Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca²⁺ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity. actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions     

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts

Thus far, determining the relative contribution of Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and Ca²⁺-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca²⁺, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO₄/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO₄/mol RLC, respectively, within 2.5 min. Ca²⁺ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation     

Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.     

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.     

Cyclic AMP- and calmodulin-dependent phosphorylation of 21 and 26 kda proteins in axoneme is a prerequisite for SAAF-induced motile activation in ascidian spermatozoa

Sperm activating and -attracting factor (SAAF), derived from the egg of the ascidian Ciona, activates sperm motility through adenosine 3':5'-cyclic monophosphate (cAMP)-synthesis. A demembranated preparation of intact immotile sperm without SAAF was shown to require cAMP for reactivation. However, a demembranated preparation of intact motile sperm treated with SAAF did not require cAMP for reactivation, suggesting that cAMP is a prerequisite factor for SAAF-dependent activation of sperm motility. Furthermore, a cAMP-dependent protein kinase (PKA) inhibitor, H-89, was found to inhibit sperm motility. During in vivo or in vitro activation of sperm motility by SAAF or cAMP, a 26 kDa axonemal protein and 21 kDa dynein light chain were phosphorylated, respectively, suggesting the involvement of PKA-dependent phosphorylation of these proteins in sperm activation. The calmodulin antagonist, W-7, and an inhibitor of calmodulin-dependent myosin light chain kinase, ML-7, also inhibited the activation of sperm motility. Inhibition was reversed by the addition of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Demembranated preparations of immotile sperm in the presence of W-7 or ML-7 were reactivated by cAMP, suggesting that calmodulin participated in sperm activation and that cAMP synthesis was followed by activation of a calmodulin-dependent mechanism.     

Phosphorylation of the small heat shock-related protein, HSP20, in vascular smooth muscles is associated with changes in the macromolecular associations of HSP20

Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of a small heat shock-related protein, HSP20. We hypothesized that phosphorylation of HSP20 in vascular smooth muscles is associated with alterations in the macromolecular associations of HSP20. Treatment of bovine carotid artery smooth muscles with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and the adenylate cyclase activator, forskolin, led to increases in the phosphorylation of HSP20 and dissociation of macromolecular aggregates of HSP20. However, 3-isobutyl-1-methylxanthine and forskolin treatment of a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation, human umbilical artery smooth muscle, did not result in increases in the phosphorylation of HSP20 or to dissociation of macromolecular aggregates. HSP20 can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (PKA) in both carotid and umbilical arteries and this phosphorylation of HSP20 is associated with dissociation of macromolecular aggregates of HSP20. Activation of cyclic nucleotide-dependent signaling pathways does not lead to changes in the macromolecular associations of another small heat shock protein, HSP27. Interestingly, the myosin light chains (MLC20) are in similar fractions as the HSP20, and phosphorylation of HSP20 is associated with changes in the macromolecular associations of MLC20. These data suggest that increases in the phosphorylation of HSP20 are associated with changes in the macromolecular associations of HSP20. HSP20 may regulate vasorelaxation through a direct interaction with specific contractile regulatory proteins.     

cAMP-mediated modulation of signal transduction of epidermal growth factor (EGF) receptor systems in human epidermoid carcinoma A431 cells. Depression of EGF-dependent diacylglycerol production and EGF receptor phosphorylation.

While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Phosphorylation of phospholipase C-gamma by cAMP-dependent protein kinase

The mechanism by which cAMP modulates the activity of phosphoinositide-specific phospholipase C (PLC) was studied. Elevation of cAMP inhibited both basal and norepinephrine-stimulated phosphoinositide breakdown in C6Bu1 cells which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of C6Bu1 cells with cAMP-elevating agents (cholera toxin, isobutylmethylxanthine, forskolin, and 8-bromo-cAMP) increased serine phosphate in PLC-gamma, but the phosphate contents in PLC-beta and PLC-delta were not changed. In addition, cAMP-dependent protein kinase selectively phosphorylated purified PLC-gamma among the three isozymes and added a single phosphate at serine. The serine phosphorylation, nevertheless, did not affect the activity of PLC-gamma in vitro. We propose, therefore, that the phosphorylation of PLC-gamma by cAMP-dependent protein kinase alters its interaction with putative modulatory proteins and leads to its inhibition.     

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.     

