Phosphatidylinositol 3,4,5-trisphosphate: an early mediator of insulin-stimulated sodium transport in A6 cells

2004 ◽  
Vol 287 (2) ◽  
pp. F319-F328 ◽  
Author(s):  
Nicolas Markadieu ◽  
Daniel Blero ◽  
Alain Boom ◽  
Christophe Erneux ◽  
Renaud Beauwens
Keyword(s):  
Sodium Transport ◽  
Cell Monolayer ◽  
Sodium Reabsorption ◽  
Na Channel ◽  
Insulin Stimulation ◽  
Cell Monolayers ◽  
A6 Cells ◽  
Cell Extracts ◽  
Pkb Phosphorylation ◽  
Stimulation Of

Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP3 and PI( 3 , 4 )P2 but not PI( 4 , 5 )P2 stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP3 levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP3 was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP3 also increased PKB phosphorylation. Taken together, the present results establish that the d-3-phosphorylated phosphoinositides PIP3 and PI( 3 , 4 )P2 mediate the effect of insulin on sodium transport across A6 cell monolayers.

scholarly journals Inhibition of insulin-stimulated hydrogen peroxide production prevents stimulation of sodium transport in A6 cell monolayers

2009 ◽  
Vol 296 (6) ◽  
pp. F1428-F1438 ◽  
Author(s):  
Nicolas Markadieu ◽  
Raphaël Crutzen ◽  
Alain Boom ◽  
Christophe Erneux ◽  
Renaud Beauwens
Keyword(s):  
Sodium Transport ◽  
Regulatory Subunit ◽  
Insulin Stimulation ◽  
Subsequent Increase ◽  
Kinase Activation ◽  
Cell Monolayers ◽  
Intracellular Ros ◽  
Exogenous Addition ◽  
Subsequent Rise ◽  
Stimulation Of

Insulin-stimulated sodium transport across A6 cell (derived from amphibian distal nephron) monolayers involves the activation of a phosphatidylinositol (PI) 3-kinase. We previously demonstrated that exogenous addition of H2O2 to the incubation medium of A6 cell monolayers provokes an increase in PI 3-kinase activity and a subsequent rise in sodium transport (Markadieu N, Crutzen R, Blero D, Erneux C, Beauwens R. Am J Physiol Renal Physiol 288: F1201–F1212, 2005). We therefore questioned whether insulin would produce an intracellular burst of H2O2 leading to PI 3-kinase activation and subsequent increase in sodium transport. An acute production of reactive oxygen species (ROS) in A6 cells incubated with the oxidation-sensitive fluorescent probe 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate was already detected after 2 min of insulin stimulation. This fluorescent signal and the increase in sodium transport were completely inhibited in monolayers incubated with peggylated catalase, indicating that H2O2 is the main intracellular ROS produced upon insulin stimulation. Similarly, preincubation of monolayers with different chelators of either superoxide (O2•−; nitro blue tetrazolium, 100 μM) or H2O2 (50 μM ebselen), or blockers of NADPH oxidase (Nox) enzymes (diphenyleneiodonium, 5 μM; phenylarsine oxide, 1 μM and plumbagin, 30 μM) prevented both insulin-stimulated H2O2 production and insulin-stimulated sodium transport. Furthermore, diphenyleneiodonium pretreatment inhibited the recruitment of the p85 PI 3-kinase regulatory subunit in an anti-phosphotyrosine immunoprecipitate in insulin-stimulated cells. In contrast, PI-103, an inhibitor of class IA PI 3-kinase, inhibited insulin-stimulated sodium transport but did not significantly reduce insulin-stimulated H2O2 production. Taken together, our data suggest that insulin induces an acute burst of H2O2 production which participates in an increase in phosphatidylinositol 3,4,5- trisphosphate production and subsequently stimulation of sodium transport.

scholarly journals Expression of epithelial Na channels in Xenopus oocytes.

