Abstract
The purpose of this study was to elucidate the mechanism by which acetylcholine (ACh) promotes prostacyclin (PGI2) production in cultured coronary endothelial cells (CEC) of the rabbit heart. ACh-induced production of PGI2, measured as immunoreactive 6-keto-PGF1 α, was enhanced by increasing the extracellular calcium (Ca++) concentration and reduced by Ca++ depletion. The receptor-operated Ca++channel blocker SK&F96365, but not the voltage-dependent Ca++ channel blockers verapamil or nifedipine, attenuated ACh-induced 6-keto-PGF1 α production and the associated rise in cytosolic Ca++. Thapsigargin, which depleted Ca++ accumulation from the intracellular Ca++ store, did not prevent the ACh-induced rise in cytosolic Ca++. In the absence of extracellular Ca++, ACh and ATP increased cytosolic Ca++ but did not alter 6-keto-PGF1 α production. In permeabilized CEC, guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) but not ACh enhanced 6-keto-PGF1 α synthesis. ACh increased 6-keto-PGF1 α production in the presence of GTP-γ-S. These effects of GTP-γ-S were attenuated by guanosine 5′-O-(2-thiotriphosphate). In the absence of extracellular Ca++, ACh or ATP increased cytosolic Ca++ in cells permeabilized with β-escin and loaded with GTP-γ-S; this effect was attenuated by guanosine 5′-O-(2-thiotriphosphate). The effect of ATP but not ACh to mobilize intracellular Ca++ or increase 6-keto-PGF1 α was inhibited by pertussis toxin. The phospholipase C inhibitor D609, which attenuated ACh- and ATP-induced mobilization of intracellular Ca++, did not alter 6-keto-PGF1 α production. The NO synthase inhibitor N-monomethyl-arginine also failed to alter ACh-induced 6-keto-PGF1 α synthesis. These data suggest that, in CEC of the rabbit heart, ACh stimulates prostacyclin production via a pertussis toxin-insensitive G protein and by increasing the influx of extracellular Ca++ through a G protein-independent receptor-operated Ca++ channel.
Footnotes
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Send reprint requests to: Dr. Kafait U. Malik, Professor of Pharmacology, Department of Pharmacology, College of Medicine, University of Tennessee, The Health Science Center, Memphis, TN 38163.
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↵1 This study was supported by United States Public Health Service-National Institutes of Health Grant 19134-23 from the National Heart, Lung and Blood Institute. This work was presented in part at the Annual FASEB Meeting, April 1994, Anaheim, CA.
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↵2 Current address: Department of Medicine, Section of Cardiology, West Virginia University Health Sciences Center, P.O. Box 9157, Morgantown, WV 26506.
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↵3 Current address: Department of Pharmacology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6260.
- Abbreviations:
- ACh
- acetylcholine
- PGI2
- prostacyclin
- Ca++
- extracellular calcium
- cGMP
- cyclic guanosine monophosphate
- CEC
- coronary endothelial cells
- ROCC
- receptor-operated Ca++ channel
- AA
- arachidonic acid
- D609
- tricyclodecan-9-yl-xanthogenate
- NO
- nitric oxide
- FCCP
- carbonyl cyanide p-(tri-fluoromethoxy)phenyl-hydrazone
- ATP
- adenosine-5′-triphosphate
- PLC
- phospholipase C
- BSS
- balanced salt solution
- HBSS
- Hanks’ BSS
- mAChR
- muscarinic acetylcholine receptor
- Received November 18, 1996.
- Accepted March 31, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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