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Phospholipase A2Activities Are Decreased during Early but Increased during Late Phases of Sepsis in Rat Heart
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
75, 165–169 (1998)
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Phospholipase A2 Activities Are Decreased during Early but Increased during Late Phases of Sepsis in Rat Heart1 Li-Jia Tong, M.D., Ph.D., Lin-Wang Dong, M.D., Ph.D., Hseng-Kuang Hsu, B.S.,2 and Maw-Shung Liu, D.D.S., Ph.D.3 Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104 Submitted for publication September 11, 1997
Background. Changes in the activities of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in the rat heart during early hyperdynamic and late hypodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of cardiac dysfunction during sepsis. Methods. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. PLA2 activity was measured based on the rate of hydrolysis of 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine. Results. The results show that under physiological conditions, sPLA2 and cPLA2 activities were time and protein dependent. The optimal Ca2/ concentrations for sPLA2 and cPLA2 activities were 3 mM and 40 mM, respectively. During sepsis, sPLA2 activity was decreased by 25% (P õ 0.01) during early phase while it was increased by 49% (P õ 0.01) during late phase of sepsis. Similarly, cPLA2 activity was decreased by 23% (P õ 0.01) during early sepsis while it was increased by 60% (P õ 0.01) during late sepsis. Conclusions. Since PLA2 functions to maintain cell membrane integrity and function, a biphasic change in sPLA2 and cPLA2 activities may contribute to the development of the two cardiodynamically distinct phases during the progression of sepsis. q 1998 Academic Press
Key Words: phospholipase A2 ; heart; sepsis; hyperdynamic phase; hypodynamic phase; membrane enzyme; membrane receptor.
INTRODUCTION
Phospholipase A2 (PLA2) catalyzes the hydrolysis of phospholipid molecules at the sn-2 position to liberate free fatty acids and lysophospholipids. Physiologically, 1 This work was supported by HL-30080 from National Heart, Lung and Blood Institute, and GM-31664 from National Institue of General Medical Sciences, National Institutes of Health. 2 Recipient of a fellowship award from Taiwan (NSC-34089F). 3 To whom reprint requests should be addressed.
PLA2 functions to maintain the integrity and dynamics of cell membranes [1–3]. Pathologically, PLA2 has been implicated to be an important etiological factor for the pathogenesis of various diseases including shock and sepsis [3–5]. In septic patients, plasma PLA2 activity was elevated by 22-fold and the elevation in plasma PLA2 correlated well with the severity of organ dysfunction and the mortality [6]. In animal studies, circulating PLA2 activity was increased by 11-fold after endotoxin injection and the increase in PLA2 activity paralleled the decrease in mean arterial blood pressure [5, 7]. At the tissue level, PLA2 activity was activated by 71–82% in canine myocardium following endotoxin administration [8, 9] and the activation in PLA2 activity was responsible in part for the dysfunction of the Na/Ca2/ exchanger and the ATP-dependent Ca2/ transport of sarcolemma (SL) [10, 11] as well as the Ca2/-induced Ca2/ release of sarcoplasmic reticulum (SR) [12]. PLA2s are a diverse class of enzymes with regard to structure, function, regulation, localization, and role of divalent metal ions [1–3]. Generally, PLA2 enzymes are classified into three major categories: (1) secretory PLA2 (sPLA2), a low molecular weight enzyme which requires a millimolar concentration of Ca2/ to express full enzymatic activity and localizes in the membrane fraction; (2) cytosolic PLA2 (cPLA2), a high molecular weight enzyme which requires a submicromolar concentration of Ca2/ for optimal activity and localizes in the cytosolic compartment; and (3) Ca2/-independent PLA2 (iPLA2), which is plasmalogen substrate-specific. The sPLA2 is further divided into two subgroups based on its tissue distribution: Group I PLA2 , which is found mostly in pancrease, and Group II PLA2 , which is distributed almost exclusively in nonpancreatic tissues [1, 2, 13]. Recent progress in the studies of PLA2 has indicated that cPLA2 , unlike sPLA2 , plays a unique regulatory role in the lipid-mediated signal transduction pathway [1, 2]. Since data available in the literature regarding changes in PLA2 activity in the myocardium as well as in the circulating system [6–9] during shock and sepsis are limited only to sPLA2 , and since cPLA2 gene transcript is expressed in cardiac tissue [14], it is conceivable that cPLA2 activity may also be modified in the myocardium during sepsis. Accordingly, the 0022-4804/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Effects of incubation time and protein concentration on sPLA2 and cPLA2 activities in control rat heart. PLA2 activity was measured as described under Methods except that either incubation time or protein concentration was varied as shown on the abscissa. Values are means { SEM of three experiments.
either 40 mM or 3 mM free Ca2/ (prepared by buffering CaCl2 with EGTA according to Biosoft EqCal software program). The reaction was initiated by the addition of enzyme preparation containing 0.8 mg protein, allowed to proceed for 10 min at 377C, and then terminated by the addition of 100 ml of 5% Triton X-100 containing 40 mM EDTA. The products of the PLA2 reaction were extracted with 10 vol of acidic hexane (hexane containing 0.1% acetic acid) by vortexing for 20 s after 200 mg of anhydrous Na2SO4 was added. An aliquot (2.5 ml) of the hexane phase containing the reaction product, [114 C]oleate, was carefully removed and the radioactivity was then determined. The enzymatic activity was calculated based on the rate of hydrolysis of 14C-labeled substrate. The activity obtained in the presence of 3 mM free Ca2/ after correcting that obtained in the absence of Ca2/ was designated as sPLA2 activity. The activity obtained in the presence of 40 mM free Ca2/ after correcting that obtained in the absence of Ca2/ was designated as cPLA2 activity. Protein assay and statistical analysis. Protein content of heart homogenates was determined by the method of Lowry et al. [19]. The statistical analysis of the data was performed using one-way analysis of variance followed by Student-Newman-Keuls tests. A P value of less than 0.05 was accepted as statistically significant. Materials. L-3-phosphatidylcholine, 1-palmitoyl-2-[1-14C]oleoyl (58 mCi/mmol), was obtained from Amersham Life Science. L-a-phosphatidylcholine, b-oleoyl-g-palmitoyl, was purchased from Sigma Chemical Co. Other chemicals and reagents were of analytical grade.
