Endothelin Increases Myofilament Ca2+ Sensitivit in a-Toxin-Permeabilized Rabbit Mesenteric Artery

coccus a-toxin-permeabilized vascular smooth muscle. Rabbit small mesenteric arteries permeabilized with ar-toxin were mounted for isometric or isotonic force recording or were processed for determination of myosin light chain (MLC) phosphorylation levels. Addition of 100 nM ET-1 plus 10 ,uM GTP significantly enhanced myofilament Ca2' sensitivity as compared with the addition of Ca2' alone (EC50, 0.47 jgM Ca2' for Ca2' alone and 0.13 ,uM Ca2' for ET-1 plus GTP). This enhanced sensitivity was reversed by GDPf3S. ET-1-induced contractions were relaxed at a constant [Ca2"] by the addition of 30 ,uM cAMP or cGMP, demonstrating a direct effect of the cyclic nucleotides on contractile regulation. Inhibition of protein kinase C activity by 100 nM staurosporine relaxed ET-1 plus GTP-induced contractions, and pretreatment with 40 uM chelerythrine inhibited the ET-1 plus GTP increase in force. At 0.32 juM Ca2', steady-state levels of shortening velocity were not increased by ET-1 plus GTP, although steady-state levels of MLC phosphorylation were significantly enhanced. The ET-1-induced increase in MLC phosphorylation was not altered by changes in [Ca2+], whereas the shortening velocity was Ca21 dependent, suggesting that the increase MLC phosphorylation level may be the result of protein kinase C, rather than MLC kinase, activation. These results are consistent with the hypothesis that ET-1 increases myofilament Ca2' sensitivity by a G protein-dependent pathway and subsequent activation of protein kinase C. We propose that protein kinase C increases myofilament Ca2' sensitivity during ET-1 stimulation either by phosphorylation of a thin-filament regulatory protein or by downregulation of the MLC phosphatase. (Circulation Research 1992;71:951-959)