255
Effect of Ethanol on Platelet Phospholipase A2 Christopher D. Stubbs* and Raphael Rubin Department of Pathology and Cel~ Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Platelet aggregation is known to be inhibited by ethanol, and this has been suggested to be one of the attenuating effects of ethanol in cardiovascular disease. Recent studies have implicated an inhibition of phospholipase A2 induced arachidonic acid release, since the production of prostanoids that are formed from arachidonic acid and are involved in the aggregation process has been shown to be diminished by ethanol. Phospholipase A2 is found in platelets in both a cytosolic form, from where it may translocate to the plasma membrane to release arachidonic acid, and in a secretory form which is released extracellularly upon activation. In the present study, the effect of ethanol on the secretion of phospholipase A2 and on its activity was determined. It was found that ethanol inhibited phospholipase As secretion but not its activity. By contrast, the activity of the cytosolic form of phospholipase A2 was inhibited by ethanol. Lipids 27, 255-260 (1992).
There is considerable evidence that ethanol exerts an inhibitory effect on platelet aggregation (1-8), and it is possible that this could contribute to its attenuating effect in cardiovascular disease (reviewed in ref. 9). A number of studies have shown that ethanol inhibits the synthesis of prostanoids including thromboxane A2, which is involved in the aggregation process (1-7). These findings would imply that ethanol reduces the level of arachidonic acid release Indeed addition of exogenous arachidonic acid is known to alleviate the effect of ethanol (ag., see ref. 6). The release of arachidonic acid may be due to the action of one or both of two enzymes, phospholipase C and phospholipase A 2. Phospholipase C activation would lead to the release of inositol 1,4,5-triphosphate and diacylglycerol. Inositol 1,4,5-triphosphate then induces an increase in intracellular Ca 2+, and the diacylglycerol activates the regulatory protein kinase C. Arachidonic acid is then formed by the breakdown of diacylglycerol by diglyceride lipase However, this pathway accounts for only a minor proportion of arachidonic acid release The major source of arachidonic acid in platelets is phospholipids in which arachidonic acid resides in the sn-2 position and is released by the action of phospholipase A 2 (ag., see ref. 10,11). Recently, an inhibition by ethanol of the release of arachidonic acid has been demonstrated, and an effect on phospholipase A2 was thereby implicated (12,13). There is evidence for existence of more than one form of phospholipase A2 in platelets (discussed in recent reviews, see ref. 14,15). Two types of phospholipase A2 have been recognized, a cytosolic form of 90 kDa molecular weight (16-20) and a secretable, 14 kDa "type II" form (21-26; see *To whom correspondence should be addressed at Room 271 JAH, Thomas Jefferson University, 1020 Locust Street, Philadelphia,PA 19107. Abbreviations: ATP, adenosine triphosphate; C6-NBD-PC, 1-palmitoyl-2-N-(7-nitrobenzc~2-oxa-1,3-diazol-4-yl)amino)hexanoylglycerophosphocholine; EGTA, ethyleneglycol-bis([~-an~oethyl ether)-N,N,N~,N'tetraacetic acid; HA, hexanoic acid; HEPES, N-[2-hydroxyethyl]piperazine~N'-[2-ethanesulfonicacid].
also reviews 14,15). The cytosolic form can be translocated to the membrane surface as a result of an increase in intracellular Ca 2+, and it appears to be identical to a number of unknown membrane associated forms of the enzyme (27). There is also evidence for arachidonoyl phospholipid species specificity of the cytosolic form of phospholipase A2 in the platelet (16,19,28}. However, platelet phospholipase A2 can also hydrolyze other species (18,29-31), and if pre-labeled glycerol is used, all phosphatidylcholine (PC) species were found to be hydrolyzed to similar extents (29, 30). A lack of selectivity is also consistent with the observation that a partially purified rabbit platelet phospholipase A 2 did not show acyl-chain selectivity (32). Although in a recent study it was found that while the arachidonoyl PC species were hydrolyzed more rapidly than the linoleoyl PC species, in a mixture of the two species both were equally hydrolyzed (18). In spite of these latter observations, the release of the fatty acids from stimulated platelets, determined as mass changes in individual molecular species, does appear to show some arachidonoyl phospholipid species selectivity (16). An arachidonic acid selective phospholipase A2 has recently been cloned (27). Taken together, above studies suggest that the phospholipase A2, which is selective for arachidonate phospholipid species, may be compartmentalized in a manner which optimizes the release of arachidonic acid upon stimulation. This would fit in with the other observation that selectivity was best observed with minimal perturbation of the membranes (33). This may also imply that there may exist more than one cytosolic form of phospholipase A 2. The secretable form of phospholipase A2 is located in agranules (21,25), as was shown by recent immunochemicallabelling studies (23). Detailed characteristics of the secretory phospholipase A2, including substrate specificit:~ Ca 2+ requirement, heat stability, and pH optimum, have been described (22,26). Studies in which phospholipase A2 from rat and human platelets were compared have revealed that the level of secretion, or activity, of the rat phosph~ lipase A2 is much greater (22). At present there is no evidence for any fundamental differences between the rat and human phospholipase A 2 and it is entirely possible that observed differences in activity are due to the presence of an endogenous inhibitor. While ethanol has been shown to inhibit the release of arachidonic acid from stimulated platelets (12,13), it is not known whether the effect is specific for the cytosolic form of the enzyme To investigate the locus of ethanol action, the effects of ethanol on both forms of phospholipase A2 were examined. Rat platelets were used since the secretory phospholipase A2 is more readily obtained. The results revealed that ethanol inhibited the secretion of phospholipase A2 but not its activity. By contrast, the cytosolic phosph~ lipase A2 was inhibited, although the effect was not specific to arachidonate.