present study was undertaken to test our hypothesis that both cPLA2 and sPLA2 activities in the rat heart are altered during the progression of sepsis. METHODS Animal model. Male Sprague-Dawley rats weighing from 270 to 320 g were used. All animals were fasted overnight with free access to water. They were divided into three groups: control, early sepsis, and late sepsis. Sepsis was induced by cecal ligation and puncture (CLP) as described by Wichterman et al. [15] with modification. Under halothane anesthesia, a laparotomy was performed, and the cecum was ligated with a 3-0 silk ligature and punctured twice with an 18-gauge needle. The cecum was then returned to the peritoneal cavity and the abdomen was closed in two layers. Control rats were sham-operated (a laparotomy was performed and the cecum was manipulated but neither ligated nor punctured). All animals were resuscitated with 4 ml/100 g body weight of normal saline at the completion of surgery and also at 7 h postsurgery. Animals were fasted but had free access to water after operative procedures. Hearts were removed from septic and control animals 9 or 18 h postoperation under chloralose and urethane anesthesia and were then used for the preparation of homogenate for the assay of PLA2 activity. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The mortality rates were 0% for control, 15% for early sepsis, and 36% for late sepsis. Previous experiments show that septic rat heart was in hyperdynamic state during early sepsis while it was in hypodynamic state during late sepsis [16, 17]. Assay of PLA2 activity. PLA2 activity was assayed by the method of Katsumata et al. [18] with modification. Heart tissues obtained from control and septic rats were cut into small chunks and then homogenized with 10 vol of 100 mM glycine-NaOH buffer (pH 7.6) containing 50 mg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 10 mg/ml soybean trypsin inhibitor, 100 mg/ml aprotinin in a Tekma Tissumizer (Model SDT) at half maximum speed for 4 1 10 s. The homogenate was filtered through a cell strainer (70 mm) followed by centrifugation at 700g for 10 min to remove nuclei and cell debris. The resulting supernatant was then used for the assay of PLA2 activiy. PLA2 activity was assayed using 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine as a labeled substrate. The 14C-labeled and nonlabeled phosphatidylcholines were prepared with absolute ethanol, and the final ethanol concentration in the reaction mixture was 2%. The reaction mixture in a final volume of 0.5 ml contained 100 mM 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine with a radioactivity of approximately 150,000 cpm, 100 mM glycine-NaOH (pH 7.6), 5 mg bovine serum albumin, 5 mM sodium deoxycholate, and
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RESULTS
Figure 1 shows the effect of incubation time and protein concentration on sPLA2 and cPLA2 activities in control rat heart. sPLA2 activity was increased linearly with incubation time for up to 15 min, and with protein concentration for up to 1.2 mg/0.5 ml tested (Fig. 1A). Similar results were also obtained for cPLA2 (Fig. 1B). These data indicate that both secretory and cytosolic PLA2 activities were time and protein dependent. Figure 2 depicts the effect of different concentrations of Ca2/ on sPLA2 and cPLA2 activities in control rat heart. sPLA2 and cPLA2 activities were increased with increasing concentrations of Ca2/ (Fig. 2). There was a hyperbolic relationship between enzyme activities and Ca2/ concentrations for both sPLA2 and cPLA2 . The maximal Ca2/ concentration for sPLA2 was 3 mM (Fig.
FIG. 2. Effects of different concentrations of Ca2/ on sPLA2 and cPLA2 activities in control rat heart. PLA2 activity was assayed as described under Methods except that Ca2/ concentration was varied as shown on the abscissa. Values are means { SEM of three experiments.
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FIG. 3. Effects of different concentrations of phosphatidylcholine on sPLA2 and cPLA2 activities in control rat heart. PLA2 activity was assayed as described under Methods except that phosphatidylcholine concentration was varied as shown on the abscissa. Values are means { SEM of three experiments.
2A) while that for cPLA2 was 40 mM (Fig. 2B). These results indicate that Ca2/ was required for the full expression of both sPLA2 and cPLA2 activities with optimal concentrations being in the millimolar range for sPLA2 and submicromolar range for cPLA2 . Figure 3 shows the effect of different concentrations of substrate, phosphatidylcholine, on sPLA2 and cPLA2 activities in control rat heart. sPLA2 and cPLA2 activities were increased with increasing concentrations of phosphatidylcholine and reached plateau at 100 mM and thereafter (Fig. 3). These data indicate that sPLA2 and cPLA2 activities were saturable at 100 mM substrate concentration. Based on the findings presented in Figs. 1–3, an optimal assay condition consisting of 10 min incubation time, 0.8 mg/0.5 ml enzyme protein, 100 mM phosphatidylcholine, and appropriate concentration of Ca2/ (3 mM for sPLA2 ; 40 mM for cPLA2) was adapted for subsequent studies in comparing PLA2 activities among control, early septic, and late septic experiments. Figure 4 depicts changes in sPLA2 and cPLA2 activities in rat heart during different phases of sepsis. As shown in Fig. 4A, sPLA2 activity was decreased by 25% (P õ 0.01) during early sepsis (9 h after CLP) while it was increased by 49% (P õ 0.01) during late sepsis (18 h after CLP). As shown in Fig. 4B, cPLA2 activity was reduced by 23% (P õ 0.01) during early phase while it was enhanced by 60% (P õ 0.01) during late phase of sepsis. These data demonstrate that both sPLA2 and cPLA2 activities in the rat heart were decreased during early hyperdynamic while they were increased during late hypodynamic phases of sepsis.
