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Endothelin-induced prostacyclin production in rat aortic endothelial cells is meditated by protein kinase C
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 263–268 © 1999 Harcourt Brace & Co. Ltd Article no. plef.1999.0034
Endothelin-induced prostacyclin production in rat aortic endothelial cells is meditated by protein kinase C G. K. Oriji Department of Biology, College of Science and Health, William Paterson University, Wayne, NJ 07470, USA and Hypertension-Endocrine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Summary Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10–9 M) produced a 10-fold increase in PGI2 release. Pretreatment with 10–9 M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10–9 M) or cyclooxygenase (indomethacin) (10–9M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.
INTRODUCTION Endothelin (ET) is a 21-amino acid peptide, initially purified from the culture medium of porcine aortic endothelial cells.1 Endothelin type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC) is coupled to a phosphatidyl-inositol-specific phospholipase C (PI-PLC).2 ETA also activates dihydropyridine-insensitive calcium channels and cells challenged with endothelin-1 (ET-1), but not endothelin-3 (ET-3), and produces prostaglandin E2 (PGE2) in a time- and concentration-dependent manner.2 However, the events linking agonist occupancy of receptor and accumulation of PGE2 were not delineated. ET has been shown to augment PGE2 production in various cell types.3–8 However, the signaling pathway involved is poorly understood. Indeed, it is not known whether ET stimulation of eicosanoid production is a sequential, stepwise event subsequent to agonist stimulation of phospholipase A2 (PLA2) by PI-PLC or a G-protein coupling event between receptor and PLA2. Furthermore, Received 17 December 1998 Accepted 19 January 1999 Correspondence to: G. K. Oriji, Department of Biology, College of Science and Health, William Paterson University, Wayne, NJ 07470, USA. Tel.: +1 973 720-3445; Fax: +1 973 720 2338
the requirement for protein kinase C (PKC) to facilitate PLA2 activation has not been carefully delineated. ET is known to activate PLA2, resulting in the production of PGE2, PGI2 or thromboxane A2, depending upon the type of cell.5,6,9–12 PLA2 is activated when phosphorylated at certain serine or threonine positions.13 PKC is known to phosphorylate protein at serine or threonine positions.14 Therefore, PKC may phosphorylate PLA2 which may lead to an increase in prostaglandin (PG) synthesis. Therefore, the aim of the present study is to determine if the PKC signaling pathway is stimulated by endothelin in rat aortic endothelial cells. MATERIALS AND METHODS Isolation of rat aortic endothelial cells Male rats (250–300 g) were anesthetized by i.p. injection of sodium pentobarbital (40 mg/kg) and the thoracic aortas were quickly removed and placed in a petri dish containing oxygenated warm Krebs–Henseleit buffer (KHB; NaCl 116 mM, KCl 5.4 mM, CaCl2–2H2O 2.5 mM, KH2PO4 4.80 mM, MgSO4 1.2 mM, dextrose 21.43 mM, NaHCO3 25.00 mM) at 37°C, aerated with 95% O2 + 5% CO2, pH=7.4. The thoracic aortas were cleaned of surrounding fat and placed in calcium and magnesium263
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free Hanks Buffer Salt Solution (HBSS). After washing the aorta three times with HBSS, the aorta was cut open longitudinally (washed twice with HBSS) and placed in an enzyme solution containing 5 mg collagenase/5 ml HBSS and allowed to incubate in a shaking water bath at 37°C for 30 min. The aorta was removed from the solution and washed five times with HBSS. The adventitia and intima were removed with forceps and the aorta was washed with HBSS twice. Then, the aorta was minced into tiny pieces and washed twice with M199. The tissue was resuspended in a 5 ml enzyme solution (sterile) containing M199, collagenase (5 mg/ml), elastase (0.25 mg/ml), soybean trypsin inhibitor (4 mg/ml), and crystallized bovine serum albumin (2 mg/ml). The tissue was allowed to incubate for two 90 min periods in fresh enzyme solution in a shaker bath at 37°C. At the end of the digestion periods, the smooth muscle cells were spun down in a centrifuge and resuspended in 5 ml HBSS. This washing process was repeated a further two times to remove enzyme and the cells were resuspended in 10 ml of M199 media containing antibiotics and 20% fetal bovine serum (FBS) and plated in a culture dish. The media were replaced 24 h later to remove debris and unattached cells. At 2 day intervals, the media were replaced with M199 containing antibiotics and 10% FBS to feed the cells. Primary cell cultures reached confluent within 10–14 days and were passaged. Passaging requires dislodging the cells from the plate with 1 ml of 0.12% trypsin in HBSS. Experiments were performed by plating cells in 12-well plates at 1 × 106 cm2, grown to subconfluent after 3–4 days. Passages 3 through 7 were used for the experiments. This protocol was modified from Ives et al.15 PGI2 was assayed by radioimmunoassay kit with a specific antibody for 6-keto-PGF1α (Kit # RPA 515; Amersham, Arlington Heights, IL, USA). PKC activity was determined in either cytosol or membrane fractions by an enzyme assay (Kit #RPN 77, Amersham, Arlington Heights, IL, USA). Cytosol and membrane fractions were prepared as per Eulalia et al.16 Experimental protocol
Protocol 1 This experiment was performed to determine the effects of different doses (0.5 × 10–9 M, 10–9 M and 2 × 10–9 M) of either ET or Phorbol 12, 13-dibutyrate (PDBu), a PKC activator, on PGI2 release. Rat aortic endothelial cells were stimulated with ET for 30 min and the perfusate was collected to measure PGI2 release (n=6 for each dose of agonist). Protocol 2 This experiment was performed to determine the effects of pretreatment with either a cyclooxygenase inhibitor
(indomethacin) or inhibitors of PLA2 (either 7,7, dimethyleicosadienoic acid [DEDA] or trifluoromethyl ketone analogue [AACOCF3]) on ET- and PDBu-induced PGI2 release. 1 nM of either indomethacin, AACOCF3 or DEDA was added to the cell wells for 30 min before the addition of either ET or PDBu; the perfusate was collected for 30 min after the addition of either ET or PDBu to determine PGI2 release (n=6 for each inhibitor and each agonist).
