Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes

Activation of K-Cl cotransport is associated with activation ofmembrane-bound serine/threonine protein phosphatases (S/T-PPases). Wecharacterize red blood cell S/T-PPases and K-Clcotransport activity regarding protein phosphatase inhibitors andresponse to changes in ionic strength and cell size. Proteinphosphatase type 1 (PP1) activity is highly sensitive to calyculin A(CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 ± 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 ± 10% per 100 meq/l). Increasesin membrane PP2A activity (270 ± 77% per 100 mosM) result fromvolume increases and also correlate with increases in K-Cl cotransportactivity (420 ± 47% per 100 mosM). The characteristics ofmembrane-associated PP1 and PP2A are consistent with a role for bothphosphatases in K-Cl cotransport activation in human erythrocytes.

     

Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase

Endothelial monolayer hyperpermeability is regulated by a myosin light chain phosphorylation (MLCP)-dependent contractile mechanism. In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125(FAK)), and monolayer hyperpermeability. Microscopic examination of CalA-treated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction.     

Microtubule disassembly increases endothelial cell barrier dysfunction: role of MLC phosphorylation

Endothelial cell (EC) barrier regulation is critically dependent on cytoskeletal components (microfilaments and microtubules). Because several edemagenic agents induce actomyosin-driven EC contraction tightly linked to myosin light chain (MLC) phosphorylation and microfilament reorganization, we examined the role of microtubule components in bovine EC barrier regulation. Nocodazole or vinblastine, inhibitors of microtubule polymerization, significantly decreased transendothelial electrical resistance in a dose-dependent manner, whereas pretreatment with the microtubule stabilizer paclitaxel significantly attenuated this effect. Decreases in transendothelial electrical resistance induced by microtubule disruption correlated with increases in lung permeability in isolated ferret lung preparations as well as with increases in EC stress fiber content and MLC phosphorylation. The increases in MLC phosphorylation were attributed to decreases in myosin-specific phosphatase activity without significant increases in MLC kinase activity and were attenuated by paclitaxel or by several strategies (C3 exotoxin, toxin B, Rho kinase inhibition) to inhibit Rho GTPase. Together, these results suggest that microtubule disruption initiates specific signaling pathways that cross talk with microfilament networks, resulting in Rho-mediated EC contractility and barrier dysfunction.     

Rho kinase signaling pathways during stretch in primary alveolar epithelia

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.     

Okadaic acid induces phosphorylation and translocation of myosin phosphatase target subunit 1 influencing myosin phosphorylation, stress fiber assembly and cell migration in HepG2 cells

It was determined that the myosin phosphatase (MP) activity and content of myosin phosphatase target subunit 1 (MYPT1) were correlated in subcellular fractions of human hepatocarcinoma (HepG2) cells. In control cells MYPT1 was localized in the cytoplasm and in the nucleus, as determined by confocal microscopy. Treatment of HepG2 cells with 50 nM okadaic acid (OA), a cell-permeable phosphatase inhibitor, induced several changes: 1) a marked redistribution of MYPT1 to the plasma membrane associated with an increased level of phosphorylation of MYPT1 at Thr695. Both effects showed only a slight influence with the Rho-kinase inhibitor, Y-27632; 2) an increase in phosphorylation of MYPT1 at Thr850 associated with its accumulation in the perinuclear region and nucleus. These effects were markedly reduced by Y-27632; 3) an increased phosphorylation of the 20 kDa myosin II light chain at Ser19 associated with an increased location of myosin II at the cell center. These effects were partially counteracted by Y-27632; 4) an increase in stress fiber formation and a decrease in cell migration, both OA-induced effects were blocked by Y-27632. In HepG2 lysates, OA (5-100 nM) did not affect MP activity but inhibited PP2A activity. These results indicate that OA induces differential phosphorylation and translocation of MYPT1, dependent on PP2A and, to varying extents, on ROK. These changes are associated with an increased level of myosin II phosphorylation and attenuation of hepatic cell migration.     

Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions.We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.     

Molecular characterization of myosin phosphatase in endothelium

The phosphorylation status of myosin light chain (MLC) is regulated by both MLC kinases and type 1 Ser/Thr phosphatase (PPase 1), MLC phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (CS1β) towards myosin depends on its associated regulatory subunit, namely myosin PPase targeting subunit 1 (MYPT1). Our previously published data strongly suggested the involvement of MLCP in endothelial cell (EC) barrier regulation. In this study, our new data demonstrate that inhibition of MLCP by either CS1β or MYPT1 siRNA-based depletion results in significant attenuation of purine nucleotide (ATP and adenosine)-induced EC barrier enhancement. Consistent with the data, thrombin-induced EC F-actin stress fiber formation and permeability increase were attenuated by the ectopic expression of constitutively active (C/A) MYPT1. The data demonstrated for the first time direct involvement of MLCP in EC barrier enhancement/protection. Cloning of MYPT1 in human pulmonary artery EC (HPAEC) revealed the presence of two MYPT1 isoforms, long and variant 2 (V2) lacking 56 amino acids from 553 to 609 of human MYPT1 long, which were previously identified in HeLa and HEK 293 cells. Our data demonstrated that in Cos-7 cells ectopically expressed EC MYPT1 isoforms co-immunoprecipitated with intact CS1β suggesting the importance of PPase 1 activity for the formation of functional complex of MYPT1/CS1β. Interestingly, MYPT1 V2 shows decreased binding affinity compared to MYPT1 long for radixin (novel MLCP substrate and a member of ERM family proteins). These results suggest functional difference between EC MYPT1 isoforms in the regulation of MLCP activity and cytoskeleton.     

