Molecular mechanisms involved in the synergistic activation of soluble guanylyl cyclase by YC-1 and nitric oxide in endothelial cells.
YC-1 is a direct activator of soluble guanylyl cyclase (sGC) and sensitizes the enzyme for activation by nitric oxide (NO) and CO. Because the potentiating effect of YC-1 on NO-induced cGMP formation in platelets and smooth muscle cells has been shown to be substantially higher than observed with the purified enzyme, the synergism between heme ligands and YC-1 is apparently more pronounced in intact cells than in cell-free systems. Here, we investigated the mechanisms underlying the synergistic activation of sGC by YC-1 and NO in endothelial cells. Stimulation of the cells with YC-1 enhanced cGMP accumulation up to approximately 100-fold. The maximal effect of YC-1 was more pronounced than that of the NO donor DEA/NO (approximately 20-fold increase in cGMP accumulation) and markedly diminished in the presence of L-N(G)-nitroarginine, EGTA, or oxyhemoglobin. Because YC-1 did not activate endothelial NO synthase, the pronounced effect of YC-1 on cGMP accumulation was apparently caused by a synergistic activation of sGC by YC-1 and basal NO. The effect of YC-1 was further enhanced by addition of DEA/NO, resulting in a approximately 160-fold stimulation of cGMP accumulation. Thus, YC-1 increased the NO-induced accumulation of cGMP in intact cells by approximately 8-fold. Addition of endothelial cell homogenate increased the stimulatory effect of YC-1 on NO-activated purified sGC from 1.2- to 3.7-fold. This effect was not observed with heat-denatured homogenates, suggesting that a heat-labile factor present in endothelial cells potentiates the effect of YC-1 on NO-activated sGC.
Schmidt
K
K
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Graz, Austria. kurt.schmidt@kfunigraz.ac.at
Schrammel
A
A
Koesling
D
D
Mayer
B
B
D000818:Animals; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004357:Drug Synergism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism*; D007191:Indazoles / Q000494:pharmacology*; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D013552:Swine
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007191:Indazoles; D020030:Nitric Oxide Donors; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D006162:Guanylate Cyclase; D006152:Cyclic GMP