Modulation of bradykinin receptor ligand binding affinity and its coupled G-proteins by nitric oxide.
To determine whether nitric oxide (NO) can modulate bradykinin (BK) signaling pathways, we treated endothelial cells with an NO donor, S-nitrosoglutathione (GSNO), to determine its effect(s) on G-proteins (Gi and Gq) that are coupled to the type II kinin (BK2) receptor. Radioligand binding assays and Western analyses showed that GSNO (10-500 microM, 0-72 h) did not alter the expression of BK2 receptor, Gi, or Gq. However, GSNO caused a 6-fold increase in basal cGMP production and decreased high affinity BK bindings sites and GTPase activity by 74 and 85%, respectively. The cGMP analogue, dibutyryl-cGMP, also inhibited BK-stimulated GTPase activity by 74% suggesting that some of the effects of NO may be mediated through activation of guanylyl cyclase. The NO synthase inhibitor, Nomega-monomethyl-L-arginine, inhibited endogenous NO synthase activity and cGMP production by 91 and 76%, respectively, but increased BK-stimulated GTPase activity by 61%. To determine which G-proteins are affected by NO, we performed GTP binding assays with [35S]GTPgammaS followed by immunoprecipitation with specific G-protein antisera. Both GSNO and dibutyryl-cGMP increased basal G-protein GTP binding activities by 18-26%. However, GSNO decreased BK-stimulated Galphai2, Galphai3, and Galphaq/11 GTP binding activity by 93, 61, and 90%, respectively, whereas epinephrine-stimulated Galphas GTP binding activity was unaffected. These results suggest that NO can modulate BK signaling pathways by selectively inhibiting G-proteins of the Gi and Gq family.
Miyamoto
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Cardiovascular Division, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachussets 02115, USA.
Laufs
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Pardo
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Liao
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D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000096:biosynthesis; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis / Q000037:antagonists & inhibitors*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D008024:Ligands; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology; D018002:Receptors, Bradykinin / Q000032:analysis*; D026422:S-Nitrosoglutathione
D008024:Ligands; D009603:Nitroso Compounds; D018002:Receptors, Bradykinin; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D006160:Guanosine Triphosphate; D019001:Nitric Oxide Synthase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D005978:Glutathione; D006152:Cyclic GMP