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The initial hypercardiodynamic state was characterized by elevations in heart rate (HR), cardiac output (CO), and /dP/dtmax ; and no changes in mean arterial blood pressure (MABP) and left ventricular end diastolic pressure (LVEDP). The hypocardiodynamic state during late phase of sepsis was characterized by decreases in HR, CO, /dP/dtmax , 0dP/dtmax , and MABP; and an increase in LVEDP [16, 17]. In the present study, we found that sPLA2 and cPLA2 activities in the rat heart were decreased during early phase while they were increased during late phase of sepsis. To our knowledge, this is the first report illustrating a biphasic change in sPLA2 and cPLA2 activities in the heart during the progression of sepsis. Furthermore, the direction of changes of sPLA2 and cPLA2 activities was inversely correlated with that of cardiodynamic states during different phases of sepsis. Biological membranes are composed of a phospholipid bilayer in which proteins are embedded. These membranes provide a suitable microenvironment for each protein/subcellular organelle to fulfill its specific function [2]. PLA2 , by virtue of its catalytic ability in hydrolyzing the fatty acyl moieties of membrane phospholipids, would be detrimental to the proper functioning of suborganelles when its catalytic activity is activated. We have reported previously that in cardiac SL, the stoichiometry of Na/-Ca2/ exchanger was altered from 3 Na/ for 1 Ca2/ to 2 Na/ for 1 Ca2/ in canine heart after endotoxin injection, and furthermore, the alteration in the stoichiometry of Na/-Ca2/ exchanger was associated with a modification of membrane lipid microenvironment in response to PLA2 activation [11]. Other membrane-associated enzyme systems such as the ATP-dependent Ca2/ transport of canine cardiac SL were impaired during endotoxin shock and the impairment was in part due to the activation of PLA2 activity [10]. In addition to affecting protein moieties situated in the SL, PLA2 activation also plays an important role in modifying the proper functioning of other suborganelles embedded in the SR. The ryano-
DISCUSSION
Using the CLP-induced septic rat model identical to that employed in this study, we have demonstrated previously that septic rat heart undergoes a biphasic change during the progression of sepsis: an initial hyper- and a subsequent hypodynamic state [16, 17].
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FIG. 4. Changes in sPLA2 and cPLA2 activities in the rat heart during different phases of sepsis. PLA2 activity was assayed as described under Methods. Vertical bars indicate standard errors of the mean. Number of experiments is shown in the parentheses of each column. C, control; ES, early sepsis; LS, late sepsis.
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dine-sensitive Ca2/ release channel of SR was found to be impaired in dog heart following endotoxin administration, and the impairment was associated with PLA2 activation [12]. Based on these reports, it is apparent that PLA2 activation is an important etiological factor contributing to the derangement of the specific functions of various suborganelles embedded in different membrane fractions during endotoxin shock. Thus, our finding that sPLA2 and cPLA2 are activated during late phase of sepsis may explain why myocardium is in hypodynamic state during late sepsis. In contrast, the inactivation of sPLA2 and cPLA2 during early stage of sepsis, as found in this study, may be a significant factor enabling various membrane-associated proteins to perform their specific optimal functions such as overexpression of b- and a-adrenergic receptors [17, 20] that eventually propel myocardium to the hyperdynamic state during early phase of sepsis. The underlying mechanism responsible for the inactivation and the activation of sPLA2 during early and late phases of sepsis, respectively, in the rat heart is not clearly understood. Little is known about the regulation of sPLA2 activity except for its requirement for millimolar concentrations of Ca2/ [2]. Recent evidence suggests that the activation in sPLA2 activity induced by lipopolysaccharide (LPS) is regulated at the transcriptional level. Oka and Arita [21] found that in vitro incubation of cultured rat astrocytes increased sPLA2 (Group II) activity and the increase in sPLA2 activity paralleled increases in enzyme protein and mRNA levels. Tan et al. [22] found that in vivo injection of LPS increased Group II PLA2 activity and its mRNA level in rat ileum. Lauritzen et al. [23] reported that the level of sPLA2 (Group II) mRNA was increased significantly in rat spleen, lung, kidney, liver, and brain after an iv challenge of LPS. Using a CLP-induced septic rat model, we have found recently that sPLA2 (Group II) activity was increased progressively in the liver during the progression of sepsis [24]. Furthermore the increases in Group II PLA2 activity correlated with concomitant increases in the protein level, the mRNA abundance, and the transcription rate of Group II PLA2 mRNA [24]. Further studies are needed in order to disclose whether or not the biphasic expression of sPLA2 during the two hemodynamically distinct phases of sepsis in the rat heart is regulated by a transcriptional mechanism. The recently described intracellular PLA2 , namely cPLA2 , has added a new dimension to the study of phospholipases [1–3]. cPLA2 is an ideal candidate for performing a regulatory role in the generation of arachidonate-derived signaling molecules because of its fatty acid preference in the sn-2 position. In nonstimulated cells, most of the cPLA2 is localized in the cytosol with a small amount associated with the membrane. When stimulated, cPLA2 is translocated to the membrane in response to a rise in intracellular Ca2/ [1–3]. Since no information is available regarding sepsis-induced quantitative changes in the cytosolic-free Ca2/ concentration in cardiomyocytes, the role of Ca2/ in regulating
