Characterization of a B2-bradykinin receptor in human glomerular podocytes

Abstract

We characterized bradykinin (BK) receptors in a human line of glomerular visceral epithelial cells (hGVEC) transfected by the SV40 virus. [3H]BK bound specifically in a manner consistent with a single high-affinity site. Scatchard analysis yielded dissociation constant and maximum binding values of 0.28 +/- 0.04 nM and 76.6 +/- 4.9 fmol/mg, respectively. Competition binding studies with selective BK type 2 (Hoe-140) receptor antagonist and type 1 ([des-Arg9]BK) receptor agonist showed that hGVEC only expressed type 2 receptors, and this was confirmed by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay. BK stimulated intracellular calcium ion concentration ([Ca2+]i) release in a dose-dependent manner with a threshold at 1 nM. Hoe-140, in contrast with [des-Arg9]BK, abolished this effect. [Ca2+]i stimulation was also inhibited by thapsigargin, an inhibitor of Ca(2+)-adenosinetriphosphatase. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid attenuated but did not suppress the [Ca2+]i peak. These results associated with the stimulatory effect of BK on inositol phosphate production indicated that [Ca2+]i stimulation was produced both by [Ca2+] mobilization from its intracellular stores and by [Ca2+] entry into the cells. In conclusion, hGVEC express specific type 2 BK receptors that enable specific BK-induced responses.