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.

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 Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation.

1995 ◽  
Vol 130 (3) ◽  
pp. 613-627 ◽  
Author(s):  
Z M Goeckeler ◽  
R B Wysolmerski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Isometric Contraction ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Isometric Tension ◽  
Stress Fibers ◽  
Light Chains ◽  
Myosin Light Chains

The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F-actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.

Immunoglobulin light chain alters mesangial cell calcium homeostasis

1997 ◽  
Vol 272 (3) ◽  
pp. F319-F324 ◽  
Author(s):  
L. Zhu ◽  
G. A. Herrera ◽  
C. R. White ◽  
P. W. Sanders
Keyword(s):  
Calcium Homeostasis ◽  
Light Chain ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Calcium Influx ◽  
Light Chains ◽  
Kappa Light Chain ◽  
Immunoglobulin Light Chains ◽  
Acetoxymethyl Ester ◽  
Cell Calcium

This study examined the hypothesis that certain immunoglobulin light chains directly altered mesangial cell calcium homeostasis. Intracellular Ca2+ concentration (intracellular [Ca2+]) signaling was determined in suspensions of rat mesangial cells using the acetoxymethyl ester of fura 2 with a calcium removal/replacement protocol. Pretreatment of cultured rat mesangial cells with a glomerulopathic kappa-light chain (gle) produced reversible dose- and time-dependent attenuation of ATP- and thrombin-evoked [Ca2+] transients (189 +/- 24 vs. 126 +/- 10 nM, P < 0.05 with ATP; 198 +/- 5 vs. 117 +/- 3 nM, P < 0.05 with thrombin) and capacitative calcium influx (199 +/- 14 vs. 142 +/- 17 nM, P < 0.05 for ATP; 252 +/- 19 vs. 198 +/- 18 nM, P < 0.05 for thrombin). Mesangial cells treated with gle and supplemented with myo-inositol (450 microM) did not demonstrate the attenuation of the ATP-evoked [Ca2+] transient and capacitative calcium influx. Gle also decreased mean [Ca2+] transient (80 +/- 7 vs. 56 +/- 1 nM, P < 0.05) and capacitative calcium influx (306 +/- 10 vs. 241 +/- 4 nM, P < 0.05) in response to thapsigargin, a Ca2+-adenosinetriphosphatase inhibitor. This inhibition was not reversed by exogenous myo-inositol. Another kappa-light chain (10 microg/ml) did not affect mesangial cell calcium signaling. Deranged mesangial cell calcium homeostasis by certain light chains may play a central pathogenetic role in glomerulosclerosis associated with deposition of immunoglobulin light chains.

scholarly journals Myosin Light Chain Kinase Binding and Transiting along and between Actin Filaments and Stress Fibers

2013 ◽  
Vol 104 (2) ◽  
pp. 649a
Author(s):  
Kevin C. Facemyer ◽  
Ruby Sukhraj ◽  
Feng Hong ◽  
Christine R. Cremo ◽  
Josh E. Baker
Keyword(s):  
Light Chain ◽  
Actin Filaments ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Stress Fibers

scholarly journals Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin α5 in the glomerular basement membrane

2003 ◽  
Vol 161 (1) ◽  
pp. 187-196 ◽  
Author(s):  
Yamato Kikkawa ◽  
Ismo Virtanen ◽  
Jeffrey H. Miner
Keyword(s):  
Cell Adhesion ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Basement Membranes ◽  
Domain Specific ◽  
Integrin Α3β1 ◽  
Glomerular Capillaries

In developing glomeruli, laminin α5 replaces laminin α1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin α5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin α5 domains VI through I fused to the human laminin α1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 −/− background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 −/− glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin α5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the α5 G domain essential for mesangial cell adhesion to α5LG3-5. Finally, in vitro studies showed that integrin α3β1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin α5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin α5 in the GBM.

