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Biochemical Characterization of the Stimulatory Effects of Halothane and Propofol on Purified Brain Protein Kinase C

Hemmings, Hugh C. Jr., MD, PhD; Adamo, Anna I. B. RMT; Hoffman, Mary M. MS

Anesthetic Actions and Outcomes
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Halothane and propofol stimulate activation of protein kinase C (PKC) in the presence of physiologically relevant lipid bilayer vesicles in vitro.The mechanism of this stimulation was characterized by analyzing the effects of halothane and propofol on the activation of purified rat brain PKC by its three essential activators, phosphatidylserine, diacylglycerol, and Ca2+, each of which is known to interact with the regulatory domain. Clinically relevant concentrations of halothane (2.4 vol%) and propofol (200 micro Meter) increased the Vmax without affecting the Km for phosphorylation of the artifical substrate histone H1 by PKC, and increased the sensitivity of PKC to activation by phosphatidylserine, diacylglycerol, and Ca2+. Halothane reduced the EC50 values for phosphatidylserine from 18 +/- 2.5 to 11 +/- 0.6 mol% (P < 0.05), for diacylglycerol from 1.6 +/- 0.3 to 0.87 +/- 0.2 mol% (P < 0.05) and for free Ca2+ from 4.5 +/- 1.0 to 2.8 +/- 0.4 micro Meter (P < 0.05). Propofol reduced the EC50 values for phosphatidylserine from 18 +/- 1.9 to 11 +/- 1.2 mol% (P < 0.01), for diacylglycerol from 2.5 +/- 0.3 to 1.2 +/- 0.4 mol% (P < 0.01) and for free Ca2+ from 2.8 +/- 0.7 to 1.9 +/- 0.2 micro Meter (P < 0.05). The IC50 values for inhibition of PKC activity by the regulatory domain-specific PKC inhibitor sphingosine were increased from 20 +/- 1.5 to 26 +/- 0.6 micro Meter (P < 0.001) by halothane and from 24 +/- 4.8 to 34 +/- 4.8 micro Meter (P < 0.05) by propofol. These data suggest that halothane and propofol stimulate brain PKC activity by stabilizing its active conformation through an interaction with its regulatory domain. Given the diverse role of PKC in physiologic regulation, alterations in PKC activity may be a relevant mechanism for general anesthetic effects.

(Anesth Analg 1995;81:1216-22)

Departments of Anesthesiology (Hemmings, Adamo) and Pharmacology, Cornell University Medical College, New York, New York (Hemmings, Hoffman).

Section Editor: John H. Tinker.

Supported in part by a Foundation for Anesthesia Education and Research/BOC Anesthesiology Young Investigator Award and a Cornell Scholar Award in biomedical Science to HCH.

Presented in part at the Annual Meeting of the American Society of Anesthesiologists, San Francisco, CA, October 1994, and at the Annual Meeting of the Society for Neuroscience, Miami, FL, November 1994.

Accepted for publication June 21, 1995.

Address correspondence and reprint requests to Hugh C. hemmings, Jr., Department of Anesthesiology, Box 50, LC-208, Cornell University Medical College, 525 East 68th St., New York, NY 10021.

Protein phosphorylation is an important biochemical mechanism in the regulation of many aspects of neuronal function [1]. A variety of evidence has implicated protein kinase C (PKC)-mediated protein phosphorylation as a target for general anesthetic effects in the central nervous system. First, PKC is involved in the regulation of several synaptic processes known to be affected by general anesthetics, including neurotransmitter release [2], ion channel activity [3], and neurotransmitter receptor desensitization [4]. Second, biochemical studies have demonstrated general anesthetic effects on PKC activation in vitro. However, the results of these studies have been contradictory, in that halothane has been found to either stimulate [5-7] or inhibit [8] PKC activity. A previous report from this laboratory described stimulation of PKC activity by general anesthetics when assayed with a physiologically relevant lipid bilayer preparation, although the mechanism of this effect was not determined [7]. The present study was designed to characterize the stimulatory effect of halothane and propofol on purified brain PKC activation in vitro as an approach to identifying the biochemical mechanism of this anesthetic-enzyme interaction.

