Transport properties of toad kidney epithelia in culture

Perkins, F. M.; Handler, Jerome S.

doi:10.1152/ajpcell.1981.241.3.c154

Cited by 154 publications

(46 citation statements)

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“…Short-circuit current (SCC) techniques were used to determine net ion flux (19,22). Cells grown on Transwell inserts were placed in a modified Ussing chamber and monitored as previously described (3).…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells

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Froelich

Vlahos

et al. 1998

American Journal of Physiology-Endocrinology and Metabolism

109

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. Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E611-E617, 1998.-Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulinstimulated sodium transport in a dose-dependent manner (IC 50 Ϸ 6 µM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.amiloride-sensitive sodium channel; insulin signaling; receptor tyrosine kinase; renal epithelia INSULIN INCREASES SODIUM REABSORPTION in dogs and humans, and this effect appears to be manifested predominantly on the renal distal nephron (21, 10). Models of the distal nephron, toad urinary bladder (Bufo marinus) and the A6 cell line (derived from Xenopus laevis kidney), have been used to demonstrate a direct effect of insulin on amiloride-sensitive sodium transport (12,14). Patch-clamp electrophysiology has indicated that insulin causes an increase in the open probability (P O ) of the apical sodium channel (20). However, blocker-induced noise analysis demonstrated that insulin induces an increase in active sodium channel density in the apical membrane (2, 4, 11). In addition, insulin increases the apical membrane area (11). These results suggest that insulin may induce the insertion of sodium channels into the apical membrane, a hypothesis supported by our study demonstrating that brefeldin A, an inhibitor of secretion, partially inhibits insulin-stimulated sodium transport (9).The first step in insulin signaling is the binding of the hormone to its receptor, followed by autophosphorylation of the receptor and subsequent activation of the receptor tyrosine kinase (IRTK). IRTK has several substrates, including the insulin receptor substrate (IRS) proteins (33). The IRS proteins mediate some of the pleiotropic effects of insulin through interactions with proteins containing SH2 domains, including phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme, a heterodimer, phosphorylates the D-3 position of the myo-inositol ring of phosphatidylinositols (PtdIns; 17). PI 3-kinase is required for several insulin-mediated effects, including the translocation of the insulinresponsive glucose transporter (GLUT-4) to the plasma membrane of adipocytes (7) and skeletal muscle (34), and ...

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Section: Methodsmentioning

confidence: 99%

Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells

Record

Froelich

Vlahos

et al. 1998

American Journal of Physiology-Endocrinology and Metabolism

109

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“…Cultured epithelial cells derived from the distal segments of the kidney of Xenopus laevis (A6) form flat monolayers. When grown on permeable supports Na + channels are expressed in the apical membranes (Perkins and Handler, 1981) which in concert with the basolateral Na+-K+-ATPase enable transepithelial Na + transport. As other distal epithelia of the kidney, the apical membrane has a negligible water permeability in the absence of anfidiuretic hormone.…”

Section: Introductionmentioning

confidence: 99%

Regulatory volume decrease in cultured kidney cells (A6): role of amino acids.

Smet

Simaels

Declercq

et al. 1995

The Journal of General Physiology

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Volume regulation was studied in A6 epithelia grown on permeable supports by measuring cell thickness (To) while simultaneously recording short circuit current (Ix) and tmalsepithelial conductance (Gt). Lowering the tonicity of the basolateral solution (Wb) from 250 or 215 to 140 mOsm/kg elicited a rapid rise in Tc followed by a regulation of the cell volume towards control. This decrease in Tc displays the characteristics of the regulatory volume decrease (RVD). Upon restoring the isoosmotic conditions, T~ decreased rapidly below its control value. A post RVD regulatory volume increase (RVI) as described for other cell types was not observed. The subsequent reduction of the basolateral osmolality increased Tc to the level recorded at the end of the first hypoosmotic pulse. Because cell content was not altered during the isoosmotic period the second hypoosmotic challenge was isotonic with the cell and did therefore not evoke an RVD. However, the cell did not lose its ability to volume regulate since an RVD could be elicited by further reduction of 1rb from 140 to 100 mOsm/kg. The possibility of an involvement of amino acids in the RVD was tested. The amount of amino acids in the cell as well as excreted in the bath was determined by amino acid analysis. Millimolar concentrations of threonine, serine, alanine, glutamate, glycine and aspartate were found in the cell extract. The cellular amino acid concentration was 28.8 -+ 0.4 mM. The amounts of glycine, aspartate and glutamate excreted from the cell during the hypotonic treatment were significantly larger than in control conditions. The excretion of these amino acids during hypotonicity decreased the cellular amino acid concentration by 8.4 -+ 0.2 mM. This quantity cannot completely account for the RVD during the first hypotonic challenge. The addition of glycine, aspartate and glutamate to the bathing solutions, although used at concentrations higher than intracellularly, did not reduce RVD. On the contrary, this maneuver increased the amplitude of the RVD following both hypoosmotic pulses. This result suggests a stimulatory role of the amino acids on the processes responsible for the RVD.

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“…This expression of hormone action is initiated only after a latent period of 60-90 min, and is dependent on mRNA (4) and protein synthesis (5). Experiments in intact tissue and cultured amphibian cells suggest that the aldosterone-induced increase in electrogenic sodium transport can be viewed as involving an early phase lasting [6][7][8][9][10][11][12] h, and a delayed or chronic phase, that persists as long as agonist is present. The early phase is characterized by a gradual increase in apical membrane conductance for sodium, due to activation of amiloride-sensitive sodium This work has previously been reported in preliminary form.…”

Section: Introductionmentioning

confidence: 99%