Volume and agonist-induced regulation of myosin light-chain phosphorylation in glomerular mesangial cells

1993 ◽  
Vol 264 (3) ◽  
pp. F421-F426 ◽  
Author(s):  
M. Takeda ◽  
T. Homma ◽  
M. D. Breyer ◽  
N. Horiba ◽  
R. L. Hoover ◽  
...  
Keyword(s):  
Cell Volume ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Mesangial Cells ◽  
Hyperosmotic Stress ◽  
Relative Distribution ◽  
Glomerular Mesangial Cells ◽  
Relative Contribution ◽  
Phosphopeptide Mapping ◽  
Volume Decrease

We investigated whether cell volume decrease per se can activate intracellular mechanisms leading to mesangial cell contraction. For this purpose, we applied hyperosmotic stress to cultured glomerular mesangial cells and examined the effects on phosphorylation of myosin light chain (MLCP). Compared with control cells, hyperosmotic stress (390 mosmol/kg) attained by either NaCl or raffinose significantly increased MLCP to 140.7 +/- 7.0% (n = 5) and 134.8 +/- 7.7% (n = 4), respectively, in parallel with a decrease in the cell volume. This increase was comparable to that achieved by the following agonists: arginine vasopressin (AVP, 100 nM; n = 5) and endothelin-1 (ET, 10 nM; n = 5). By using two-dimensional tryptic phosphopeptide mapping, contribution of myosin light-chain kinase (MLCK) and protein kinase C (PKC) to the observed phosphorylation was examined by identifying phosphorylation at serine-19 (by MLCK) and at serine-1 or serine-2 (by PKC). Under resting conditions, relative distribution of phosphorylation between MLCK and PKC sites was 60.1 +/- 8.4 and 39.9 +/- 8.4%. The relative contribution by these enzymes remained similar during hyperosmotic stress or agonist stimulation. Since cytosolic Ca2+ concentration ([Ca2+]i) is an important determinant of MLCP, we also examined [Ca2+]i in these settings. While AVP and ET-induced a characteristic transient spike in [Ca2+]i, hyperosmotic stress caused a gradual and modest increase in [Ca2+]i. These studies show that, in mesangial cells, reduction in cell volume induces MLCP through mechanisms distinct from those involved in agonist-induced events.

scholarly journals Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase

2002 ◽  
Vol 367 (2) ◽  
pp. 517-524 ◽  
Author(s):  
Jing Ti DENG ◽  
Cindy SUTHERLAND ◽  
David L. BRAUTIGAN ◽  
Masumi ETO ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Kinase Assay ◽  
Myosin Light Chain Phosphatase ◽  
Phosphatase Inhibitors ◽  
Phosphatase Inhibitor ◽  
Integrin Linked Kinase ◽  
Phosphopeptide Mapping

Integrin-linked kinase (ILK) has been implicated in Ca2+- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr→Ala mutation at Thr38 of CPI-17 and Thr57 of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr38 of CPI-17 or Thr57 of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr38 or Thr57 respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr38Ala and PHI-1-Thr57Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca2+ sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr38Ala and PHI-1-Thr57Ala treated with ILK in the presence of adenosine 5′-[γ-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.

scholarly journals α1-Adrenoceptor-mediated phosphorylation of myosin in rat-tail arterial smooth muscle

1997 ◽  
Vol 327 (3) ◽  
pp. 669-674 ◽  
Author(s):  
Mitsuo MITA ◽  
P. Michael WALSH
Keyword(s):  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Contractile Response ◽  
Bathing Medium ◽  
Adrenoceptor Agonist ◽  
Phosphoamino Acid Analysis ◽  
Phosphopeptide Mapping ◽  
Rat Tail

The mechanism of α1-adrenoceptor-mediated contraction was investigated in helical strips of the rat-tail artery. Muscle strips with the endothelium removed contracted in response to the α1-adrenoceptor agonist cirazoline, with half-maximal contraction at 0.23 μM. The contractile response to a submaximal concentration of cirazoline (0.3 μM) was biphasic, with a rapid phasic component peaking at approx. 30 s, followed by sustained tonic contraction. Phosphorylation of the 20 kDa light chain of myosin (LC20) in response to 0.3 μM cirazoline was also biphasic and closely matched the time-course of contraction. Resting LC20 phosphorylation levels were 0.22±0.06 mol of Pi/mol of LC20 (n = 3) and reached a maximum of 0.58±0.08 mol of Pi/mol of LC20 (n = 3). Phosphopeptide mapping and phosphoamino acid analysis revealed that LC20 phosphorylation occurred exclusively at serine-19. The sustained phase of contraction was eliminated by removal of extracellular Ca2+ and the phasic response was eliminated by depletion of endogenous Ca2+ stores. Both phases of the contractile response were restored by re-addition of Ca2+ to the bathing medium. LC20 phosphorylation and both phases of the contractile response to 0.3 μM cirazoline were inhibited by the myosin light-chain kinase inhibitor ML-9 (30 μM). Resting LC20 phosphorylation, however, was unaffected by ML-9. Finally, both phasic and tonic responses to 0.3 μM cirazoline were partially inhibited by chloroethylclonidine (50 μM), suggesting the involvement of both α1A and α1B adrenoceptors in these contractile responses.

