Effect of Na + flux inhibitors on induction of c-fos, c-myc, and ODC genes during cell cycle

The role of Na + transport systems in the mitogenic signal induced by growth factors was studied, and it was shown that two Na + transport systems contribute to the early increase in cytoplasmic Na + in response to serum growth factors, namely the amiloride-sensitive Na+/H+ antiport and the bumetanide-sensitive Na+/K+/Cl- cotransport. Bumetanide or amiloride, when added separately, inhibited part of the increase in cytoplasmic Na +, as a response to the addition of serum to quiescent BALB/c mouse 3T3 fibroblasts. Each drug also suppressed part of the stimulation of the ouabain-sensitive Rb + influx, which was controlled by intracellular Na +. However, when both drugs were added together with serum growth factors, a complete inhibition of the early increase in [Na +], and subsequently a complete blockage of Na+/K+ pump stimulation was obtained. Amiloride or bumetanide, when added separately, only partially inhibited DNA synthesis induced by serum, 24% and 8% respectively. However, when both drugs were added together, at the time of serum addition to the quiescent cells, cell entry into S-phase was completely inhibited. To investigate the mode of cell-cycle inhibition, analysis was done of the possible role of early Na + fluxes in the mitogenic signal transduced from cell membrane receptors to the nucleus. The effects of the two drugs amiloride and bumetanide on induction of three genes--c-fos, c-myc, and ornithin decarboxylase (ODC)--was measured during cell transition through the G1-phase. Amiloride and bumetanide, when added separately or in combination, did not inhibit the induction of c-fos, c-myc, and ODC mRNAs. These results suggest that stimulation of Na + fluxes by serum growth factors is essential for cell transition into the S-phase of cell cycle, but it plays no apparent role in the growth factor signal transduced from the cell surface to the interior of the cell, as manifested by c-fos, c-myc, and ODC genes induction.     

Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts.     

Regulation of intracellular pH during the G1/S-phase transition of the neuroblastoma cell cycle

Changes in active K+ and Na+ influx during the cell cycle of neuroblastoma (clone Neuro-2A) have suggested activation of an Na+, H+ exchange system during the G1/S-phase transition. Here we report that pHi, measured by the digitonin null-point method, is constant during G1-phase and the G1/S-phase transition and decreases in early S-phase. In addition pHi is shown to be most sensitive to the diuretic amiloride in the G1/S-phase transition, in agreement with the ion influx data. It is concluded from these data, that pHi is tightly regulated during the early cell cycle phases by the Na+, H+ exchange system, in particular during the G1/S-phase transition.     

Amiloride inhibition of DNA synthesis and immunoglobulin production by activated human peripheral blood mononuclear cells is independent of sodium/hydrogen antiport

Activation of sodium/proton (Na+/H+) antiport activity has been shown to occur as an early event in mitogenesis. Because amiloride inhibits Na+/H+ antiport activity, it is hypothesized that mitogenesis may be inhibited by amiloride. In this work, we examined the effect of amiloride on DNA synthesis as measured by [3H]thymidine uptake and immunoglobulin (Ig) production as measured by an ELISA system in human peripheral blood mononuclear cells (PBM). Amiloride at 100 microM concentration inhibited irradiated Raji cell (*R)-activated and phytohemagglutinin-P (PHA-P)-stimulated DNA synthesis by 50 +/- 11% and 72 +/- 12%, respectively. IgG production was inhibited by 71% at 100 microM amiloride concentration in *R-activated PBM. This concentration of amiloride inhibited Na+/H+ antiport activity by 92%. Because amiloride is known to inhibit other pre-replicative cellular functions such as protein synthesis, we used an amiloride analogue, dimethylamiloride, which inhibited Na+/H+ antiport activity by 90% at a concentration of 1 microM without inhibition of PBM Ig or DNA synthesis. Furthermore, neither PHA-P nor *R-stimulated PBM demonstrated an intracellular alkalinization even after 6 hr of stimulation. Similarly, T cell-enriched or B cell-enriched populations did not show intracellular alkalinization after PHA-P or *R activation. Thus, it appears that Na+/H+ antiport activation is not an early event in PBM mitogenesis. The inhibition of mitogenesis by amiloride may be due to abrogation of premitotic events such as protein synthesis.     