1990 ◽  
Vol 96 (1) ◽  
pp. 23-46 ◽  
Author(s):  
L G Palmer ◽  
I Corthesy-Theulaz ◽  
H P Gaeggeler ◽  
J P Kraehenbuhl ◽  
B Rossier
Keyword(s):  
Sedimentation Coefficient ◽  
High Sensitivity ◽  
Sucrose Density Gradient ◽  
Na Channel ◽  
Channel Activity ◽  
Membrane Expression ◽  
Cell Monolayers ◽  
A6 Cells ◽  
Epithelial Na Channels ◽  
Confluent Cell

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride-sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.

scholarly journals Insulin stimulation of Akt/PKB phosphorylation in the placenta of preeclampsia patients

2011 ◽  
Vol 129 (6) ◽  
pp. 387-391 ◽  
Author(s):  
Gustavo Dias Ferreira ◽  
Rafael Bueno Orcy ◽  
Sérgio Hofmeister Martins-Costa ◽  
José Geraldo Lopes Ramos ◽  
Ilma Simoni Brum ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Systemic Disease ◽  
Cross Sectional Study ◽  
University Hospital ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Stimulation ◽  
Cross Sectional ◽  
Pkb Phosphorylation ◽  
Stimulation Of

CONTEXT AND OBJECTIVE: Preeclampsia is a multi-systemic disease and one of the most frequent severe health problems during pregnancy. Binding of insulin triggers phosphorylation and activates cytoplasmic substrates such as phosphatidylinositol 3 kinase (PI3K). Phosphorylation of membrane phosphoinositide 2 (PIP2) to phosphoinositide 3 (PIP3) by PI3K starts Akt/PKB activation. Defects in phosphorylation of the insulin receptor and its substrates have an important role in insulin resistance. Studies have shown that insulin resistance is associated with preeclampsia and its pathophysiology. The aim here was to investigate insulin stimulation of the Akt/PKB pathway in the placenta, in normal and preeclampsia parturients. DESIGN AND SETTING: Cross-sectional study in a tertiary public university hospital. METHODS: Placentas were collected from 12 normal and 12 preeclampsia patients. These were stimulated and analyzed using Western blot to quantify the Akt/PKB phosphorylation. RESULTS: The insulin stimulation was confirmed through comparing the stimulated group (1.14 ± 0.10) with the non-stimulated group (0.91 ± 0.08; P < 0.001). The phosphorylation of Akt/PKB did not differ between the placenta of the normal patients (1.26 ± 0.16) and those of the preeclampsia patients (1.01 ± 0.11; P = 0.237). CONCLUSIONS: In vitro insulin stimulation of the human placenta has been well established. There was no difference in Akt/PKB phosphorylation, after stimulation with insulin, between placentas of normal and preeclampsia patients. Nevertheless, it cannot be ruled out that the Akt/PKB signaling pathway may have a role in the pathophysiology of preeclampsia, since the substrates of Akt/PKB still need to be investigated.

scholarly journals Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

1996 ◽  
Vol 16 (2) ◽  
pp. 577-583 ◽  
Author(s):  
K H Holt ◽  
B G Kasson ◽  
J E Pessin
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Stimulation ◽  
Anion Exchange Column ◽  
Cell Extracts ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Stimulation Of

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

Basolateral P2X4-like receptors regulate the extracellular ATP-stimulated epithelial Na+ channel activity in renal epithelia

2007 ◽  
Vol 292 (6) ◽  
pp. F1734-F1740 ◽  
Author(s):  
Yanjun Zhang ◽  
Daniel Sanchez ◽  
Julia Gorelik ◽  
David Klenerman ◽  
Max Lab ◽  
...  
Keyword(s):  
Sodium Transport ◽  
Purinergic Receptors ◽  
Morphological Changes ◽  
Extracellular Atp ◽  
Sodium Reabsorption ◽  
Na Channel ◽  
Renal Epithelia ◽  
Stimulatory Action ◽  
Sodium Conductance ◽  
Basolateral Side