Protocol 3 This experiment was performed to determine the effects of pretreatment with different doses (10–9 M and 10–3 M) of PKC inhibitors: 1-(5 isoquinolinesulfonylmethyl) piperazine (H7), staurosporine or 1-(5-isoquinolinesulfonyl) piperazine (CL) on ET induced PGI2 release. 10–9 M or 10–3 M of one of the three PKC inhibitors was added to the cell wells for 30 min before ET was added; the perfusate was collected for 30 min after the addition of ET for PGI2 determination (n=6 for each dose of inhibitor). Drugs and chemicals Drugs used in this study were endothelin, phorbol 12, 13dibutyrate, indomethacin, trifluoromethyl ketone analogue, 7,7, dimethyleicosadienoic acid (DEDA), 1-(5-isoquinolinemethyl) piperazine (H7) and staurosporine (Calbiochem Inc., San Diego, CA, USA) and 1-(5-isoquinolinesulfonyl) piperazine (CL) (Sigma Chemical Company, St Louis, MO, USA). Statistical analysis All data were generated with paired controls. Values were expressed as mean±S.E.M. A two way analysis of variance was used for comparisons within experiments. A value of P < 0.05 was considered significant.
RESULTS Effects of different doses of either ET or PDBu on PGI2 release Both ET and PDBu produced significant and dose-dependent increases in PGI2 release (Fig. 1). The response to ET (2 × 10–9 M) was 15% greater than the response to the same dose of PDBu. Effects of pretreatment with indomethacin or DEDA or AACOCF3 on either ET- or PDBu-induced PGI2 release ET-induced PGI2 release was significantly blocked by pretreatment with either a cyclooxygenase inhibitor (indomethacin) (96% inhibition) or different inhibitors of PLA2 (DEDA [100% inhibition] or AACOCF3 [97% inhibition]).
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 263–268
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PKC inhibits ET-induced PGI2 production
Fig. 1 The effects of different doses of (A) ET and (B) PDBu on prostacyclin release in rat aortic endothelial cells. *P < 0.01 vs control.
Also, PDBu-induced PGI2 release was significantly blocked by pretreatment with either a cyclooxygenase inhibitor (indomethacin) (100% inhibition) or different inhibitors of PLA2 (DEDA [97% inhibition] or AACOCF3 [100% inhibition]) (Fig. 2). These results show that inhibition of either cyclooxygenase or PLA2 markedly decreased both ET- and PDBu-induced PGI2 release. Effects of pretreatment with different doses of PKC inhibitors on ET-induced PGI2 release ET-induced PGI2 release was inhibited 71–75% by 10–9 M, and 96–99% by 10–3 M pretreatment doses of any of three different PKC inhibitors, that is, H7, staurosporine or 1-(5-isoquinolinesulfonyl) piperazine (CL) (Fig. 3). In preliminary experiments, ET-induced PGI2 release was inhibited 84–87% by 10–6 M doses of any of three different PKC inhibitors (data not shown). These results confirm that PKC inhibition completely blocked PGI2 release. The PKC enzyme assay showed activation, that is, translocation of PKC from the cytosol to the membrane fraction in endothelial cells that were treated with either ET or PDBu. Translocation was inhibited by pretreatment with PKC inhibitors and unchanged by PDD (Fig. 4). PKC © 1999 Harcourt Brace & Co. Ltd
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Fig. 2 The effects of indomethacin, AACOCF3 or DEDA (10–9 M) on ET-induced prostacyclin release in rat aortic endothelial cells. Endothelial cells were pretreated with one of the prostacyclin inhibitors for 30 min before the addition of (A) ET or (B) PDBu. *P < 0.01 vs agonist alone.
inhibitors or PDD alone did not affect translocation of PKC (Fig. 4). DISCUSSION In this study, we found that (1) ET induced PGI2 release in rat aortic endothelial cells, (2) activation of PKC by PDBu increased PGI2 release, (3) both ET- and PDBu-induced PGI2 release were inhibited via inhibitors of either cyclooxygenase or PLA2, (4) ET induced PGI2 release was inhibited by different inhibitors of PKC and (5) ET caused activation of PKC, that is, translocation of PKC from cytosol to membrane fractions. ET is a very potent vasoconstrictor in porcine coronary arteries.1 Vasoconstriction was shown to be accompanied by an increase in the release of PGE2, but not PGI2.17 PGE2 and PGI2 are potent vasodilators in most vascular beds;18 ET has been shown to release both PGE2 and PGI2 from perfused rat kidney.19 The mechanism for this ET-induced PGI2 release appears to be mediated by PKC. The biologically active phorbol ester, PDBu, causes PKC translocation from the cytosol to the cell membrane and increased
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Fig. 3 Effects of two different doses: (A) 10–9 M and (B) 10–3 M of PKC inhibitors on ET-induced prostacyclin release in rat aortic endothelial cells. Endothelial cells were pretreated with either vehicle or PKC inhibitors for 30 min prior to ET treatment. *P < 0.01 vs ET alone.
