Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation.

We investigated whether myosin light chain phosphatase activity changes during nitric oxide-induced relaxation of contracted intact carotid media and how changes in phosphatase activity mediate this relaxation. We also investigated one mechanism for regulating this phosphatase. Myosin phosphatase activity, myosin light chain phosphorylation, guanosine 3',5'-cyclic monophosphate (cGMP) concentration, and phosphorylation of the inhibitory protein CPI-17 were all assayed in homogenates of one carotid media ring at each time point during nitric oxide-induced relaxation. The application of sodium nitroprusside to histamine-contracted media caused rapid declines in light chain phosphorylation and force. These were temporally correlated with a rapid elevation of cGMP and a large transient increase in myosin phosphatase activity. During the early response to nitroprusside, when force declined, increases in myosin phosphatase activity, concurrent with cGMP-mediated decreases in calcium and myosin light chain kinase activity, could accelerate light chain dephosphorylation. CPI-17 was dephosphorylated upon application of nitroprusside at the same time that myosin phosphatase activity increased, suggesting that the removal of inhibition by phospho-CPI-17 contributed to the increase in myosin phosphatase activity. After 20 min of nitroprusside, myosin phosphatase activity had declined to basal levels, however low force was sustained. Additional light chain phosphorylation-independent mechanisms may be involved in sustaining the relaxation.     

M-RIP targets myosin phosphatase to stress fibers to regulate myosin light chain phosphorylation in vascular smooth muscle cells

Vascular smooth muscle cell contraction and relaxation are directly related to the phosphorylation state of the regulatory myosin light chain. Myosin light chains are dephosphorylated by myosin phosphatase, leading to vascular smooth muscle relaxation. Myosin phosphatase is localized not only at actin-myosin stress fibers where it dephosphorylates myosin light chains, but also in the cytoplasm and at the cell membrane. The mechanisms by which myosin phosphatase is targeted to these loci are incompletely understood. We recently identified myosin phosphatase-Rho interacting protein as a member of the myosin phosphatase complex that directly binds both the myosin binding subunit of myosin phosphatase and RhoA and is localized to actin-myosin stress fibers. We hypothesized that myosin phosphatase-Rho interacting protein targets myosin phosphatase to the contractile apparatus to dephosphorylate myosin light chains. We used RNA interference to silence the expression of myosin phosphatase-Rho interacting protein in human vascular smooth muscle cells. Myosin phosphatase-Rho interacting protein silencing reduced the localization of the myosin binding subunit to stress fibers. This reduction in stress fiber myosin phosphatase-Rho interacting protein and myosin binding subunit increased basal and lysophosphatidic acid-stimulated myosin light chain phosphorylation. Neither cellular myosin phosphatase, myosin light chain kinase, nor RhoA activities were changed by myosin phosphatase-Rho interacting protein silencing. Furthermore, myosin phosphatase-Rho interacting protein silencing resulted in marked phenotypic changes in vascular smooth muscle cells, including increased numbers of stress fibers, increased cell area, and reduced stress fiber inhibition in response to a Rho-kinase inhibitor. These data support the importance of myosin phosphatase-Rho interacting protein-dependent targeting of myosin phosphatase to stress fibers for regulating myosin light chain phosphorylation state and morphology in human vascular smooth muscle cells.     

Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca²⁺-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.     

Targeting by myosin phosphatase-RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase-rho interacting protein, that directly binds to both RhoA and the myosin-binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase-rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase-rho interacting protein was required for RhoA/ROCK-mediated myosin phosphatase regulation. Myosin phosphatase-rho interacting protein silencing prevented LPA-mediated myosin-binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase-rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M-RIP lead to loss of stress fiber-associated RhoA, suggesting that myosin phosphatase-rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber.     