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the biphasic expression of cPLA2 activity in the myocardium during different phases of spesis is not certain. Rodewald et al. [25] found that in vitro incubation of LPS with human peripheral blood leukocytes increased cPLA2 activity and its corresponding mRNA level. Further studies are warranted to reveal whether or not the sepsis-induced biphasic expression of cPLA2 activity in the rat heart during the two distinct cardiodynamic states are regulated at the transcriptional level. To date, no information is available in regard to the relative role of cPLA2 versus sPLA2 in affecting various membrane-associated enzyme/receptor systems. sPLA2 has been speculated to function either as a digestive enzyme or in the maintenance of all membrane homeostasis because it has no fatty acid preference at the sn2 position [26]. cPLA2 , however, may play an even more important role in regard to the maintenance of membrane integrity in the myocardium because its translocation from cytosol to the membrane takes place at 1007 –1005 M Ca2/ [27], a physiological range that fluctuates during the systole and the diastole [28]. In addition, the fact that cPLA2 activity is subjected to modulation by protein kinaes [29] and that kinase-mediated phosphorylation has been reported to be an etiological factor responsible for the derangement of various membrane-associated enzyme/receptor systems during endotoxin shock [4, 10, 11], it is suggested that cPLA2 may play an important role in the pathogenesis of cardiac dysfunction during shock. REFERENCES 1. Roberts, M. F. Phospholipases: Structural and functional motifs for working at an interface. FASEB J. 10: 1159, 1996. 2. Van Belsen, M., and Van der Vusse, G. J. Phospholipase-A2dependent signalling in the heart. Cardiovasc. Res. 30: 518, 1995. 3. Vernon, L. P., and Bell, J. D. Membrane structure, toxins and phospholipase A2 activity. Pharmacol. Ther. 54: 269, 1992. 4. Liu, M. S. Mechanisms of myocardial membrane alterations in endotoxin shock: Roles of phospholipase and phosphorylation. Circ. Shock 30: 43, 1990. 5. Vadas, P., and Pruzanski, W. Induction of group II phospholipase A2 expression and pathogenesis of the sepsis syndrome. Circ. Shock 39: 160, 1993. 6. Guidet, B., Piat, O., Masliak, J., Barakett, V., Maury, E., Bereziat, G., and Offenstadt, G. Secretory non-pancreatic phospholipase A2 in severe sepsis: Relation to endotoxin, cytokines and thromboxane B2 . Infection 24: 103, 1996. 7. Vadas, P., and Hay, J. B. Involvement of circulating phospholipase A2 in the pathogenesis of the hemodynamic changes in endotoxin shock. Can. J. Physiol. Pharmacol. 61: 561, 1983. 8. Liu, M. S., Kang, G. F., and Ghosh, S. Activation of phospholipases A1 and A2 in heart, liver, and blood during endotoxin shock. J. Surg. Res. 45: 472, 1988. 9. Liu, M. S., and Takeda, H. Endotoxin-induced stimulation on phospholipase A activities in dog hearts. Biochem. Med. 28: 62, 1982. 10. Liu, M. S., and Wu, L. L. Heart sarcolemmal Ca2/ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2/ transport. Mol. Cell. Biochem. 112: 135, 1992. 11. Liu, M. S., and Xuan, Y. T. Mechanisms of endotoxin-induced
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12.
13. 14.
15.
16.
17.
18.
19.
20.
21.
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impairment in Na/-Ca2/ exchange in canine myocardium. Am. J. Physiol. 251: R1078, 1986. Wu, L. L., and Liu, M. S. Altered ryanodine receptor of canine cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock. J. Surg. Res. 53: 82, 1992. Dennis, E. A. Diversity of group types, regulation, and function of phospholipase A2 . J. Biol. Chem. 269: 13057, 1994. Sharp, J. D., and White, D. L. Cytosolic PLA2 : mRNA levels and potential for transcriptional regulation. J. Lipid. Mediat. 8: 183, 1993. Wichterman, K. A., Baue, A. E., and Chaudry, I. H. Sepsis and septic shock: A review of laboratory models and a proposal. J. Surg. Res. 29: 189, 1980. Tang, C., Hsu, H. K., Chen, X. Y., and Liu, M. S. Externalization and internalization of (Na/ / K/)-ATPase in rat heart during different phases of sepsis. Circ. Shock 41: 19, 1993. Tang, C., and Liu, M. S. Initial externalization followed by internalization of b-adrenergic receptors in rat heart during sepsis. Am. J. Physiol. 270: R254, 1996. Katsumata, M., Gupta, C., and Goldman, A. S. A rapid assay for activity of phospholipase A2 using radioactive substrate. Anal. Biochem. 154: 676, 1986. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265, 1951. Wu, L. L., Tang, C., and Liu, M. S. Hyper- and hypo-cardiodynamic states are associated with externalization and internalization, respectively, of a-adrenergic receptors in rat heart during sepsis. Shock 7: 318, 1997. Oka, S., and Arita, H. Inflammatory factors stimulate expres-
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22.
23.
24.
25.
26.
27.
28. 29.
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sion of group II phospholipase A2 in rat cultured astrocytes. J. Biol. Chem. 266: 9956, 1991. Tan, X. D., Wang, H., Gonzalez-Crussi, F. X., Chang, H., Gonzalez-Crussi, F., and Hsueh, W. Platelet-activating factor and endotoxin increase the enzyme activity and gene expression of type II phospholipase A2 in the rat intestine. J. Immunol. 156: 2985, 1996. Lauritzen, I., Heurteaux, C., and Lazdunski, M. Expression of group II phospholipase A2 in rat brain after severe forebrain ischemia and in endotoxin shock. Brain Res. 651: 353, 1994. Dong, L. W., Yang, J., Tong, L. J., Hsu, H. K., and Liu, M. S. Group II phospholipase A2 gene expression is transcriptionally regulated in rat liver during the progression of sepsis. Am. J. Physiol. 273: G706, 1997. Rodewald, E., Tibes, U., Maass, G., and Scheuer, W. Induction of cytosolic phospholipase A2 in human leukocytes by lipopolysaccharide. Eur. J. Biochem. 223: 743, 1994. Lin, L. L., Waitmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269, 1993. Clark, J. D., Lin, L. L., Kutz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopf, J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2/-dependent translocation domain with homology to PKC and GAP. Cell 65: 1043, 1991. Opie, L. H. The Heart: Physiology and Metabolism. New York: Raven Press, 1991. Pp. 127–146. Lin, L. L., Lin, A. Y., and Knopf, J. L. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc. Natl. Acad. Sci. USA 89: 6147, 1992.