enzyme activity in astroglia.20 Conversely, an inactive phorbol ester, such as 4-alpha-phorbol, 12,13-Didecanoate (PDD) does not produce these effects;20–22 PDD does not increase PG production in intact piglet parietal cortex.23 In this study, PDBu increased PGI2 release significantly,
although, this increase in PGI2 release was somewhat less than that produced by ET alone. In addition, ET-induced PGI2 release was markedly decreased by three different PKC inhibitors: H7, staurosporine and 1-(5-isoquinolinesulfonyl) piperazine (CL). Although, H7 is also a potent inhibitor of adenosine 3′,5′-cyclic monophosphatedependent protein kinase,24 it seems unlikely that this mechanism is involved in the inhibition of PGI2 release observed in this study. In intact piglet parietal cortex, stimulation of either adenylate cyclase with isoproterenol25 or guanylate cyclase with arginine-containing compounds26 or glutamate27 did not increase cerebrospinal fluid levels of PGs. The mechanism by which ET causes increased PGI2 release is not known with certainty, but it appears to involve PKC since activation of PLA2, via either ET or PDBu-induced PGI2 release was blocked by pretreatment with inhibitors of either PLA2 (DEDA or AACOCF3) or cyclooxygenase (indomethacin). However, phorbol esters have been shown to cause arachidonic acid release in cultured rat astrocytes.28 There are several ways by which PKC could activate PLA2: PKC could phosphorylate PLA2 and directly activate this enzyme;29–31 PKC could phosphorylate and thereby inactivate a protein that normally inhibits PLA2, e.g. lipocortins;32–35 or PKC could increase intracellular calcium and activate PLA2 by this mechanism.36 Several studies have shown that prostanoids are able to influence the tone of cerebral arteries and arterioles.37–39 Indeed, PG synthesis is altered in various pathological conditions and causes profound modifications in tissue blood flow. Therefore, it is important to understand the mechanisms involved in PG release. In the aorta, PGI2 may be important as either a regulator of vascular tone
Fig. 4 Effects of ET or PDBu alone or PKC inhibitors plus ET on activation of PKC in rat aortic endothelial cells. Endothelial cells were pretreated with either vehicle or PKC inhibitors (10–9 M) for 30 min prior to ET (10–9 M) treatment. *P < 0.001 vs PKC inhibitors plus ET (10–9 M).
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PKC inhibits ET-induced PGI2 production
or a modulator of the vascular responses to certain neurotransmitters. In summary, ET activates PKC which activates PLA2 which liberates arachidonic acid which increases PGI2 production and release in rat aortic endothelial cells.
ACKNOWLEDGMENTS The author thanks Mrs Margaret Hill and Mr John Tate for their excellent technical assistance.
REFERENCES 1. Yanagisawa M., Kurihara H., Kimura S. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 333: 411–415. 2. Wilkes B. M., Ruston A. S., Mento P. et al. Characterization of endothelin 1 receptor and signal transduction mechanisms in rat medullary interstitial cells. Am J Physiol 1991; 260 (Renal Fluid Electrolyte Physiol 29): F579–F589. 3. Abdel-Latif A. A., Zhang Y., Yousufzai S. Y. Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in rabbit iris sphincter smooth muscle: activation of phospholipase A2. Curr Eye Res 1991; 3: 259–265. 4. Fukunaga M., Ochi S., Takama T. et al. Endothelin-1 stimulates prostaglandin E2 production in an extracellular calciumindependent manner in cultured rat mesangial cells. Am J Hyperten 1991; 4: 137–143. 5. Resink T. J., Scott-Burden T., Bühler F. R. Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1989; 158: 279–286. 6. Simonson M. S., Dunn M. J. Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates b-adrenergicmediated cyclic adenosine monophosphate accumulation. J Clin Invest 1990; 85: 790–797. 7. Huribal M., Cunningham M. E., D’Aiuto M. L., Pleban W. E., McMillen M. A. Endothelin-1, prostaglandin F2 alpha, and corpus luteum – the crisis of lysis. Endocrinology 1996; 3: 5189–5190. 8. Coroneos E., Kester M., Thomas P., Dunn M. J. Endothelin regulates PGE2 formation in rat mesangial cells through induction of prostaglandin endoperoxide synthase-2. Adv Prostaglandin Thromboxane Leukot Res 1995; 3: 117–119. 9. DeNucci G., Thomas R., D’Orleans-Juste P. et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci USA 1988; 85: 9797–9800. 10. Ihara M., Noguchi K., Saeki T. et al. Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor. Life Sci 1992; 50: 247–255. 11. Husain S., Abdel-Latif A. A. Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in cultured human ciliary muscle cells: activation of phospholipase A2. Exp Eye Res 1997; 1: 73–81. 12. Husain S., Abdel-Latif A. A. Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM2 cells. J Lipid Mediat Cell Signal 1996; 1: 147–155. 13. Qiu Z., De Carvalho M., Leslie C. Regulation of phospholipase A2 activation by phosphorylation in mouse peritoneal. J Biol Chem 1993; 266: 24506–24513.
