Serum stimulates the Na+,K+ pump in quiescent fibroblasts by increasing Na+ entry.

Abstract

Two ionophores (monensin and gramnicidin) that carry Na+ into MT3 cells markedly enhance the rate of 86Rb+ uptake. Ouabain prevents both ionophores from increasing RVb+ uptake, indicating that the ionophores activate the Na+,K+ pump. Measurements of 8sRb+ uptake and cell Na+ and R+ over a range of monensin concentrations show that the activit of the Na+,KQ pump in M3 cells is limited by the supply of internal Na+ and is extremely sensitive to small changes in internal Na+. Serum ridly enhance the rate of UNa upake and net Na+ entry when Na+ exit is inhibited b ouabain. At 03 pg/ml, monensin increases the rate of get Na+ and activates the Na+ + Dump by the same degree as serum. lie stimulation of sfbw uptake by serum or the ion s has an absolute requirement for external Na+. Thus, serum appears to stimulate the NaQ+ pump in quiescent M3 cells by increasing its supply of Na+.Fibroblasts in culture undergo arrest in the GI/Go phase of the cell cycle when the medium becomes depleted of growth faiors or essential nutrients (1-3). Addition of fresh serum or purified growth factors to quiescent cultures sets in motion a complex array of events which precede DNA synthesis and cell division (4).One of the more striking changes occurring in the plasma membrane of quiescent fibroblasts soon after the replenishment of growth factors is the activation of the Na+,K+ pump which in quiescent 3T3 cells increases about 3fold within minutes after the addition of fresh serum (5, 6). The purified mitogens, multiplication stimulating activity and fibroblast-derived growth factor, stimulate the Na+,K+ pump in quiescent cultures of chicken embryo fibroblasts (7) and 317 cells (8), respectively. Ouabain reversibly inhibits the Na+,K+ pump in fibroblasts and blocks protein and DNA synthesis, which appear to depend on the level of intracellular K+ (9). Significantly, fibroblasts arrested in Go increase their K+ content when growth is initiated (5, 6) and cell K+ and '*Rb+ uptake fall when the fibroblasts stop growing at confluency (10). Transformed fibroblasts, which have a characteristically high rate of proliferation, likewise display a higher rate of Na+,K+ pumping (11,12).This report addresses the mechanism by which serum and growth factors rapidly modulate the Na+,K+ pump in fibroblasts. We propose the following model of pump regulation: (i) there is a Na+ channel in the plasma membrane of fibroblasts; (a) growth factors increase Na+ entry via the channel; (iO) the activity of the Na+,K+ pump in fibroblasts is limited by the supply of internal Na+; and (iv) growth factors stimulate the pump by activating the Na+ channel, thereby supplying the pump with more Na+. Our study of Li+ transport (13) duced the concentration of Na+ in the medium to 50mM and was done to increase the specific activity of the isotope. Ouabain was present at 1 mM to prevent Na+ from exiting via the Na+,K+ pump. After a 5-min incubation at 370C in a humidified atmosphere of 10% C02/90% air in 2 ml of medium, 0.100 ml of 22Na+ containing about 3 X 106 cp...

Secondary effect of aldosterone on Na-KATPase activity in the rabbit cortical collecting tubule.

“…(35) using a toad kidney cell culture have also demonstrated an amil-sensitive rise in Na-K ATPase activity following 18-h exposure to steroids. This phenomenon of activation of Na-K ATPase subsequent to an increased entry of Na into the cell has also been observed in fibroblasts (36,37), neuroblastoma (38), and HeLa cells (39).…”

Regulation of Sodium and Potassium Transport in Phytohemagglutinin-Stimulated Human Blood Lymphocytes

“…Na and K transport in red cells is regulated primarily by the internal Na concentration (15,18). This mechanism for the regulation of pump rate is probably relevant to nucleated cells also (19). Accurate measurement of lymphocyte Na concentration was complicated by the low cell Na concentration relative to the medium Na concentration since PHAtreated lymphocytes cannot be dispersed to wash them free of the suspending medium.…”

“…First, lymphocyte Na is increased about 35% or 6 mmol/liter cell water after PHA treatment at a time when active transport is increased 1.5-to 2-fold. Second, the cell Na concentration of 14-15 mmol/liter cell water is near the Km for the Na, K-ATPase of the lymphocyte membrane and thus an increase in cell Na from [15][16][17][18][19][20][21] mmol/liter cell water would lead to a significant increase in pump activity. A 6-mM increase in Na heightens the Na, K-ATPase activity -35%, whereas active transport nearly doubles.…”

Intracellular Na+:K+ ratios in human cancer cells as revealed by energy dispersive x-ray microanalysis.

“…Although several experimental approaches have been attempted to test this assumption, both in vivo and in vitro (3,8,9,21,24), the evidence obtained has been mostly circumstantial and indirect . Some authors have even refuted the basic concept .…”