The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein.
The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001-0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, gamma-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-alpha-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of G(i)-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca2+ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G(i)-protein.
Manolopoulos
V G
VG
University of Wisconsin Medical School, Milwaukee Clinical Campus at Sinai Samaritan Medical Center, 53201, USA.
Fenton
J W
JW
Lelkes
P I
PI
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000313:Adrenal Medulla / Q000098:blood supply*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D010450:Endopeptidases; D004730:Endothelium, Vascular / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D008833:Microcirculation / Q000378:metabolism; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D018179:Receptors, Thrombin / Q000187:drug effects / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D010566:Virulence Factors, Bordetella
D037361:Adenylate Cyclase Toxin; D018179:Receptors, Thrombin; D010566:Virulence Factors, Bordetella; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010450:Endopeptidases; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; D002118:Calcium