,000 phosphoprotein was identified as MARCKS by immunoprecipitation (vide infra). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left. + and - indicate the presence and absence of halothane, respectively, and duplicate lanes of each sample are shown.
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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 (Mr= 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 Mrof 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.
Figure 2. Effects of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was treated as described in the legend to figure 1 and subjected to immunoprecipitation with an antibody to MARCKS, a specific substrate for PKC, as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (Mr= 83,000; arrow) was stimulated by 2.4 vol% halothane (lanes 3, 4) compared to control (lanes 1, 2; plus 10 micro Meter [Ala286]CaMKII281-302), and was inhibited by 10 micro Meter PKC19-36 (lanes 5, 6).
Figure 2. Effects of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was treated as described in the legend to figure 1 and subjected to immunoprecipitation with an antibody to MARCKS, a specific substrate for PKC, as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (Mr= 83,000; arrow) was stimulated by 2.4 vol% halothane (lanes 3, 4) compared to control (lanes 1, 2; plus 10 micro Meter [Ala286]CaMKII281-302), and was inhibited by 10 micro Meter PKC19-36 (lanes 5, 6).
Figure 2. Effects of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was treated as described in the legend to figure 1 and subjected to immunoprecipitation with an antibody to MARCKS, a specific substrate for PKC, as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (Mr= 83,000; arrow) was stimulated by 2.4 vol% halothane (lanes 3, 4) compared to control (lanes 1, 2; plus 10 micro Meter [Ala286]CaMKII281-302), and was inhibited by 10 micro Meter PKC19-36 (lanes 5, 6).
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Table 1. Effects of Halothane on MARCKS Phosphorylation in Lysed and Intact Synaptosomes
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Table 1. Effects of Halothane on MARCKS Phosphorylation in Lysed and Intact Synaptosomes
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A dose-response analysis of the effect of halothane on the phosphorylation of the Mr= 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.
Figure 3. Dose-response analysis of the effect of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. Assays were carried out in the presence of increasing amounts of halothane (with 1%(vol/vol) dimethylsulfoxide included in all assays to allow the addition of addition of small volumes of halothane) as described in methods and materials. All assays contained 1.2 mM CaCl2and 10 micro Meter [Ala sup 286]CaMKII281-302. MARCKS phosphorylation was quantified by PhosphorImager analysis of the Mr= 83,000 phosphoprotein identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and are reported in arbituary units (a.u.). Data are the mean of triplicate determinations; error bars indicate the SD.
Figure 3. Dose-response analysis of the effect of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. Assays were carried out in the presence of increasing amounts of halothane (with 1%(vol/vol) dimethylsulfoxide included in all assays to allow the addition of addition of small volumes of halothane) as described in methods and materials. All assays contained 1.2 mM CaCl2and 10 micro Meter [Ala sup 286]CaMKII281-302. MARCKS phosphorylation was quantified by PhosphorImager analysis of the Mr= 83,000 phosphoprotein identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and are reported in arbituary units (a.u.). Data are the mean of triplicate determinations; error bars indicate the SD.
Figure 3. Dose-response analysis of the effect of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. Assays were carried out in the presence of increasing amounts of halothane (with 1%(vol/vol) dimethylsulfoxide included in all assays to allow the addition of addition of small volumes of halothane) as described in methods and materials. All assays contained 1.2 mM CaCl2and 10 micro Meter [Ala sup 286]CaMKII281-302. MARCKS phosphorylation was quantified by PhosphorImager analysis of the Mr= 83,000 phosphoprotein identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and are reported in arbituary units (a.u.). Data are the mean of triplicate determinations; error bars indicate the SD.
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Effect of Halothane on MARCKS Phosphorylation in Intact Synaptosomes
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.
Figure 4. Effect of halothane on the phosphorylation of MARCKS in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min, followed by a 60-s incubation in the absence or presence of 2.4 vol% halothane at 37 degrees C before termination of the reaction. MARCKS was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (arrow) was stimulated by halothane in the absence (lanes 1–4) or presence (lanes 5–8) of 2 micro Meter okadaic acid.
Figure 4. Effect of halothane on the phosphorylation of MARCKS in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min, followed by a 60-s incubation in the absence or presence of 2.4 vol% halothane at 37 degrees C before termination of the reaction. MARCKS was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (arrow) was stimulated by halothane in the absence (lanes 1–4) or presence (lanes 5–8) of 2 micro Meter okadaic acid.