MATERIALS AND METHODS Materials. Male Sprague-Dawley rats (100-200 g) were obtained from Charles River Laboratories (St. Constant, Quebec, Canada). Thrombin, prostacyclin, indomethacin, LIPIDS, Voi. 27, no. 4 (1992)
256
C.D. STUBBS AND R. RUBIN and arachidonic acid were obtained from Sigma (St. Louis, MO); lipid standards and 1-palmitoyl-2-N-(7-nitrobenzo-2oxa-l,3-diazol-4-yl)amino)hexanoyl-glycerophosphocholine (C6-NBD-PC) was from Avanti Polar Lipids (Birmingham, AL); 6-(N-(7-nitrobenzo-2-oxa-l,3-diazol-4-yl)amino) hexanoic acid) was from Molecular Probes (Eugene, OR). The radiolabeled lipids [5,6,8,9,11,12,14,15-3H]arachi donic acid (206 Ci/mmol),l-stearoyl-2-[5,6,8,9,11,12,14,153H]arachidonoyl-sn-glycero-3-phosphocholine (120 Ci/ mmol) and 1-palmitoyl-2-[1-14C]oleoyl-sn-glycero-3-phosphocholine (53.8 mCi/mmol) were from Amersham International (Arlington Height, IL). Solvents and other reagents were from Fisher Scientific (Pittsburg, PA). Platelet preparation. Blood was taken by cardiac puncture from rats anesthetized by nembutol. The blood was anticoagulated with 0.38% (w/v) sodium citrate and centrifuged at 140 X g for 20 rain at room temperature. The platelet-rich plasma was further centrifuged at 600 >
Effect of Ethanol on Platelet Phospholipase A2 Christopher D. Stubbs* and Raphael Rubin Department of Pathology and Cel~ Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Platelet aggregation is known to be inhibited by ethanol, and this has been suggested to be one of the attenuating effects of ethanol in cardiovascular disease. Recent studies have implicated an inhibition of phospholipase A2 induced arachidonic acid release, since the production of prostanoids that are formed from arachidonic acid and are involved in the aggregation process has been shown to be diminished by ethanol. Phospholipase A2 is found in platelets in both a cytosolic form, from where it may translocate to the plasma membrane to release arachidonic acid, and in a secretory form which is released extracellularly upon activation. In the present study, the effect of ethanol on the secretion of phospholipase A2 and on its activity was determined. It was found that ethanol inhibited phospholipase As secretion but not its activity. By contrast, the activity of the cytosolic form of phospholipase A2 was inhibited by ethanol. Lipids 27, 255-260 (1992).
There is considerable evidence that ethanol exerts an inhibitory effect on platelet aggregation (1-8), and it is possible that this could contribute to its attenuating effect in cardiovascular disease (reviewed in ref. 9). A number of studies have shown that ethanol inhibits the synthesis of prostanoids including thromboxane A2, which is involved in the aggregation process (1-7). These findings would imply that ethanol reduces the level of arachidonic acid release Indeed addition of exogenous arachidonic acid is known to alleviate the effect of ethanol (ag., see ref. 6). The release of arachidonic acid may be due to the action of one or both of two enzymes, phospholipase C and phospholipase A 2. Phospholipase C activation would lead to the release of inositol 1,4,5-triphosphate and diacylglycerol. Inositol 1,4,5-triphosphate then induces an increase in intracellular Ca 2+, and the diacylglycerol activates the regulatory protein kinase C. Arachidonic acid is then formed by the breakdown of diacylglycerol by diglyceride lipase However, this pathway accounts for only a minor proportion of arachidonic acid release The major source of arachidonic acid in platelets is phospholipids in which arachidonic acid resides in the sn-2 position and is released by the action of phospholipase A 2 (ag., see ref. 10,11). Recently, an inhibition by ethanol of the release of arachidonic acid has been demonstrated, and an effect on phospholipase A2 was thereby implicated (12,13). There is evidence for existence of more than one form of phospholipase A2 in platelets (discussed in recent reviews, see ref. 14,15). Two types of phospholipase A2 have been recognized, a cytosolic form of 90 kDa molecular weight (16-20) and a secretable, 14 kDa "type II" form (21-26; see *To whom correspondence should be addressed at Room 271 JAH, Thomas Jefferson University, 1020 Locust Street, Philadelphia,PA 19107. Abbreviations: ATP, adenosine triphosphate; C6-NBD-PC, 1-palmitoyl-2-N-(7-nitrobenzc~2-oxa-1,3-diazol-4-yl)amino)hexanoylglycerophosphocholine; EGTA, ethyleneglycol-bis([~-an~oethyl ether)-N,N,N~,N'tetraacetic acid; HA, hexanoic acid; HEPES, N-[2-hydroxyethyl]piperazine~N'-[2-ethanesulfonicacid].
