scholarly journals PDGF receptor-β modulates metanephric mesenchyme chemotaxis induced by PDGF AA

2009 ◽  
Vol 296 (2) ◽  
pp. F406-F417 ◽  
Author(s):  
Jill M. Ricono ◽  
Brent Wagner ◽  
Yves Gorin ◽  
Mazen Arar ◽  
Andrius Kazlauskas ◽  
...  
Keyword(s):  
Cell Migration ◽  
Kidney Development ◽  
Receptor Activation ◽  
Ros Generation ◽  
Pdgf Receptor ◽  
Metanephric Mesenchyme ◽  
Wild Type ◽  
Intracellular Ca ◽  
Β Receptor ◽  
In Cells

PDGF B chain or PDGF receptor (PDGFR)-β-deficient (−/−) mice lack mesangial cells. To study responses of α- and β-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and −/− mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for −/− MMCs. The mechanism by which PDGFR-β inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular Ca2+ and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of −/− MMCs with the wild-type β-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of Ca2+ signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular Ca2+ concentration, suggesting that ROS act as upstream mediators of Ca2+ in suppressing PDGF AA-induced migration. These data indicate that ROS and Ca2+ generated by active PDGFR-β play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs. During kidney development, PDGFR β-mediated ROS generation and Ca2+ influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme.

scholarly journals Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

2004 ◽  
Vol 279 (44) ◽  
pp. 46242-46252 ◽  
Author(s):  
Larry E. Wagner ◽  
Wen-Hong Li ◽  
Suresh K. Joseph ◽  
David I. Yule
Keyword(s):  
Splice Variants ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Igm Antibody ◽  
Temporal Properties ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Spatio Temporal ◽  
In Cells

Regulation of Ca2+release through inositol 1,4,5-trisphosphate receptors (InsP3R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca2+signals. In this study, regulation of Ca2+release by phosphorylation of type 1 InsP3R (InsP3R-1) was investigated by constructing “phosphomimetic” charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP3R-1 splice variants. Ca2+release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP3R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP3R-1, InsP3-induced Ca2+release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca2+release through the S2- S1589E/S1755E InsP3R-1 was enhanced ∼8-fold over wild type and ∼50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP3R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca2+release was enhanced ∼3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca2+release. The sensitivity of Ca2+release in cells expressing S2+ S1755E InsP3R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP3R-1. In contrast, mutation of S2+ S1589E InsP3R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP3R-1 resulted in robust Ca2+oscillations when cells were stimulated with concentrations of α-IgM antibody that were threshold for stimulation in S2- wild type InsP3R-1-expressing cells. However, at higher concentrations of α-IgM antibody, Ca2+oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP3R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.

ITD-Flt3 Enhances Migration of Hematopoietic Cells through Aberrant CXCR4 Signaling Pathways Overlapping but Functionally Distinct From SDF1/CXCR4 Signaling Axis in Normal Hematopoietic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2383-2383
Author(s):  
Seiji Fukuda ◽  
Satomi Mori ◽  
Mariko Abe ◽  
Seiji Yamaguchi ◽  
Louis M. Pelus
Keyword(s):  
Cell Migration ◽  
Signaling Pathways ◽  
Cross Talk ◽  
Hematopoietic Cells ◽  
Leukemia Cells ◽  
Wild Type ◽  
Transcription Analysis ◽  
Normal Cells ◽  
Cxcr4 Signaling ◽  
In Cells

