Growth factor activation of an amiloride-sensitive Na+/H+ exchange system in quiescent fibroblasts: coupling to ribosomal protein S6 phosphorylation.

Abstract

Chinese hamster lung fibroblast cells (CC139)enter the GO/G1 nonproliferative state after serum deprivation.In this report, we show that reinitiation ofDNA synthesis by serum or the combination of purified human thrombin and insulin (1-10 j.g/ml) is preceded by very early stimulation ofionic fluxes (Na+/ Rb+) and protein phosphorylation (27,000 daltons, 62,000 daltons, and the ribosomal S6 proteins). The potentiating action of insulin on thrombin-stimulated DNA synthesis is also observed on thrombin-stimulated Na+ influx, Rb+ influx, and protein S6 phosphorylation. Moreover, we demonstrate that CC139 cells possess a Na+/ H+ exchange system sensitive to amiloride. Half-maximal inhibition of growth factor-activated Na+ influx and Na+-dependent H+ efflux is obtained with 3-10 ,uM amiloride. Two lines of evidence indicate that the extrusion of H+ via the activation of the Na+/H+ exchanger is coupled to protein S6 phosphorylation: serum-stimulated phosphorylation is blocked by (i) amiloride at a concentration that abolishes serum-stimulated Na+ influx and (ii) protonophores that acidify the cell interior. The present data support the idea that the regulation of intracellular pH is a key event in the mechanism of growth factor action.Control ofeukaryotic cell proliferation is among the major problems in biology that remain poorly understood. Polypeptide growth factors and hormones have been shown to play a crucial role in the regulation of cell division (1, 2). However, the molecular mechanisms by which these regulatory peptides act to stimulate cell metabolism, protein synthesis, DNA replication, and subsequent cell division are largely unknown.Marked alterations in ionic fluxes are one of the earliest biochemical events detected after addition of serum or purified growth factors to arrested cells (3). Stimulation of Na+ influx seems ubiquitous; it has been observed in various cell types activated for cell division (3)(4)(5)(6)(7). Closely associated with the increase in Na+ uptake is activation of the Na+/K+ pump leading to increased K+ influx (8).Protein phosphorylation is another rapid biochemical change associated with growth factor action (9-11).In light of these different observations, an obvious question is that of the relationship between these early events and mitogen action. Indeed, it is not known whether the monovalent ionic fluxes represent one ofthe initial triggers ofmitogen action or secondary events to early metabolic activation. It is also not clear whether protein phosphorylation is coupled to growth factor-activated Na+ influx.To approach these questions, we have used a Chinese hamster lung fibroblast line, CC139, capable of growing in a chemically defined medium (12) and reversibly entering the nonproliferative GO/G1 state (13). From this line, we have derived stable variants altered either in metabolic pathways (13,14) or in growth factor dependence for DNA synthesis (15). We now report that CC139 cells possess an amiloride-sensitive Na+/H+ exchange system that is activated by growth fa...

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

“…In 1982, Pouysségur et al described a growth factor-activated, amiloride-sensitive Na + /H + transporter [137]. Employing an elegant functional complementation approach with a functionally NHE-deficient cell line, this group went on to clone the first NHE in 1989 and named it NHE1 [148].…”

“…The mature plasmalemmal NHE1 protein is both N-and O-glycosylated, although glycosylation is not required for transport function [61,33]. NHE1 is highly sensitive to amiloride, the first NHE1 inhibitor identified [137,103]. Subsequently, more selective and potent inhibitors have been developed including lipophilic amiloride derivatives (e.g.…”

The chemistry, physiology and pathology of pH in cancer

“…The pH sensors involved in cancer disease progression have proved to be more challenging to identify, possibly because of difficulties in distinguishing bona fide pH sensors from the plethora of proteins that bind H þ ions [2]. Cells sensing an alkaline pH i have been shown to proliferate [61], enter the cell cycle [62,63], differentiate [64], migrate [65,66], reduce apoptosis [67] and clastogenesis [68], and undergo malignant transformation [69,70]-events that are critical in cancer formation and metastasis. Considering the complexity of these processes, the observed pH i sensitivity may involve a number of H þ -binding molecular switches.…”

Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.

“…Since the PDGF-induced increase in Na + influx is blocked by amiloride, Na + /H + exchange may represent a significant pathway for the entry of Na + into 3T3 cells stimulated by PDGF. The stimulation of Na + uptake by PDGF via the Na + /H + antiport could result from at least two alternative possibilities (Pouyssegur et al, 1982;Frelin et al, 1983;Burns and Rozengurt, 1983). First, PDGF could directly activate the membrane-bound antiporter leading to an increase in Na + for H + exchange and subsequent increase in intracellular pH.…”

“…One of the earliest events that takes place after the mitogenic stimulation of Swiss 3T3 cells is an increase in the rate of Na + influx into the cells (Smith and Rozengurt, 1978;Mendoza et al, 1980aMendoza et al, , 1980b, which is mediated by a Na + /H + antiport system, driven by the electrochemical Na + gradient across the plasma membrane (Schuldiner and Rozengurt, 1982). An early stimulation of Na + influx also occurs in a variety of other cell types induced to proliferate (Koch and Leffert, 1979;Mendoza et al, 1980a;Moolenaar et al, 1981Moolenaar et al, , 1982Rothenberg et al, 1982;Pouyssegur et al, 1982;Owen and Villereal, 1983). Because Na + entry is at least partly coupled to the exit of H +, the stimulation of Na + entry may lead to intracellular alkalinisation, which may play a crucial role in the mechanism of cellular stimulation in various systems (Nuccitelli and Deamer, 1982).…”