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)

Role of myosin light-chain phosphorylation and microtubules in stress fiber morphology in cultured mesangial cells

1985 ◽  
Vol 249 (2) ◽  
pp. F227-F235 ◽  
Author(s):  
J. I. Kreisberg ◽  
M. A. Venkatachalam ◽  
R. A. Radnik ◽  
P. Y. Patel
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Mesangial Cells ◽  
Shape Change ◽  
Stress Fiber ◽  
Model Systems ◽  
Stress Fibers ◽  
Intracellular Camp ◽  
Fiber Morphology ◽  
Physiological Basis

Induced elevations in intracellular cAMP caused remarkable shape changes in cultured mesangial cells that were prevented by prostaglandin E2 (PGE2). The purpose of this study was to determine the morphological and physiological basis for these alterations. Coincident with elevated levels of cAMP and shape change there was rapid dissolution of stress fibers. Cytochalasin B treatment also caused shape change and stress fiber dissolution. Stress fibers were visualized by fluorescence microscopy after appropriate staining. In such cells, microtubules were intact. PGE2 prevented stress fiber loss as well as shape change if it was included in the medium from the outset, or restored stress fibers and normal morphology within 30 min if it was added following shape change. Agents that depolymerize microtubules also prevented both stress fiber loss and shape change. Coincident with stress fiber loss and shape change induced by elevations of cAMP, there was a decrease in the rate of [32P]-orthophosphate incorporation into myosin light chain; this was also prevented or reversed by PGE2. Remarkably, PGE2 alone caused an increase in the rate of [32P]orthophosphate incorporation into myosin light chain in cells that were otherwise untreated. Phosphorylation of the light chain of myosin may be an important factor in the maintenance of stress fiber morphology. The results of this study point out complex interrelationships between microtubules, and stress fibers in the maintenance of cell shape needing further study. The model systems that are presented seem suited for such investigation.

Inhibition of myosin light chain phosphatase during Ca(2+)-independent vasocontraction

1993 ◽  
Vol 265 (5) ◽  
pp. C1319-C1324 ◽  
Author(s):  
H. Itoh ◽  
A. Shimomura ◽  
S. Okubo ◽  
K. Ichikawa ◽  
M. Ito ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Water Soluble ◽  
Two Dimensional ◽  
Kinase C ◽  
Rat Thoracic Aorta ◽  
Phosphopeptide Mapping

Phorbol 12,13-dibutyrate (PDB) induced a sustained contraction of rat thoracic aorta strip in Ca(2+)-free buffer without significant change in intracellular free Ca2+ concentration. NKH477, a water-soluble forskolin derivative, markedly relaxed the PDB-induced contraction. The PDB-induced contraction was associated with the phosphorylation of 20-kDa myosin light chain (MLC). Two-dimensional phosphopeptide mapping of 20-kDa MLC revealed that approximately 90% of the phosphopeptides was derived from an MLC kinase-catalyzed reaction and approximately 10% was due to phosphorylation by protein kinase C. NKH477 inhibited the PDB-induced phosphorylation of 20-kDa MLC. MLC phosphatase activity of intact aorta strips was inhibited by the treatment with PDB, and the inhibition was recovered by the application of NKH477. These results suggest that the regulation of MLC phosphatase in vascular smooth muscle may play important roles in the PDB-induced contraction and the NKH477-induced relaxation in Ca(2+)-free buffer.

scholarly journals Heparin inhibits Ca2+/calmodulin-dependent kinase II activation and c-fos induction in mesangial cells

1998 ◽  
Vol 330 (2) ◽  
pp. 651-657 ◽  
Author(s):  
Tiho MIRALEM ◽  
M. Douglas TEMPLETON
Keyword(s):  
Phorbol Ester ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Mesangial Cells ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Cell Components ◽  
Fos Expression