Abstract Abstract 2383 Poster Board II-360 Elevated expression of CXCR4, a receptor for SDF1, with Internal Tandem Duplication of Flt3 (ITD-Flt3) is an indicator of poor prognosis in patients with acute myeloid leukemia. We previously showed that ITD-Flt3 enhances migration of hematopoietic cells to SDF1, suggesting that ITD-Flt3 may facilitate dissemination of leukemia cells by modulating SDF1/CXCR4 signaling and that blocking this functional cross-talk between ITD-Flt3 and CXCR4 pathways may have therapeutic benefit. While identification of selective pathways in cells transformed by ITD-Flt3, which are distinct from normal cells is crucial to develop therapeutic agents without hematopoietic toxicity, the mechanisms responsible for aberrant migration induced by ITD-Flt3 are not known. We now demonstrate the existence of CXCR4 signaling pathways regulated by ITD-Flt3 that are distinct from normal CXCR4 signaling using genome wide transcription analysis. Ectopic expression of ITD-Flt3 in Ba/F3 cells enhances random cell migration and modulates expression of 1,675 genes out of 41,174 genes (4.1%) examined compared to control cells lacking ITD-Flt3. ITD-Flt3 down-regulated CXCR4 mRNA by 55% compared to control and modulated 36 additional transcripts implicated in cell migration and localization (0.09%, P<0.05), further substantiating a role of ITD-Flt3 in cell migration and suggesting that enhanced migration was not dependent on CXCR4. Coordinately, ectopic ITD-Flt3 significantly enhanced cell migration to SDF1 compared to cells expressing wild-type Flt3 despite reduction in CXCR4. In contrast, in primary HPC, enhanced migration induced by ITD-Flt3 was inhibited by conditional deletion of CXCR4 in Cre-ERTM-CXCR4fl/fl mice cells or by incubation with the selective CXCR4 antagonist AMD3100. Analysis of gene expression in control and ITD-Flt3 expressing Ba/F3 cells migrating to SDF1 indicated that SDF1 modulates 1,647 (4.0%) of 41,174 genes analyzed in cells expressing ITD-Flt3, of which 1,190 genes (2.5%) were regulated by SDF1 exclusively in ITD-Flt3 cells, such as neurofibromatosis type 1 and p27kip1. SDF1 regulated 457 genes (1.1%) in both cells expressing wild-type Flt3 and ITD Flt3, whereas, 384 (0.5%) transcripts were regulated by SDF1 in wild-type Flt3 expressing cells but not in ITD-Flt3 cells. Genes associated with transcription, cytoskeleton, ubiquitination, kinase and iron transport were functionally clustered both in ITD-Flt3 and wild-type Flt3 cells migrating to SDF1. In contrast, genes involved in apoptosis, cell cycle, glycosylation and dephosphorylation were significantly enriched in ITD-Flt3 but not in wild-type Flt3 cells, suggesting that ITD-Flt3 generates CXCR4 signaling pathways that are qualitatively different from normal cells. In addition, 69 genes (0.17%) were down-regulated by SDF1 in ITD-Flt3 cells but up-regulated in control cells or vice versa, including Rho-associated coiled-coil forming kinase 1 (ROCK1), which has been linked with leukocyte transendothelial migration, cell motility and tumor cell invasion. While baseline expression of ROCK1 was not affected by the presence of ITD-Flt3, ROCK1 was up-regulated 251% by SDF1 in control cells but down-regulated by 67% in ITD-Flt3 cells. The ROCK antagonist Y27632 significantly decreased migration of control Ba/F3 cells lacking ITD-Flt3 to SDF1 (77 ± 6% inhibition, P<0.01), but had no effect on SDF1-induced migration of ITD-Flt3 cells, suggesting that an increase of ROCK1 is required for normal CXCR4 signaling, while enhanced migration to SDF1 induced by ITD-Flt3 is independent of ROCK system. Our data provide additional evidence for functional cross-talk between SDF1/CXCR4 and ITD-Flt3 signaling pathways. Inhibition of aberrant migration to SDF1 induced by ITD-Flt3 by blocking CXCR4 suggests that antagonizing CXCR4 may inhibit dissemination of hematopoietic cells expressing ITD-Flt3. ITD-Flt3 regulates overlapping but functionally distinct pathways down-stream of SDF1/CXCR4 compared to those in cells without ITD-Flt3. Genes differentially regulated by SDF1 specifically in ITD-Flt3 cells may represent key targets regulating aberrant migration by ITD-Flt3 in response to SDF1 to prevent unnecessary dissemination and invasion of ITD-Flt3+ acute leukemia cells without affecting normal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Src tyrosine kinase mediates platelet-derived growth factor BB-induced and redox-dependent migration in metanephric mesenchymal cells

2014 ◽  
Vol 306 (1) ◽  
pp. F85-F97 ◽  
Author(s):  
Brent Wagner ◽  
Yves Gorin
Keyword(s):  
Cell Migration ◽  
Growth Factor ◽  
Tyrosine Kinase ◽  
Wound Closure ◽  
Platelet Derived Growth Factor ◽  
Ros Generation ◽  
Pdgf Receptor ◽  
Src Tyrosine Kinase ◽  
Akt Phosphorylation ◽  
And Migration

The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild-type and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-l-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2′,7′-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4.

scholarly journals Morphological Insights into the Origin of Glomerular Endothelial and Mesangial Cells and Their Precursors

