Endothelin-1-Induced Phosphorylation of the 20-kDa Myosin Light Chain and Caldesmon in Porcine Coronary Artery Smooth Muscle.

Abstract

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

Endothelin-1–Induced Enhancement of Coronary Smooth Muscle Contraction via MAPK-Dependent and MAPK-Independent [Ca 2+ ] i Sensitization Pathways

“…24,44 This is also supported by a study showing that ET-1 induces caldesmon phosphorylation in porcine coronary artery. 45 However, a clear relation between the ET-1-induced caldesmon phosphorylation and the smooth muscle contraction could not be conclusively demonstrated in that study. 45 Small concentrations of PGF 2␣ (10 Ϫ7 mol/L) alone caused cell contraction that was associated with a significant increase in [ 29 -31 Although the enhanced PGF 2␣ contraction in cells pretreated with ET-1 was not associated with additional increases in [Ca 2ϩ ] i , it seemed to require extracellular Ca 2ϩ because it was completely inhibited by verapamil.…”

“…45 However, a clear relation between the ET-1-induced caldesmon phosphorylation and the smooth muscle contraction could not be conclusively demonstrated in that study. 45 Small concentrations of PGF 2␣ (10 Ϫ7 mol/L) alone caused cell contraction that was associated with a significant increase in [ 29 -31 Although the enhanced PGF 2␣ contraction in cells pretreated with ET-1 was not associated with additional increases in [Ca 2ϩ ] i , it seemed to require extracellular Ca 2ϩ because it was completely inhibited by verapamil. Also, the enhancement of PGF 2␣ contraction by ET-1 seems to involve PKC because (1) direct activation of PKC by PMA caused similar enhancement of PGF 2␣ contraction and (2) the enhancement of PGF 2␣ -induced contraction by ET-1 or PMA was inhibited by the PKC inhibitor calphostin C. Thus, the enhanced PGF 2␣ -induced contraction in tissues pretreated with ET-1 seems to require both Ca 2ϩ and PKC, which raises the possibility that it involves a Ca 2ϩ -dependent PKC isoform.…”

Interaction between endothelin and vasodilators in the human internal mammary artery.

“…In addition, GTN has been shown to reverse completely the ET-induced phosphorylation of myosin light chain in coronary artery smooth muscle [52]. However, despite the fact that nitroprusside-induced relaxation in the human IMA has been studied [53], the effect of GTN on ET-1-induced contraction has not been investigated.…”

Smooth muscle function and myosin polymerization

“…Review of the literature reveals that 10-20% of MLC phosphorylation is regularly observed in relaxed smooth muscle with no active tension (Gerthoffer and Murphy, 1983;Silver and Stull, 1984;Hathaway and Haeberle, 1985;Ratz et al, 1989;Ekmehag and Hellstrand, 1989;Fischer and Pfitzer, 1989;Adam et al, 1990;Abe et al, 1991;Washabau et al, 1994;Taggart et al, 1997;Mehta et al, 1998;Chitano et al, 2005). The fact that a significant portion of MLC could be phosphorylated without causing tension development may be related to a threshold of MLC phosphorylation that must be overcome during muscle activation (Warshaw et al, 1990), perhaps due to a cooperative mechanism suggested by Rembold et al (2004).…”