Dephosphorylation of myosin by the catalytic subunit of a type-2 phosphatase produces relaxation of chemically skinned uterine smooth muscle

It is now well-established that phosphorylation of the 20,000-dalton light chain of smooth muscle myosin (LC20) is a prerequisite for muscle contraction. However, the relationship between myosin dephosphorylation and muscle relaxation remains controversial. In the present study, we utilized a highly purified catalytic subunit of a type-2, skeletal muscle phosphoprotein phosphatase (protein phosphatase 2A) and a glycerinated smooth muscle preparation to determine if myosin dephosphorylation, in the presence of saturating calcium and calmodulin, would cause relaxation of contracted uterine smooth muscle. Addition of the phosphatase catalytic subunit (0.28 microM) to the muscle bath produced complete relaxation of the muscle. The phosphatase-induced relaxation could be reversed by adding to the muscle bath either purified, thiophosphorylated, chicken gizzard 20,000-dalton myosin light chains or purified, chicken gizzard myosin light chain kinase. Incubation of skinned muscles with adenosine 5'-O-(thiotriphosphate) prior to the addition of phosphatase resulted in the incorporation of 0.93 mol of PO4/mol of LC20 and prevented phosphatase-induced relaxation. Under all of the above conditions, changes in steady-state isometric force were associated with parallel changes in myosin light chain phosphorylation over a range of phosphorylation extending from 0.01 to 0.97 mol of PO4/mol of LC20. We found no evidence that dephosphorylation of contracted uterine smooth muscles, in the presence of calcium and calmodulin, could produce a latch-state where isometric force was maintained in the absence of myosin light chain phosphorylation. These results show that phosphorylation or dephosphorylation of the 20,000-dalton myosin light chain is adequate for the regulation of contraction or relaxation, respectively, in glycerinated uterine smooth muscle.     

Myosin regulatory light chain diphosphorylation slows relaxation of arterial smooth muscle

The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC(20)) at Ser(19) by Ca(2+)/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca(2+)-independent phosphorylation at both Ser(19) and Thr(18) by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr(18) on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser(19) and Thr(18) was achieved by treatment with adenosine 5'-O-(3-thiotriphosphate) in the presence of Ca(2+), which induced stoichiometric thiophosphorylation at Ser(19), followed by microcystin (phosphatase inhibitor) in the absence of Ca(2+), which induced phosphorylation at Thr(18). Phosphorylation at Thr(18) had no effect on steady-state force induced by Ser(19) thiophosphorylation. However, phosphorylation of Ser(19) or both Ser(19) and Thr(18) to comparable stoichiometries (0.5 mol of P(i)/mol of LC(20)) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t(½) values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser(19)) and diphosphorylated LC(20), respectively. We conclude that phosphorylation at Thr(18) decreases the rates of LC(20) dephosphorylation and smooth muscle relaxation compared with LC(20) phosphorylated exclusively at Ser(19). These effects of LC(20) diphosphorylation, combined with increased Ser(19) phosphorylation (Ca(2+)-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension.     

Telokin expression and the effect of hypoxia on its phosphorylation status in smooth muscle cells from small and large pulmonary arteries

Small pulmonary arteries (SPA), <500 microm diameter of the cat, constrict when exposed to hypoxia, whereas larger arteries (large pulmonary arteries; LPA), >800 microm diameter, show little or no response. It is unknown why different contractile responses occur within the same vascular bed, but activator or repressor proteins within the smooth muscle cell (SMC) can modify myosin phosphatase and myosin light chain kinase (MLCK), thereby influencing the phosphorylation state of myosin light chain (MLC) and ultimately, contraction. Telokin, a protein with a sequence identical to the COOH-terminal domain of MLCK, is expressed in smooth muscle where in its phosphorylated state it inhibits myosin phosphatase, binds to unphosphorylated myosin, and helps maintain smooth muscle relaxation. We measured telokin mRNA and telokin protein in smooth muscle from different diameter feline pulmonary arteries and sought to determine whether changes in the phosphorylation status of telokin and MLC occurred during hypoxia. In pulmonary arteries, telokin expression varied inversely with artery diameter, but cerebral arteries showed neither telokin protein nor telokin mRNA. Although telokin and MLC were distributed uniformly throughout the SPA muscle cell cytoplasm, they were not colocalized. During hypoxia, telokin dephosphorylated, and MLC became increasingly phosphorylated in SPA SMC, whereas in LPA SMC there was no change in either telokin or MLC phosphorylation. When LPA SMC were exposed to phenylephrine, MLC phosphorylation increased with no change in telokin phosphorylation. These results suggest that in SPA, phosphorylated telokin may help maintain relaxation under unstimulated conditions, whereas in LPA, telokin's function remains undetermined.     