Figure 4. Effect of halothane on the phosphorylation of MARCKS in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min, followed by a 60-s incubation in the absence or presence of 2.4 vol% halothane at 37 degrees C before termination of the reaction. MARCKS was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (arrow) was stimulated by halothane in the absence (lanes 1–4) or presence (lanes 5–8) of 2 micro Meter okadaic acid.
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Effects of Halothane on Calcium sup 2+-dependent Synapsin 1 Phosphorylation in Intact Synaptosomes
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 (Mr= 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).
Figure 5. Effect of halothane on the phosphorylation of synapsin 1 in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min and incubated for 15 s with control buffer, KCl, or ionomycin (all in the presence of 1 mM CaCl2) in the absence or presence of 2.4 vol% halothane at 37 degrees C. Synapsin 1 (1a and 1b) was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of synapsin 1 (arrows) was not affected by halothane under basal conditions (control) or when stimulated by 30 mM KCl-evoked depolarization (KCl). However, halothane enhanced synapsin 1 phosphorylation stimulated by 2 micro Meter ionomycin (Iono).
Figure 5. Effect of halothane on the phosphorylation of synapsin 1 in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min and incubated for 15 s with control buffer, KCl, or ionomycin (all in the presence of 1 mM CaCl2) in the absence or presence of 2.4 vol% halothane at 37 degrees C. Synapsin 1 (1a and 1b) was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of synapsin 1 (arrows) was not affected by halothane under basal conditions (control) or when stimulated by 30 mM KCl-evoked depolarization (KCl). However, halothane enhanced synapsin 1 phosphorylation stimulated by 2 micro Meter ionomycin (Iono).
Figure 5. Effect of halothane on the phosphorylation of synapsin 1 in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min and incubated for 15 s with control buffer, KCl, or ionomycin (all in the presence of 1 mM CaCl2) in the absence or presence of 2.4 vol% halothane at 37 degrees C. Synapsin 1 (1a and 1b) was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of synapsin 1 (arrows) was not affected by halothane under basal conditions (control) or when stimulated by 30 mM KCl-evoked depolarization (KCl). However, halothane enhanced synapsin 1 phosphorylation stimulated by 2 micro Meter ionomycin (Iono).
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Table 2. Effects of Halothane on Synapsin 1 Phosphorylation in Intact Synaptosomes
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Table 2. Effects of Halothane on Synapsin 1 Phosphorylation in Intact Synaptosomes
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Effects of Halothane on cAMP-dependent Endogenous Protein Phosphorylation in Synaptic Membranes
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 Mr[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 Mr= 48,000 that was not observed in its absence; the phosphorylation of this protein was not affected by halothane.
Figure 6. Effects of halothane on endogenous cyclic AMP-dependent protein phosphorylation in rat synaptosomal membranes. The membrane fraction from rat cerebrocortical synaptosomes was incubated under phosphorylating conditions in the absence of CaCl2for 60 s at 37 degrees C with [gamma-sup 32 p]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter PKI4-24, lanes 5–12 contained 10 micro Meter 8-bromo-cyclic AMP (8BrcA), and lanes 9–12 contained 10 micro Meter 8BrcA and 2 micro Meter okadaic acid (OA). The most prominent cyclic AMP-stimulated phosphoproteins are synapsin 1a, 1b, 2a, and 2b (Mr= 80,000, 75,000, 72,000, and 57,000, respectively; arrows). Halothane slightly potentiated the phosphorylation of several cyclic AMP-stimulated phosphoproteins (lanes 5–8), an effect that was prevented by the inclusion of OA (lanes 9–12), and inhibited the phosphorylation of a protein of Mr[approximately equal] 40,000 (lower arrow). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left.
Figure 6. Effects of halothane on endogenous cyclic AMP-dependent protein phosphorylation in rat synaptosomal membranes. The membrane fraction from rat cerebrocortical synaptosomes was incubated under phosphorylating conditions in the absence of CaCl2for 60 s at 37 degrees C with [gamma-sup 32 p]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter PKI4-24, lanes 5–12 contained 10 micro Meter 8-bromo-cyclic AMP (8BrcA), and lanes 9–12 contained 10 micro Meter 8BrcA and 2 micro Meter okadaic acid (OA). The most prominent cyclic AMP-stimulated phosphoproteins are synapsin 1a, 1b, 2a, and 2b (Mr= 80,000, 75,000, 72,000, and 57,000, respectively; arrows). Halothane slightly potentiated the phosphorylation of several cyclic AMP-stimulated phosphoproteins (lanes 5–8), an effect that was prevented by the inclusion of OA (lanes 9–12), and inhibited the phosphorylation of a protein of Mr[approximately equal] 40,000 (lower arrow). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left.
