Alpha-1A Adrenergic Receptor Stimulation with Phenylephrine Promotes Arachidonic Acid Release by Activation of Phospholipase D in Rat-1 Fibroblasts: Inhibition by Protein Kinase A 1 YING RUAN, 2 HONG KAN, 3 JEAN-HUGUES PARMENTIER, SOGHRA FATIMA, LEE F. ALLEN and KAFAIT U. MALIK Department of Pharmacology, College of Medicine, The University of Tennessee, Memphis, Memphis, Tennessee (Y.R., H.K., J.-H.P., S.F., K.U.M.) and Huntsman Cancer Institute, Departments of Medicine/Oncological Sciences, The University of Utah Health Sciences Center, Salt Lake City, Utah (L.F.A.) Accepted for publication October 20, 1997 This paper is available online at http://www.jpet.org ABSTRACT This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation of alpha adrenergic receptor (AR), and its modula- tion by cyclic adenosine 3',5'-monophosphate (cAMP) in Rat-1 fibroblasts (R-1Fs) transfected with the alpha-1A, alpha-1B or alpha-1D AR. PHE increased AA release and also caused a marked accumulation of cAMP in R-1Fs expressing the alpha-1 AR subtypes, but not in those transfected with vector alone. PHE also enhanced phospholipase D (PLD), but not phospho- lipase A 2 (PLA 2 ) activity. The increase in PHE-induced AA re- lease, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A  alpha-1B  al- pha-1D AR. The effect of PHE to increase AA release was attenuated by C 2 -ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing the alpha-1A AR. Forskolin, which activates adenylyl cyclase, increased cAMP accumulation and inhibited PHE-induced AA release and PLD activity in alpha-1A-AR– expressing R-1Fs. 8-(4-chloro- phenyl-thio)-cAMP, a nonhydrolyzable analog of cAMP, also attenuated the rise in AA release and PLD activity elicited by PHE in these cells. In contrast, SQ 22536, an adenylyl cyclase inhibitor, and KT 5720, a protein kinase A inhibitor, increased PHE-induced AA release and PLD activity in R-1Fs expressing the alpha-1A AR. These data suggest that the alpha-1A, al- pha-1B and alpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing the alpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release via protein kinase A. The adrenergic transmitter norepinephrine produces a wide variety of biological actions, including AA release for prostaglandin synthesis, via activation of distinct types of AR, e.g., alpha ARs in the kidney, spleen and blood vessels (Malik, 1988), beta-1 ARs in the heart (Shaffer and Malik, 1982) and beta-2 ARs in the lung (bronchial smooth muscle) (Lew et al., 1992). Pharmacological, radioligand binding and molecular cloning studies have led to the further character- ization of subtypes of alpha-1, alpha-2 and beta ARs (Bylund, 1992; Minneman and Esbenshade, 1994; Graham et al., 1996; Lands et al., 1967; Frielle et al., 1987; Kobilka et al., 1987; Emorine et al., 1989). Alpha-1 ARs initially were subclassi- fied into alpha-1A and alpha-1B ARs based on differences in their binding profiles of alpha AR antagonists and the ability to selectively block various biological responses mediated via Received for publication August 12, 1997. 1 This study was supported by USPHS-NIH grant 19134 –22 from the Na- tional Heart, Lung and Blood Institute. This work was presented in part at the Annual FASEB Meeting, April 1996, Washington, DC. 2 A postdoctoral trainee, supported by the USPHS grant HL 07641; Lipid/ Lipoprotein Metabolism and Cardiovascular Disease. Current affiliation: De- partment of Pharmacology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6260. 3 Current affiliation: Department of Medicine, Section of Cardiology, West Virginia University Health Sciences Center, P.O. Box 9157, Morgantown, WV 26506. ABBREVIATIONS: AA, arachidonic acid; AR, adrenergic receptor; BSA, bovine serum albumin; cAMP, adenosine 3'5'-cyclic monophosphate; cpt-cAMP, 8-(4-chlorophenyl-thio)-cAMP; DAG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; EGTA, ethylene glycol bis(- aminoethyl ether)-N,N,N',N'-tetraacetic acid; HBSS, Hanks’ balanced salt solution; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; IBMX, 3-isobutyl-1-methylxanthine; MAG, monoacylglycerol; PA, phosphatidic acid; PEt, phosphatidyl ethanol; PHE, phenylephrine; PKA, protein kinase A; PKC, protein kinase C; PLA 2 , phospholipase A 2 ; PLC, phospholipase C; PLD, phospholipase D; PMSF, phenylmethylsulfonyl fluoride; PPH, phosphatidate phosphohydrolase; RHC 80267, 1,6-bis-(cyclohexyloximino-carbonylamino)-hexane; R-1F, Rat-1 fibroblasts; TLC, thin-layer chromatography. 0022-3565/98/2842-0576$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 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