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JR985273
Phospholipase A2 Activities Are Decreased during Early but Increased during Late Phases of Sepsis in Rat Heart1 Li-Jia Tong, M.D., Ph.D., Lin-Wang Dong, M.D., Ph.D., Hseng-Kuang Hsu, B.S.,2 and Maw-Shung Liu, D.D.S., Ph.D.3 Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104 Submitted for publication September 11, 1997
Background. Changes in the activities of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in the rat heart during early hyperdynamic and late hypodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of cardiac dysfunction during sepsis. Methods. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. PLA2 activity was measured based on the rate of hydrolysis of 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine. Results. The results show that under physiological conditions, sPLA2 and cPLA2 activities were time and protein dependent. The optimal Ca2/ concentrations for sPLA2 and cPLA2 activities were 3 mM and 40 mM, respectively. During sepsis, sPLA2 activity was decreased by 25% (P õ 0.01) during early phase while it was increased by 49% (P õ 0.01) during late phase of sepsis. Similarly, cPLA2 activity was decreased by 23% (P õ 0.01) during early sepsis while it was increased by 60% (P õ 0.01) during late sepsis. Conclusions. Since PLA2 functions to maintain cell membrane integrity and function, a biphasic change in sPLA2 and cPLA2 activities may contribute to the development of the two cardiodynamically distinct phases during the progression of sepsis. q 1998 Academic Press
Key Words: phospholipase A2 ; heart; sepsis; hyperdynamic phase; hypodynamic phase; membrane enzyme; membrane receptor.
INTRODUCTION
Phospholipase A2 (PLA2) catalyzes the hydrolysis of phospholipid molecules at the sn-2 position to liberate free fatty acids and lysophospholipids. Physiologically, 1 This work was supported by HL-30080 from National Heart, Lung and Blood Institute, and GM-31664 from National Institue of General Medical Sciences, National Institutes of Health. 2 Recipient of a fellowship award from Taiwan (NSC-34089F). 3 To whom reprint requests should be addressed.
PLA2 functions to maintain the integrity and dynamics of cell membranes [1–3]. Pathologically, PLA2 has been implicated to be an important etiological factor for the pathogenesis of various diseases including shock and sepsis [3–5]. In septic patients, plasma PLA2 activity was elevated by 22-fold and the elevation in plasma PLA2 correlated well with the severity of organ dysfunction and the mortality [6]. In animal studies, circulating PLA2 activity was increased by 11-fold after endotoxin injection and the increase in PLA2 activity paralleled the decrease in mean arterial blood pressure [5, 7]. At the tissue level, PLA2 activity was activated by 71–82% in canine myocardium following endotoxin administration [8, 9] and the activation in PLA2 activity was responsible in part for the dysfunction of the Na/Ca2/ exchanger and the ATP-dependent Ca2/ transport of sarcolemma (SL) [10, 11] as well as the Ca2/-induced Ca2/ release of sarcoplasmic reticulum (SR) [12]. PLA2s are a diverse class of enzymes with regard to structure, function, regulation, localization, and role of divalent metal ions [1–3]. Generally, PLA2 enzymes are classified into three major categories: (1) secretory PLA2 (sPLA2), a low molecular weight enzyme which requires a millimolar concentration of Ca2/ to express full enzymatic activity and localizes in the membrane fraction; (2) cytosolic PLA2 (cPLA2), a high molecular weight enzyme which requires a submicromolar concentration of Ca2/ for optimal activity and localizes in the cytosolic compartment; and (3) Ca2/-independent PLA2 (iPLA2), which is plasmalogen substrate-specific. The sPLA2 is further divided into two subgroups based on its tissue distribution: Group I PLA2 , which is found mostly in pancrease, and Group II PLA2 , which is distributed almost exclusively in nonpancreatic tissues [1, 2, 13]. Recent progress in the studies of PLA2 has indicated that cPLA2 , unlike sPLA2 , plays a unique regulatory role in the lipid-mediated signal transduction pathway [1, 2]. Since data available in the literature regarding changes in PLA2 activity in the myocardium as well as in the circulating system [6–9] during shock and sepsis are limited only to sPLA2 , and since cPLA2 gene transcript is expressed in cardiac tissue [14], it is conceivable that cPLA2 activity may also be modified in the myocardium during sepsis. Accordingly, the 0022-4804/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Effects of incubation time and protein concentration on sPLA2 and cPLA2 activities in control rat heart. PLA2 activity was measured as described under Methods except that either incubation time or protein concentration was varied as shown on the abscissa. Values are means { SEM of three experiments.
either 40 mM or 3 mM free Ca2/ (prepared by buffering CaCl2 with EGTA according to Biosoft EqCal software program). The reaction was initiated by the addition of enzyme preparation containing 0.8 mg protein, allowed to proceed for 10 min at 377C, and then terminated by the addition of 100 ml of 5% Triton X-100 containing 40 mM EDTA. The products of the PLA2 reaction were extracted with 10 vol of acidic hexane (hexane containing 0.1% acetic acid) by vortexing for 20 s after 200 mg of anhydrous Na2SO4 was added. An aliquot (2.5 ml) of the hexane phase containing the reaction product, [114 C]oleate, was carefully removed and the radioactivity was then determined. The enzymatic activity was calculated based on the rate of hydrolysis of 14C-labeled substrate. The activity obtained in the presence of 3 mM free Ca2/ after correcting that obtained in the absence of Ca2/ was designated as sPLA2 activity. The activity obtained in the presence of 40 mM free Ca2/ after correcting that obtained in the absence of Ca2/ was designated as cPLA2 activity. Protein assay and statistical analysis. Protein content of heart homogenates was determined by the method of Lowry et al. [19]. The statistical analysis of the data was performed using one-way analysis of variance followed by Student-Newman-Keuls tests. A P value of less than 0.05 was accepted as statistically significant. Materials. L-3-phosphatidylcholine, 1-palmitoyl-2-[1-14C]oleoyl (58 mCi/mmol), was obtained from Amersham Life Science. L-a-phosphatidylcholine, b-oleoyl-g-palmitoyl, was purchased from Sigma Chemical Co. Other chemicals and reagents were of analytical grade.