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14. Taylor C., Marshal I. Calcium and inositol 1,4,5-triphosphate receptors: a complex relationship. Oxford: Elsevier Science Publishers, 1992; 403–407. 15. Ives H. E., Schultz G. S., Galardy R. E., Jamieson J. D. Preparation of functional smooth muscle cells from the rabbit aorta. J Exp Med 1978; 148: 1400–1413. 16. Eulalia B., Campbell A. K., Rapoport R. M. Protein kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats. Eur J Pharm 1992; 227: 343–348. 17. Suzuki S., Suzuki A., Kajikuri J., Itoh T. Endothelin-1-induced prostaglandin E2 production: modulation of contractile response to endothelin-1 in porcine coronary artery. Eur J Pharmacol 1992; 217: 97–100. 18. Campbell W. B. Lipid-derived autacoids: eicosanoids and platelets-activating factor. In: Gilman A. G., Rall T. W., Nies A. S., Taylor P (Eds). Goodman and Gilman’s The Pharmacological Basis of Therapeutics. New York: Pergamon Press, 1990; 600. 19. Trybulec M., Dudeck R. R., Gryglewski R. R. Effects of endothelin-1 and endothelin-3 on the release of prostanoids from isolated perfused rat kidney. J Cardiovasc Pharmacol 1991; 17: S229. 20. Neary J. R., Norenberg L. O. B., Norenberg M. D. Protein kinase C in primary astrocyte cultures: cytoplasmic localization and translocation by a phorbol ester. J Neurochem 1988; 50: 1179–1184. 21. Gao G., Serrero G. Phospholipase A2 is a differentiation dependent enzymatic activity for adipogenic cell line and adipocyte precursors in primary culture. J Biol Chem 1988; 263: 16645–16651. 22. Shoyab M., Todaro G. J. Specific high affinity cell membrane receptors for biologically active phorbol and ingenol esters. Nature 1980; 288: 451–455. 23. Busija D. W., Leffler C. W. Effects of phorbol esters on pial arteriolar diameter and brain production of prostanoids in piglets. Circ Res 1991; 69: 1253–1258. 24. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinoline sulfonamides, novel potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 1984; 25: 5036–5041. 25. Busija D. W., Heistad D. D. Factors involved in physiological regulation of cerebral blood flow. Rev Physiol Biochem Pharmacol 1984; 101: 161–211. 26. Busija D. W., Wagerle L. C., Pourcyrous M., Leffler C. W. Acetylcholine dramatically increases prostanoid synthesis in piglet parietal cortex. Brain Res 1988; 439: 122–126. 27. Busija D. W., Leffler C. S. Dilator effects of amino acid neurotransmitters on piglet pial arterioles. Am J Physiol 1989; 257: H1200–H1203. 28. Hartung H-P., Toyka K. V. Phorbol diester TPA elicits prostaglandin E. release from cultured rat astrocytes. Brain Res 1987; 417: 347–349. 29. Nishizuka Y. Turnover of inositol phospholipids and signal transduction. Science 1984; 225: 1365–1370. 30. Sano K., Takai Y., Yamanishi J., Nishizuka Y. A role of calciumactivated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions. J Biol Chem 1983; 258: 2010–2013. 31. Bocckino S. B., Blackmore P. F., Exton J. H. Stimulation of 1,2diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II. J Biol Chem 1985; 260: 14201–14207. 32. Rothhut B., Russo-Marie F., Wood J., DiRosa M., Flower R. J. Further characterization of the glucocorticoid-induced antiphospholipase protein renocortin. Biochem Biophys Res Commun 1983; 117: 878–884.
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33. Hirata F. The regulation of lipomodulin, a phospholipase inhibitory protein, in rabbit neutrophils by phosphorylation. J Biol Chem 1981; 256: 7730–7733. 34. Hirata F., Matsuda K., Notsu Y., Hattori T., delCarmine R. Phosphorylation at a tyrosine residue of lipomodulin in mitogen-stimulated murine thymocytes. Proc Natl Acad Sci 1984; 81: 4717–4721. 35. Touqui L., Rothhut B., Shaw A. M., Fradin A., Vargaftig B. B., Russo-Marie F. Platelet activation – a role for a 40 K antiphospholipase A2 protein indistinguishable from lipocortin. Nature 1986; 321: 177–180. 36. Debbaghi A., Hidi R., Vargaftig B. B., Touqui L. Inhibition of
phospholipase A2 activity in guinea pig eosinophilis by human recombinant IL-1 beta. J Immunol 1992; 149: 1374–1380. 37. Armstead W. M., Mirro R., Busija D. W., Leffler C. W. Permissive role of prostanoids in acetylcholine-induced cerebral constriction. J Pharmacol Exp Ther 1989; 251: 1012–1019. 38. Busija D. W., Leffler C. W. Eicosanoid synthesis elicited by norepinephrine in piglet parietal cortex. Brain Res 1987; 403: 243–248. 39. Pourcyrous M., Leffler C. W., Busija D. W. Postasphyxial increases in prostaglandins in cerebrospinal fluid in piglets. Pediatr Res 1988; 24: 229–232.
