EVIDENCE FOR EXISTENCE OF 2 DISTINCT BRADYKININ RECEPTORS ON RAT MESANGIAL CELLS

We investigated the possible presence of bradykinin (BK) B1 receptor on rat mesangial cells (MC) by binding studies and by the effect of the B1 agonist des-Arg9-BK on intracellular calcium concentration ([Ca2+]i) and DNA synthesis in comparison with the effects of BK. Binding studies demonstrated specific, saturable binding for des-Arg9-[H-3]BK inhibited by B1 but not by B2 antagonists. Scatchard analysis revealed a single class of B1 binding site with a maximum density of 15 fmol/mg protein and an affinity of 8.7 +/- 2.4 nM. Saturation and competition studies of I-125-[Tyr0]BK demonstrated the presence of two classes of B2 binding sites [dissociation constant (K(d)) = 0.1 and 4 nM, respectively]. On fura-2-loaded adherent MC, both des-Arg9-BK and BK induced a biphasic increase (a transient enhancement followed by a sustained phase) in [Ca2+]i, both in primary culture and in cloned MC. Both the transient and sustained phases of [Ca2+]i induced by des-Arg9-BK were dose dependent, whereas BK induced a transient dose-dependent rise in [Ca2+]i, but the sustained phase remained constant. The increases in [Ca2+]i induced by des-Arg9-BK and BK were specifically abolished by B1 and B2 receptor antagonists, respectively, and showed homologous but not heterologous desensitization. Des-Arg9-BK and BK induced a significant proliferation (tested by cell counting and [H-3]thymidine incorporation) of quiescent MC. Furthermore, the effects of des-Arg9-BK and BK were additive on Ca2+ mobilization but not on mitogenesis. The mitogenic effects were enhanced on the addition of insulin (5 mug/ml) by over 85% and were prevented by prior treatment with des-Arg9-[Leu8]BK and [D-Arg,Hyp3,D-Phe7]BK, respectively. The results provide evidence for the coexistence of B1 and B2 receptors on MC and indicate a reinforcement of the effects of BK and des-Arg9-BK by insulin.