Molecular mechanism of telokin-mediated disinhibition of myosin light chain phosphatase and cAMP/cGMP-induced relaxation of gastrointestinal smooth muscle

Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca(2+)-activated contraction and Ca(2+) sensitization of force induced by a GST-MYPT1(654-880) fragment inhibiting myosin light chain phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with telokin in SM cells. We conclude that the mechanism of action of phospho-telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.     

Preservation of cGMP-induced relaxation of pulmonary veins of fetal lambs exposed to chronic high altitude hypoxia: role of PKG and Rho kinase

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.     

Dynamic regulation of myosin light chain phosphorylation by Rho-kinase

Myosin light chain (MLC) phosphorylation plays important roles in various cellular functions such as cellular morphogenesis, motility, and smooth muscle contraction. MLC phosphorylation is determined by the balance between activities of Rho-associated kinase (Rho-kinase) and myosin phosphatase. An impaired balance between Rho-kinase and myosin phosphatase activities induces the abnormal sustained phosphorylation of MLC, which contributes to the pathogenesis of certain vascular diseases, such as vasospasm and hypertension. However, the dynamic principle of the system underlying the regulation of MLC phosphorylation remains to be clarified. Here, to elucidate this dynamic principle whereby Rho-kinase regulates MLC phosphorylation, we developed a mathematical model based on the behavior of thrombin-dependent MLC phosphorylation, which is regulated by the Rho-kinase signaling network. Through analyzing our mathematical model, we predict that MLC phosphorylation and myosin phosphatase activity exhibit bistability, and that a novel signaling pathway leading to the auto-activation of myosin phosphatase is required for the regulatory system of MLC phosphorylation. In addition, on the basis of experimental data, we propose that the auto-activation pathway of myosin phosphatase occurs in vivo. These results indicate that bistability of myosin phosphatase activity is responsible for the bistability of MLC phosphorylation, and the sustained phosphorylation of MLC is attributed to this feature of bistability.     

Phosphatase inhibition with okadaic acid does not alter the relationship between force and myosin light chain phosphorylation in permeabilized smooth muscle

The phosphatase inhibitor, okadaic acid, has been used to test the hypothesis that myosin light chain phosphatase activity plays a central role in latchbridge formation in smooth muscle. In the permeabilized rabbit portal vein there is a non-linear relationship between myosin light chain phosphorylation and force production such that maximum force output occurs with about 50% phosphorylation. Treatment of the muscle with okadaic acid does not change this relationship even though there is a profound inhibition of phosphatase activity. The data suggest that dephosphorylation of the myosin light chain while the myosin is in the force producing state does not account for the high force output with low levels of light chain phosphorylation in smooth muscle.     

Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age

We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cdelta) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cdelta contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.     

Atrial natriuretic peptide inhibits the agonist-induced increase in extent of myosin light chain phosphorylation in aortic smooth muscle

The effect of atrial natriuretic peptide (ANP) on angiotensin II- and histamine-induced contraction and muscle light chain phosphorylation was examined in strips of rabbit aorta smooth muscle. Preincubation of strips with 10(-7) M ANP prior to addition of either agonist inhibits both the increase in extent of myosin light chain phosphorylation and the contractile response to either 5 x 10(-8) M angiotensin II or 10(-5) M histamine without inhibiting the agonist-induced increase in the intracellular free Ca2+ concentration. Furthermore, in muscle strips precontracted with either angiotensin II or histamine, addition of ANP leads to a prompt relaxation and a prompt decrease in the extent of myosin light chain phosphorylation. These data argue that ANP uncouples the initial agonist-induced Ca2+ transient from the increase in extent of myosin light chain phosphorylation either by inhibiting the Ca2+-dependent activation of myosin light chain kinase or stimulating the activity of a phosphoprotein phosphatase capable of bringing about the rapid dephosphorylation of phosphorylated myosin light chains.     