present study was undertaken to test our hypothesis that both cPLA2 and sPLA2 activities in the rat heart are altered during the progression of sepsis. METHODS Animal model. Male Sprague-Dawley rats weighing from 270 to 320 g were used. All animals were fasted overnight with free access to water. They were divided into three groups: control, early sepsis, and late sepsis. Sepsis was induced by cecal ligation and puncture (CLP) as described by Wichterman et al. [15] with modification. Under halothane anesthesia, a laparotomy was performed, and the cecum was ligated with a 3-0 silk ligature and punctured twice with an 18-gauge needle. The cecum was then returned to the peritoneal cavity and the abdomen was closed in two layers. Control rats were sham-operated (a laparotomy was performed and the cecum was manipulated but neither ligated nor punctured). All animals were resuscitated with 4 ml/100 g body weight of normal saline at the completion of surgery and also at 7 h postsurgery. Animals were fasted but had free access to water after operative procedures. Hearts were removed from septic and control animals 9 or 18 h postoperation under chloralose and urethane anesthesia and were then used for the preparation of homogenate for the assay of PLA2 activity. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The mortality rates were 0% for control, 15% for early sepsis, and 36% for late sepsis. Previous experiments show that septic rat heart was in hyperdynamic state during early sepsis while it was in hypodynamic state during late sepsis [16, 17]. Assay of PLA2 activity. PLA2 activity was assayed by the method of Katsumata et al. [18] with modification. Heart tissues obtained from control and septic rats were cut into small chunks and then homogenized with 10 vol of 100 mM glycine-NaOH buffer (pH 7.6) containing 50 mg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 10 mg/ml soybean trypsin inhibitor, 100 mg/ml aprotinin in a Tekma Tissumizer (Model SDT) at half maximum speed for 4 1 10 s. The homogenate was filtered through a cell strainer (70 mm) followed by centrifugation at 700g for 10 min to remove nuclei and cell debris. The resulting supernatant was then used for the assay of PLA2 activiy. PLA2 activity was assayed using 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine as a labeled substrate. The 14C-labeled and nonlabeled phosphatidylcholines were prepared with absolute ethanol, and the final ethanol concentration in the reaction mixture was 2%. The reaction mixture in a final volume of 0.5 ml contained 100 mM 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine with a radioactivity of approximately 150,000 cpm, 100 mM glycine-NaOH (pH 7.6), 5 mg bovine serum albumin, 5 mM sodium deoxycholate, and
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RESULTS
Figure 1 shows the effect of incubation time and protein concentration on sPLA2 and cPLA2 activities in control rat heart. sPLA2 activity was increased linearly with incubation time for up to 15 min, and with protein concentration for up to 1.2 mg/0.5 ml tested (Fig. 1A). Similar results were also obtained for cPLA2 (Fig. 1B). These data indicate that both secretory and cytosolic PLA2 activities were time and protein dependent. Figure 2 depicts the effect of different concentrations of Ca2/ on sPLA2 and cPLA2 activities in control rat heart. sPLA2 and cPLA2 activities were increased with increasing concentrations of Ca2/ (Fig. 2). There was a hyperbolic relationship between enzyme activities and Ca2/ concentrations for both sPLA2 and cPLA2 . The maximal Ca2/ concentration for sPLA2 was 3 mM (Fig.
FIG. 2. Effects of different concentrations of Ca2/ on sPLA2 and cPLA2 activities in control rat heart. PLA2 activity was assayed as described under Methods except that Ca2/ concentration was varied as shown on the abscissa. Values are means { SEM of three experiments.
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FIG. 3. Effects of different concentrations of phosphatidylcholine on sPLA2 and cPLA2 activities in control rat heart. PLA2 activity was assayed as described under Methods except that phosphatidylcholine concentration was varied as shown on the abscissa. Values are means { SEM of three experiments.
2A) while that for cPLA2 was 40 mM (Fig. 2B). These results indicate that Ca2/ was required for the full expression of both sPLA2 and cPLA2 activities with optimal concentrations being in the millimolar range for sPLA2 and submicromolar range for cPLA2 . Figure 3 shows the effect of different concentrations of substrate, phosphatidylcholine, on sPLA2 and cPLA2 activities in control rat heart. sPLA2 and cPLA2 activities were increased with increasing concentrations of phosphatidylcholine and reached plateau at 100 mM and thereafter (Fig. 3). These data indicate that sPLA2 and cPLA2 activities were saturable at 100 mM substrate concentration. Based on the findings presented in Figs. 1–3, an optimal assay condition consisting of 10 min incubation time, 0.8 mg/0.5 ml enzyme protein, 100 mM phosphatidylcholine, and appropriate concentration of Ca2/ (3 mM for sPLA2 ; 40 mM for cPLA2) was adapted for subsequent studies in comparing PLA2 activities among control, early septic, and late septic experiments. Figure 4 depicts changes in sPLA2 and cPLA2 activities in rat heart during different phases of sepsis. As shown in Fig. 4A, sPLA2 activity was decreased by 25% (P õ 0.01) during early sepsis (9 h after CLP) while it was increased by 49% (P õ 0.01) during late sepsis (18 h after CLP). As shown in Fig. 4B, cPLA2 activity was reduced by 23% (P õ 0.01) during early phase while it was enhanced by 60% (P õ 0.01) during late phase of sepsis. These data demonstrate that both sPLA2 and cPLA2 activities in the rat heart were decreased during early hyperdynamic while they were increased during late hypodynamic phases of sepsis.