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Endothelin-induced prostacyclin production in rat aortic endothelial cells is meditated by protein kinase C G. K. Oriji Department of Biology, College of Science and Health, William Paterson University, Wayne, NJ 07470, USA and Hypertension-Endocrine Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Summary Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10–9 M) produced a 10-fold increase in PGI2 release. Pretreatment with 10–9 M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10–9 M) or cyclooxygenase (indomethacin) (10–9M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.
INTRODUCTION Endothelin (ET) is a 21-amino acid peptide, initially purified from the culture medium of porcine aortic endothelial cells.1 Endothelin type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC) is coupled to a phosphatidyl-inositol-specific phospholipase C (PI-PLC).2 ETA also activates dihydropyridine-insensitive calcium channels and cells challenged with endothelin-1 (ET-1), but not endothelin-3 (ET-3), and produces prostaglandin E2 (PGE2) in a time- and concentration-dependent manner.2 However, the events linking agonist occupancy of receptor and accumulation of PGE2 were not delineated. ET has been shown to augment PGE2 production in various cell types.3–8 However, the signaling pathway involved is poorly understood. Indeed, it is not known whether ET stimulation of eicosanoid production is a sequential, stepwise event subsequent to agonist stimulation of phospholipase A2 (PLA2) by PI-PLC or a G-protein coupling event between receptor and PLA2. Furthermore, Received 17 December 1998 Accepted 19 January 1999 Correspondence to: G. K. Oriji, Department of Biology, College of Science and Health, William Paterson University, Wayne, NJ 07470, USA. Tel.: +1 973 720-3445; Fax: +1 973 720 2338
the requirement for protein kinase C (PKC) to facilitate PLA2 activation has not been carefully delineated. ET is known to activate PLA2, resulting in the production of PGE2, PGI2 or thromboxane A2, depending upon the type of cell.5,6,9–12 PLA2 is activated when phosphorylated at certain serine or threonine positions.13 PKC is known to phosphorylate protein at serine or threonine positions.14 Therefore, PKC may phosphorylate PLA2 which may lead to an increase in prostaglandin (PG) synthesis. Therefore, the aim of the present study is to determine if the PKC signaling pathway is stimulated by endothelin in rat aortic endothelial cells. MATERIALS AND METHODS Isolation of rat aortic endothelial cells Male rats (250–300 g) were anesthetized by i.p. injection of sodium pentobarbital (40 mg/kg) and the thoracic aortas were quickly removed and placed in a petri dish containing oxygenated warm Krebs–Henseleit buffer (KHB; NaCl 116 mM, KCl 5.4 mM, CaCl2–2H2O 2.5 mM, KH2PO4 4.80 mM, MgSO4 1.2 mM, dextrose 21.43 mM, NaHCO3 25.00 mM) at 37°C, aerated with 95% O2 + 5% CO2, pH=7.4. The thoracic aortas were cleaned of surrounding fat and placed in calcium and magnesium263
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free Hanks Buffer Salt Solution (HBSS). After washing the aorta three times with HBSS, the aorta was cut open longitudinally (washed twice with HBSS) and placed in an enzyme solution containing 5 mg collagenase/5 ml HBSS and allowed to incubate in a shaking water bath at 37°C for 30 min. The aorta was removed from the solution and washed five times with HBSS. The adventitia and intima were removed with forceps and the aorta was washed with HBSS twice. Then, the aorta was minced into tiny pieces and washed twice with M199. The tissue was resuspended in a 5 ml enzyme solution (sterile) containing M199, collagenase (5 mg/ml), elastase (0.25 mg/ml), soybean trypsin inhibitor (4 mg/ml), and crystallized bovine serum albumin (2 mg/ml). The tissue was allowed to incubate for two 90 min periods in fresh enzyme solution in a shaker bath at 37°C. At the end of the digestion periods, the smooth muscle cells were spun down in a centrifuge and resuspended in 5 ml HBSS. This washing process was repeated a further two times to remove enzyme and the cells were resuspended in 10 ml of M199 media containing antibiotics and 20% fetal bovine serum (FBS) and plated in a culture dish. The media were replaced 24 h later to remove debris and unattached cells. At 2 day intervals, the media were replaced with M199 containing antibiotics and 10% FBS to feed the cells. Primary cell cultures reached confluent within 10–14 days and were passaged. Passaging requires dislodging the cells from the plate with 1 ml of 0.12% trypsin in HBSS. Experiments were performed by plating cells in 12-well plates at 1 × 106 cm2, grown to subconfluent after 3–4 days. Passages 3 through 7 were used for the experiments. This protocol was modified from Ives et al.15 PGI2 was assayed by radioimmunoassay kit with a specific antibody for 6-keto-PGF1α (Kit # RPA 515; Amersham, Arlington Heights, IL, USA). PKC activity was determined in either cytosol or membrane fractions by an enzyme assay (Kit #RPN 77, Amersham, Arlington Heights, IL, USA). Cytosol and membrane fractions were prepared as per Eulalia et al.16 Experimental protocol
Protocol 1 This experiment was performed to determine the effects of different doses (0.5 × 10–9 M, 10–9 M and 2 × 10–9 M) of either ET or Phorbol 12, 13-dibutyrate (PDBu), a PKC activator, on PGI2 release. Rat aortic endothelial cells were stimulated with ET for 30 min and the perfusate was collected to measure PGI2 release (n=6 for each dose of agonist). Protocol 2 This experiment was performed to determine the effects of pretreatment with either a cyclooxygenase inhibitor
(indomethacin) or inhibitors of PLA2 (either 7,7, dimethyleicosadienoic acid [DEDA] or trifluoromethyl ketone analogue [AACOCF3]) on ET- and PDBu-induced PGI2 release. 1 nM of either indomethacin, AACOCF3 or DEDA was added to the cell wells for 30 min before the addition of either ET or PDBu; the perfusate was collected for 30 min after the addition of either ET or PDBu to determine PGI2 release (n=6 for each inhibitor and each agonist).