Regulation of MDCK cell-substratum adhesion by RhoA and myosin light chain kinase after ATP depletion

2004 ◽  
Vol 286 (3) ◽  
pp. C693-C707 ◽  
Author(s):  
Priya Prahalad ◽  
Ignacio Calvo ◽  
Holly Waechter ◽  
Jeffrey B. Matthews ◽  
Anna Zuk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Light Chain ◽  
Actin Filaments ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Cell Attachment ◽  
Mdck Cells ◽  
Atp Depletion ◽  
Collagen I ◽  
Stress Fibers

The attachment of epithelial cells to the extracellular matrix substratum is essential for their differentiation and polarization. Despite this, the precise adhesion mechanism and its regulation are poorly understood. In the kidney, an ischemic insult causes renal tubular epithelial cells to detach from the basement membrane, even though they remain viable. To understand this phenomenon, and to probe the regulation of epithelial cell attachment, we used a model system consisting of newly adherent Madin-Darby canine kidney (MDCK) cells subjected to ATP depletion to mimic ischemic injury. We found that MDCK cells detach from collagen I after 60 min of ATP depletion but reattach when resupplied with glucose. Detachment is not caused by degradation or endocytosis of β1-integrins, which mediate attachment to collagen I. Basal actin filaments and paxillin-containing adhesion complexes are disrupted by ATP depletion and quickly reform on glucose repletion. However, partial preservation of basal actin by overexpression of constitutively active RhoA does not significantly affect cell detachment. Furthermore, Y-27632, an inhibitor of the RhoA effector Rho-kinase, does not prevent reattachment of cells on glucose addition, even though reformation of central stress fibers and large adhesion complexes is blocked. In contrast, reattachment of ATP-depleted cells and detachment of cells not previously subjected to ATP depletion are prevented by ML-7, an inhibitor of myosin light chain kinase (MLCK). We conclude that initial adherence of MDCK cells to a collagen I substratum is mediated by peripheral actin filaments and adhesion complexes regulated by MLCK but not by stress fibers and adhesion complexes controlled by RhoA.

scholarly journals The molecular organization of myosin in stress fibers of cultured cells.

1986 ◽  
Vol 102 (1) ◽  
pp. 200-209 ◽  
Author(s):  
G Langanger ◽  
M Moeremans ◽  
G Daneels ◽  
A Sobieszek ◽  
M De Brabander ◽  
...  
Keyword(s):  
Light Chain ◽  
Cultured Cells ◽  
Staining Pattern ◽  
Central Zone ◽  
Molecular Organization ◽  
Stress Fibers ◽  
General Distribution ◽  
Immunogold Staining ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

scholarly journals Fibronectin localization in the rat glomerulus.

1980 ◽  
Vol 87 (3) ◽  
pp. 691-696 ◽  
Author(s):  
P J Courtoy ◽  
Y S Kanwar ◽  
R O Hynes ◽  
M G Farquhar
Keyword(s):  
Epithelial Cells ◽  
Mesangial Cell ◽  
Cultured Cells ◽  
Mesangial Cells ◽  
Basement Membranes ◽  
Frozen Sections ◽  
Mesangial Matrix ◽  
Capillary Loop ◽  
Important Constituent

Fibronectin (FN) has been localized in the rat glomerulus using indirect immunolabeling. It was demonstrated in frozen sections by immunofluorescence, in sections of fixed kidneys by both peroxidase and ferritin-labeled antibodies, and in isolated glomerular basement membranes (GBM) with ferritin-labeled antibodies. Complementary and convergent results were obtained with these approaches. FN was most abundant in the mesangial matrix where it was especially concentrated at the interface between the endothelial and mesangial cells. In the peripheral capillary loop, FN was also detected in the laminae rarae (interna and externa) of the GBM--i.e., between the endothelial and epithelial cells, respectively, and the GBM. These findings indicate that FN is an important constituent of the glomerulus, and they are compatible with the assumption that, in the glomerulus, as in cultured cells, FN is involved in cell-to-cell (mesangial-mesangial, mesangial-endothelial) and cell-to-substrate (mesangial cell-mesangial matrix, epithelium-GBM, endothelium-GBM) attachment.

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