INVOLVEMENT OF BOTULINUM C-3-SENSITIVE GTP-BINDING PROTEINS IN ALPHA(1)-ADRENOCEPTOR SUBTYPES MEDIATING CA2+-SENSITIZATION

The mechanisms of alpha(1)-adrenoceptor agonist-mediated sensitization of the contractile apparatus of smooth muscle to Ca2+ were studied in beta-escin-permeabilized thoracic arterial smooth muscle of rabbit. Addition of norepinephrine (10 mu M) plus guanosine 5'-triphosphate (GTP, 50 eta M) significantly enhanced Ca2+ sensitivity as compared with the addition of 0.3 mu M Ca2+ alone (pCa6.5). In beta-escin-skinned smooth muscle of chloroethylclonidine-treated tissues, the enhancement of Ca2+-contraction produced by norepinephrine or clonidine was completely inhibited by guanosine 5'-0-(beta-thiodiphosphate) (GDP beta-S, 1 mM). In addition, Clostridium botulinum C-3, which inactivates low molecular weight GTP-binding protein families, abolished norepinephrine- or clonidine-induced Ca2+-sensitization, but did not affect clonidine-induced translocation of protein kinase C to the membrane. The norepinephrine-enhanced Ca2+ sensitivity was partially reversed by a pretreatment with a selective myosin light chain kinase inhibitor (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9,10-tetrahydro-8,11-epoxy,1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926, 500 nM), but those of clonidine and in the chloroethylclonidine-treated tissues norepinephrine were not. These results suggest that Ca2+-sensitization produced by the activation of the alpha(1)-adrenoceptor subtypes is linked via a low molecular weight GTP-binding protein (Rho), and the regulations of phosphorylation in contractile elements.