Protocol 3 This experiment was performed to determine the effects of pretreatment with different doses (10–9 M and 10–3 M) of PKC inhibitors: 1-(5 isoquinolinesulfonylmethyl) piperazine (H7), staurosporine or 1-(5-isoquinolinesulfonyl) piperazine (CL) on ET induced PGI2 release. 10–9 M or 10–3 M of one of the three PKC inhibitors was added to the cell wells for 30 min before ET was added; the perfusate was collected for 30 min after the addition of ET for PGI2 determination (n=6 for each dose of inhibitor). Drugs and chemicals Drugs used in this study were endothelin, phorbol 12, 13dibutyrate, indomethacin, trifluoromethyl ketone analogue, 7,7, dimethyleicosadienoic acid (DEDA), 1-(5-isoquinolinemethyl) piperazine (H7) and staurosporine (Calbiochem Inc., San Diego, CA, USA) and 1-(5-isoquinolinesulfonyl) piperazine (CL) (Sigma Chemical Company, St Louis, MO, USA). Statistical analysis All data were generated with paired controls. Values were expressed as mean±S.E.M. A two way analysis of variance was used for comparisons within experiments. A value of P < 0.05 was considered significant.
RESULTS Effects of different doses of either ET or PDBu on PGI2 release Both ET and PDBu produced significant and dose-dependent increases in PGI2 release (Fig. 1). The response to ET (2 × 10–9 M) was 15% greater than the response to the same dose of PDBu. Effects of pretreatment with indomethacin or DEDA or AACOCF3 on either ET- or PDBu-induced PGI2 release ET-induced PGI2 release was significantly blocked by pretreatment with either a cyclooxygenase inhibitor (indomethacin) (96% inhibition) or different inhibitors of PLA2 (DEDA [100% inhibition] or AACOCF3 [97% inhibition]).
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PKC inhibits ET-induced PGI2 production
Fig. 1 The effects of different doses of (A) ET and (B) PDBu on prostacyclin release in rat aortic endothelial cells. *P < 0.01 vs control.
Also, PDBu-induced PGI2 release was significantly blocked by pretreatment with either a cyclooxygenase inhibitor (indomethacin) (100% inhibition) or different inhibitors of PLA2 (DEDA [97% inhibition] or AACOCF3 [100% inhibition]) (Fig. 2). These results show that inhibition of either cyclooxygenase or PLA2 markedly decreased both ET- and PDBu-induced PGI2 release. Effects of pretreatment with different doses of PKC inhibitors on ET-induced PGI2 release ET-induced PGI2 release was inhibited 71–75% by 10–9 M, and 96–99% by 10–3 M pretreatment doses of any of three different PKC inhibitors, that is, H7, staurosporine or 1-(5-isoquinolinesulfonyl) piperazine (CL) (Fig. 3). In preliminary experiments, ET-induced PGI2 release was inhibited 84–87% by 10–6 M doses of any of three different PKC inhibitors (data not shown). These results confirm that PKC inhibition completely blocked PGI2 release. The PKC enzyme assay showed activation, that is, translocation of PKC from the cytosol to the membrane fraction in endothelial cells that were treated with either ET or PDBu. Translocation was inhibited by pretreatment with PKC inhibitors and unchanged by PDD (Fig. 4). PKC © 1999 Harcourt Brace & Co. Ltd
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Fig. 2 The effects of indomethacin, AACOCF3 or DEDA (10–9 M) on ET-induced prostacyclin release in rat aortic endothelial cells. Endothelial cells were pretreated with one of the prostacyclin inhibitors for 30 min before the addition of (A) ET or (B) PDBu. *P < 0.01 vs agonist alone.
inhibitors or PDD alone did not affect translocation of PKC (Fig. 4). DISCUSSION In this study, we found that (1) ET induced PGI2 release in rat aortic endothelial cells, (2) activation of PKC by PDBu increased PGI2 release, (3) both ET- and PDBu-induced PGI2 release were inhibited via inhibitors of either cyclooxygenase or PLA2, (4) ET induced PGI2 release was inhibited by different inhibitors of PKC and (5) ET caused activation of PKC, that is, translocation of PKC from cytosol to membrane fractions. ET is a very potent vasoconstrictor in porcine coronary arteries.1 Vasoconstriction was shown to be accompanied by an increase in the release of PGE2, but not PGI2.17 PGE2 and PGI2 are potent vasodilators in most vascular beds;18 ET has been shown to release both PGE2 and PGI2 from perfused rat kidney.19 The mechanism for this ET-induced PGI2 release appears to be mediated by PKC. The biologically active phorbol ester, PDBu, causes PKC translocation from the cytosol to the cell membrane and increased
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Fig. 3 Effects of two different doses: (A) 10–9 M and (B) 10–3 M of PKC inhibitors on ET-induced prostacyclin release in rat aortic endothelial cells. Endothelial cells were pretreated with either vehicle or PKC inhibitors for 30 min prior to ET treatment. *P < 0.01 vs ET alone.