2003 ◽  
Vol 51 (2) ◽  
pp. 141-150 ◽  
Author(s):  
Jill M. Ricono ◽  
Yi-Chun Xu ◽  
Mazen Arar ◽  
Dong-chan Jin ◽  
Jeffrey L. Barnes ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Kidney Development ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Rat Kidney ◽  
Temporal Distribution ◽  
Metanephric Mesenchyme ◽  
Early Stages ◽  
Glomerular Capillary ◽  
In Cells

Glomerular endothelial and mesangial cells may originate from the metanephric mesenchyme. We used the MAb Thy1.1, a mesangial cell marker in the adult rat kidney, and rat endothelial cell markers MAb RECA-1, MAb PECAM-1 (CD31), and MAb Flk-1 as potential markers to characterize the spatial and temporal distribution of mesangial and endothelial cell precursors during nephrogenesis in the rat. At early stages of glomerulogenesis, RECA-1- and Thy1.1-positive cells were detected in the metanephric blastema at 14 days post conception (dpc) embryos and 15 dpc, respectively, with Thy1.1 expression in cells surrounding the ureteric bud. At 17 and 18 dpc, both RECA-1- and Thy1.1-positive cells were found in the cleft of the S-shaped bodies and in the capillary loops of maturing glomeruli. Double staining for BrdU, a marker of proliferation, and for RECA-1 or BrdU and Thy1.1 also localize in the cleft of S-shaped bodies and in glomerular capillary loops at later stages of development. PDGFRβ co-localizes in cells expressing endothelial or mesangial markers. The data suggest that endothelial and mesangial cell precursors share common markers during the course of glomerulogenesis and that full differentiation of these cells occurs at late stages of glomerular maturation. Thy1.1- and RECA-1-positive cells may be derived from the metanephric blastemal cells at early stages of kidney development. A sub-population of these Thy1.1- or RECA-1-positive cells may be precursors that can migrate into the cleft of comma and S-shaped bodies and proliferate in situ to form glomerular capillary tufts.

Giα3 protein-coupled dopamine D3 receptor-mediated inhibition of renal NHE3 activity in SHR proximal tubular cells is a PLC-PKC-mediated event

2004 ◽  
Vol 287 (5) ◽  
pp. F1059-F1066 ◽  
Author(s):  
Rui Pedrosa ◽  
Pedro Gomes ◽  
Ulrich Hopfer ◽  
Pedro A. Jose ◽  
Patrício Soares-da-Silva
Keyword(s):  
Spontaneously Hypertensive Rat ◽  
Calcium Ionophore ◽  
Receptor Activation ◽  
Inhibitory Effects ◽  
Atpase Inhibitor ◽  
Spontaneously Hypertensive ◽  
P38 Inhibitor ◽  
Intracellular Ca ◽  
Proximal Tubular ◽  
In Cells

This study evaluated the transduction pathway associated with type 3 Na+/H+ exchanger (NHE3) activity-induced inhibition during dopamine D3 receptor activation in immortalized renal proximal tubular epithelial cells from the spontaneously hypertensive rat. The dopamine D3 receptor agonist 7-OH-DPAT decreased NHE3 activity, which was prevented by the D2-like receptor antagonist S-sulpiride, pertussis toxin (PTX; overnight treatment), and the PKC inhibitor chelerythrine, but not by cholera toxin (overnight treatment), the MAPK inhibitor PD-098059, or the p38 inhibitor SB-203580. The PKA inhibitor H-89 abolished the inhibitory effects of forskolin on NHE3 activity, but not that of 7-OH-DPAT. The phospholipase C (PLC) inhibitor U-73122 prevented the inhibitory effects of 7-OH-DPAT, whereas PDBu and 7-OH-DPAT increased PLC activity and reduced NHE3 activity; downregulation of PKC abolished the inhibitory effects of both PDBu and 7-OH-DPAT on NHE activity. The inhibition of NHE3 activity by GTPγS and the prevention of the effect of 7-OH-DPAT by PTX suggest an involvement of a Gi/o protein coupled to the dopamine D3 receptor. Indeed, the 7-OH-DPAT-induced decrease in NHE3 activity was abolished in cells treated overnight with the anti-Giα3 antibody, but not in cells treated with antibodies against Gq/11, Gsα, Gβ, and Giα1,2 proteins. The calcium ionophore A-23187 and the Ca2+-ATPase inhibitor thapsigargin increased intracellular Ca2+ but did not affect NHE3 activity. However, the inhibitory effects of PDBu and 7-OH-DPAT on NHE3 activity were completely abolished by A-23287 and thapsigargin. It is concluded that inhibition of NHE3 activity by dopamine D3 receptors coupled to Giα3 proteins is a PLC-PKC-mediated event, modulated by intracellular Ca2+.

