Identification of amiloride-sensitive Na+/H+ exchange in rat NB2 node lymphoma cells. Stimulation by 12-O-tetradecanoyl-phorbol-13-acetate.
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.
Too
C K
CK
Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Walker
A
A
Murphy
P R
PR
Cragoe
E J
EJ
Jacobs
H K
HK
Friesen
H G
HG
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008223:Lymphoma / Q000378:metabolism* / Q000473:pathology; D010703:Phorbol Esters / Q000494:pharmacology; D011388:Prolactin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D051381:Rats; D017923:Sodium-Hydrogen Exchangers; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002352:Carrier Proteins; D010703:Phorbol Esters; D047428:Protein Kinase Inhibitors; D017923:Sodium-Hydrogen Exchangers; C053695:phorbol 12,13,20-triacetate; D000584:Amiloride; D011388:Prolactin; D013755:Tetradecanoylphorbol Acetate; C039614:ethylisopropylamiloride