Myosin light chain kinase A is activated by cGMP-dependent and cGMP-independent pathways

Stimulation of Dictyostelium cells with the chemoattractant cAMP results in transient phosphorylation of the myosin regulatory light chain (RLC). We show that myosin light chain kinase A (MLCK-A) is responsible for RLC phosphorylation during chemotaxis, and that MLCK-A itself is transiently phosphorylated on threonine-166, dramatically increasing its catalytic activity. MLCK-A activation during chemotaxis is highly responsive to cellular cGMP levels and the cGMP-binding protein GbpC. MLCK-A- cells have a partial cytokinesis defect, and do not phosphorylate RLC in response to concanavalin A (conA), but cells lacking cGMP or GbpC divide normally and phosphorylate in response to conA. Thus MLCK-A is activated by a cGMP/GbpC-independent mechanism activated during cytokinesis or by conA, and a cGMP/GbpC-dependent pathway during chemotaxis.     

Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase

Microinjection of the catalytic subunit of cAMP-dependent protein kinase (A-kinase) into living fibroblasts or the treatment of these cells with agents that elevate the intracellular cAMP level caused marked alterations in cell morphology including a rounded phenotype and a complete loss of actin microfilament bundles. These effects were transient and fully reversible. Two-dimensional gel electrophoresis was used to analyze the changes in phosphoproteins from cells injected with A-kinase. These experiments showed that accompanying the disassembly of actin microfilaments, phosphorylation of myosin light chain kinase (MLCK) increased and concomitantly, the phosphorylation of myosin P- light chain decreased. Moreover, inhibiting MLCK activity via microinjection of affinity-purified antibodies specific to native MLCK caused a complete loss of microfilament bundle integrity and a decrease in myosin P-light chain phosphorylation, similar to that seen after injection of A-kinase. These data support the idea that A-kinase may regulate microfilament integrity through the phosphorylation and inhibition of MLCK activity in nonmuscle cells.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

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.     

RhoA inactivation is crucial to manganese-induced astrocyte stellation

Actin depolymerization through Rho GTPases or exogenous mechanical tension has been suggested as a key determinant for the formation of astrocyte stellation. Rho GTPases function as switching molecules to converge both extracellular and intracellular signals in regulation of cytoskeletal organization. Their involvement in manganese-induced astrocyte stellation was assessed. The disruption of cytoskeletal architecture by manganese indicated the decreased activity of RhoA. Pharmacological and biochemical approaches revealed the inactivation of RhoA by manganese. This inactivation was partly through the down-regulation of guanine nucleotide exchange factor phosphorylation. Furthermore, the dephosphorylation of myosin light chain and cofilin through the inactivated RhoA effectors synergistically destabilized actin stress fibers. We conclude that manganese regulates cytoskeletal organization in astrocytes by modulating the activity of p115RhoGEF and RhoA.     

Failure to implicate cAMP in transduction in lobster olfactory receptor cells

The possible role of adenosine 3',5'-cyclic monophosphate (cAMP)in olfactory transduction in the spiny lobster was investigatedusing radioimmunoassay of cAMP and intracellular recording.Application of forskolin or 1-isobutyl-3-methylxanthine increasedcAMP levels in intact sensilla containing the chemoreceptiveouter dendritic segments of the lobster olfactory receptor cell,thereby demonstrating adenylate cyclase and phosphodiesteraseactivity in the sensilla. A complex odor mixture and identifiedexcitatory odor molecules failed to stimulate the productionof cAMP, however In intracellular recordings, superfusion ofthe outer dendritic segments with forskolin, 1-isobutyl-3-methylxanthineand cyclic nucleotide analogs had no direct effect on odor-responsivecells. These compounds did cause infrequent enhancements (sixof 42 cells) of odor-evoked receptor potentials, but processesother than transduction are the most likely causes of this effect.We conclude that cAMP-dependent transduction mechanisms areunlikely to mediate most odor responses in lobsters, in contrastto transduction mechanisms in amphibians and rats.     

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca²⁺/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca²⁺ sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.     

Signaling to myosin regulatory light chain in sarcomeres

Myosin regulatory light chain (RLC) phosphorylation in skeletal and cardiac muscles modulates Ca(2+)-dependent troponin regulation of contraction. RLC is phosphorylated by a dedicated Ca(2+)-dependent myosin light chain kinase in fast skeletal muscle, where biochemical properties of RLC kinase and phosphatase converge to provide a biochemical memory for RLC phosphorylation and post-activation potentiation of force development. The recent identification of cardiac-specific myosin light chain kinase necessary for basal RLC phosphorylation and another potential RLC kinase (zipper-interacting protein kinase) provides opportunities for new approaches to study signaling pathways related to the physiological function of RLC phosphorylation and its importance in cardiac muscle disease.     

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