Ten isoforms of PKC have been identified in mammalian tissues. These isoforms differ in their tissue distribution, regional distribution in brain, subcellular localization, and sensitivity to various activators [9,10]. The conventional PKC isoforms (alpha, beta I, beta II, and gamma, which are abundant in mammalian brain) are activated by the lipid second messenger sn-1,2-diacylglycerol, phospholipids, such as phosphatidylserine, and Ca2+[11]. The structure of PKC consists of an amino-terminal regulatory domain, which includes specific sites that bind diacylglycerol, phosphatidylserine, and Ca2+, and a carboxylterminal catalytic domain, which contains the adenosine triphosphate (ATP)-binding and catalytic sites [11]. Physiologic activation of PKC occurs when extracellular signals activate receptors coupled to stimulation of phospholipase C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-triphosphate, a second messenger that activates intracellular Ca2+ release, and 1,2-diacyl-sn-glycerol, a second messenger that activates PKC by binding to specific sites in the regulatory domain of PKC. Diacylglycerol binding increases the affinity of other sites in the regulatory domain for the cofactors Ca2+ and phosphatidylserine, facilitates translocation of PKC from the cytosol to the plasma membrane, where PKC is activated by phosphatidylserine present in the membrane and diacylglycerol formed by phospholipase C [10-12]. Activated PKC then phosphorylates specific substrate proteins, and thereby regulates their physiologic functions [1]. Activation of the conventional isoforms of PKC requires interaction of the enzyme with two substrates (ATP and a phosphorylatable substrate protein such as histone) and four essential cofactors (phosphatidylserine, diacylglycerol, Ca (2+) and Mg2+) [13]. The phosphorylatable substrates commonly used to assay PKC in vitro are polycationic peptides and proteins, which can themselves alter the cofactor requirements of PKC by affecting enzyme aggregation [14]. In the present study of the effects of halothane and propofol on the cofactor dependence of PKC activation in vitro [7], histone H1 was used as the protein substrate, since it has minimal effects on enzyme aggregation and consequently requires all four physiologic cofactors [14].

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Methods

PKC was purified to >80% homogeneity from rat forebrain as described [15] and stored in liquid N2. This preparation consists of a mixture of the alpha, beta I, beta II, and gamma isoforms of conventional Ca2+-dependent PKC [16]. Initial rate assays of PKC were performed at 30 degrees C to minimize enzyme inactivation as described previously [7]. All reactions contained 50 mM N-2-hydroxyethylpiperazine-N prime-2-ethanesulfonic acid, pH 7.4 with NaOH, 1 mM EGTA, 10 mM MgCl2, 20 micro gram/mL bovine serum albumin (fraction V; Baker, Phillipsburg, NJ), 0.1 mM dithiothreitol, 100 micro Meter [gamma-(32) P]ATP (100-250 cpm/pmol; DuPont-New England Nuclear, Boston, MA), and 0.1-1 micro gram/mL purified PKC. The standard assay also contained 0.2 mg/mL (9.3 micro Meter) lysine-rich histone H1 (histone HL; Worthington Biochemical, Freehold, NJ) as protein substrate. Small unilamellar lipid vesicles were freshly prepared by mixing solutions of lipids in CHCl3, drying under N2, hydration in 20 mM Tris (pH 7.4), and sonication for 5 min. The standard assay contained vesicles composed of 20 micro Meter bovine brain L-alpha-phosphatidylserine (Avanti Polar Lipids, Alabaster, AL), 80 micro Meter chicken egg L-alpha-phosphatidylcholine (Avanti Polar Lipids), and 2 micro Meter 1,2-dioleoyl-sn-glycerol (Avanti Polar Lipids) [17], as well as 1.5 mM CaCl2 and 1 mM EGTA (free [Ca2+] = 500 micro Meter). The reaction mixture, containing all assay components except ATP, was equilibrated with halothane or propofol for 5 min, after which reactions were initiated by the addition of ATP. After a 5-min incubation, reactions were terminated by the addition of 10 micro Liter glacial acetic acid. Histone phosphorylation was determined by the phosphocellulose paper method as described [7].

Enzyme kinetic analysis and EC50 determinations were performed by independently varying the concentrations of histone H1 or phosphatidylserine, diacylglycerol, and CaCl2, respectively. In experiments to determine the EC50 for activation by Ca2+, CaCl2 concentration was varied and free Ca2+ concentrations were calculated as described [18]. In experiments to determine the EC50 for activation of PKC by phosphatidylserine, the total lipid concentration was maintained at 102 micro Meter by adjusting the phosphatidylcholine concentration. Assays to determine the IC50 values for sphingosine (Sigma, St. Louis, MO) were performed using standard assay conditions and varying concentrations of sphingosine (5-50 micro Meter) in the absence or presence of anesthetics [19].

Reactions in the presence of halothane (thymol-free; Halocarbon Products, North Augusta, SC) were performed by the method of Blanck [20] as described previously [7]. This method allows the use of a small reaction volume by the addition of liquid halothane directly to the closed reaction vessel. Vapor phase halothane concentrations were determined by gas chromatography [21]. Reactions in the presence of propofol were performed in open vials using 5% (vol/vol) ethanol as a drug vehicle as described [7].