Like vascular smooth-muscle cells, rat mesangial cells (RMCs) display an anti-mitogenic response to heparin. In particular, heparin partially suppresses the ability of quiescent RMCs to enter the cell cycle and induce c-fos expression. When the mitogenic stimulus is serum, phorbol ester or platelet-derived growth factor, this response appears to result from the ability of heparin to suppress activation of the extracellular-signal-regulated kinase family of mitogen-activated protein kinases. However, we have also shown that heparin suppresses c-fos expression in response to ionophores such as ionomycin, an event independent of mitogen-activated protein kinase [Miralem, Wang, Whiteside and Templeton (1996) J. Biol. Chem.271, 17100-17106]. Here we identify this second heparin-sensitive pathway as involving Ca2+/calmodulin-dependent kinase (CaMK) II. Ionomycin (100 nM) caused a transient rise in intracellular Ca2+ concentration ([Ca2+]i) in quiescent RMCs to 386±55 nM, with an increase in CaMK II activity that peaked 30 s later. The accumulation of c-fos mRNA that ensued 30 min later was prevented when the increase in [Ca2+]i was prevented with the intracellular Ca2+ chelator, 1,2-bis-(2-aminophenyoxy)ethane-N,N,Nʹ,Nʹ-tetra-acetic acid. The broad-specificity CaMK inhibitor, KT 5926, inhibited ionomycin-dependent c-fos induction at a concentration at which it was without effect on induction by serum or phorbol ester. The CaMK II-specific inhibitor, KN-93, likewise inhibited c-fos induction by ionomycin, but not by serum or phorbol ester. ML-7, an inhibitor of the CaMK-related myosin light-chain kinase (MLCK), was without effect. Heparin (1 μg/ml) suppressed ionomycin-dependent c-fos induction. It was without effect on [Ca2+]i, but inhibited the development of autonomous CaMK II activity. However, when heparin was added to the CaMK II assay solution in vitro, it was without effect on autonomous activity. Furthermore, heparin did not prevent full activation of CaMK II by the Ca2+-calmodulin complex in vitro. Heparin did not affect myosin light-chain phosphorylation or RMC contraction, processes mediated by MLCK. We conclude that ionomycin induces c-fos in RMCs through the CaMK II pathway, and that heparin prevents CaMK II activation by an indirect process mediated by other cell components. Heparin does not affect activation of the closely related CaMK, MLCK.

Collagen type I enhances endothelin-mediated contraction and induces nonproliferating phenotype in mesangial cells

1996 ◽  
Vol 270 (6) ◽  
pp. F960-F970 ◽  
Author(s):  
T. Miralem ◽  
C. I. Whiteside ◽  
D. M. Templeton
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Collagen Type ◽  
Mesangial Cells ◽  
Collagen Gel ◽  
Collagen Type I ◽  
Cell Number ◽  
Type I ◽  
Total Dna ◽  
Glomerular Extracellular Matrix

Accumulation of glomerular extracellular matrix is a characteristic accompaniment of mesangial cell proliferation in progressive renal disease. We examined how growth on several matrices affected the proliferative phenotype of cultured rat mesangial cells. Compared with growth on plastic, Matrigel, or mesangial matrix, collagen type I caused a decreased cell number at 72 h, decreased total DNA per culture, and a decrease in the incorporation of [3H]thymidine during S phase in cells released from quiescence. These antiproliferative and antimitogenic effects of collagen type I required growth on a collagen gel; soluble collagen or collagen fragments were without effect. Because a number of agents elicit both proliferative and contractile responses in mesangial cells, we examined the effect of growth on collagen on contractility. Compared with plastic, cells grown on collagen type I were more contractile, showed a higher Ca2+ signal in response to endothelin, and responded to endothelin with a more rapid myosin light-chain kinase-dependent phosphorylation of myosin light chain. We conclude that growth on a collagen type I gel uncouples contractility from a proliferative response in mesangial cells, suppressing proliferation while enhancing contraction and Ca2+ signaling in response to endothelin.

scholarly journals Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

2011 ◽  
Vol 22 (5) ◽  
pp. 634-650 ◽  
Author(s):  
Elisabeth T. Barfod ◽  
Ann L. Moore ◽  
Benjamin G. Van de Graaf ◽  
Steven D. Lidofsky
Keyword(s):  
Cell Volume ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Membrane Dynamics ◽  
Cell Swelling ◽  
Membrane Turnover ◽  
Osmotic Swelling ◽  
Volume Recovery

 The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.

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.

Ca(2+)-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment

1995 ◽  
Vol 268 (1) ◽  
pp. C21-C29 ◽  
Author(s):  
S. Morimoto ◽  
Y. Ogawa
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Aortic Smooth Muscle ◽  
Skinned Smooth Muscle ◽  
Acidic Conditions ◽  
Mlc20 Phosphorylation ◽  
Phosphopeptide Mapping ◽  
High Level ◽  
Triton X

After an acidic treatment in the presence of ADP, Triton X-100-skinned rabbit aortic smooth muscle strips were found to develop a large sustained, Ca(2+)-insensitive tension when returned to a relaxing solution with neutral pH. The presence of ADP during treatment was essential for the manifestation of the Ca(2+)-insensitive contraction. This contraction was reversibly eliminated by withdrawal of MgATP or addition of vanadate and was found to be accompanied by an extraordinarily high level of 20-kDa myosin light-chain (MLC20) phosphorylation. The rate constant for dephosphorylation of MLC20 in treated strips was about one-twenty-fifth that in untreated control, when determined after removal of Ca2+, Mg2+, and ATP. Two-dimensional phosphopeptide mapping of tryptic digests of MLC20 showed that most incorporated phosphate was in the peptides which would be phosphorylated by myosin light-chain kinase. These results provide strong evidence that ADP inactivates myosin light-chain phosphatase under acidic conditions.

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