Involvement of botulinum C3-sensitive GTP-binding proteins in α1-adrenoceptor subtypes mediating Ca2+-sensitization

The mechanisms of α₁-adrenoceptor agonist-mediated sensitization of the contractile apparatus of smooth muscle to Ca²⁺ were studied in β-escin-permeabilized thoracic artreal smooth muscle of rabbit. Addition of norepinephrine (10 μM) plus guanosine 5′-triphosphate (GTP, 50 μM) significantly enhanced Ca²⁺ sensitivity as compared with the addition of 0.3 μM Ca²⁺ alone (pCa6.5). In β-escin-skinned smooth muscle of chloroethylclonidine-treated tissues, the enhancement of Ca²⁺-contraction produced by norepinephrine or clonidine was completely inhibited by guanosine 5′-O-(β-thiodiphosphate) (GDPβ-S, 1 mM). In addition, Clostridium botulinum C₃, which inactivates low molecular weight GTP-binding protein families, abolished norepinephrine- or clonide-induced Ca²⁺-sensitization, but did not affect clonidine-induced 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 α₁-adrenoceptor subtypes is linked via a low molecular weight GTP-binding protein (Rho), and the regulations of phosphorylation in contractile elements.