Data were analyzed using a graded concentration-response and enzyme kinetics computer program (PHARM/PCS Pharmacologic Calculation System, v. 4.2). Cooperativity was assessed by fitting concentration-response data by computer analysis (SigmaPlot for Windows, v. 1.01) to a modified Hill Equation definedby Equation 1 where y is the measured PKC activity, a and b are the asymptotic minimum and maximum activity values, x is the concentration of the activator, k is the EC50 of the activator and n is the Hill coefficient [22]. Results are presented as mean values +/- SD. Statistical significance was assessed by Student's two-tailed unpaired t-test.

Protein concentrations were determined by the bicinchoninic acid method [23] (Pierce Chemical Co., Rockford, IL) using bovine serum albumin as a standard.

These studies were approved by the Cornell University Medical College Institutional Animal Care and Use Committee.

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Results

Previous studies demonstrated a stimulatory effect of both halothane and propofol on the activation of purified brain PKC [7] using a physiologically relevant lipid bilayer vesicle preparation [17]. A kinetic analysis of this effect was performed under identical assay conditions using histone H1 as a substrate. Halothane was studied at a concentration of 2.4 vol%, which is near its EC50 (concentration that produces 50% maximal effect) for activation of PKC at 30 degrees C (2.2 vol%) [7], and is equivalent to approximate 3 minimum alveolar anesthetic concentration in the rat [7]. Propofol was studied at a concentration of 200 micro Meter, which is near its EC50 for activation of PKC (240 micro Meter) [7], and is 2.4 times the Cp50 i (concentration at which 50% of subjects did not respond to incision) in humans [24]. Double-reciprocal plots of PKC activity versus histone H1 concentration in the absence or presence of anesthetics resulted in linear plots Figure 1. Both halothane and propofol increased the Vmax (maximal velocity) for histone H1 phosphorylation by PKC without affecting apparent Km (Michaelis constant) values for histone H1 Table 1.

Figure 1

Figure 1

Table 1

Table 1

The mechanism of the anesthetic-induced stimulation of PKC activity was investigated further by determining the effects of halothane and propofol on the sensitivity of PKC to activation by its second messenger diacylglycerol and its cofactor phosphatidylserine. Both halothane Figure 2 and propofol Figure 3 decreased the EC50 values for the activation of PKC by phosphatidylserine or diacylglycerol Table 1.

Figure 2

Figure 2

Figure 3

Figure 3

PKC was essentially inactive in the absence of phosphatidylserine; however, halothane and propofol stimulated PKC activity in the absence of diacylglycerol. Both anesthetics produced a leftward shift in the concentration-effect curves for phosphatidylserine and diacylglycerol. There was also an increase in the efficacy for PKC activation by diacylglycerol, but no change in the efficacy of PKC activation by phosphatidylserine. In a separate series of experiments, higher concentrations of phosphatidylserine (80 vol%) saturated PKC activation and prevented further activation by halothane (54 +/- 4.8 pmol/min versus 56 +/- 3.7 pmol/min in the absence of presence of 2.4 vol% halothane, respectively; n = 3), although the stimulatory effect of propofol was not saturated (36 +/- 0.7 pmol/min versus 46 +/- 3.3 pmol/min in the absence or presence of 200 micro Meter propofol; P < 0.01; n = 3). An increase in the apparent cooperativity of phosphatidylserine-dependent activation was indicated by the increased slopes of the concentration-effect curves for PKC activation by halothane (Hill coefficient increase from 3.3 to 5.4) and propofol (Hill coefficient increase from 3.0 to 4.0). There was no effect of halothane or propofol on the cooperativity of the response to diacylglycerol (Hill coefficients 0.8-1.2 for all curves).

Both halothane and propofol decreased the EC50 values for the activation of PKC by Ca2+ under standard assay conditions Figure 4, Table 1. both anesthetics produced significant activation of PKC over the range of free Ca2+ concentrations tested. Higher concentrations of free Ca2+ did not activate PKC further (data no shown). PKC activity in the absence of Ca2+ was not affected significantly by halothane or propofol, although the ethanol vehicle increased basal Ca2+-independent PKC activity.

Figure 4

Figure 4

Sphingosine is an inhibitor of PKC activation by diacylglycerol and phorbol esters and an inhibitor of phorbol ester binding to PKC [19]. Sphingosine produced a dose-dependent inhibition of PKC activation by phosphatidylserine and diacylglycerol. The potency of sphingosine as a PKC inhibitor was reduced by halothane or propofol, both of which increased the IC50 (concentration that produces 50% inhibition of the maximal effect) values of sphingosine for inhibition of PKC Table 1.