Role of myosin light chain phosphorylation in the regulation of cytokinesis

Phosphorylation of regulatory light chain (RMLC) of myosin II at Ser19/Thr18 is likely to play important roles in controlling the morphological changes seen during cell division of cultured mammalian cells. Phosphorylation of RMLC regulates the activity of myosin II, an essntial motor for cytokinesis, and phosphorylation of RMLC shows dramatic changes during mitosis. Two exzymes, myosin phosphatase and kinase, control phosphorvlation of RMLC. Myosin phosphatase is activated during mitosis, apparently as a result of mitosis-specific phosphorylation of the myosin phosphatase targeting subunit (MYPT). This activation of myosin phosphatase is likely to result in RMLC dephosphorylation, causing the disassemly of stress fibers and focal adhesions during prophase. The phosphorylation of MYPT is lost in cyotokinesis, which would decrease myosin phosphatase activity. At the same time, ROCK (Rho-kinase) probably phosphorylates MYPT at its inhibitory sites, further decreasing the activity of myosin phosphatase. These changes in MYPT phosphorylation would raise RMLC phosphorylation, leading to the activation of myosin II for cyotokinesis. RMLC phosphorylation is also regulated by several RMLC kinases including ROCK (Rho-kinase), MLCK and citron kinase, all of which are localized at cleavage furrows. Future studies should examine whether these multiple kinases are redundant or whether they control distinct aspects of cell division.     

The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ

The mammalian MYPT family consists of the products of five genes, denoted MYPT1, MYPT2, MBS85, MYPT3 and TIMAP, which function as targeting and regulatory subunits to confer substrate specificity and subcellular localization on the catalytic subunit of type 1δ protein serine/threonine phosphatase (PP1cδ). Family members share several conserved domains, including an RVxF motif for PP1c binding and several ankyrin repeats that mediate protein–protein interactions. MYPT1, MYPT2 and MBS85 contain C-terminal leucine zipper domains involved in dimerization and protein–protein interaction, whereas MYPT3 and TIMAP are targeted to membranes via a C-terminal prenylation site. All family members are regulated by phosphorylation at multiple sites by various protein kinases; for example, Rho-associated kinase phosphorylates MYPT1, MYPT2 and MBS85, resulting in inhibition of phosphatase activity and Ca²⁺ sensitization of smooth muscle contraction. A great deal is known about MYPT1, the myosin targeting subunit of myosin light chain phosphatase, in terms of its role in the regulation of smooth muscle contraction and, to a lesser extent, non-muscle motile processes. MYPT2 appears to be the key myosin targeting subunit of myosin light chain phosphatase in cardiac and skeletal muscles. MBS85 most closely resembles MYPT2, but little is known about its physiological function. Little is also known about the physiological role of MYPT3, although it is likely to target myosin light chain phosphatase to membranes and thereby achieve specificity for substrates involved in regulation of the actin cytoskeleton. MYPT3 is regulated by phosphorylation by cAMP-dependent protein kinase. TIMAP appears to target PP1cδ to the plasma membrane of endothelial cells where it serves to dephosphorylate proteins involved in regulation of the actin cytoskeleton and thereby control endothelial barrier function. With such a wide range of regulatory targets, MYPT family members have been implicated in diverse pathological events, including hypertension, Parkinson’s disease and cancer.     

Myosin light chain phosphatase. Effect on the activation and relaxation of gizzard smooth muscle skinned fibers

