The role of the small heat shock proteins, HSP20 and HSP27, in cyclic nucleotide-dependent vasorelaxatio

Abstract

Vascular smooth muscle tone is controlled by a balance between the contractile and relaxation cellular signaling pathways. The signaling events leading to contraction include Ca2 + -dependent myosin light chain phosphorylation. The active relaxation of smooth muscle occurs through activation of cyclic nucleotide-dependent signaling pathways. In this study, the effect of activation of cyclic nucleotide signaling pathways in two muscles, bovine carotid and human umbilical artery smooth muscle, on serotonin-induced contraction was compared and correlated with phosphorylation events. Stimulation with-3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and forskolin, an adenylate cyclase activator, inhib~ted serotonin-induced contraction in bovine carotid artery smooth muscle but not in human umbilical artery sm~oth muscle. In carotid artery smooth muscle,. inhibition of contraction was associated with increases in the phosphorylation of HSP20. ln umbilical artery smooth muscle, the lack of inhibition of contraction was associated with a slow increase in the phosphorylation of HSP20. The delayed.increase in the phosphorylation ofHSP20 was,-associated with a delayed decrease in ~orce in the umbilical artery smooth muscle. In both carotid and umbilical artery smooth muscle contracted with serotonin and relaxed with IBMX/forskolin, the rate of relaxation corresponded to the rate of phosphorylation ofHSP20. The site on HSP20 molecule that is phosphorylated during cyclic nucleotide-dependent relaxation (serine16 ) was identified. Synthetic peptide analogs, with various modifications of the serine16 site, were introduced into transiently permeabilized muscles. Phosphorylated HSP20 peptides inhibited contractile responses, while non-phosphorylatable HSP20 peptides augmented contractile responses. Additionally, HSP20 may be regulated by interactions with another small heat shock protein, HSP27. More specifically, the phosphorylated · HSP27 was found to inhibit the phosphorylation of HSP20 by PKA _in vitro.· Finally, stimulation with IBMX/forskolin did not inhibit serotonin-induced myosin light chain phosphorylation or oxygen consumption in bovine carotid artery smooth muscle. Taken together these data suggest that activation of cyclic nucleotide-dependent signaling pathways mediate vascular smooth muscle relaxation via increases in the phosphorylation of HSP20. Our results indicate that the mechanisms of cyclic nucieotide-dependent relaxation are independent of the Ca2+ -dependent phosphorylation of myosin light chains and activation of the actomyosin ATPase, i.e. crossbridge cycling.