enzyme activity in astroglia.20 Conversely, an inactive phorbol ester, such as 4-alpha-phorbol, 12,13-Didecanoate (PDD) does not produce these effects;20–22 PDD does not increase PG production in intact piglet parietal cortex.23 In this study, PDBu increased PGI2 release significantly,
although, this increase in PGI2 release was somewhat less than that produced by ET alone. In addition, ET-induced PGI2 release was markedly decreased by three different PKC inhibitors: H7, staurosporine and 1-(5-isoquinolinesulfonyl) piperazine (CL). Although, H7 is also a potent inhibitor of adenosine 3′,5′-cyclic monophosphatedependent protein kinase,24 it seems unlikely that this mechanism is involved in the inhibition of PGI2 release observed in this study. In intact piglet parietal cortex, stimulation of either adenylate cyclase with isoproterenol25 or guanylate cyclase with arginine-containing compounds26 or glutamate27 did not increase cerebrospinal fluid levels of PGs. The mechanism by which ET causes increased PGI2 release is not known with certainty, but it appears to involve PKC since activation of PLA2, via either ET or PDBu-induced PGI2 release was blocked by pretreatment with inhibitors of either PLA2 (DEDA or AACOCF3) or cyclooxygenase (indomethacin). However, phorbol esters have been shown to cause arachidonic acid release in cultured rat astrocytes.28 There are several ways by which PKC could activate PLA2: PKC could phosphorylate PLA2 and directly activate this enzyme;29–31 PKC could phosphorylate and thereby inactivate a protein that normally inhibits PLA2, e.g. lipocortins;32–35 or PKC could increase intracellular calcium and activate PLA2 by this mechanism.36 Several studies have shown that prostanoids are able to influence the tone of cerebral arteries and arterioles.37–39 Indeed, PG synthesis is altered in various pathological conditions and causes profound modifications in tissue blood flow. Therefore, it is important to understand the mechanisms involved in PG release. In the aorta, PGI2 may be important as either a regulator of vascular tone
Fig. 4 Effects of ET or PDBu alone or PKC inhibitors plus ET on activation of PKC in rat aortic endothelial cells. Endothelial cells were pretreated with either vehicle or PKC inhibitors (10–9 M) for 30 min prior to ET (10–9 M) treatment. *P < 0.001 vs PKC inhibitors plus ET (10–9 M).
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PKC inhibits ET-induced PGI2 production
or a modulator of the vascular responses to certain neurotransmitters. In summary, ET activates PKC which activates PLA2 which liberates arachidonic acid which increases PGI2 production and release in rat aortic endothelial cells.
ACKNOWLEDGMENTS The author thanks Mrs Margaret Hill and Mr John Tate for their excellent technical assistance.
REFERENCES 1. Yanagisawa M., Kurihara H., Kimura S. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 333: 411–415. 2. Wilkes B. M., Ruston A. S., Mento P. et al. Characterization of endothelin 1 receptor and signal transduction mechanisms in rat medullary interstitial cells. Am J Physiol 1991; 260 (Renal Fluid Electrolyte Physiol 29): F579–F589. 3. Abdel-Latif A. A., Zhang Y., Yousufzai S. Y. Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in rabbit iris sphincter smooth muscle: activation of phospholipase A2. Curr Eye Res 1991; 3: 259–265. 4. Fukunaga M., Ochi S., Takama T. et al. Endothelin-1 stimulates prostaglandin E2 production in an extracellular calciumindependent manner in cultured rat mesangial cells. Am J Hyperten 1991; 4: 137–143. 5. Resink T. J., Scott-Burden T., Bühler F. R. Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1989; 158: 279–286. 6. Simonson M. S., Dunn M. J. Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates b-adrenergicmediated cyclic adenosine monophosphate accumulation. J Clin Invest 1990; 85: 790–797. 7. Huribal M., Cunningham M. E., D’Aiuto M. L., Pleban W. E., McMillen M. A. Endothelin-1, prostaglandin F2 alpha, and corpus luteum – the crisis of lysis. Endocrinology 1996; 3: 5189–5190. 8. Coroneos E., Kester M., Thomas P., Dunn M. J. Endothelin regulates PGE2 formation in rat mesangial cells through induction of prostaglandin endoperoxide synthase-2. Adv Prostaglandin Thromboxane Leukot Res 1995; 3: 117–119. 9. DeNucci G., Thomas R., D’Orleans-Juste P. et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci USA 1988; 85: 9797–9800. 10. Ihara M., Noguchi K., Saeki T. et al. Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor. Life Sci 1992; 50: 247–255. 11. Husain S., Abdel-Latif A. A. Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in cultured human ciliary muscle cells: activation of phospholipase A2. Exp Eye Res 1997; 1: 73–81. 12. Husain S., Abdel-Latif A. A. Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM2 cells. J Lipid Mediat Cell Signal 1996; 1: 147–155. 13. Qiu Z., De Carvalho M., Leslie C. Regulation of phospholipase A2 activation by phosphorylation in mouse peritoneal. J Biol Chem 1993; 266: 24506–24513.