Opposite effect of cAMP signaling in endothelial barriers of different origin

cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined. cAMP signaling was stimulated by the adenosine receptor agonist 5'-N-(ethylcarboxamido)-adenosine (NECA), the -adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imitated by the PKA activator Sp-cAMPS. Under cAMP/PKA-dependent stimulation, isometric force and MLC phosphorylation were reduced in monolayers of either cell type, due to an activation of MLC phosphatase. In CEC but not in AEC, FSK induced delocalization of VE-cadherin and paxillin from cellular adhesion complexes as indicated by cell fractionation and immunofluorescence microscopy. In conclusion, decline in contractile activation and isometric force contribute to cAMP/PKA-mediated stabilization of barrier function in AEC. In CEC, this stabilizing effect is overruled by cAMP-induced disintegration of cell adhesion structures. endothelial cell adhesion; endothelial permeability; isometric force; myosin light chain kinase; myosin light chain phosphatase     

Regulation of endothelial cell gap formation and barrier dysfunction: Role of myosin light chain phosphorylation

Endothelial cell (EC) contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. We tested the hypothesis that phosphorylation of regulatory myosin light chains (MLC) by Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) is critical to EC barrier dysfunction elicited by thrombin. Thrombin stimulated a rapid (<15 sec) increase in [Ca²⁺]_i which preceded maximal MLC phosphorylation (60 sec) with a 6 to 8-fold increase above constitutive levels of phosphorylated MLC. Dramatic cellular shape changes indicative of contraction and gap formation were observed at 5 min with maximal increases in albumin permeability occurring by 10 min. Neither the Ca²⁺ ionophore, A23187, nor phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), alone or in combination, produced MLC phosphorylation. The combination was synergistic, however, in stimulating EC contraction/gap formation and barrier dysfunction (3 to 4-fold increase). Down-regulation or inhibition of PKC activity attenuated thrombin-induced MLC phosphorylation (～40% inhibition) and both thrombin- and PMA-induced albumin clearance (～50% inhibition). Agents which augmented [cAMP]_i partially blocked thrombin-induced MLC phosphorylation (～50%) and completely inhibited both thrombin- and PMA-induced EC permeability (100% inhibition). Furthermore, cAMP produced significant reduction in the basal levels of constitutive MLC phosphorylation. Finally, MLCK inhibition (with either ML-7 or KT 5926) or Ca²⁺/calmodulin antagonism (with either trifluoperazine or W-7) attenuated thrombin-induced MLC phosphorylation and barrier dysfunction. These results suggest a model wherein EC contractile events, gap formation and barrier dysfunction occur via MLCK-dependent and independent mechanisms and are significantly modulated by both PKC and cAMP-dependent protein kinase A activities. © 1995 Wiley-Liss, Inc.     

Phosphatase 2A is involved in endothelial cell microtubule remodeling and barrier regulation

We have recently shown that microtubule (MT) inhibitor, nocodazole (2-5 microM) significantly increases endothelial cells (EC) actomyosin contraction and permeability indicating the importance of MT in maintaining the EC barrier (Verin et al. [2001]: Cell Mol Physiol 281:L565-L574). Okadaic acid (OA, 2-5 nM), a powerful inhibitor of protein phosphatase 2A (PP2A), significantly potentiates the effect of submaximal concentrations of nocodazole (50-200 nM) on transendothelial electrical resistance (TER) suggesting the involvement of PP2A activity in the MT-mediated EC barrier regulation. Immunofluorescent staining of EC revealed that in control cells PP2A distributes in a pattern similar to MT. Consistent with these results, we demonstrated that significant amounts of PP2A were present in MT-enriched EC fractions indicating tight association of PP2A with MT in endothelium. Treatment of EC with OA leads to disappearance of MT-like PP2A staining suggesting dissociation of PP2A from the MT network. Next, we examined the effect of PP2A inhibition on phosphorylation status of MT-associated protein tau, which in its unphosphorylated form promotes MT assembly. OA caused significant increases in tau phosphorylation confirming that tau is a substrate for PP2A in endothelium. Immunofluorescent experiments demonstrated that the OA-induced increases in tau phosphorylation strongly correlated with translocation of phospho-tau to cell periphery and disassembly of peripheral MT. These results suggest the involvement of PP2A-mediated tau dephosphorylation in alteration of EC MT structure and highlight the potential importance of PP2A in the regulation of EC the MT cytoskeleton and barrier function.     

