Abstract
This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation ofalpha adrenergic receptor (AR), and its modulation 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 phospholipase A2 (PLA2) activity. The increase in PHE-induced AA release, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A >alpha-1B > alpha-1D AR. The effect of PHE to increase AA release was attenuated by C2-ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing thealpha-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-chlorophenyl-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 thealpha-1A AR. These data suggest that thealpha-1A, alpha-1B andalpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing thealpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release viaprotein kinase A.
Footnotes
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Send reprint requests to: Kafait U. Malik, Ph.D., D.Sc., Professor of Pharmacology, College of Medicine, The University of Tennessee, Memphis, 874 Union Avenue, Memphis, TN 38163.
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↵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 1996, Washington, DC.
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↵2 A postdoctoral trainee, supported by the USPHS grant HL 07641; Lipid/Lipoprotein Metabolism and Cardiovascular Disease. Current affiliation: Department of Pharmacology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6260.
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↵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(β-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
- PLA2
- phospholipase A2
- 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
- Received August 12, 1997.
- Accepted October 20, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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