Endothelin-1 (ET), a potent vasoconstrictor, induces a sustained in crease in the phosphorylation level of the 20-kDa myosin light chain (MLC) in por cine coronary artery strips. ET also induces late phosphorylation of caldesmon, which is mimicked by 12-deoxyphorbol 13-isobutyrate, but not by 60 mM KC1. Nitroglycerin, a vasorelaxant, completely reverses the ET-induced phosphorylation of MLC, but not that of caldesmon. These results suggest an important regulatory role of MLC phos phorylation in ET-induced contraction.Endothelin-1 (ET) is one of the most potent vasoconstrictors to be discovered (1). Our pre vious studies (2-4) as well as observations by other investigators (5) demonstrate that ET receptor activation in vascular smooth muscle leads to the activation of phospholipase C and Ca 2+ channels, resulting in an increase in the intracellular free Ca 2+ concentration and the activation of protein kinase C. However, it is not known how ET-1 activates the contractile mechanism following the generation of the second messengers. To understand in more detail the mechanism of the ET-induced con traction of vascular smooth muscle, phos phorylation changes of the proteins thought to be involved in the regulation of contraction were explored in porcine coronary smooth muscle. Particularly, we studied the time-de pendent changes in the extent of the Ca 2+ and calmodulin-dependent phosphorylation of 20-kDa myosin light chain (MLC), which is known to serve as a specific signal for initiat ing cross bridge cycling (6), and phosphoryla tion of caldesmon, a calmodulin-binding, thin filament-associated protein (7,8).Porcine right coronary arteries were dis sected and carefully cleared of adhering con nective tissue (2, 4). Transverse arterial strips of approximately 2-mm width were prepared for the experiments. Strips were mounted in 10-ml static muscle chambers and equilibrated in modified Krebs-Henseleit bicarbonate buf fer containing 10-5 M phentolamine and 10-6 M atropin (9) aerated with 95% 02/5% CO2 at 37°C. Tension was isometrically measured with a Nihon Kohden TB-611T force-displace ment transducer and displayed on a Nihon Kohden WT-647G recorder. The tension in re sponse to agonist stimulation was expressed as a percent of the maximal response to KCl (110 mM). To measure the phosphorylation of 20
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