© 1999 Harcourt Brace & Co. Ltd
267
14. Taylor C., Marshal I. Calcium and inositol 1,4,5-triphosphate receptors: a complex relationship. Oxford: Elsevier Science Publishers, 1992; 403–407. 15. Ives H. E., Schultz G. S., Galardy R. E., Jamieson J. D. Preparation of functional smooth muscle cells from the rabbit aorta. J Exp Med 1978; 148: 1400–1413. 16. Eulalia B., Campbell A. K., Rapoport R. M. Protein kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats. Eur J Pharm 1992; 227: 343–348. 17. Suzuki S., Suzuki A., Kajikuri J., Itoh T. Endothelin-1-induced prostaglandin E2 production: modulation of contractile response to endothelin-1 in porcine coronary artery. Eur J Pharmacol 1992; 217: 97–100. 18. Campbell W. B. Lipid-derived autacoids: eicosanoids and platelets-activating factor. In: Gilman A. G., Rall T. W., Nies A. S., Taylor P (Eds). Goodman and Gilman’s The Pharmacological Basis of Therapeutics. New York: Pergamon Press, 1990; 600. 19. Trybulec M., Dudeck R. R., Gryglewski R. R. Effects of endothelin-1 and endothelin-3 on the release of prostanoids from isolated perfused rat kidney. J Cardiovasc Pharmacol 1991; 17: S229. 20. Neary J. R., Norenberg L. O. B., Norenberg M. D. Protein kinase C in primary astrocyte cultures: cytoplasmic localization and translocation by a phorbol ester. J Neurochem 1988; 50: 1179–1184. 21. Gao G., Serrero G. Phospholipase A2 is a differentiation dependent enzymatic activity for adipogenic cell line and adipocyte precursors in primary culture. J Biol Chem 1988; 263: 16645–16651. 22. Shoyab M., Todaro G. J. Specific high affinity cell membrane receptors for biologically active phorbol and ingenol esters. Nature 1980; 288: 451–455. 23. Busija D. W., Leffler C. W. Effects of phorbol esters on pial arteriolar diameter and brain production of prostanoids in piglets. Circ Res 1991; 69: 1253–1258. 24. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinoline sulfonamides, novel potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 1984; 25: 5036–5041. 25. Busija D. W., Heistad D. D. Factors involved in physiological regulation of cerebral blood flow. Rev Physiol Biochem Pharmacol 1984; 101: 161–211. 26. Busija D. W., Wagerle L. C., Pourcyrous M., Leffler C. W. Acetylcholine dramatically increases prostanoid synthesis in piglet parietal cortex. Brain Res 1988; 439: 122–126. 27. Busija D. W., Leffler C. S. Dilator effects of amino acid neurotransmitters on piglet pial arterioles. Am J Physiol 1989; 257: H1200–H1203. 28. Hartung H-P., Toyka K. V. Phorbol diester TPA elicits prostaglandin E. release from cultured rat astrocytes. Brain Res 1987; 417: 347–349. 29. Nishizuka Y. Turnover of inositol phospholipids and signal transduction. Science 1984; 225: 1365–1370. 30. Sano K., Takai Y., Yamanishi J., Nishizuka Y. A role of calciumactivated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions. J Biol Chem 1983; 258: 2010–2013. 31. Bocckino S. B., Blackmore P. F., Exton J. H. Stimulation of 1,2diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II. J Biol Chem 1985; 260: 14201–14207. 32. Rothhut B., Russo-Marie F., Wood J., DiRosa M., Flower R. J. Further characterization of the glucocorticoid-induced antiphospholipase protein renocortin. Biochem Biophys Res Commun 1983; 117: 878–884.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 263–268
268
Oriji
33. Hirata F. The regulation of lipomodulin, a phospholipase inhibitory protein, in rabbit neutrophils by phosphorylation. J Biol Chem 1981; 256: 7730–7733. 34. Hirata F., Matsuda K., Notsu Y., Hattori T., delCarmine R. Phosphorylation at a tyrosine residue of lipomodulin in mitogen-stimulated murine thymocytes. Proc Natl Acad Sci 1984; 81: 4717–4721. 35. Touqui L., Rothhut B., Shaw A. M., Fradin A., Vargaftig B. B., Russo-Marie F. Platelet activation – a role for a 40 K antiphospholipase A2 protein indistinguishable from lipocortin. Nature 1986; 321: 177–180. 36. Debbaghi A., Hidi R., Vargaftig B. B., Touqui L. Inhibition of
phospholipase A2 activity in guinea pig eosinophilis by human recombinant IL-1 beta. J Immunol 1992; 149: 1374–1380. 37. Armstead W. M., Mirro R., Busija D. W., Leffler C. W. Permissive role of prostanoids in acetylcholine-induced cerebral constriction. J Pharmacol Exp Ther 1989; 251: 1012–1019. 38. Busija D. W., Leffler C. W. Eicosanoid synthesis elicited by norepinephrine in piglet parietal cortex. Brain Res 1987; 403: 243–248. 39. Pourcyrous M., Leffler C. W., Busija D. W. Postasphyxial increases in prostaglandins in cerebrospinal fluid in piglets. Pediatr Res 1988; 24: 229–232.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 263–268
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