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The initial hypercardiodynamic state was characterized by elevations in heart rate (HR), cardiac output (CO), and /dP/dtmax ; and no changes in mean arterial blood pressure (MABP) and left ventricular end diastolic pressure (LVEDP). The hypocardiodynamic state during late phase of sepsis was characterized by decreases in HR, CO, /dP/dtmax , 0dP/dtmax , and MABP; and an increase in LVEDP [16, 17]. In the present study, we found that sPLA2 and cPLA2 activities in the rat heart were decreased during early phase while they were increased during late phase of sepsis. To our knowledge, this is the first report illustrating a biphasic change in sPLA2 and cPLA2 activities in the heart during the progression of sepsis. Furthermore, the direction of changes of sPLA2 and cPLA2 activities was inversely correlated with that of cardiodynamic states during different phases of sepsis. Biological membranes are composed of a phospholipid bilayer in which proteins are embedded. These membranes provide a suitable microenvironment for each protein/subcellular organelle to fulfill its specific function [2]. PLA2 , by virtue of its catalytic ability in hydrolyzing the fatty acyl moieties of membrane phospholipids, would be detrimental to the proper functioning of suborganelles when its catalytic activity is activated. We have reported previously that in cardiac SL, the stoichiometry of Na/-Ca2/ exchanger was altered from 3 Na/ for 1 Ca2/ to 2 Na/ for 1 Ca2/ in canine heart after endotoxin injection, and furthermore, the alteration in the stoichiometry of Na/-Ca2/ exchanger was associated with a modification of membrane lipid microenvironment in response to PLA2 activation [11]. Other membrane-associated enzyme systems such as the ATP-dependent Ca2/ transport of canine cardiac SL were impaired during endotoxin shock and the impairment was in part due to the activation of PLA2 activity [10]. In addition to affecting protein moieties situated in the SL, PLA2 activation also plays an important role in modifying the proper functioning of other suborganelles embedded in the SR. The ryano-
DISCUSSION
Using the CLP-induced septic rat model identical to that employed in this study, we have demonstrated previously that septic rat heart undergoes a biphasic change during the progression of sepsis: an initial hyper- and a subsequent hypodynamic state [16, 17].
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FIG. 4. Changes in sPLA2 and cPLA2 activities in the rat heart during different phases of sepsis. PLA2 activity was assayed as described under Methods. Vertical bars indicate standard errors of the mean. Number of experiments is shown in the parentheses of each column. C, control; ES, early sepsis; LS, late sepsis.
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dine-sensitive Ca2/ release channel of SR was found to be impaired in dog heart following endotoxin administration, and the impairment was associated with PLA2 activation [12]. Based on these reports, it is apparent that PLA2 activation is an important etiological factor contributing to the derangement of the specific functions of various suborganelles embedded in different membrane fractions during endotoxin shock. Thus, our finding that sPLA2 and cPLA2 are activated during late phase of sepsis may explain why myocardium is in hypodynamic state during late sepsis. In contrast, the inactivation of sPLA2 and cPLA2 during early stage of sepsis, as found in this study, may be a significant factor enabling various membrane-associated proteins to perform their specific optimal functions such as overexpression of b- and a-adrenergic receptors [17, 20] that eventually propel myocardium to the hyperdynamic state during early phase of sepsis. The underlying mechanism responsible for the inactivation and the activation of sPLA2 during early and late phases of sepsis, respectively, in the rat heart is not clearly understood. Little is known about the regulation of sPLA2 activity except for its requirement for millimolar concentrations of Ca2/ [2]. Recent evidence suggests that the activation in sPLA2 activity induced by lipopolysaccharide (LPS) is regulated at the transcriptional level. Oka and Arita [21] found that in vitro incubation of cultured rat astrocytes increased sPLA2 (Group II) activity and the increase in sPLA2 activity paralleled increases in enzyme protein and mRNA levels. Tan et al. [22] found that in vivo injection of LPS increased Group II PLA2 activity and its mRNA level in rat ileum. Lauritzen et al. [23] reported that the level of sPLA2 (Group II) mRNA was increased significantly in rat spleen, lung, kidney, liver, and brain after an iv challenge of LPS. Using a CLP-induced septic rat model, we have found recently that sPLA2 (Group II) activity was increased progressively in the liver during the progression of sepsis [24]. Furthermore the increases in Group II PLA2 activity correlated with concomitant increases in the protein level, the mRNA abundance, and the transcription rate of Group II PLA2 mRNA [24]. Further studies are needed in order to disclose whether or not the biphasic expression of sPLA2 during the two hemodynamically distinct phases of sepsis in the rat heart is regulated by a transcriptional mechanism. The recently described intracellular PLA2 , namely cPLA2 , has added a new dimension to the study of phospholipases [1–3]. cPLA2 is an ideal candidate for performing a regulatory role in the generation of arachidonate-derived signaling molecules because of its fatty acid preference in the sn-2 position. In nonstimulated cells, most of the cPLA2 is localized in the cytosol with a small amount associated with the membrane. When stimulated, cPLA2 is translocated to the membrane in response to a rise in intracellular Ca2/ [1–3]. Since no information is available regarding sepsis-induced quantitative changes in the cytosolic-free Ca2/ concentration in cardiomyocytes, the role of Ca2/ in regulating