Figure 6. Effects of halothane on endogenous cyclic AMP-dependent protein phosphorylation in rat synaptosomal membranes. The membrane fraction from rat cerebrocortical synaptosomes was incubated under phosphorylating conditions in the absence of CaCl2for 60 s at 37 degrees C with [gamma-sup 32 p]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter PKI4-24, lanes 5–12 contained 10 micro Meter 8-bromo-cyclic AMP (8BrcA), and lanes 9–12 contained 10 micro Meter 8BrcA and 2 micro Meter okadaic acid (OA). The most prominent cyclic AMP-stimulated phosphoproteins are synapsin 1a, 1b, 2a, and 2b (Mr= 80,000, 75,000, 72,000, and 57,000, respectively; arrows). Halothane slightly potentiated the phosphorylation of several cyclic AMP-stimulated phosphoproteins (lanes 5–8), an effect that was prevented by the inclusion of OA (lanes 9–12), and inhibited the phosphorylation of a protein of Mr[approximately equal] 40,000 (lower arrow). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left.
×
Discussion
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 Mrvalues 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 Mr= 83,000 protein is identified as MARCKS and the Mr= 46,000 protein as GAP-43; the identity of the Mr= 83,000 protein as MARCKS was confirmed by immunoprecipitation. The Mr= 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 Mr= 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 Mr= 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.
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Figure 1. Effects of halothane on endogenous Calcium2+-dependent protein phosphorylation in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was incubated under phosphorylating conditions with 1.2 mM CaCl2for 60 sec at 37 degrees Celsius with [gamma-sup 32 Phosphorus]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter [Ala286]CaMKII281-302, lanes 5–8 contained 10 micro Meter PKC19-36, and lanes 9–12 contained 2 micro Meter okadaic acid (OA). Halothane significantly stimulated the phosphorylation of proteins of M sub r = 83,000, Mr= 47,000, and Mr= 46,000 (arrows), which was inhibited by PKC19-36 and slightly potentiated by okadaic acid. The Mr= 83,000 phosphoprotein was identified as MARCKS by immunoprecipitation (vide infra). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left. + and - indicate the presence and absence of halothane, respectively, and duplicate lanes of each sample are shown.
Figure 1. Effects of halothane on endogenous Calcium2+-dependent protein phosphorylation in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was incubated under phosphorylating conditions with 1.2 mM CaCl2for 60 sec at 37 degrees Celsius with [gamma-sup 32 Phosphorus]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter [Ala286]CaMKII281-302, lanes 5–8 contained 10 micro Meter PKC19-36, and lanes 9–12 contained 2 micro Meter okadaic acid (OA). Halothane significantly stimulated the phosphorylation of proteins of M sub r = 83,000, Mr= 47,000, and Mr= 46,000 (arrows), which was inhibited by PKC19-36 and slightly potentiated by okadaic acid. The Mr= 83,000 phosphoprotein was identified as MARCKS by immunoprecipitation (vide infra). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left. + and - indicate the presence and absence of halothane, respectively, and duplicate lanes of each sample are shown.
Figure 1. Effects of halothane on endogenous Calcium2+-dependent protein phosphorylation in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was incubated under phosphorylating conditions with 1.2 mM CaCl2for 60 sec at 37 degrees Celsius with [gamma-sup 32 Phosphorus]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter [Ala286]CaMKII281-302, lanes 5–8 contained 10 micro Meter PKC19-36, and lanes 9–12 contained 2 micro Meter okadaic acid (OA). Halothane significantly stimulated the phosphorylation of proteins of M sub r = 83,000, Mr= 47,000, and Mr= 46,000 (arrows), which was inhibited by PKC19-36 and slightly potentiated by okadaic acid. The Mr= 83,000 phosphoprotein was identified as MARCKS by immunoprecipitation (vide infra). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left. + and - indicate the presence and absence of halothane, respectively, and duplicate lanes of each sample are shown.
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Figure 2. Effects of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was treated as described in the legend to figure 1 and subjected to immunoprecipitation with an antibody to MARCKS, a specific substrate for PKC, as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (Mr= 83,000; arrow) was stimulated by 2.4 vol% halothane (lanes 3, 4) compared to control (lanes 1, 2; plus 10 micro Meter [Ala286]CaMKII281-302), and was inhibited by 10 micro Meter PKC19-36 (lanes 5, 6).
Figure 2. Effects of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was treated as described in the legend to figure 1 and subjected to immunoprecipitation with an antibody to MARCKS, a specific substrate for PKC, as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (Mr= 83,000; arrow) was stimulated by 2.4 vol% halothane (lanes 3, 4) compared to control (lanes 1, 2; plus 10 micro Meter [Ala286]CaMKII281-302), and was inhibited by 10 micro Meter PKC19-36 (lanes 5, 6).