also reviews 14,15). The cytosolic form can be translocated to the membrane surface as a result of an increase in intracellular Ca 2+, and it appears to be identical to a number of unknown membrane associated forms of the enzyme (27). There is also evidence for arachidonoyl phospholipid species specificity of the cytosolic form of phospholipase A2 in the platelet (16,19,28}. However, platelet phospholipase A2 can also hydrolyze other species (18,29-31), and if pre-labeled glycerol is used, all phosphatidylcholine (PC) species were found to be hydrolyzed to similar extents (29, 30). A lack of selectivity is also consistent with the observation that a partially purified rabbit platelet phospholipase A 2 did not show acyl-chain selectivity (32). Although in a recent study it was found that while the arachidonoyl PC species were hydrolyzed more rapidly than the linoleoyl PC species, in a mixture of the two species both were equally hydrolyzed (18). In spite of these latter observations, the release of the fatty acids from stimulated platelets, determined as mass changes in individual molecular species, does appear to show some arachidonoyl phospholipid species selectivity (16). An arachidonic acid selective phospholipase A2 has recently been cloned (27). Taken together, above studies suggest that the phospholipase A2, which is selective for arachidonate phospholipid species, may be compartmentalized in a manner which optimizes the release of arachidonic acid upon stimulation. This would fit in with the other observation that selectivity was best observed with minimal perturbation of the membranes (33). This may also imply that there may exist more than one cytosolic form of phospholipase A 2. The secretable form of phospholipase A2 is located in agranules (21,25), as was shown by recent immunochemicallabelling studies (23). Detailed characteristics of the secretory phospholipase A2, including substrate specificit:~ Ca 2+ requirement, heat stability, and pH optimum, have been described (22,26). Studies in which phospholipase A2 from rat and human platelets were compared have revealed that the level of secretion, or activity, of the rat phosph~ lipase A2 is much greater (22). At present there is no evidence for any fundamental differences between the rat and human phospholipase A 2 and it is entirely possible that observed differences in activity are due to the presence of an endogenous inhibitor. While ethanol has been shown to inhibit the release of arachidonic acid from stimulated platelets (12,13), it is not known whether the effect is specific for the cytosolic form of the enzyme To investigate the locus of ethanol action, the effects of ethanol on both forms of phospholipase A2 were examined. Rat platelets were used since the secretory phospholipase A2 is more readily obtained. The results revealed that ethanol inhibited the secretion of phospholipase A2 but not its activity. By contrast, the cytosolic phosph~ lipase A2 was inhibited, although the effect was not specific to arachidonate.
MATERIALS AND METHODS Materials. Male Sprague-Dawley rats (100-200 g) were obtained from Charles River Laboratories (St. Constant, Quebec, Canada). Thrombin, prostacyclin, indomethacin, LIPIDS, Voi. 27, no. 4 (1992)
256
C.D. STUBBS AND R. RUBIN and arachidonic acid were obtained from Sigma (St. Louis, MO); lipid standards and 1-palmitoyl-2-N-(7-nitrobenzo-2oxa-l,3-diazol-4-yl)amino)hexanoyl-glycerophosphocholine (C6-NBD-PC) was from Avanti Polar Lipids (Birmingham, AL); 6-(N-(7-nitrobenzo-2-oxa-l,3-diazol-4-yl)amino) hexanoic acid) was from Molecular Probes (Eugene, OR). The radiolabeled lipids [5,6,8,9,11,12,14,15-3H]arachi donic acid (206 Ci/mmol),l-stearoyl-2-[5,6,8,9,11,12,14,153H]arachidonoyl-sn-glycero-3-phosphocholine (120 Ci/ mmol) and 1-palmitoyl-2-[1-14C]oleoyl-sn-glycero-3-phosphocholine (53.8 mCi/mmol) were from Amersham International (Arlington Height, IL). Solvents and other reagents were from Fisher Scientific (Pittsburg, PA). Platelet preparation. Blood was taken by cardiac puncture from rats anesthetized by nembutol. The blood was anticoagulated with 0.38% (w/v) sodium citrate and centrifuged at 140 X g for 20 rain at room temperature. The platelet-rich plasma was further centrifuged at 600 >
