THE MONOMERIC G-PROTEINS RAC1 AND OR CDC42 ARE REQUIRED FOR THE INHIBITION OF VOLTAGE-DEPENDENT CALCIUM CURRENT BY BRADYKININ/

Although regulation of voltage-dependent calcium current (I-Ca,I-V) by neurotransmitters is a ubiquitous mechanism among nerve cells, the si gnaling pathways involved are not well understood. We have determined previously that in a neuroblastoma-glioma hybrid cell line (NG108-15), the heterotrimeric G-protein G(13) mediates the inhibition of I-Ca,I- V produced by bradykinin (BK) via an unknown mechanism. Various report s indicate that G(13) can couple to RhoA, Rac1, and Cdc42, which are c losely related members of the Rho family of monomeric G-proteins. We h ave investigated their role as signaling intermediates in the pathway used by BK to inhibit I-Ca,I-V. Using immunoblot analysis and the PCR, we found evidence that RhoA, Rad, and Cdc42 all are expressed in NG10 8-15 cells. Intracellularly perfused recombinant Rho-GDI (an inhibitor of guanine nucleotide exchange specific for the Rho family) attenuate d the inhibition of I-Ca,I-V by BK. These findings indicate that activ ation of RhoA, Rac1, or Cdc42 may be required for the response to BK. To determine whether any of these monomeric G-proteins mediate the res ponse to BK, we have intracellularly applied blocking antibodies speci fic for each of the candidate proteins. Only the anti-Rac1 antibody bl ocked the response to BK. In parallel experiments, peptides correspond ing to the C-terminal regions of Rac1 and Cdc42 blocked the same respo nse. These data indicate a novel functional contribution of Rac1 and p ossibly also of Cdc42 to the inhibition of I-Ca,I-V by neurotransmitte rs.