INVOLVEMENT OF HETEROTRIMERIC GTP-BINDING PROTEIN AND RHO-PROTEIN, BUT NOT PROTEIN-KINASE-C, IN AGONIST-INDUCED CA2-PIG VAS-DEFERENS( SENSITIZATION OF SKINNED MUSCLE OF GUINEA)

We studied the involvement of protein kinase C (PKC) and a small GTP-b inding protein (G-protein), rho, in receptor-mediated Ca2+ sensitizati on of the contractile apparatus of smooth muscle of guinea pig vas def erens. In beta-escin-permeabilized smooth muscle strips, norepinephrin e (NE) in the presence of GTP caused further contraction of the prepar ations at a constant Ca2+ level (Ca2+ sensitization). Prazosin and GDP (beta)S, a nonhydrolyzable GDP analogue, inhibited NE-induced Ca2+ sen sitization, indicating an alpha-1 adrenoceptor/G-protein mediated resp onse. GTP alone (>10 mu M) and GTP(gamma)S, a nonhydrolyzable GTP anal ogue, also induced Ca2+ sensitization. Pretreatment of preparations wi th C3 exoenzyme of Clostridium botulinum, which is known to ADP-ribosy late rho family proteins, with NAD resulted in complete inhibition of NE- and GTP (GTP(gamma)S)-induced Ca2+ sensitization. AIF(4)(-), which activates heterotrimeric G-, but not small G-protein also induced Ca2 + sensitization. interestingly, AIF(4)(-)-induced Ca2+ sensitization w as inhibited by not only GDP(beta)S but also C3-treatment, suggesting that activation of heterotrimeric GTP-binding protein precedes activat ion of rho protein. On the other hand, phorbol 12,13-dibutyrate, like NE, also induced Ca2+ sensitization. The sensitization was inhibited b y PKC(19-31), a PKC inhibitor peptide. However, PKC(19-31) did not hav e any effect on NE- or AIF(4)(-)-induced Ca2+ sensitization. These res ults suggested that NE induces Ca2+ sensitization of the contractile a pparatus of smooth muscle of guinea pig vas deferens by activation of alpha,-adrenoceptor-coupled heterotrimeric G-protein, which in turn ac tivates rho protein, and that PKC has little, if any role in the sensi tization under the present experimental conditions.