Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta.
Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter, the intracellular signalling molecules mediating this effect of insulin have not been elucidated. Treatment of 3T3-L1 fibroblasts with insulin increased total 86Rb+ (K+) uptake from 0.8 +/- 0.04 to 1.02 +/- 0.05 nmol.mg-1.protein-1.min-1 (p < 0.005). These changes in K+ flux, though small, can alter the membrane potential. Uptake occurred through both the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter and both were stimulated by insulin. Interestingly, when bumetanide was used to inhibit the Na+/K+/2Cl- cotransporter prior to insulin action, no increase in 86Rb+ uptake via the Na+/K(+)-ATPase was observed. The structurally distinct phosphatidylinositol 3-kinase inhibitors wortmannin (50-200 nmol/l) and LY294002 (50 mumol/l) attenuated both total insulin-stimulated 86Rb+ uptake as well as uptake via the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter. Neither the inhibitor of p70.S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098059 (50 mumol/l), had any effect on insulin's stimulation of K+ influx. A 10 mumol/l concentration of the protein kinase C (PKC) inhibitor bisindolylmaleimide attenuated insulin action but at 1 mumol/l it was ineffective, suggesting involvement of the atypical PKC-zeta isoform. We conclude that insulin-stimulated K+ uptake in 3T3-L1 fibroblasts appears to involve concerted regulation of both the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter and we show for the first time that this process is signalled via a pathway involving phosphatidylinositol 3-kinase and PKC-zeta.
Sweeney
G
G
Programme in Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Somwar
R
R
Ramlal
T
T
Martin-Vasallo
P
P
Klip
A
A
D016475:3T3 Cells / Q000187:drug effects* / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011188:Potassium / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases / Q000037:antagonists & inhibitors; D012414:Rubidium Radioisotopes; D020123:Sirolimus / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002352:Carrier Proteins; D002867:Chromones; D004791:Enzyme Inhibitors; D007211:Indoles; D007328:Insulin; D007527:Isoenzymes; D008301:Maleimides; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011188:Potassium; D011493:Protein Kinase C; D019893:Ribosomal Protein S6 Kinases; D012414:Rubidium Radioisotopes; D020123:Sirolimus; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C088060:bisindolylmaleimide