scholarly journals Mechanisms of cGMP-dependent mesangial-cell relaxation: a role for myosin light-chain phosphatase activation

2000 ◽  
Vol 346 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Guadalupe TORRECILLAS ◽  
María L. DÍEZ-MARQUÉS ◽  
Carmen GARCÍA-ESCRIBANO ◽  
Ricardo J. BOSCH ◽  
Diego RODRÍGUEZ-PUYOL ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Cell Labelling ◽  
Myosin Light Chain Phosphatase ◽  
Kinase C ◽  
Phosphatase Activation ◽  
Mlc Phosphorylation

Although the cGMP-dependent relaxation of contractile cells seems to depend on the ability of the cyclic nucleotide to interfere with intracellular calcium, this does not appear to be the only mechanism involved. The present experiments were designed to analyse alternative mechanisms, trying to test the hypothesis that cGMP could relax rat mesangial cells by activating myosin light-chain phosphatase (MLC-PP), with the subsequent dephosphorylation of myosin light chain (MLC). The effect of a cGMP analogue, dibutyryl cGMP (dbcGMP), on angiotensin II-(AII) and PMA-induced MLC phosphorylation (MLCP) was tested, in the presence of calyculin A (CA), an inhibitor of MLC-PP. MLCP was measured, after cell labelling with 32P, by immunoprecipitation. dbcGMP prevented the increased MLCP induced by AII or PMA, and this inhibition was blocked by CA. dbcGMP also increased the MLC dephosphorylation observed in cells incubated with AII and in which MLC kinase and protein kinase C activities were blocked. The AII-elicited increased intracellular calcium concentration was only partially inhibited by dbcGMP. These results suggest that the cGMP-induced mesangial-cell relaxation could be due, at least partially, to the stimulation of MLC-PP.

scholarly journals Mechanisms of cGMP-dependent mesangial-cell relaxation: a role for myosin light-chain phosphatase activation

2000 ◽  
Vol 346 (1) ◽  
pp. 217 ◽  
Author(s):  
Guadalupe TORRECILLAS ◽  
María L. DÍEZ-MARQUÉS ◽  
Carmen GARCÍA-ESCRIBANO ◽  
Ricardo J. BOSCH ◽  
Diego RODRÍGUEZ-PUYOL ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Mesangial Cell ◽  
Myosin Light Chain Phosphatase ◽  
Phosphatase Activation

scholarly journals Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle

2003 ◽  
Vol 369 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Naohisa NIIRO ◽  
Yasuhiko KOGA ◽  
Mitsuo IKEBE
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphatase ◽  
Kinase C ◽  
Agonist Stimulation ◽  
Myosin Binding ◽  
Mlc Phosphorylation

The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both α-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.

Vasoactive agents affect mesangial cell adhesion

1986 ◽  
Vol 251 (4) ◽  
pp. C505-C511 ◽  
Author(s):  
J. I. Kreisberg ◽  
M. A. Venkatachalam
Keyword(s):  
Cell Adhesion ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Mesangial Cell ◽  
Cultured Cells ◽  
Mesangial Cells ◽  
Stress Fibers ◽  
Vasoactive Agents ◽  
Substrate Adhesion ◽  
Adhesive Contacts