scholarly journals Role of adenosine A1 and A3 receptors in regulation of cardiomyocyte homeostasis after mitochondrial respiratory chain injury

2005 ◽  
Vol 288 (6) ◽  
pp. H2792-H2801 ◽  
Author(s):  
Vladimir Shneyvays ◽  
Dorit Leshem ◽  
Tova Zinman ◽  
Liaman K. Mamedova ◽  
Kenneth A. Jacobson ◽  
...  
Keyword(s):  
Respiratory Chain ◽  
Sodium Azide ◽  
Adenosine Receptor ◽  
Mitochondrial Respiratory Chain ◽  
Receptor Activation ◽  
Atp Level ◽  
Intracellular Ca ◽  
Mitochondrial Respiratory ◽  
In Cells

Activation of either the A1 or the A3 adenosine receptor (A1R or A3R, respectively) elicits delayed cardioprotection against infarction, ischemia, and hypoxia. Mitochondrial contribution to the progression of cardiomyocyte injury is well known; however, the protective effects of adenosine receptor activation in cardiac cells with a respiratory chain deficiency are poorly elucidated. The aim of our study was to further define the role of A1R and A3R activation on functional tolerance after inhibition of the terminal link of the mitochondrial respiratory chain with sodium azide, in a state of normoxia or hypoxia, compared with the effects of the mitochondrial ATP-sensitive K+ channel opener diazoxide. Treatment with 10 mM sodium azide for 2 h in normoxia caused a considerable decrease in the total ATP level; however, activation of adenosine receptors significantly attenuated this decrease. Diazoxide (100 μM) was less effective in protection. During treatment of cultured cardiomyocytes with hypoxia in the presence of 1 mM sodium azide, the A1R agonist 2-chloro- N6-cyclopentyladenosine was ineffective, whereas the A3R agonist 2-chloro- N6-iodobenzyl-5′- N-methylcarboxamidoadenosine (Cl-IB-MECA) attenuated the decrease in ATP level and prevented cell injury. Cl-IB-MECA delayed the dissipation in the mitochondrial membrane potential during hypoxia in cells impaired in the mitochondrial respiratory chain. In cells with elevated intracellular Ca2+ concentration after hypoxia and treatment with NaN3 or after application of high doses of NaN3, Cl-IB-MECA immediately decreased the elevated intracellular Ca2+ concentration toward the diastolic control level. The A1R agonist was ineffective. This may be especially important for the development of effective pharmacological agents, because mitochondrial dysfunction is a leading factor in the pathophysiological cascade of heart disease.

scholarly journals A cardioprotective role for platelet-activating factor through NOS-dependent S-nitrosylation

2008 ◽  
Vol 294 (6) ◽  
pp. H2775-H2784 ◽  
Author(s):  
Peter J. Leary ◽  
Surender Rajasekaran ◽  
R. Ray Morrison ◽  
Elaine I. Tuomanen ◽  
Thomas K. Chin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Diseases ◽  
Ischemia Reperfusion ◽  
Platelet Activating Factor ◽  
Receptor Activation ◽  
Protective Effects ◽  
Loss Of Function ◽  
Wild Type ◽  
Intracellular Ca ◽  
Paf Receptor

Controversy exists as to whether platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, can actually protect the heart from postischemic injury. To determine whether endogenous activation of the PAF receptor is cardioprotective, we examined postischemic functional recovery in isolated hearts from wild-type and PAF receptor-knockout mice. Postischemic function was reduced in hearts with targeted deletion of the PAF receptor and in wild-type hearts treated with a PAF receptor antagonist. Furthermore, perfusion with picomolar concentrations of PAF improved postischemic function in hearts from wild-type mice. To elucidate the mechanism of a PAF-mediated cardioprotective effect, we employed a model of intracellular Ca2+ overload and loss of function in nonischemic ventricular myocytes. We found that PAF receptor activation attenuates the time-dependent loss of shortening and increases in intracellular Ca2+ transients in Ca2+-overloaded myocytes. These protective effects of PAF depend on nitric oxide, but not activation of cGMP. In addition, we found that reversible S-nitrosylation of myocardial proteins must occur in order for PAF to moderate Ca2+ overload and loss of myocyte function. Thus our data are consistent with the hypothesis that low-level PAF receptor activation initiates nitric oxide-induced S-nitrosylation of Ca2+-handling proteins, e.g., L-type Ca2+ channels, to attenuate Ca2+ overload during ischemia-reperfusion in the heart. Since inhibition of the PAF protective pathway reduces myocardial postischemic function, our results raise concern that clinical therapies for inflammatory diseases that lead to complete blockade of the PAF receptor may eliminate a significant, endogenous cardioprotective pathway.

