calyculin-a and Hypertension

The role of Ca(2+) sensitization induced by a Ca(2+)-independent myosin light chain kinase (MLCK) in hypertension has not been determined. The aim of this study was to clarify the role of possible Ca(2+)-independent MLCK activity in hypertension.. We compared increases in contractile force and phosphorylation of myosin light chain (MLC) evoked by calyculin A, a phosphatase inhibitor, in β-escin-permeabilized mesenteric arteries at pCa 9.0 between spontaneously hypertensive rat (SHR) and Wistar Kyoto rat (WKY). We found that there was no detectable phosphorylation of MLC at pCa 9.0, but that the administration of 1 μM calyculin A gradually increased force and mono- and di-phosphorylation of MLC. This contraction was inhibited by staurosporine but not by wortmannin, Y-27632, or calphostin-C. The calyculin A-induced contraction was significantly greater in the SHR than in the WKY and was associated with an increase in mono- and di-phosphorylation of MLC. SM-1, a zipper-interacting protein kinase (ZIPK)-inhibiting peptide, significantly inhibited the amplitude of the calyculin A-induced contraction and di-phosphorylation. Total ZIPK expression (54 + 32 kDa) was greater in the SHR than in the WKY. Phosphorylation of myosin phosphatase target subunit at Thr(697), but not at Thr(855), was consistently stronger in the SHR than in the WKY in calyculin A-treated tissues at pCa 9.0.. Our results suggest that Ca(2+)-independent MLCK activity is enhanced in the SHR, and that ZIPK plays, at least in part, an important role as a candidate for this kinase in rat mesenteric arteries.

Topics: Analysis of Variance; Animals; Apoptosis Regulatory Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Death-Associated Protein Kinases; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypertension; Male; Marine Toxins; Mesenteric Arteries; Muscle Proteins; Muscle, Smooth, Vascular; Myosin Light Chains; Myosin-Light-Chain Kinase; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Phosphatase 1; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Up-Regulation; Vasoconstriction; Vasoconstrictor Agents

We compared baseline and protein kinase A (PKA)-dependent troponin I (TnI) phosphorylation in 32Pi-labeled left ventricular myocytes from hearts of 26-wk spontaneously hypertensive rats (SHR) and Wistar-Kyoto controls (WKY). TnI phosphorylation was normalized to myosin light chain 2 phosphorylation, which was invariant. There was no difference in baseline TnI phosphorylation in SHR and WKY, but stimulation with isoproterenol, norepinephrine plus prazosin, forskolin, chloroadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine caused a greater increase in TnI phosphorylation in the SHR than in the WKY. This was observed both in the presence and absence of the phosphatase inhibitor calyculin A; thus the differences in TnI phosphorylation between SHR and WKY are not due to decreased phosphatase activity in the SHR. After stimulation of the beta-adrenergic pathway, phospholamban phosphorylation was not different in SHR and WKY, indicating that the observed differences may be specific for PKA phosphorylation of TnI. The increased PKA-dependent TnI phosphorylation in the SHR resulted in decreased Ca2+ sensitivity of actomyosin adenosinetriphosphatase activity as compared with the WKY. We conclude that increased PKA-dependent TnI phosphorylation in the SHR may contribute to the impaired response to sympathetic stimulation.

Topics: 1-Methyl-3-isobutylxanthine; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; 8-Bromo Cyclic Adenosine Monophosphate; Adrenergic beta-Agonists; Animals; Calcium; Calcium-Binding Proteins; Cells, Cultured; Colforsin; Cyclic AMP-Dependent Protein Kinases; Electric Stimulation; Enzyme Inhibitors; Heart; Heart Ventricles; Hypertension; Isoproterenol; Marine Toxins; Myocardial Contraction; Myocardium; Myosin Light Chains; Norepinephrine; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Prazosin; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Troponin I