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 (V(m)) and intracellular Ca(2+) 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 activating bradykinin receptor type 2 (B(2)R) and Ca(2+)-dependent Cl(-) channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 μM). The BK-induced Ca(2+) signal was blocked by the B(2)R inhibitor HOE 140. [Des-Arg(9)]-bradykinin, an activator of B(1)R, had no effect on intracellular Ca(2+). NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K(+) 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 BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca(2+) 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 μM) completely inhibited the BK-induced Ca(2+) signaling. UTP, another activator of the PLC-mediated Ca(2+) 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 B(2)R signaling pathway, NP may also positively interact in pathological conditions caused by BK.
Department of Physiology, School of Medicine, Croatian Institute for Brain Research, University of Zagreb, Zagreb, Croatia.
Marina
M
Dobrivojević
Aleksandra
A
Sinđić
Bayram
B
Edemir
Stefanie
S
Kalweit
Wolf-Georg
WG
Forssmann
Jochen R
JR
Hirsch
D001896:Boron Compounds; D001920:Bradykinin / Q000031:analogs & derivatives / Q000494:pharmacology*; D065094:Bradykinin B2 Receptor Antagonists; D002227:Carbazoles / Q000494:pharmacology; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000037:antagonists & inhibitors; D008564:Membrane Potentials / Q000187:drug effects; D045265:Natriuretic Peptides / Q000494:pharmacology*; D009579:Nitrobenzoates / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D020710:RGS Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D001896:Boron Compounds; D065094:Bradykinin B2 Receptor Antagonists; D002227:Carbazoles; D018118:Chloride Channels; D004963:Estrenes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D045265:Natriuretic Peptides; D009579:Nitrobenzoates; D026902:Potassium Channel Blockers; D047428:Protein Kinase Inhibitors; D011760:Pyrrolidinones; D020710:RGS Proteins; D013873:Thionucleotides; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C073601:KT 5823; C083763:8-bromoguanosino-3',5'-cyclic monophosphorothioate; C046722:bradykinin, des-Arg(1)-; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; C065679:icatibant; C109986:2-aminoethoxydiphenyl borate; D010738:Type C Phospholipases; D006152:Cyclic GMP; D001920:Bradykinin