New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.     

Inhibition of endothelial barrier dysfunction by P21-activated kinase-1

We investigated the activity of P21-activated kinase-1 (Pak1) on myosin light chain phosphorylation and on thrombin-induced barrier dysfunction in human endothelial cells (HMEC). HMEC were infected with recombinant adenoviruses that express constitutively active Pak1, LacZ, wild-type, and a mutant myosin regulatory light chain, mMLC20 (Thr18Ala, Ser19Ala). Expression of the recombinant Pak1 mediated by adenovirus in HMEC was regulated. Active Pak1 induced dephosphorylation of MLC20 in HMEC, but not in smooth muscle cells. Active Pak1 significantly inhibited thrombin-induced endothelial barrier dysfunction. Expression of the unphosphorylatable MLC20 also inhibited thrombin-induced endothelial barrier dysfunction. Constitutively active Pak1 associated with phosphatase 2A and induced a post-translational modification of the phosphatase. Our data provide novel evidence indicating that Pak1 regulates endothelial barrier function through activation of phosphatase 2A.     

Thrombin-induced endothelial barrier disruption in intact microvessels: role of RhoA/Rho kinase-myosin phosphatase axis

Endothelial hyperpermeability is regulated by a myosin light chain-2 (MLC2) phosphorylation-dependent contractile mechanism. Thrombin is a potent inducer of hyperpermeability of cultured monolayers of endothelial cells (ECs) via Rho kinase-mediated MLC2-phosphorylation. The aim of the present study was to investigate the effects of thrombin on in situ endothelial morphology and barrier integrity. Cytoskeletal dynamics, regions of paracellular flux, and MLC2-phosphorylation of ECs were visualized by digital three-dimensional imaging microscopy of pressurized rat kidney arterioles. Myosin phosphatase targeting subunit (MYPT1)-phosphorylation was used as a surrogate marker for Rho kinase activity. Thrombin induced the formation of F-actin filaments in ECs in situ and rounding of the ECs in the absence of obvious formation of gaps between ECs. These changes were accompanied by an increase in MLC2 phosphorylation and a decrease in barrier integrity. In vitro analysis revealed that Rho kinase activity on F-actin filaments was associated with a contractile response that enhanced opening of the barrier. Rho kinase activity was not detectable on F-actin filaments induced by histamine, an inducer of a more transient hyperpermeability response. Inhibition of the myosin phosphatase mimicked the effects of thrombin on barrier function. The thrombin-induced changes in in situ MLC2 phosphorylation and barrier function were Rho kinase dependent. These data demonstrate a direct effect of thrombin on EC morphology and barrier integrity in intact microvessels. Furthermore, they establish an important contribution of enhanced Rho kinase activity to the development of prolonged but not transient types of endothelial barrier dysfunction.     

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.     

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.     

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.     

Mildly oxidized low density lipoprotein induces contraction of human endothelial cells through activation of Rho/Rho kinase and inhibition of myosin light chain phosphatase.

Mildly oxidized low density lipoprotein (mox-LDL) is critically involved in the early atherogenic responses of the endothelium and increases endothelial permeability through an unknown signal pathway. Here we show that (i) exposure of confluent human endothelial cells (HUVEC) to mox-LDL but not to native LDL induces the formation of actin stress fibers and intercellular gaps within minutes, leading to an increase in endothelial permeability; (ii) mox-LDL induces a transient decrease in myosin light chain (MLC) phosphatase that is paralleled by an increase in MLC phosphorylation; (iii) phosphorylated MLC stimulated by mox-LDL is incorporated into stress fibers; (iv) cytoskeletal rearrangements and MLC phosphorylation are inhibited by C3 transferase from Clostridium botulinum, a specific Rho inhibitor, and Y-27632, an inhibitor of Rho kinase; and (v) mox-LDL does not increase intracellular Ca(2+) concentration. Our data indicate that mox-LDL induces endothelial cell contraction through activation of Rho and its effector Rho kinase which inhibits MLC phosphatase and phosphorylates MLC. We suggest that inhibition of this novel cell signaling pathway of mox-LDL could be relevant for the prevention of atherosclerosis.     

PHI-1 interacts with the catalytic subunit of myosin light chain phosphatase to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force in avian smooth muscle

In avian smooth muscles, GTPgammaS produces a Rho kinase mediated increase in PHI-1 phosphorylation and force, but whether this correlation is causal is unknown. We examined the effect of phosphorylated PHI-1 (P-PHI-1) on force and myosin light chain (MLC(20)) phosphorylation at a constant [Ca(2+)]. P-PHI-1, but not PHI-1, increased MLC(20) phosphorylation and force, and phosphorylation of PHI-1 increased the interaction of PHI-1 with PP1c. Microcystin induced a dose-dependent reduction in the binding of PHI-1 to PP1c. These results suggest PHI-1 inhibits myosin light chain phosphatase by interacting with the active site of PP1c to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force.     

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.