Contractile Responses and Myosin Phosphorylation in Reconstituted Fibers of Smooth Muscle Cells From the Rat Cerebral Artery

Abstract

ABSTRACT-String-shaped reconstituted smooth muscle fibers were prepared in rectangular wells by thermal gelation of a mixed solution of collagen and cultured smooth muscle cells derived from the rat cerebral artery. The fibers contracted in response to KCl, 5-hydroxytryptamine (5-HT), noradrenaline, endothelin-1, endothelin-2, angiotensin II, prostaglandin F 2= and prostaglandin E 2 . 5-HT-induced contraction was partially inhibited by the L-type voltage-dependent Ca 2+ channel inhibitor nifedipine, putative non-selective cationic channel inhibitor SKF96365 and intracellular Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), and completely abolished by the myosin light chain kinase inhibitor ML-9. The fibers pre-contracted by 5-HT were completely relaxed by the Rho kinase inhibitor Y-27632, serine /threonine kinase inhibitor staurosporine, 8-bromo cyclic GMP and papaverine, and partially relaxed by dibutyryl cyclic AMP. Moreover, 5-HT as well as endothelin-1 and KCl enhanced 20-kDa myosin light chain phosphorylation in the fibers. These results suggested that the characteristics of contraction of the fibers reflect typical contractilities of vascular smooth muscle tissues. This technique will allow us to directly address questions relating to heterogeneity of receptor mechanisms and intracellular pathways of vascular smooth muscle contraction as a function of vessel type.

Angiotensin II–Induced Vascular Dysfunction Is Mediated by the AT 1A Receptor in Mice

“…However, responses to 5-HT were markedly enhanced in the AT 1A ϩ/ϩ mice (but not the AT 1A Ϫ/Ϫ mice) after Ang II infusion ( Figure 4A). Because 5-HT can cause contraction through RhoA/Rho-kinase signaling, 20,21 we repeated the 5-HT concentration-response experiment after a 30-minute incubation with the Rho-kinase inhibitor Y-27632 (1 mol/L) and found that it attenuated the response to 5-HT in Ang IIinfused mice (and Ang II-infused AT 1A Ϫ/Ϫ mice; Figure 4A). Y-27632 also significantly reduced 5-HT contraction in the vehicle-infused AT 1A ϩ/ϩ and AT 1A Ϫ/Ϫ mice, confirming the importance of Rho-kinase in 5-HT-induced contraction.…”