Skinned cells of chicken gizzard were used to study the effect of a smooth muscle phosphatase (SMP-IV) on activation and relaxation of tension. SMP-IV has previously been shown to dephosphorylate light chains on myosin. When this phosphatase was added to submaximally Ca2+-activated skinned cells, tension increased while phosphorylation of myosin light chains decreased. In contrast, when the myosin phosphatase was added to cell bundles activated in the absence of Ca2+ by a Ca2+-insensitive myosin light chain kinase, tension and phosphorylation of the myosin light chains both decreased. These data suggest that Ca2+ inhibits the deactivation of tension even when myosin light chains are dephosphorylated to a low level. Furthermore, comparison of Ca2+-activated cells caused to relax in CTP, in the presence or absence of Ca2+, shows that cells in the presence of Ca2+ do not relax completely, whereas in the absence of Ca2+ cells completely relax. Solutions containing Ca2+ and CTP, however, are incapable of generating tension from the resting state. Endogenous myosin light chain kinase is not active in solutions containing CTP and dephosphorylation of myosin light chains occurs in CTP solutions both in the presence and absence of Ca2+. These data imply that Ca2+ inhibits relaxation even though myosin light chains are dephosphorylated. These data are consistent with a model wherein an obligatory Ca2+-activated myosin light chain phosphorylation is followed by a second Ca2+ activation process for further tension development or maintenance.     

Molecular aspects of arterial smooth muscle contraction: focus on Rho

The vascular smooth muscle cell is a highly specialized cell whose primary function is contraction and relaxation. It expresses a variety of contractile proteins, ion channels, and signalling molecules that regulate contraction. Upon contraction, vascular smooth muscle cells shorten, thereby decreasing the diameter of a blood vessel to regulate the blood flow and pressure. Contractile activity in vascular smooth muscle cells is initiated by a Ca(2+)-calmodulin interaction to stimulate phosphorylation of the light chain of myosin. Ca(2+)-sensitization of the contractile proteins is signaled by the RhoA/Rho-kinase pathway to inhibit the dephosphorylation of the light chain by myosin phosphatase, thereby maintaining force. Removal of Ca(2+) from the cytosol and stimulation of myoson phosphatase initiate the relaxation of vascular smooth muscle.     

Role of Rho kinases in PKG-mediated relaxation of pulmonary arteries of fetal lambs exposed to chronic high altitude hypoxia

An increase in Rho kinase (ROCK) activity is implicated in chronic hypoxia-induced pulmonary hypertension. In the present study, we determined the role of ROCKs in cGMP-dependent protein kinase (PKG)-mediated pulmonary vasodilation of fetal lambs exposed to chronic hypoxia. Fourth generation pulmonary arteries were isolated from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days and from control ewes. In vessels constricted to endothelin-1, 8-bromoguanosine-cGMP (8-Br-cGMP) caused a smaller relaxation in chronically hypoxic (CH) vessels compared with controls. Rp-8-Br-PET-cGMPS, a PKG inhibitor, attenuated relaxation to 8-Br-cGMP in control vessels to a greater extent than in CH vessels. Y-27632, a ROCK inhibitor, significantly potentiated 8-Br-cGMP-induced relaxation of CH vessels and had only a minor effect in control vessels. The expression of PKG was increased but was not accompanied with an increase in the activity of the enzyme in CH vessels. The expression of type II ROCK and activity of ROCKs were increased in CH vessels. The phosphorylation of threonine (Thr)696 and Thr850 of the regulatory subunit MYPT1 of myosin light chain phosphatase was inhibited by 8-Br-cGMP to a lesser extent in CH vessels than in controls. The difference was eliminated by Y-27632. These results suggest that chronic hypoxia in utero attenuates PKG-mediated relaxation in pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity. Increased ROCK activity may inhibit PKG action through increased phosphorylation of MYPT1 at Thr696 and Thr850.     

Roles of myosin phosphatase during Drosophila development

Myosins are a superfamily of actin-dependent molecular motor proteins, among which the bipolar filament forming myosins II have been the most studied. The activity of smooth muscle/non-muscle myosin II is regulated by phosphorylation of the regulatory light chains, that in turn is modulated by the antagonistic activity of myosin light chain kinase and myosin light chain phosphatase. The phosphatase activity is mainly regulated through phosphorylation of its myosin binding subunit MYPT. To identify the function of these phosphorylation events, we have molecularly characterized the Drosophila homologue of MYPT, and analyzed its mutant phenotypes. We find that Drosophila MYPT is required for cell sheet movement during dorsal closure, morphogenesis of the eye, and ring canal growth during oogenesis. Our results indicate that the regulation of the phosphorylation of myosin regulatory light chains, or dynamic activation and inactivation of myosin II, is essential for its various functions during many developmental processes.