Regulation of Vascular Smooth Muscle Contraction. The Roles of Ca2+, Protein Kinase C ant Myosin Light Chain Phosphatase.

Ca2+-dependent myosin light chain (MLC) phosphorylation is an important step in the initiation of smooth muscle contraction. However, MLC phosphorylation alone cannot account for all aspects of contractile regulation, suggesting the involvement of other elements. In this article we present evidence obtained from Triton X-100 detergent skinned and intact tissue which demonstrates that vascular smooth muscle contraction can be initiated by a Ca2+-dependent mechanism that does not require prior MLC phosphorylation. We show that Ca2+ can initiate contractions supported by cytidine triphosphate (CTP) and that these contractions are inhibited by calmodulin antagonists, suggesting a Ca2+–calmodulin dependence of force distinct from that for MLC phosphorylation. Evidence is presented to demonstrate that carotid medial fibers contain a mitogen-activated protein (MAP) kinase which is activated by Ca2+ and may catalyze caldesmon phosphorylation. Based in part on our results and those of other investigators, we propose that direct Ca2+–calmodulin binding to caldesmon or phosphorylation of caldesmon by a Ca2+-dependent MAP kinase disinhibits caldesmon. Disinhibition of caldesmon allows an inherent basal level of actin-activated myosin ATPase activity to be expressed. The result is the slow development of force.Key words: mitogen-activated protein kinase, caldesmon, Triton X-100, detergent-skinned fibers, cytidine triphosphate, calmodulin.

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Purified rabbit brain protein kinase C relaxes skinned vascular smooth muscle and phosphorylates myosin light chain

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(87)90089-0 ◽

1987 ◽

Vol 254 (1) ◽

pp. 136-141 ◽

Cited By ~ 44

Author(s):

Masaki Inagaki ◽

Hisayuki Yokokura ◽

Takeo Itoh ◽

Yuichi Kanmura ◽

Hirosi Kuriyama ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Rabbit Brain ◽

Brain Protein ◽

Kinase C

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Phorbol ester-induced contraction in chemically skinned vascular smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1986.251.3.c356 ◽

1986 ◽

Vol 251 (3) ◽

pp. C356-C361 ◽

Cited By ~ 126

Author(s):

M. Chatterjee ◽

M. Tejada

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Phorbol Ester ◽

Light Chain ◽

Myosin Light Chain ◽

Phorbol Esters ◽

Myosin Light Chain Phosphorylation ◽

Kinase C

We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.

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Phosphatidylinositol 3-kinase modulates vascular smooth muscle contraction by calcium and myosin light chain phosphorylation-independent and -dependent pathways

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00497.2003 ◽

2004 ◽

Vol 286 (2) ◽

pp. H657-H666 ◽

Cited By ~ 25

Author(s):

Xiaoling Su ◽

Elaine M. Smolock ◽

Kristi N. Marcel ◽

Robert S. Moreland

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Muscle Contraction ◽

Myosin Light Chain ◽

Smooth Muscle Contraction ◽

Pi3 Kinase ◽

Kinase C ◽

Dependent Inhibition ◽

Mlc Phosphorylation

Regulation of smooth muscle contraction involves a number of signaling mechanisms that include both kinase and phosphatase reactions. The goal of the present study was to determine the role of one such kinase, phosphatidylinositol (PI)3-kinase, in vascular smooth muscle excitation-contraction coupling. Using intact medial strips of the swine carotid artery, we found that inhibition of PI3-kinase by LY-294002 resulted in a concentration-dependent decrease in the contractile response to both agonist stimulation and membrane depolarization-dependent contractions and a decrease in Ca2+-dependent myosin light chain (MLC) phosphorylation, the primary step in the initiation of smooth muscle contraction. Inhibition of PI3-kinase also depressed phorbol dibutyrate-induced contractions, which are not dependent on either Ca2+ or MLC phosphorylation but are dependent on protein kinase C. To determine the Ca2+-dependent site of action of PI3-kinase, we determined the effect of several inhibitors of calcium metabolism on LY-294002-dependent inhibition of contraction. These inhibitors included nifedipine, SK&F-96365, and caffeine. Only SK&F-96365 blocked the LY-294002-dependent inhibition of contraction. Interestingly, all compounds blocked the LY-294002-dependent inhibition of MLC phosphorylation. Our results suggest that activation of PI3-kinase is involved in a Ca2+- and MLC phosphorylation-independent pathway for contraction likely to involve protein kinase C. In addition, our results also suggest that activation of PI3-kinase is involved in Ca2+-dependent signaling at the level of receptor-operated calcium channels.

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Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2+ entry and Ca2+ sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697

Biochemical Journal ◽

10.1042/bj20050237 ◽

2005 ◽

Vol 389 (3) ◽

pp. 763-774 ◽

Cited By ~ 141

Author(s):

David P. Wilson ◽

Marija Susnjar ◽

Enikő Kiss ◽

Cindy Sutherland ◽

Michael P. Walsh

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Kinase Inhibitors ◽

Thromboxane A2 ◽

Myosin Light Chain Phosphatase ◽

Kinase C

The signal transduction pathway whereby the TxA2 (thromboxane A2) mimetic U-46619 activates vascular smooth muscle contraction was investigated in de-endothelialized rat caudal artery. U-46619-evoked contraction was inhibited by the TP receptor (TxA2 receptor) antagonist SQ-29548, the ROK (Rho-associated kinase) inhibitors Y-27632 and H-1152, the MLCK (myosin light-chain kinase) inhibitors ML-7, ML-9 and wortmannin, the voltagegated Ca2+-channel blocker nicardipine, and removal of extracellular Ca2+; the protein kinase C inhibitor GF109203x had no effect. U-46619 elicited Ca2+ sensitization in α-toxin-permeabilized tissue. U-46619 induced activation of the small GTPase RhoA, consistent with the involvement of ROK. Two downstream targets of ROK were investigated: CPI-17 [protein kinase C-potentiated inhibitory protein for PP1 (protein phosphatase type 1) of 17 kDa], a myosin light-chain phosphatase inhibitor, was not phosphorylated at the functional site (Thr-38); phosphorylation of MYPT1 (myosin-targeting subunit of myosin light-chain phosphatase) was significantly increased at Thr-855, but not Thr-697. U-46619-evoked contraction correlated with phosphorylation of the 20 kDa light chains of myosin. We conclude that: (i) U-46619 induces contraction via activation of the Ca2+/calmodulin/MLCK pathway and of the RhoA/ROK pathway; (ii) Thr-855 of MYPT1 is phosphorylated by ROK at rest and in response to U-46619 stimulation; (iii) Thr-697 of MYPT1 is phosphorylated by a kinase other than ROK under resting conditions, and is not increased in response to U-46619 treatment; and (iv) neither ROK nor protein kinase C phosphorylates CPI-17 in this vascular smooth muscle in response to U-46619.

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