Endocrinology, Vol 121, 1503-1511, Copyright © 1987 by Endocrine Society

ARTICLES

Identification of amiloride-sensitive Na+/H+ exchange in rat NB2 node lymphoma cells. Stimulation by 12-O-tetradecanoyl-phorbol-13-acetate

CK Too, A Walker, PR Murphy, EJ Cragoe Jr, HK Jacobs and HG Friesen
Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.

Stimulation of mitogenesis in rat Nb2 node lymphoma cells by human (h) PRL was inhibited by inhibitors of Na+/H+ exchange (viz. amiloride and its analogs) and inhibitors of protein kinases (isoquinolinesulfonamide derivatives). The most potent were ethylisopropylamiloride (EP-Am) (IC50, 13 microM) and H-7, selective for protein kinase C (IC50, 23 microM), suggesting the possible involvement of Na+/H+ exchange and protein kinase C in mediating Nb2 cell mitogenesis. In the presence of hPRL, the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to activate the Na+/H+ antiporter as well as protein kinase C in other cell systems, enhanced the hPRL-stimulated Nb2 cell mitogenesis. TPA alone caused a dose- and time-dependent stimulation of H+ efflux in stationary cultures of Nb2 cells but had no effect on cell growth. From 25-100 nM TPA, the increase in the rate of H+ efflux was detectable by 3 min, reached a maximum by 15 min, and was sustained 30 min after the addition of TPA. The TPA-stimulated H+ efflux was dependent on extracellular Na+ and was almost completely inhibited after a 10 min preincubation with 25 microM EP-Am. TPA also increased the intracellular pH (pHi) of stationary cultures of Nb2 cells from 7.29 +/- 0.02 (n = 8) to a maximum of 7.45 +/- 0.03 (n = 10). The most rapid and greatest response was observed with 40 nM TPA which gave a detectable increase in pHi within 1 min and reached a maximum alkalinization by 6 min. Higher concentrations of TPA had no additional effect. The nontumor promoter phorbol 12,13,20-triacetate (PTA), either alone or in the presence of hPRL, had no effect on Nb2 cell proliferation or on H+ efflux or pHi in Hb2 cells. The TPA-induced increase in pHi was Na+- dependent and was inhibited by EP-Am and H-7. A preincubation with EP- Am (25 microM) for 5-10 min abolished the TPA-induced increase in pHi whereas prolonged incubation with H-7 (50 microM) for up to 3 h was required to decrease the stimulatory effect of TPA by approximately 50%. Although activation of the Na+/H+ exchange system is clearly an early consequence of the action of TPA on Nb2 cells, the failure of TPA to stimulate Nb2 cell proliferation suggests that stimulation of Na+/H+ exchange and protein kinase C activity are not sufficient to generate a mitogenic response in these cells.

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