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

Abstract

lin 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 PIP 3 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 PIP 3 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 PIP 3 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 PIP 3 also increased PKB phosphorylation. Taken together, the present results establish that the D-3-phosphorylated phosphoinositides PIP3 and PI(3,4)P 2 mediate the effect of insulin on sodium transport across A6 cell monolayers.

Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides

“…As expected, overexpression of active PI3-K with ENaC in CHO cells increased channel activity (79,86,87). Similarly, addition of membrane-permeant PIP 3 increased Na ϩ absorption across renal epithelia, and overexpression of a PIP 3 -phosphatase, phosphatase and TENsin homolog deleted on chromosome 10, attenuated the natriferic effects of insulin and exogenous PIP 3 (47). Activation of PI3-K, moreover, with H 2 O 2 provoked a rise in Na ϩ transport (48).…”

“…Several laboratories have subsequently confirmed the observations that aldosterone activates PI3-K in renal epithelia and that active PI3-K is necessary for increases in Na ϩ transport in response to aldosterone (22,86,90). Two other natriferic factors, insulin and antidiuretic hormone (ADH), also activate PI3-K in epithelia and increase Na ϩ absorption mediated by ENaC in a PI3-K-sensitive manner (20,47,48,59). Importantly, insulin, while targeting basolateral receptors, quickly increases apical membrane PIP 3 levels to affect activity of ENaC in this latter membrane (5).…”

“…Our rationale is that ENaC quickly runs down in settings such as excised inside-out patches and activation of PLC, where membrane PIP 2 levels rapidly decline in response to uninhibited hydrolysis and/or washout (39,44,47,88,95). In contrast, in settings, such as whole cell and excised, outside-out patch configurations, where PIP 2 levels are expected to be relatively constant, ENaC does not run down (6,79,87,88).…”

“…The converse of increasing (above starting levels) open probability linked to elevations in PIP 2 levels has not been established. Indeed, increasing cellular PIP 2 levels by direct addition of a permeant form of this phosphatidylinositide failed to increase Na ϩ absorption mediated by ENaC in renal epithelia (47). Moreover, chronic overexpression of PI(4)5-K, which is a phospholipid kinase that promotes PIP 2 synthesis, increased membrane levels of ENaC heterologously expressed in CHO cells rather than the channel's open probability (77) (see below).…”

Synthesis and Biological Activity of PTEN-Resistant Analogues of Phosphatidylinositol 3,4,5-Trisphosphate

“…19 The physiological function of the 3-PT-and 3-MP-PtdIns(3,4,5)P 3 analogues was examined in A6 cell monolayers, a renal epithelium model that expresses epithelial sodium channels (ENaC). 20 ENaC activity is the rate-limiting step of the sodium transport and is stimulated by insulin.…”