Studies of insulin action on the amphibian oocyte plasma membrane using NMR, electrophysiological and ion flux techniques

Abstract

Insulin (0.1–10 μM) reinitiates the meiotic divisions in Rana oocytes and produces a 14–20 mV negative-going hyperpolarization of the plasma membrane as well as a 0.25 unit increase in intracellular pH during the first 90 min. During hyperpolarization, the Na + conductance of the membrane decreases by 40–50% with a concomitant increase in 22Na + uptake from the medium. The increased uptake of Na + during a period of decreasing Na + conductance is apparently due to an increase in fluid phase turnover associated with insulin-mediated endocytosis. Both membrane hyperpolarization and increase in pH i are Na +-dependent and are blocked by the serine proteinase inhibitor, phenylmethylsulfonyl fluoride. The membrane potential of the prophase oocyte has a significant electrogenic component with potential but not conductance sensitive to glycosides and substitution of Li + for Na +. Insulin hyperpolarizes Li + or glycoside-treated oocytes whereas glycosides do not affect insulin-hyperpolarized oocytes. [ 3H]Ouabain binding by the plasma membrane of the untreated oocyte shows at least two K +-sensitive components ( K d = 42 and 2000 nM) linked to inhibition of the Na + pump. Insulin-treated oocytes show a single class of intermediate-affinity ouabain sites ( K d = 490 nM) which appear to result from insulin-induced internalization of membrane-bound ouabain. [ 125I]Insulin binding to the plasma membrane shows a class of high-affinity sites ( K d = 87 nM) with 40–50 pump sites per insulin-binding site. Our results suggest that insulin-induced mediator peptides stimulate Na +-H + exchange resulting in an increase in intracellular pH and Na + uptake concomitant with an increase in receptor-mediated endocytosis and a decrease in Na + conductance and associated membrane hyperpolarization. The net result appears to be a down-regulation of the Na + pump which together with a decrease in Na + conductance may divert high-energy phosphate compounds from cation regulation to anabolic processes of meiosis.