We investigated the role of myosin light chain kinase (MLCK) phosphorylation in regulating the sensitivity of vascular smooth muscle myosin light chain (MLC) phosphorylation to intracellular Ca2+ concentration ([Ca2+]i). 32PO4-loaded swine carotid arteries were stimulated with histamine or high K+, MLCK was isolated, and the relative phosphorylation of tryptic peptides was measured. In nonlabeled tissues, we measured [Ca2+]i with aequorin, MLCK activity ratio, MLC phosphorylation, and force. A comparison of MLCK phosphorylation on peptide A (mol P in site A/mol MLCK) and MLCK activity ratio showed an inverse relation, suggesting that MLCK site A phosphorylation can regulate the Ca2+ sensitivity of MLCK. MLCK site A phosphorylation and MLCK activity ratio depended on [Ca2+]i. Histamine stimulation yielded greater MLC phosphorylation than high K+ stimulation over a range of [Ca2+]i; however, there were no apparent stimulus-dependent differences in MLCK phosphorylation, suggesting that stimulus-dependent differences in the Ca2+ sensitivity of MLC phosphorylation are not based on differences in MLCK phosphorylation. We also determined whether MLCK phosphorylation was involved in adenosine 3',5'-cyclic monophosphate-mediated relaxation. In histamine-contracted tissues, forskolin decreased [Ca2+]i, MLC phosphorylation, and force. MLCK phosphorylation decreased to an extent consistent with the decrease in [Ca2+]i. In KCl-stimulated tissues, forskolin did not alter [Ca2+]i or increase MLCK phosphorylation but forskolin did decrease MLC phosphorylation. Thus, in swine carotid artery, MLCK phosphorylation appears to be regulated exclusively by Ca2+ and plays little role in stimulus-dependent differences in Ca2+ sensitivity of MLC phosphorylation or in mediating forskolin-induced relaxation.
Nitrovasodilators are hypothesized to induce smooth muscle relaxation by their metabolism to nitric oxide, which then activates soluble guanylyl cyclase, increases [cGMP], and activates cGMP-dependent protein kinase. cGMP-dependent phosphorylation is then proposed to decrease intracellular [Ca2+] ([Ca2+]i) and to reduce the Ca(2+)-sensitivity of contraction. We hypothesized that one component of decreased Ca(2+)-sensitivity, reduced Ca(2+)-sensitivity of MLC phosphorylation, was due to phosphorylation of myosin light chain kinase (MLCK) on the peptide site A. In the swine carotid artery, histamine (10 microM) stimulation increased aequorin-estimated [Ca2+]i, MLCK site A phosphorylation, MLC phosphorylation, and force. Subsequent addition of 100 microM nitroglycerin (NTG) or 100 microM sodium nitroprusside (NP) to histamine-stimulated tissues increased [cGMP], decreased both MLC phosphorylation and force, but did not significantly alter [cAMP], [Ca2+]i, or MLCK site A phosphorylation. Addition of NTG and NP alone to unstimulated tissues increased MLCK site A phosphorylation, but did not alter [Ca2+]i. In tissues preincubated with NP, subsequent histamine contraction was slowed compared with controls, however, this slowed rate of contraction appeared to result from an attenuation of histamine-dependent increases in [Ca2+]i. These data suggest that, in swine carotid artery, nitrovasodilators can decrease the Ca(2+)-sensitivity of MLC phosphorylation without increasing MLCK site A phosphorylation. Nitrovasodilators, per se, can induce site A MLCK phosphorylation, potentially by cGMP dependent activation of cAMP-dependent protein kinase.
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