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the biphasic expression of cPLA2 activity in the myocardium during different phases of spesis is not certain. Rodewald et al. [25] found that in vitro incubation of LPS with human peripheral blood leukocytes increased cPLA2 activity and its corresponding mRNA level. Further studies are warranted to reveal whether or not the sepsis-induced biphasic expression of cPLA2 activity in the rat heart during the two distinct cardiodynamic states are regulated at the transcriptional level. To date, no information is available in regard to the relative role of cPLA2 versus sPLA2 in affecting various membrane-associated enzyme/receptor systems. sPLA2 has been speculated to function either as a digestive enzyme or in the maintenance of all membrane homeostasis because it has no fatty acid preference at the sn2 position [26]. cPLA2 , however, may play an even more important role in regard to the maintenance of membrane integrity in the myocardium because its translocation from cytosol to the membrane takes place at 1007 –1005 M Ca2/ [27], a physiological range that fluctuates during the systole and the diastole [28]. In addition, the fact that cPLA2 activity is subjected to modulation by protein kinaes [29] and that kinase-mediated phosphorylation has been reported to be an etiological factor responsible for the derangement of various membrane-associated enzyme/receptor systems during endotoxin shock [4, 10, 11], it is suggested that cPLA2 may play an important role in the pathogenesis of cardiac dysfunction during shock. REFERENCES 1. Roberts, M. F. Phospholipases: Structural and functional motifs for working at an interface. FASEB J. 10: 1159, 1996. 2. Van Belsen, M., and Van der Vusse, G. J. Phospholipase-A2dependent signalling in the heart. Cardiovasc. Res. 30: 518, 1995. 3. Vernon, L. P., and Bell, J. D. Membrane structure, toxins and phospholipase A2 activity. Pharmacol. Ther. 54: 269, 1992. 4. Liu, M. S. Mechanisms of myocardial membrane alterations in endotoxin shock: Roles of phospholipase and phosphorylation. Circ. Shock 30: 43, 1990. 5. Vadas, P., and Pruzanski, W. Induction of group II phospholipase A2 expression and pathogenesis of the sepsis syndrome. Circ. Shock 39: 160, 1993. 6. Guidet, B., Piat, O., Masliak, J., Barakett, V., Maury, E., Bereziat, G., and Offenstadt, G. Secretory non-pancreatic phospholipase A2 in severe sepsis: Relation to endotoxin, cytokines and thromboxane B2 . Infection 24: 103, 1996. 7. Vadas, P., and Hay, J. B. Involvement of circulating phospholipase A2 in the pathogenesis of the hemodynamic changes in endotoxin shock. Can. J. Physiol. Pharmacol. 61: 561, 1983. 8. Liu, M. S., Kang, G. F., and Ghosh, S. Activation of phospholipases A1 and A2 in heart, liver, and blood during endotoxin shock. J. Surg. Res. 45: 472, 1988. 9. Liu, M. S., and Takeda, H. Endotoxin-induced stimulation on phospholipase A activities in dog hearts. Biochem. Med. 28: 62, 1982. 10. Liu, M. S., and Wu, L. L. Heart sarcolemmal Ca2/ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2/ transport. Mol. Cell. Biochem. 112: 135, 1992. 11. Liu, M. S., and Xuan, Y. T. Mechanisms of endotoxin-induced
srga
TONG ET AL.: PHOSPHOLIPASE A2 IN RAT HEART DURING SEPSIS
12.
13. 14.
15.
16.
17.
18.
19.
20.
21.
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impairment in Na/-Ca2/ exchange in canine myocardium. Am. J. Physiol. 251: R1078, 1986. Wu, L. L., and Liu, M. S. Altered ryanodine receptor of canine cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock. J. Surg. Res. 53: 82, 1992. Dennis, E. A. Diversity of group types, regulation, and function of phospholipase A2 . J. Biol. Chem. 269: 13057, 1994. Sharp, J. D., and White, D. L. Cytosolic PLA2 : mRNA levels and potential for transcriptional regulation. J. Lipid. Mediat. 8: 183, 1993. Wichterman, K. A., Baue, A. E., and Chaudry, I. H. Sepsis and septic shock: A review of laboratory models and a proposal. J. Surg. Res. 29: 189, 1980. Tang, C., Hsu, H. K., Chen, X. Y., and Liu, M. S. Externalization and internalization of (Na/ / K/)-ATPase in rat heart during different phases of sepsis. Circ. Shock 41: 19, 1993. Tang, C., and Liu, M. S. Initial externalization followed by internalization of b-adrenergic receptors in rat heart during sepsis. Am. J. Physiol. 270: R254, 1996. Katsumata, M., Gupta, C., and Goldman, A. S. A rapid assay for activity of phospholipase A2 using radioactive substrate. Anal. Biochem. 154: 676, 1986. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265, 1951. Wu, L. L., Tang, C., and Liu, M. S. Hyper- and hypo-cardiodynamic states are associated with externalization and internalization, respectively, of a-adrenergic receptors in rat heart during sepsis. Shock 7: 318, 1997. Oka, S., and Arita, H. Inflammatory factors stimulate expres-
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22.
23.
24.
25.
26.
27.
28. 29.
169
sion of group II phospholipase A2 in rat cultured astrocytes. J. Biol. Chem. 266: 9956, 1991. Tan, X. D., Wang, H., Gonzalez-Crussi, F. X., Chang, H., Gonzalez-Crussi, F., and Hsueh, W. Platelet-activating factor and endotoxin increase the enzyme activity and gene expression of type II phospholipase A2 in the rat intestine. J. Immunol. 156: 2985, 1996. Lauritzen, I., Heurteaux, C., and Lazdunski, M. Expression of group II phospholipase A2 in rat brain after severe forebrain ischemia and in endotoxin shock. Brain Res. 651: 353, 1994. Dong, L. W., Yang, J., Tong, L. J., Hsu, H. K., and Liu, M. S. Group II phospholipase A2 gene expression is transcriptionally regulated in rat liver during the progression of sepsis. Am. J. Physiol. 273: G706, 1997. Rodewald, E., Tibes, U., Maass, G., and Scheuer, W. Induction of cytosolic phospholipase A2 in human leukocytes by lipopolysaccharide. Eur. J. Biochem. 223: 743, 1994. Lin, L. L., Waitmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269, 1993. Clark, J. D., Lin, L. L., Kutz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopf, J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2/-dependent translocation domain with homology to PKC and GAP. Cell 65: 1043, 1991. Opie, L. H. The Heart: Physiology and Metabolism. New York: Raven Press, 1991. Pp. 127–146. Lin, L. L., Lin, A. Y., and Knopf, J. L. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc. Natl. Acad. Sci. USA 89: 6147, 1992.
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