Figure 2. Effects of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. The washed membrane fraction from lysed rat cerebrocortical synaptosomes was treated as described in the legend to figure 1 and subjected to immunoprecipitation with an antibody to MARCKS, a specific substrate for PKC, as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (Mr= 83,000; arrow) was stimulated by 2.4 vol% halothane (lanes 3, 4) compared to control (lanes 1, 2; plus 10 micro Meter [Ala286]CaMKII281-302), and was inhibited by 10 micro Meter PKC19-36 (lanes 5, 6).
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Figure 3. Dose-response analysis of the effect of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. Assays were carried out in the presence of increasing amounts of halothane (with 1%(vol/vol) dimethylsulfoxide included in all assays to allow the addition of addition of small volumes of halothane) as described in methods and materials. All assays contained 1.2 mM CaCl2and 10 micro Meter [Ala sup 286]CaMKII281-302. MARCKS phosphorylation was quantified by PhosphorImager analysis of the Mr= 83,000 phosphoprotein identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and are reported in arbituary units (a.u.). Data are the mean of triplicate determinations; error bars indicate the SD.
Figure 3. Dose-response analysis of the effect of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. Assays were carried out in the presence of increasing amounts of halothane (with 1%(vol/vol) dimethylsulfoxide included in all assays to allow the addition of addition of small volumes of halothane) as described in methods and materials. All assays contained 1.2 mM CaCl2and 10 micro Meter [Ala sup 286]CaMKII281-302. MARCKS phosphorylation was quantified by PhosphorImager analysis of the Mr= 83,000 phosphoprotein identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and are reported in arbituary units (a.u.). Data are the mean of triplicate determinations; error bars indicate the SD.
Figure 3. Dose-response analysis of the effect of halothane on the phosphorylation of MARCKS in rat synaptosomal membranes. Assays were carried out in the presence of increasing amounts of halothane (with 1%(vol/vol) dimethylsulfoxide included in all assays to allow the addition of addition of small volumes of halothane) as described in methods and materials. All assays contained 1.2 mM CaCl2and 10 micro Meter [Ala sup 286]CaMKII281-302. MARCKS phosphorylation was quantified by PhosphorImager analysis of the Mr= 83,000 phosphoprotein identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and are reported in arbituary units (a.u.). Data are the mean of triplicate determinations; error bars indicate the SD.
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Figure 4. Effect of halothane on the phosphorylation of MARCKS in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min, followed by a 60-s incubation in the absence or presence of 2.4 vol% halothane at 37 degrees C before termination of the reaction. MARCKS was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (arrow) was stimulated by halothane in the absence (lanes 1–4) or presence (lanes 5–8) of 2 micro Meter okadaic acid.
Figure 4. Effect of halothane on the phosphorylation of MARCKS in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min, followed by a 60-s incubation in the absence or presence of 2.4 vol% halothane at 37 degrees C before termination of the reaction. MARCKS was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (arrow) was stimulated by halothane in the absence (lanes 1–4) or presence (lanes 5–8) of 2 micro Meter okadaic acid.
Figure 4. Effect of halothane on the phosphorylation of MARCKS in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min, followed by a 60-s incubation in the absence or presence of 2.4 vol% halothane at 37 degrees C before termination of the reaction. MARCKS was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of MARCKS (arrow) was stimulated by halothane in the absence (lanes 1–4) or presence (lanes 5–8) of 2 micro Meter okadaic acid.
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Figure 5. Effect of halothane on the phosphorylation of synapsin 1 in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min and incubated for 15 s with control buffer, KCl, or ionomycin (all in the presence of 1 mM CaCl2) in the absence or presence of 2.4 vol% halothane at 37 degrees C. Synapsin 1 (1a and 1b) was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of synapsin 1 (arrows) was not affected by halothane under basal conditions (control) or when stimulated by 30 mM KCl-evoked depolarization (KCl). However, halothane enhanced synapsin 1 phosphorylation stimulated by 2 micro Meter ionomycin (Iono).
Figure 5. Effect of halothane on the phosphorylation of synapsin 1 in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min and incubated for 15 s with control buffer, KCl, or ionomycin (all in the presence of 1 mM CaCl2) in the absence or presence of 2.4 vol% halothane at 37 degrees C. Synapsin 1 (1a and 1b) was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of synapsin 1 (arrows) was not affected by halothane under basal conditions (control) or when stimulated by 30 mM KCl-evoked depolarization (KCl). However, halothane enhanced synapsin 1 phosphorylation stimulated by 2 micro Meter ionomycin (Iono).