Renal agenesis and hypodysplasia in ret-k- mutant mice result from defects in ureteric bud development

Development ◽  
1996 ◽  
Vol 122 (6) ◽  
pp. 1919-1929 ◽  
Author(s):  
A. Schuchardt ◽  
V. D'Agati ◽  
V. Pachnis ◽  
F. Costantini
Keyword(s):  
Kidney Development ◽  
Renal Agenesis ◽  
Critical Role ◽  
Wolffian Duct ◽  
Excretory System ◽  
Metanephric Mesenchyme ◽  
Wild Type ◽  
Ureteric Bud ◽  
Primary Defect ◽  
Newborn Mice

The c-ret gene encodes a receptor tyrosine kinase that is expressed in the Wolffian duct and ureteric bud of the developing excretory system. Newborn mice homozygous for a mutation in c-ret displayed renal agenesis or severe hypodysplasia, suggesting a critical role for this gene in metanephric kidney development. To investigate the embryological basis of these defects, we characterized the early development of the excretory system in mutant homozygotes, and observed a range of defects in the formation, growth and branching of the ureteric bud, which account for the spectrum of renal defects seen at birth. Co-culture of isolated ureteric buds and metanephric mesenchyme show that the primary defect is intrinsic to the ureteric bud. While the mutant bud failed to respond to induction by wild-type mesenchyme, mutant mesenchyme was competent to induce the growth and branching of the wild-type bud. Furthermore, the mutant metanephric mesenchyme displayed a normal capacity to differentiate into nephric tubules when co-cultured with embryonic spinal cord. These findings suggest a model in which c-ret encodes the receptor for a (yet to be identified) factor produced by the metanephric mesenchyme, which mediates the inductive effects of this tissue upon the ureteric bud. This factor appears to stimulate the initial evagination of the ureteric bud from the Wolffian duct, as well as its subsequent growth and branching.

Changes in Na+-K+-ATPase activity influence cell attachment to fibronectin

2002 ◽  
Vol 282 (2) ◽  
pp. C302-C309 ◽  
Author(s):  
Roger Belusa ◽  
Oleg Aizman ◽  
Ronnie M. Andersson ◽  
Anita Aperia
Keyword(s):  
Atpase Activity ◽  
Cell Attachment ◽  
Ion Homeostasis ◽  
Cell Types ◽  
Wild Type ◽  
Cellular Functions ◽  
Intracellular Ca ◽  
Atpase Inhibition ◽  
Sustained Increase ◽  
In Cells

Most vital cellular functions are dependent on a fine-tuned regulation of intracellular ion homeostasis. Here we have demonstrated, using COS cells that were untransfected or transfected with wild-type rat ouabain-resistant Na+-K+-ATPase, that partial inhibition of Na+-K+-ATPase has a dramatic influence on cell attachment to fibronectin. Ouabain dose-dependently decreased attachment in untransfected cells and in cells expressing wild-type Na+-K+-ATPase, but not in cells expressing ouabain-insensitive Na+-K+-ATPase, whereas inhibition of Na+-K+-ATPase by lowering extracellular K+ concentration decreased attachment in all three cell types. Thirty percent inhibition of Na+-K+-ATPase significantly attenuated attachment. Na+-K+-ATPase inhibition caused a sustained increase in the intracellular Ca2+ concentration that obscured Ca2+ transients observed in untreated cells during attachment. Inhibitors of Ca2+ transporters significantly decreased attachment, but inhibition of Na+/H+ exchanger did not. Ouabain reduced focal adhesion kinase autophosphorylation but had no effect on cell surface integrin expression. These results suggest that the level of Na+-K+-ATPase activity strongly influences cell attachment, possibly by an effect on intracellular Ca2+.

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