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Discussion

The results of a previous study showed that halothane and propofol stimulate purified brain PKC when PKC is assayed with a physiologically relevant lipid bilayer vesicle preparation in vitro [7]. This stimulatory effect was specific for PKC; Ca2+/calmodulin-dependent protein kinase II and cyclic adenosine monophosphate-dependent protein kinase, two other second messenger-regulated protein kinases that posses structurally similar catalytic domains [25], were insensitive to halothane or propofol treatment. Stimulation by anesthetics required the intact regulatory domain of PKC, which contains the Ca2+, phospholipid and diacylglycerol (or phorbol ester) binding sites [11]. The biochemical data described in the present study are consistent with an interaction between general anesthetics and the lipid and Ca2+-binding regulatory domain of PKC.

The catalytic fragment of PKC has been shown previously to exhibit Michaelis-menten kinetics with histone as a substrate [13] with a Km value of 0.12 micro gram/mL, which is similar to the values we determined for intact PKC Table 1. Enzyme kinetic analysis of the effects of halothane or propofol on the phosphorylation of histone H1 by PKC revealed an increase in the Vmax with no effect on the Km for histone H1. This finding indicates an increase in the catalytic efficiency of PKC under the assay conditions used, but no apparent effect on substrate protein binding, which suggests that the anesthetic effect is not substrate-mediated. The enhanced catalytic efficiency could be due either to a direct effect on the catalytic domain of the enzyme or to an indirect effect on catalytic activity mediated through an interaction with the regulatory domain; the latter interpretation is favored since anesthetic effects were not observed on the isolated catalytic fragment of PKC or on other protein kinases with structurally similar catalytic domains but distinct regulatory domains [7]. Additional support for this mechanism was obtained by analysis of the effects of halothane and propofol on the sensitivity of PKC to activation by its endogenous regulators.

Halothane and propofol produced leftward shifts in the concentration-effect curves for activation of PKC by phosphatidylserine, as evidenced by reductions of approximate 50% in the EC50 values of phosphatidylserine. The rate of histone H1 phosphorylation by PKC has been shown previously to be highly cooperative with respect to phosphatidylserine concentration using a mixed micelle [13,26] or lipid bilayer vesicle [22,27] assay, an effect that we also observed. The stimulatory effects of halothane and propofol were accompanied by increases in the cooperativity of phosphatidylserine-dependent PKC activation, which indicates an anesthetic effect on the nature of the interaction between PKC and phosphatidylserine at the membrane surface. The increase in cooperativity of phosphatidylserine-dependent PKC activation may be due to a change in dimensionality of the interaction between PKC and phosphatidylserine in the membrane [27].

Halothane and propofol also induced a leftward shift in the concentration-effect curves for activation of PKC by diacylglycerol, as evidenced by reductions of approximate 50% in the EC50 values of diacylglycerol. The activation of PKC by diacylglycerol is not cooperative in the mixed micelle assay [26] which we also observed using the lipid bilayer vesicle assay. The effects of halothane or propofol on PKC activation by diacylglycerol were not accompanied by a change in cooperativity.

There are noticeable differences in the control concentration-effect curves between halothane and propofol experiments. This is due to the presence of ethanol as a drug vehicle in the propofol experiments. Ethanol alone significantly reduced the Vmax of PKC and the EC50 for Ca2+, and increased basal Ca2+-independent PKC activity and the EC50 for diacylglycerol (compare propofol control with halothane control in Table 1). Despite this interference by ethanol, the effects of propofol on the activation of PKC were qualitatively comparable to those produced by halothane in the absence of ethanol.