The formation and maintenance of stress fibers in cultured mesangial cells is associated with myosin light chain phosphorylation [Kreisberg et al. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F227-F235, 1985], a biochemical indicator for activation of actin-myosin interactions. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (e.g., isoproterenol) fragment stress fibers and cause myosin light chain dephosphorylation, whereas the addition of contractile agents such as arginine vasopressin (AVP) and prostaglandin E2 (PGE2) reverses these changes. Because stress fiber development in cultured cells is correlated with tight cell to substrate adhesion, we wanted to examine whether vasoactive agents have an effect on mesangial cell adhesion. Both isoproterenol and dibutyryl cAMP (DBcAMP) reduced mesangial cell adherence as measured by a trypsin assay (% detached cells: control 11 +/- 2.4%; isoproterenol plus isobutylmethylxanthine (IBMX) = 48.3 +/- 7.4%; DBcAMP = 29.3 +/- 3.7%; DBcAMP-IBMX = 73 +/- 4.4%). The areas of focal (adhesive) contacts between the cell and substratum as observed by interference-reflexion microscopy were also reduced, being replaced by areas of greater separation (% of the surface in contact with the substratum: control = 7.4 +/- 0.8%; isoproterenol-IBMX = 2.9 +/- 1.1%). Addition of PGE2 or AVP to the incubation medium containing the cAMP-elevating agents prevented the above changes. PGE2 or AVP alone increased mesangial cell adhesion (% detached cells: control 11 +/- 2.4%; PGE2 = 6.8 +/- 0.5%; AVP = 5.1 +/- 1.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle

2001 ◽  
Vol 281 (2) ◽  
pp. G467-G478 ◽  
Author(s):  
U. D. Sohn ◽  
Weibiao Cao ◽  
Da-Chun Tang ◽  
J. T. Stull ◽  
J. R. Haeberle ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Circular Muscle ◽  
Kinase C ◽  
Pkc Inhibitors ◽  
Esophageal Sphincter ◽  
Mlc Phosphorylation

In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.

scholarly journals The involvement of protein kinase C in myosin phosphorylation and force development in rat tail arterial smooth muscle

2000 ◽  
Vol 352 (2) ◽  
pp. 573-582 ◽  
Author(s):  
Lynn P. WEBER ◽  
Minoru SETO ◽  
Yasuharu SASAKI ◽  
Karl SWÄRD ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Light Chain Phosphatase ◽  
Arterial Smooth Muscle ◽  
Kinase C ◽  
Detergent Treatment ◽  
Rat Tail

Myosin light-chain phosphorylation is the primary mechanism for activating smooth-muscle contraction and occurs principally at Ser-19 of the 20kDa light chains of myosin (LC20). In some circumstances, Thr-18 phosphorylation may also occur. Protein kinase C (PKC) can regulate LC20 phosphorylation indirectly via signalling pathways leading to inhibition of myosin light-chain phosphatase. The goal of this study was to determine the relative importance of myosin light-chain kinase (MLCK) and PKC in basal and stimulated LC20 phosphorylation in rat tail arterial smooth-muscle strips (RTA). Two MLCK inhibitors (ML-9 and wortmannin) and two PKC inhibitors (chelerythrine and calphostin C) that have different mechanisms of action were used. Results showed the following: (i) basal LC20 phosphorylation in intact RTA is mediated by MLCK; (ii) α1-adrenoceptor stimulation increases LC20 phosphorylation via MLCK and PKC; (iii) Ca2+-induced LC20 phosphorylation in Triton X-100-demembranated RTA is catalysed exclusively by MLCK, consistent with the quantitative loss of PKCs α and β following detergent treatment; (iv) very little LC20 diphosphorylation (i.e. Thr-18 phosphorylation) occurs in intact or demembranated RTA at rest or in response to contractile stimuli; and (v) the level of LC20 phosphorylation correlates with contraction in intact and demembranated RTA, although the steady-state tension–LC20 phosphorylation relationship is markedly different between the two preparations such that the basal level of LC20 phosphorylation in intact muscles is sufficient to generate maximal force in demembranated preparations. This may be due, in part, to differences in the phosphatase/kinase activity ratio, resulting from disruption of a signalling pathway leading to myosin light-chain phosphatase inhibition following detergent treatment.

Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle

1990 ◽  
Vol 259 (4) ◽  
pp. C631-C639 ◽  
Author(s):  
H. A. Singer
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Activation Mechanism ◽  
Arterial Smooth Muscle ◽  
Maximal Force ◽  
Kinase C ◽  
Mlc Phosphorylation

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms.

scholarly journals 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

2005 ◽  
Vol 389 (3) ◽  
pp. 763-774 ◽  
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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