Modulation of the Na-H antiport by insulin: interplay between protein kinase C, tyrosine kinase, and protein phosphatases.
The insulin modulation of Na-H antiport in rat hepatocytes was studied using the fluorescent, pH-sensitive intracellular probe, 2',7' bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Our data show that insulin stimulates the Na-H antiport. The dose-response of insulin effect shows a behavior typical of other insulin responses: a maximum in the physiological range (1 nM) and smaller effects at higher and lower hormone concentrations. The time-course of activation is very fast at high hormone concentrations and slow, but reaching a higher value, for the physiological concentrations (0.26 +/- 0.05 and 0.18 +/- 0.022 pH units for 1 nM and 1 microM insulin respectively). The use of phorbol, 12-myristate, 13-acetate (PMA), a potent activator of protein kinase C and its inhibitor staurosporine, and the inhibitor of tyrosine kinase erbstatin analog, suggests that both protein kinase C and tyrosine kinase could be involved in the mechanism leading to Na-H antiport activation by insulin. We suggest that the activation of the antiport involves the two pathways depending on the hormone concentration. In particular, protein kinase C would mediate the effects of high hormone concentrations, acting as a growth factor, since staurosporine fully inhibited insulin 1 microM, but only partially 1 nM effects, and tyrosine kinase would mediate the effect of insulin 1 nM and only partially 1 microM. Okadaic acid 1 microM, a potent inhibitor of protein phosphatases, mimicked the hormone effects on the antiport and abolished the different time-course due to hormone concentration, suggesting a role of kinases and phosphatases in the signal transduction. The effect of all activators was abolished by amiloride analog, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), confirming the specificity of these effects.
Incerpi
S
S
Department of Biology, University of Rome Tor Vergata, Italy.
Baldini
P
P
Bellucci
V
V
Zannetti
A
A
Luly
P
P
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D004988:Ethers, Cyclic / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D004988:Ethers, Cyclic; D007328:Insulin; D017923:Sodium-Hydrogen Exchangers; D019319:Okadaic Acid; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate