Beta Adrenergic Receptor Stimulated Prostacyclin Synthesis in Rabbit Coronary Endothelial Cells Is Mediated by Selective Activation of Phospholipase D: Inhibition by Adenosine 3959-Cyclic Monophosphate

Activation of beta adrenergic receptors in the isolated rabbit heart by catecholamines stimulates prostacyclin (PGI2) synthesis, which is inhibited by adenosine 3959-cyclic monophosphate (cAMP). The purpose of this study was to determine if activation of beta adrenergic receptors in cultured coronary endothelial cells (CEC) of rabbit heart with isoproterenol (ISOP) stimulates PGI2 synthesis and if cAMP inhibits the synthesis of this prostanoid and to investigate the underlying mechanism. Incubation of CEC with ISOP increased production of cAMP and PGI2, measured as immunoreactive cAMP and 6-keto-prostaglandin F1a, (6-keto-PGF1a), respectively. Forskolin, an activator of adenylyl cyclase, increased cAMP accumulation and inhibited ISOP-stimulated 6-keto-PGF1a synthesis. 8-(4-chlorophenylthio) cAMP also inhibited ISOP-induced 6-keto-PGF1a production. However, miconazole, an inhibitor of adenylyl cyclase, reduced cAMP accumulation and enhanced ISOP-stimulated 6-keto-PGF1a synthesis in CEC. ISOP-induced 6-keto-PGF1a synthesis was attenuated by C2-ceramide, an inhibitor of phospholipase D (PLD) by propranolol, a beta-AR antagonist that also inhibits phosphatidate phosphohydrolase and by the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267). Acetylcholine (ACh) induced 6-keto-PGF1a synthesis was also inhibited by these agents. Both ISOP and ACh increased PLD activity, which was inhibited by C2ceramide but not by RHC 80267 or propranolol. ACh but not ISOP increased phospholipase A2 activity in CEC. ISOPbut not AChinduced increase in PLD activity was attenuated by forskolin and 8-(4-chlorophenyl-thio)-adenosine 39-59-cyclic monophosphate and augmented by miconazole. These data suggest that beta adrenergic receptors activation promotes PGI2 synthesis in the CEC by selective activation of PLD and that cAMP decreases PGI2 synthesis by decreasing PLD activity. Moreover, beta adrenergic receptors activated PLD appears to be distinct from that stimulated by ACh. Sympathetic nerve stimulation or administration of norepinephrine increases synthesis of PG in various tissues, including the heart (Hedqvist, 1977; Malik and Sehic, 1990; Shaffer and Malik, 1982). The principal prostanoid synthesized in the heart during sympathetic nerve stimulation, PGI2, has cardioprotective effects ascribed to its action to inhibit platelet aggregation (Moncada et al., 1976), produce coronary vasodilation (Dusting et al., 1978), reduce norepinephrine release from sympathetic fibers and decrease myocardial contractility in response to norepinephrine (Khan and Malik, 1982; Lanier and Malik, 1985) and inhibit free radical production and ventricular arrhythmias (Kecskemeti et al., 1973). PG synthesis elicited by adrenergic transmitter in the heart is mediated by activation of beta-AR (Shaffer and Malik, 1982). AR activation in the heart also increases levels of cAMP (Kopecky et al., 1965), and cAMP in the heart mediates some cardiovascular actions of norepinephrine (Drummond and Severson, 1979). cAMP has also been reported to influence PG synthesis in several tissues and cells. For example, cAMP or agents that increase cAMP accumulation inhibit PG synthesis in ventricular myocytes elicited Received for publication November 11, 1996. 1 This study was supported by USPHS-NIH Grant 19134-22 from the National Heart, Lung and Blood Institute. This work was presented in part at the Annual FASEB Meeting, April 1994, Anaheim, CA. 2 Current address: Dr. Ying Ruan, Department of Pharmacology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6260. 3 Current address: Dr. Hong Kan, Department of Medicine, Section of Cardiology, West Virginia University Health Sciences Center, P.O. Box 9157, Morgantown, WV 26506. ABBREVIATIONS: AA, arachidonic acid; ACh, acetylcholine; AR, adrenergic receptor; cAMP, adenosine 3959-cyclic monophosphate; CEC, coronary endothelial cells; cpt-cAMP, 8-(4-chlorophenyl-thio)-cAMP; DAG, diacylglycerol; D-609, tricyclodecan-9-yl xanthogenate z K; HELSS, e-6(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2one; IBMX, 3-isobutyl-1-methyl-xanthine; ISOP, isoproterenol; PG, prostaglandins; PGI2, prostacyclin; PLA2, phospholipase A2; PLD, phospholipase D; PPH, phosphatidate phosphohydrolase; RHC 80267, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane; KHB, Krebs-Henseleit buffer; PEt, phosphatidylethanol. 0022-3565/97/2813-1038$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 281, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 281:1038–1046, 1997 1038 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from by ISOP (Ruan et al., 1996), platelets (Gerrad et al., 1977), Madin Darby Canine Kidney (MDCK) cells (Hassid, 1983) and collecting tubular cells (Teitelbaum et al., 1986). In contrast, cAMP and its analogs or forskolin increase PG synthesis in human decidua and amnion cells (Warrick et al., 1985) and human adherent synovial cells (Baker et al., 1985) but do not alter PG synthesis in vascular endothelial cells (Brotherton and Hoak, 1982; Whorton et al., 1985). In the heart, cAMP, or agents that increase cAMP accumulation also do not alter basal production of PGI2 but inhibit that elicited by stimulation of beta-AR with ISOP (Williams and Malik, 1989). These findings, together with the demonstration that agents that reduce cAMP levels increase ISOP-induced PGI2 synthesis, suggest that cAMP acts as an inhibitory modulator of beta-AR-stimulated PGI2 synthesis in the heart (Williams and Malik, 1989). PGs are synthesized in several cell types, including ventricular myocytes (Ahumada et al., 1980; Bolton et al., 1980), and coronary vascular endothelial cells (Gerritsen and Cheli, 1983; Revtyak et al., 1988). However, the site(s) of beta-AR-stimulated PGI2 synthesis and the mechanism by which cAMP inhibits PGI2 synthesis in the heart is not known. This study was undertaken to determine if activation of beta-AR with ISOP stimulates PGI2 synthesis and cAMP generated during beta-AR activation inhibits PGI2 production in CEC of rabbit heart. To elucidate the mechanism of modulation of PGI2 synthesis by cAMP, we examined the contribution of different phospholipases to PGI2 production and modification of their activity by alterations in cAMP levels in response to beta-AR activation in CEC.