Desensitization of endothelial nitric oxide synthase by receptor agonists.
Stimulation of endothelial cells with receptor agonists leads to an activation of endothelial nitric oxide synthase (eNOS) that only lasts for a short duration. A more prolonged effect, however, is observed in response to non-receptor agonists, such as Ca2+ ionophores or thapsigargin (TG). To investigate the molecular mechanisms underlying the rapid deactivation of eNOS after stimulation with receptor agonists, we measured the time courses of eNOS activation and intracellular free Ca2+ concentration ([Ca2+]i) in response to bradykinin (BK) and ATP. Incubation of porcine aortic endothelial cells with BK (1 microM) in the presence of 3 mM extracellular Ca2+ increased [Ca2+]i from 110 to 350 nM and enhanced the rate of l-[3H]citrulline formation from 0.1 to 5 fmol/min. In the absence of extracellular Ca2+, the BK-induced increase in [Ca2+]i was only marginal (from 30 to 110 nM) and not sufficient to activate eNOS. When Ca(2+) (final concentration 3 mM) was added 10 min after BK, [Ca2+]i increased to 330 nM within 3 min, but interestingly, formation of l-[3H]citrulline was not detectable. A similar phenomenon was observed with ATP, but not with Ca2+ ionophores or TG. This indicates that stimulation of endothelial cells with receptor agonists leads to desensitization of eNOS, which renders the enzyme insensitive to activation by subsequent increases in [Ca2+]i. However, when ATP was added to BK-pretreated cells or, conversely, BK to ATP-pretreated cells, activation of eNOS was comparable with that of untreated cells, suggesting that BK and ATP affect different pools of eNOS. The desensitization of eNOS was reversible, since removal of ATP or BK from the incubation buffer restored the response to the respective agonist within 20 min. In addition to the transient Ca2+ signal, desensitization of eNOS may represent a further mechanism by which endothelial cells rapidly terminate receptor-dependent NO formation.
Wagner
Sabine
S
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Groschner
Klaus
K
Mayer
Bernd
B
Schmidt
Kurt
K
D000818:Animals; D001011:Aorta; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D013552:Swine; D019284:Thapsigargin / Q000494:pharmacology
D019284:Thapsigargin; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D001920:Bradykinin; D002118:Calcium