Selected Contribution: HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid media tone

Abstract

Cyclic nucleotide-induced relaxation of maximally activated arterial smooth muscle has two phases. 1) The initial relaxation transient is typically characterized by a rapid reduction in force associated with brief reductions in myoplasmic Ca(2+) concentration ([Ca(2+)](i)) and myosin regulatory light chain (MRLC) phosphorylation on serine (Ser)-19 (Ser(19)). 2) The sustained inhibitory response is typically associated with Ser(16) phosphorylation of heat shock protein 20 (HSP20) without sustained reductions in [Ca(2+)](i) or MRLC phosphorylation. We investigated whether the extent of Ser(16)-HSP20 phosphorylation quantitatively correlated with the sustained inhibitory response. With addition of nitroglycerin to histamine-stimulated swine carotid media, the initial relaxation transient was associated with a decrease in MRLC phosphorylation without an increase in Ser(16)-HSP20 phosphorylation. During the sustained phase of nitroglycerin-induced relaxation and during force redevelopment induced by washout of nitroglycerin in the continued presence of histamine, the level of Ser(16)-HSP20 phosphorylation, but not MRLC phosphorylation, correlated with inhibition of force. Forskolin, which increases cAMP concentration, also induced a sustained inhibitory response that was associated with increases in Ser(16)-HSP20 phosphorylation without reductions in MRLC phosphorylation levels. Forskolin increased Ser(16)-HSP20 phosphorylation to a greater extent and inhibited force more completely than that observed with nitroglycerin. Increases in Ser(16)-HSP20 phosphorylation correlated with the degree of force inhibition regardless of whether the relaxation was induced by nitroglycerin or forskolin. These data are consistent with the hypothesis that Ser(16)-HSP20 phosphorylation may be a cyclic nucleotide-dependent, yet MRLC phosphorylation-independent, inhibitor of smooth muscle contractile force.

Essential role of EDHF in the initiation and maintenance of adrenergic vasomotion in rat mesenteric arteries

“…Our present results with removal of the endothelium as well as buffering of Ca 2ϩ in ECs could be taken to suggest that NO and cGMP are essential for vasomotion. Elevation of cytosolic [Ca 2ϩ ] in ECs facilitates the binding of calmodulin to an endothelial NO synthase signaling complex, thus increasing production of NO (6,11,23,25), and NO can exert vasodilatory influence either directly or via downstream activation of soluble G cyclase and cGMP accumulation in SMCs (2,3,14,19,22,29,31,38,39). Indeed, it has been suggested (35) that a rise in smooth muscle cGMP concentration, as a result of the action of NO derived from ECs, is essential to the development of vasomotion.…”

Tissue length modulates “stimulated actin polymerization,” force augmentation, and the rate of swine carotid arterial contraction

“…Cross-bridge (S19-myosin regulatory light chain) phosphorylation was determined in swine common carotid artery rings mounted isometrically at 1.0 L o and then treated to obtain forces similar to those observed in the stiffness experiments (22) described above. At goal force, rings were frozen in acetone-dry ice and homogenized, and the level of cross-bridge phosphorylation was determined by isoelectric focusing and immunoblotting, as described elsewhere (21). Three dilutions of homogenates were loaded to ensure that the enhanced chemiluminescence detection system was in the linear range (20).…”

Force augmentation and stimulated actin polymerization in swine carotid artery

“…The level of crossbridge (Ser 19 -MRLC) phosphorylation was determined in the same homogenates by isoelectric focusing and immunoblotting as described (24). Three dilutions of homogenates were loaded to ensure that the enhanced chemiluminescence detection system was in the linear range (23).…”