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 (G(i) and G(q)) that are coupled to the type II kinin (BK2) receptor. Radioligand binding assays and Western analyses showed that GSNO (10-500 mu M, 0-72 h) did not alter the expression of BK2 receptor, G(i), or G(q). 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, N-omega-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 [S-35]GTP gamma S 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 G alpha(i2%), G alpha(i3), and G alpha(q/11) GTP binding activity by 93, 61, and 90%, respectively, whereas epinephrine-stimulated G alpha(s) GTP binding activity was unaffected. These results suggest that NO can modulate BK signaling pathways by selectively inhibiting G-proteins of the G(i) and G(q) family.