Extracellular ATP initiates potent effects on sodium transport across renal epithelia through membrane-associated purinergic receptors. Dependent on the location of these receptors, ATP either inhibits or stimulates sodium reabsorption. Using A6 cells, transepithelial electrical resistance measurements, and scanning ion conductance microscopy, we have identified the purinergic receptors involved in the stimulatory action on the epithelial cell basolateral plasma membrane. Addition of the potent P2X4 receptor agonist 2-methylthio-ATP (2MeSATP) to the basolateral side of the A6 monolayer stimulated amiloride-sensitive sodium conductance and produced similar cell morphological changes to those found with ATPγS, aldosterone, or hypotonic stress. The agonist potency order determined by sodium conductance changes of the monolayer was: 2MeSATP ≥ ATPγS > CTP, a similar agonist potency profile to that of cloned P2X4 receptors but with higher sensitivity for β, γ-methylene-ATP and α,β-methylene-ATP. We further demonstrated that the ATP effect on sodium transport was potentiated by ivermectin, not blocked by suramin and PPADS, enhanced by Zn2+ but not by Cu2+, and significantly reduced but not totally inhibited by brilliant blue G. These results led us to conclude that basolateral P2X4-like receptors were involved. We suggest that there is a reciprocal purinergic system acting both at a basolateral and apical location for control of Na+ transport. This requires a mechanism within the cell that leads to either basolateral or apical ATP release to regulate renal tubular function.

Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-kinase and increase sodium transport in A6 cell monolayers

2005 ◽  
Vol 288 (6) ◽  
pp. F1201-F1212 ◽  
Author(s):  
Nicolas Markadieu ◽  
Raphaël Crutzen ◽  
Daniel Blero ◽  
Christophe Erneux ◽  
Renaud Beauwens
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Sodium Transport ◽  
Kinase Activity ◽  
Mapk Pathway ◽  
Phosphatidylinositol 3 Kinase ◽  
Cell Monolayers ◽  
Epidermal Growth ◽  
Kinase Pathway ◽  
Stimulation Of

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin stimulation of sodium transport in A6 cell monolayers. In this study, we investigate whether stimulation of the PI 3-kinase by other agents also provoked an increase in sodium transport. Both epidermal growth factor (EGF) and H2O2 provoked a rise in sodium transport that was inhibited by LY-294002, an inhibitor of PI 3-kinase activity. PI 3-kinase activity was estimated in extracts from A6 cell monolayers directly by performance of a PI 3-kinase assay. We also estimated the relative importance of the PI 3-kinase pathway by two different methods: 1) coprecipitation of the p85 regulatory subunit with anti-phosphotyrosine antibodies and 2) phosphorylation of PKB on both Ser 473 and Thr 308 residues observed by Western blotting. Since the mitogen-activated protein kinase (MAPK) pathway has also been implicated in the regulation of sodium transport, we also investigated whether this pathway is turned on by insulin, H2O2, or EGF. Phosphorylation of ERK1/2 was increased only transiently by insulin and H2O2 but quite sustainedly by EGF. Inhibitors of this pathway (U-0126 and PD-98059) failed to affect the insulin and H2O2 stimulation of sodium transport but increased substantially the stimulation induced by EGF. The latter effect was associated with an increase in PKB phosphorylation, thus suggesting that the stimulation of the MAPK pathway prevents, in part, the stimulation of the PI 3-kinase pathway in the transport of sodium stimulated by EGF.

Disinhibitory pathways for control of sodium transport: regulation of ENaC by SGK1 and GILZ

2006 ◽  
Vol 291 (4) ◽  
pp. F714-F721 ◽  
Author(s):  
Vivek Bhalla ◽  
Rama Soundararajan ◽  
Alan C. Pao ◽  
Hongyan Li ◽  
David Pearce
Keyword(s):  
Sodium Transport ◽  
Functional Significance ◽  
Early Phase ◽  
Mineralocorticoid Receptor ◽  
Tonic Inhibition ◽  
Sodium Reabsorption ◽  
Transport Regulation ◽  
Tight Epithelia ◽  
New Evidence ◽  
Stimulation Of

Regulation of ENaC occurs at several levels. The principal hormonal regulator of ENaC, aldosterone, acts through the mineralocorticoid receptor to modulate ENaC-mediated sodium transport, and considerable attention has focused on defining the components of the early phase of this response. Two genes, SGK1 and GILZ, have now been implicated in this regulation. While the functional significance of SGK1 in mediating aldosterone effects is well established, new evidence has enhanced our understanding of the mechanisms of SGK1 action. In addition, recent work demonstrates a novel role for GILZ in the stimulation of ENaC-mediated sodium transport. Interestingly, both SGK1 and GILZ appear to negatively regulate tonic inhibition of ENaC and thus use disinhibition to propagate the rapid effects of aldosterone to increase sodium reabsorption in tight epithelia.

