Dobrivojevic´M, Sin ic´A, Edemir B, Kalweit S, Forssmann WG, Hirsch JR. Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells. In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential (Vm) and intracellular Ca 2ϩ using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells

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... activating bradykinin receptor type 2 (B2R) and Ca 2ϩ -dependent Cl Ϫ channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 M). The BK-induced Ca 2ϩ signal was blocked by the B2R inhibitor HOE 140. [Des-Arg 9 ]-bradykinin, an activator of B1R, had no effect on intracellular Ca 2ϩ . NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K ϩ channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BKtriggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca 2ϩ signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 M) completely inhibited the BK-induced Ca 2ϩ signaling. UTP, another activator of the PLC-mediated Ca 2ϩ signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B2R signaling pathway, NP may also positively interact in pathological conditions caused by BK. 8-Br-cGMP; PKG; bradykinin type 2 receptor; signaling Address for reprint requests and other correspondence: J. R. Hirsch, Pharis Biotec,