Studies of insulin action on the amphibian oocyte plasma membrane using NMR, electrophysiological and ion flux techniques.
Insulin (0.1-10 microM) 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 pHi 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 (Kd = 42 and 2000 nM) linked to inhibition of the Na+ pump. Insulin-treated oocytes show a single class of intermediate-affinity ouabain sites (Kd = 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 (Kd = 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.
D000818:Animals; D002414:Cations, Monovalent; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D004553:Electric Conductivity; D005260:Female; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008564:Membrane Potentials / Q000187:drug effects; D009865:Oocytes / Q000502:physiology*; D010042:Ouabain / Q000378:metabolism; D011894:Rana pipiens; D012964:Sodium / Q000378:metabolism*; D012979:Sodium Radioisotopes; D013327:Strophanthidin / Q000494:pharmacology; D014817:Vitelline Membrane / Q000378:metabolism
D002414:Cations, Monovalent; D007328:Insulin; D012979:Sodium Radioisotopes; D010042:Ouabain; D013327:Strophanthidin; D012964:Sodium