scholarly journals Vasopressin stimulates sodium transport in A6 cells via a phosphatidylinositide 3-kinase-dependent pathway

1999 ◽  
Vol 277 (4) ◽  
pp. F575-F579 ◽  
Author(s):  
R. S. Edinger ◽  
M. D. Rokaw ◽  
J. P. Johnson
Keyword(s):  
Adenylate Cyclase ◽  
Sodium Transport ◽  
Second Messengers ◽  
Specific Inhibitor ◽  
Receptor Activation ◽  
A6 Cells ◽  
Inositol Phospholipids ◽  
A Site ◽  
Stimulation Of

The enzyme phosphatidylinositide 3-kinase (PI3K) phosphorylates the D-3 position of the inositol ring of inositol phospholipids and produces 3-phosphorylated inositides. These novel second messengers are thought to mediate diverse cellular signaling functions. The fungal metabolite wortmannin covalently binds to PI3K and selectively inhibits its activity. The role of PI3K in basal and hormone-stimulated transepithelial sodium transport was examined using this specific inhibitor. Wortmannin, 50 nM, did not affect basal, aldosterone-stimulated, or insulin-stimulated transport in A6 cells. Wortmannin completely inhibits vasopressin stimulation of transport in these cells. Vasopressin stimulates PI3K activity in A6 cells. Vasopressin stimulation of transport is also blocked by 5 μM LY-294002, a second inhibitor of PI3K. One-hour preincubation with wortmannin blocked vasopressin stimulation of protein kinase A activity in the cells. Sodium transport responses to exogenous cAMP and forskolin, which directly activates adenylate cyclase, were not affected by wortmannin. These results indicate that wortmannin inhibits vasopressin stimulation of Na+transport at a site proximal to activation of adenylate cyclase. The results suggest that PI3K may be involved in receptor activation by vasopressin.

Extracellular cGMP inhibits transepithelial sodium transport by LLC-PK1 renal tubular cells

1996 ◽  
Vol 270 (2) ◽  
pp. F283-F288 ◽  
Author(s):  
R. L. Chevalier ◽  
G. D. Fang ◽  
M. Garmey
Keyword(s):  
Sodium Transport ◽  
Short Circuit ◽  
Organic Anion ◽  
Short Circuit Current ◽  
Sodium Reabsorption ◽  
Cell Monolayers ◽  
Cyclic Monophosphate ◽  
Renal Tubular Cells ◽  
Renal Tubular ◽  
Transepithelial Sodium Transport

Both atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP) inhibit tubular sodium reabsorption by generation of guanosine 3',5'-cyclic monophosphate (cGMP). To determine the role of extracellular cGMP in this response, monolayers of porcine renal tubular LLC-PK1 cells were incubated for 5 min with ANP, SNP, cGMP, or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) (10 nM to 0.1 mM). Transepithelial sodium transport was measured as amiloride-inhibitable short-circuit current (Isc). Incubation of cell monolayers with 1 microM of ANP, cGMP, or 8-BrcGMP inhibited Isc by > 70%, as did SNP at 100 microM (P < 0.01). Adenosine 3',5'-cyclic monophosphate (0.1 mM) had no significant effect. Incubation of monolayers with 1 microM LY-83583 (an inhibitor of guanylyl cyclase), 10 microM probenecid (an organic anion transport inhibitor), or preincubation with 1 microgram/ml nocodazole (a microtubule disrupter) reduced extracellular accumulation of cGMP (P < 0.05) and abolished the SNP-mediated reduction of Isc. However, addition of these inhibitors did not affect reduction of Isc by exogenous cGMP. We conclude that SNP inhibits sodium transport by LLC-PK1 monolayers through generation of cGMP but that extrusion of cGMP out the cell is necessary for its effect.

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