Figure 5. Effect of halothane on the phosphorylation of synapsin 1 in intact rat synaptosomes. Cerebrocortical synaptosomes were prelabelled with [sup 32 p]orthosphosphate for 45 min and incubated for 15 s with control buffer, KCl, or ionomycin (all in the presence of 1 mM CaCl2) in the absence or presence of 2.4 vol% halothane at 37 degrees C. Synapsin 1 (1a and 1b) was immunoprecipitated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. The phosphorylation of synapsin 1 (arrows) was not affected by halothane under basal conditions (control) or when stimulated by 30 mM KCl-evoked depolarization (KCl). However, halothane enhanced synapsin 1 phosphorylation stimulated by 2 micro Meter ionomycin (Iono).
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Figure 6. Effects of halothane on endogenous cyclic AMP-dependent protein phosphorylation in rat synaptosomal membranes. The membrane fraction from rat cerebrocortical synaptosomes was incubated under phosphorylating conditions in the absence of CaCl2for 60 s at 37 degrees C with [gamma-sup 32 p]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter PKI4-24, lanes 5–12 contained 10 micro Meter 8-bromo-cyclic AMP (8BrcA), and lanes 9–12 contained 10 micro Meter 8BrcA and 2 micro Meter okadaic acid (OA). The most prominent cyclic AMP-stimulated phosphoproteins are synapsin 1a, 1b, 2a, and 2b (Mr= 80,000, 75,000, 72,000, and 57,000, respectively; arrows). Halothane slightly potentiated the phosphorylation of several cyclic AMP-stimulated phosphoproteins (lanes 5–8), an effect that was prevented by the inclusion of OA (lanes 9–12), and inhibited the phosphorylation of a protein of Mr[approximately equal] 40,000 (lower arrow). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left.
Figure 6. Effects of halothane on endogenous cyclic AMP-dependent protein phosphorylation in rat synaptosomal membranes. The membrane fraction from rat cerebrocortical synaptosomes was incubated under phosphorylating conditions in the absence of CaCl2for 60 s at 37 degrees C with [gamma-sup 32 p]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter PKI4-24, lanes 5–12 contained 10 micro Meter 8-bromo-cyclic AMP (8BrcA), and lanes 9–12 contained 10 micro Meter 8BrcA and 2 micro Meter okadaic acid (OA). The most prominent cyclic AMP-stimulated phosphoproteins are synapsin 1a, 1b, 2a, and 2b (Mr= 80,000, 75,000, 72,000, and 57,000, respectively; arrows). Halothane slightly potentiated the phosphorylation of several cyclic AMP-stimulated phosphoproteins (lanes 5–8), an effect that was prevented by the inclusion of OA (lanes 9–12), and inhibited the phosphorylation of a protein of Mr[approximately equal] 40,000 (lower arrow). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left.
Figure 6. Effects of halothane on endogenous cyclic AMP-dependent protein phosphorylation in rat synaptosomal membranes. The membrane fraction from rat cerebrocortical synaptosomes was incubated under phosphorylating conditions in the absence of CaCl2for 60 s at 37 degrees C with [gamma-sup 32 p]ATP in the absence or presence of 2.4 vol% halothane as described in methods and materials. Protein phosphorylation was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. Lanes 1–4 contained 10 micro Meter PKI4-24, lanes 5–12 contained 10 micro Meter 8-bromo-cyclic AMP (8BrcA), and lanes 9–12 contained 10 micro Meter 8BrcA and 2 micro Meter okadaic acid (OA). The most prominent cyclic AMP-stimulated phosphoproteins are synapsin 1a, 1b, 2a, and 2b (Mr= 80,000, 75,000, 72,000, and 57,000, respectively; arrows). Halothane slightly potentiated the phosphorylation of several cyclic AMP-stimulated phosphoproteins (lanes 5–8), an effect that was prevented by the inclusion of OA (lanes 9–12), and inhibited the phosphorylation of a protein of Mr[approximately equal] 40,000 (lower arrow). The relative molecular weights (Mr) in kilodaltons (kDa) of protein standards are indicated on the left.
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Table 1. Effects of Halothane on MARCKS Phosphorylation in Lysed and Intact Synaptosomes
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Table 1. Effects of Halothane on MARCKS Phosphorylation in Lysed and Intact Synaptosomes
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Table 2. Effects of Halothane on Synapsin 1 Phosphorylation in Intact Synaptosomes
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Table 2. Effects of Halothane on Synapsin 1 Phosphorylation in Intact Synaptosomes
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