Examination of the concentration-effect curves for activation of PKC by halothane and propofol revealed the mechanism for the observed stimulatory effects of these anesthetics in vitro. The standard lipid bilayer PKC assay described by Boni and Rando [17] that was used in this study contains 2 mol% diacylglycerol, 20 mol% phosphatidylserine, 80 mol% phosphatidylcholine, and 500 micro Meter free Ca2+. Under these conditions, the concentration of Ca2+ present in the assay is saturating, while the concentrations of diacylglycerol and phosphatidylserine present are subsaturating for PKC activation. The addition of halothane or propofol under standard assay conditions significantly reduced the requirement of PKC for these lipid cofactors, and thereby increased the activity of PKC toward its maximal value observed in the presence of saturating diacylglycerol and phosphatidylserine concentrations. Halothane-induced stimulation of PKC activity occurred in spite of saturating concentrations of either diacylglycerol or Ca2+, while saturating concentrations of phosphatidylserine prevented further stimulation by halothane. This finding suggests that the principal effect of halothane on PKC activation is mediated by an increase in the sensitivity of PKC to phosphatidylserine. This is apparently due to an increase in the affinity of the enzyme for phosphatidylserine rather than an increase in the efficacy of phosphatidylserine in activating PKC because we observed a reduction in the EC50 of phosphatidylserine and no change in the maximal activation of PKC in response to halothane or propofol. The additional activation of PKC by halothane in the presence of saturating diacylglycerol or Ca2+ concentrations is also likely due to this increase in phosphatidylserine affinity, since the assays of diacylglycerol and Ca2+ dependence were performed at a subsaturating phosphatidylserine concentration (20 mol%). Phosphatidylserine was capable of fully activating PKC in the presence of subsaturating concentrations of diacylglycerol or Ca2+, while the converse was not true. This difference explains the lesser activation of PKC by saturating diacylglycerol or Ca2+ compared to phosphatidylserine, and is consistent with the observed kinetic differences between phosphatidylserine and diacylglycerol in their activation of PKC [13].

The effects of halothane and propofol on PKC activation are not specific for a single activator, but result in increased sensitivity to activation by phosphatidylserine, diacylglycerol, and Ca2+. This suggests that these anesthetics activate PKC by stabilizing the active conformation of PKC. The mechanism of activation of PKC by halothane and propofol is comparable to the mechanism of inhibition of PKC by sphingosine, in which sphingosine competitively inhibits the activation of PKC by all three activators (phosphatidylserine, diacylglycerol, and Ca2+) through an effect on the regulatory domain of PKC [19]. The observation that halothane and propofol opposed the inhibitory effects of sphingosine suggests that general anesthetics and sphingosine may have opposite actions mediated at the same site on the regulatory domain of PKC.

In human red cells, phosphatidylserine constitutes approximately 15 mol% of the inner plasma membrane leaflet lipid [28], which is within the range of greatest sensitivity of PKC activity to phosphatidylserine concentration determined using physiologically relevant lipid bilayer preparations by others [22,27] and in the present study. It is more difficult to estimate the in vivo concentrations of diacylglycerol and Ca2+, both of which are variable and highly regulated due to their roles as intracellular second messengers. The amount of diacylglycerol generated in vivo can be estimated from the amount of phosphatidylinositol turnover (1-2 mol%), which is within the range of greatest sensitivity of PKC activity to diacylglycerol concentration observed by others [22,26] and in this study. The concentration of Ca2+ in neurons is approximate equals 200 nM in the basal unstimulated state [29], but may increase 1000-fold during depolarization [30]. This range encompasses the range of sensitivity of purified PKC activation to Ca2+ concentration in vitro. Taken together, these data obtained in vitro are consistent with the ability of halothane or propofol to stimulate PKC activity in vivo by increasing the sensitivity of PKC to endogenous phosphatidylserine, diacylglycerol and/or Ca2+ over their physiologic concentration ranges.

Alterations in PKC activity in intact cell systems have been invoked as the mechanism underlying anesthetic effects observed in PC12 cells [6], bovine endothelial cells [31], intact rat liver [32], canine tracheal smooth muscle [33], neonatal rat brain [34], and Rana pipiens tadpoles [35]. The interpretation of these studies is severely limited, however, because anesthetic effects on PKC activity, subcellular translocation, and/or down-regulation were not determined directly. In order to ascribe anesthetic effects in intact cells to an effect on PKC, it is necessary to demonstrate directly the ultimate effect of the anesthetic on PKC activity. Such experiments are necessary since pharmacologic activation of PKC can ultimately result in a reduction in PKC activity due to subcellular translocation of PKC away from a specific substrate protein or to down-regulation of PKC activity, as seen with phorbol ester PKC agonists [36].

In conclusion, this study demonstrates the ability of a volatile anesthetic (halothane) and an intravenous anesthetic (propofol) to induce significant increases in the sensitivity of purified brain PKC to its endogenous activators phosphatidylserine, diacylglycerol, and Ca2+ in vitro. The observed disparity in the effects of anesthetics on PKC activation in the absence of lipids [8] or in the presence of various lipids and substrates in vitro [7] emphasizes the importance of demonstrating an anesthetic effect on endogenous PKC activity, translocation and down-regulation in each particular system under study, preferably with endogenous substrate proteins, before it can be concluded that an observed anesthetic effect in intact cells is mediated through an effect on PKC.

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