Growth factors activate the bumetanide-sensitive Na+/K+/Cl-cotransport in hamster fibroblasts

alpha-Thrombin, a potent mitogen for the hamster fibroblast cell line CCL 39, stimulates by approximately 3-fold 86Rb+ uptake in a mutant lacking the Na+/H+ antiport activity (PS 120). The major component of this stimulated 86Rb+ (K+) uptake is a bumetanide-sensitive flux (IC50 = 0.4 microM), which accounts for 50% of total K+ uptake in Go-arrested cells and is approximately 4-fold stimulated by maximal thrombin concentrations (EC50 = 5 X 10(-4) units/ml). This bumetanide-sensitive 86Rb+ uptake represents a Na+/K+/Cl- cotransport, as indicated by its dependence on extracellular Na+ and Cl- and by the existence in PS 120 cells of a bumetanide-sensitive K+-dependent 22Na+ uptake. The stimulation reaches its maximum within 2 min, is reduced at acidic intracellular pH values (half-maximal at pHi = 6.8), and can also be induced, to a lesser extent, by EGF and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the effects of which are nearly additive. In contrast, preincubation with 12-O-tetradecanoylphorbol 13-acetate results in inhibition of thrombin- and EGF-induced stimulations, suggesting that activated protein kinase C might exert a feedback inhibitory control. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport is separated from the activation of the Na+/H+ antiport. However, activation of this cotransporter does not seem to play a major role in the mitogenic signaling pathway since its complete inhibition with bumetanide reduces only by 25-30% reinitiation of DNA synthesis.     

Activation of the Na+/H+ antiport is not required for lectin-induced proliferation of human T lymphocytes

Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes.     

Na+/H+ exchange involvement in colony-stimulating factor-1-stimulated macrophage proliferation. Evidence for a requirement during late G1 of the cell cycle but not for early growth factor responses

Na+/H+ exchange activation by growth factors is proposed to be an important early signal for mitogenesis; however, little is known of its duration and requirement during later stages of the cell cycle. Macrophage-specific colony factor (CSF-1) rapidly activates murine bone marrow-derived macrophage Na+/H+ exchange, resulting in stimulation of Na+,K(+)-ATPase activity. The response to CSF-1 is maintained for at least 24 h. Inhibition of Na+/H+ exchange with 5-N,N-dimethylamiloride prevents CSF-1-stimulated DNA synthesis and cell growth. This is unlikely to be due to cytoplasmic acidosis, but more likely reflects a requirement for Na+/H+ exchange-mediated Na+ influx. DMA addition even up to 8 h after the growth factors suppresses S-phase progression. Na+/H+ exchange appears not to be involved in the induction of other early growth factor responses (c-fos and c-myc mRNA induction and general RNA and protein synthesis). We propose that growth factor-stimulated Na+/H+ exchange late in G1 of the cell cycle is required for S-phase progression but not for certain early growth factor responses.     

Retinoblastoma susceptibility gene product (pRb) and p107 functionally separate the requirements for serum and anchorage in the cell cycle G1-phase

Growth factors and cell anchorage are both required for cell cycle G(1)-phase progression, but it is unclear whether their function is mediated through the same set of cell cycle components and whether they are both required during the same periods of time. We separately analyzed the requirements of serum and anchorage during G(1)-phase progression and found that human dermal fibroblasts as well as wild type, pRb(-/-), and p107(-/-) mouse embryonic fibroblasts needed serum (growth factors) until mid-G(1)-phase but required cell anchorage until late G(1)-phase to be competent for S-phase entry. Importantly, however, pRb/p107 double-null mouse embryonic fibroblasts lacked serum requirement in mid-G(1)-phase but still required cell anchorage until late G(1)-phase to enter S-phase. Our results indicate that pRb and p107 do not constitute the last control point for extracellular factors during G(1)-phase progression, and they functionally separate the requirements for serum and cell anchorage in terms of involved cell cycle components.     

Bumetanide and furosemide inhibited vascular endothelial cell proliferation

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl–cotransport in the mitogenic signal of vascular endothelial cell proliferation. The activity of the Na+/K+/Cl– cotransport is dramatically decreased in quiescent subconfluent cells, as compared to subconfluent cells growing in the presence of FGF. The Na+/K+/Cl– cotransport activity of quiescent subconfluent cultures deprived of FGF decreased to 6%, whereas that of quiescent cells grown to confluency was reduced to only 33% of the activity of subconfluent cells growing in the presence of FGF. The basal low activity of Na+/K+/Cl– cotransport in the quiescent subconfluent vascular endothelial cells was dramatically stimulated by FGF. In order to explore the role of the Na+/K+/Cl– cotransport in the mitogenic signal of the endothelial cells, the effect of two specific inhibitors of the cotransport -furosemide and -bumetanide was tested on cell proliferation induced by FGF. Bumetanide and furosemide inhibited synchronized cell proliferation measured by direct counting of cells and by DNA synthesis. Inhibition by fuorsemide and bumetanide was reversible; removal of these compounds completely released the cells to proliferate. These results indicate that the effect of these drugs is specific and is not due to an indirect toxic effect. This study clearly demonstrates that the FGF-induced activation of the Na+/K+/Cl– cotransport plays a role in the mitogenic signal pathway of vascular endothelial cells. © 1994 Wiley-Liss, Inc.     

Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series

We have previously characterized in Chinese hamster lung fibroblasts a growth factor activatable and amiloride-sensitive Na+/H+ antiport (Pouysségur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3935-3939). In this report, we compared the affinity of 28 analogs of amiloride for inhibition of the Na+/H+ antiport and inhibition of growth factor-induced DNA synthesis. We showed that the guanidino moiety of amiloride must be protonated to elicit inhibition of the Na+/H+ exchange. Substitutions within this moiety by methyl, phenyl, or benzyl groups reduced the activity 20- to 1000-fold. On the contrary, substitution of the proton(s) of the 5-amino group of amiloride with alkyl or alkenyl groups increases potency up to 100-fold (5-N,N-diethylamiloride has a KI of 4 X 10(-8) M). In HCO-3-free medium and at lower [Na+]0 (25 or 50 mM) to reduce competition with amiloride, we found that growth factor-stimulated DNA synthesis of G0-arrested cells is inhibited by amiloride and its analogs with the same rank order as that for Na+/H+ antiporter inhibition. Over a range of 3 logs of concentration, a tight correlation was established between IC50 for the blockade of both processes, Na+/H+ exchange and percentage of cells entering the S phase upon growth factor action. These findings indicate that, in HCO-3-free medium, the functioning of the Na+/H+ exchange system is required for growth factor-induced DNA synthesis.     

The amiloride-sensitive Na+/H+ antiport in 3T3 fibroblasts

BALB/c 3T3 fibroblasts have an amiloride-sensitive Na+ uptake mechanism which is hardly detectable under normal physiological conditions. The activity of this Na+ transport system can be increased to a large extent by treatments that decrease the internal pH such as loss of intracellular NH4+ as NH3 or incubation with nigericin in the presence of a low external K+ concentration. These treatments have made possible an analysis of the interaction of the Na+/H+ antiport with amiloride and of the external pH dependence of the system. The addition of fetal bovine serum to quiescent 3T3 cells stimulates the initial rate of the amiloride-sensitive 22Na+ uptake by only 50%. However, after treatment of the cells with ammonia or nigericin, serum produces a 40-fold stimulation of the rate of the amiloride-sensitive 22Na+ uptake. Control experiments show that serum does not stimulate the activity of the Na+/H+ antiport by an indirect mechanism involving a depolarization of the membrane or a modification of the internal Ca2+ concentration. It is suggested that some serum component directly interacts with the Na+/H+ exchanger to modify its catalytic properties.     

Fibroblast growth factor induces a transient net K+ influx carried by the bumetanide-sensitive transporter in quiescent BALB/c 3T3 fibroblasts

The bumetanide-sensitive transport system performed a net efflux of K+ in serum deprived quiescent cells. The addition of partially purified fibroblast growth factor (FGF) to G0/G1 phase 3T3 fibroblasts induced a transient net influx of K+, carried out by the bumetanide-sensitive transport system for 2-6 minutes. The stimulation of the bumetanide-sensitive K+ influx by FGF was followed by stimulation of the ouabain-sensitive K+ influx. In addition, both the bumetanide-sensitive and the ouabain-sensitive K+ influxes were found to be similarly stimulated when the G0/G1 3T3 cells were treated with insulin. These results suggest that growth factors such as FGF and insulin induce a change in the action of the bumetanide-sensitive transporter from performing net K+ efflux along its concentration gradient to an uphill transport pumping of K+ into the cell. We propose, therefore, that the bumetanide-sensitive transporter contributes to the increase in the intracellular K+ (and probable Na+) stimulated by growth factors such as FGF and insulin in early G1 phase of the cell cycle.     

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum- stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.     

Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways

The synthesis of specific protein has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr approximately 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid-G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell-cycle-dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr approximately 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase-delta. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S-phase. These data support the view that the cAMP-mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1-phase.     

Lys-bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts

The effect of Lys-bradykinin on net Na+ influx in serum-deprived cultured human fibroblasts (HSWP cells) was measured. It was found that Lys-bradykinin stimulates net Na+ influx with a K1/2 of 1 nM. When Lys-bradykinin was combined with epidermal growth factor, vasopressin and insulin, the net Na+ influx stimulated was comparable with that measured in response to 10% serum. The above combination of growth factors also was found to stimulate DNA synthesis to 92% of the serum-stimulated levels in HSWP cells and to support cell growth over a period of 6 days. In addition, it was observed that the Na+ influx stimulated by Lys-bradykinin or by the combination of four growth factors was completely inhibited by the amiloride analog benzamil. Thus Lys-bradykinin presumably stimulates the same Na+ transport system as is stimulated by serum. Finally, the present results suggest that an increase in Na+ influx always accompanies DNA synthesis in HSWP cells. On the basis of these observations, it can be hypothesized that Na+ influx is a necessary event to stimulate DNA synthesis.     

Overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene induces cell proliferation and phenotypic transformation in mouse fibroblasts

Na(+)/K(+)/Cl(-) cotransporter activity is stimulated in early G(1) phase of the cell cycle and this stimulation was shown to be an essential event in fibroblast cell proliferation. In order to elucidate further the role of the Na(+)/K(+)/Cl(-) cotransporter in cell proliferation, we overexpressed the gene encoding the Na(+)/K(+)/Cl(-) cotransporter in mouse fibroblasts, and analyzed cellular phenotypic changes. Mouse Balb/c 3T3 cells were stably transfected with the cDNA of the shark rectal gland Na(+)/K(+)/Cl(-) cotransporter gene (NKCC1), and expressed in a mammalian vector under the cytomegalovirus promoter (Balb/c-NKCC1 cells). The transfected cells exhibited up to 10-fold greater bumetanide-sensitive Rb(+) influx compared to the control cells. The Balb/c-NKCC1 cells have acquired a typical transformation phenotype indicated by: (1) Loss of contact inhibition exhibited by growth to a higher cell density in confluent cultures, and formation of cell foci; (2) proliferation in low serum concentrations; and (3) formation of cell colonies in soft agar. The control cells transfected with the NKCC1 gene inserted in the opposite orientation in the vector retained their normal phenotype. Furthermore, the two specific inhibitors of the Na(+)/K(+)/Cl(-) cotransporter activity; bumetanide and furosemide inhibited the clonogenic efficiency in the NKCC1 transfected cells. These control experiments indicate that the apparent transformation phenotype acquired by the Balb/c-NKCC1 cells was not merely associated with the process of transfection and selecting for the neomycin-resistant clones, but rather with the overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene. In order to ascertain that the regulated and normal expression of the Na(+)/K(+)/Cl(-) cotransporter control cell proliferation, the effect of bumetanide a specific inhibitor of the cotransporter, was tested on Balb/c 3T3 cell proliferation, induced by fibroblasts growth factor (FGF) and fetal calf serum (FCS). Bumetanide inhibited synchronized Balb/c 3T3 cell exit from the G(0)/G(1) arrest and entering S-phase. The inhibition was reversible, as removal of bumetanide completely released cell proliferation. Taken together, these results propose that the NKCC1 gene is involved in the control of normal cell proliferation, while its overexpression results in apparent cell transformation, in a manner similar to some protooncogenes.     

Species-specific Mn2+/Mg2+ antiport from Mg2(+)-loaded erythrocytes.

Mg2(+)-loaded rat erythrocytes performed Mn2+/Mg2+ antiport, which was nonspecifically stimulated by anions and cations. Mn2+/Mg2+ antiport was shown to operate via the Na+/Mg2+ antiporter because extracellular Na+ and Mn2+ inhibited the intracellular uptake of each other's ions competitively. Furthermore, Mn2+/Mg2+ antiport and Na+/Mg2+ antiport were identically inhibited by various amiloride derivatives. Na+/Mg2+ antiport of chicken and human erythrocytes cannot perform Mn2+/Mg2+ antiport although chicken erythrocytes took up more Mn2+ than rat erythrocytes.     

Intracellular pH controls growth factor-induced ribosomal protein S6 phosphorylation and protein synthesis in the G0→G1 transition of fibroblasts

Mitogen-induced intracellular alkalinization mediated by activation of a Na+/H+ antiporter is a common feature of eukaryotic cells stimulated to divide. A Chinese hamster fibroblast mutant (PS120) lacking Na+/H+ antiport activity (Pouysségur et al., Proc natl acad sci US 81 (1984) 4833) [42] possesses an intracellular pH (pHi) 0.2-0.3 units lower than the wild type (CCL39) and requires a more alkaline pHout (pHo) for growth. Here, we show that serum-stimulated ribosomal protein S6 phosphorylation, protein synthesis activation and DNA synthesis re-initiation are pH-regulated events that display a similar threshold pHo value (6.60) in CCL39 cells. pH-Dependencies for initiation of all three events are shifted toward higher pHo values in the mutant PS120, indicating that growth factor-induced alkalinization has a permissive effect on the pleiotypic response. However, cytoplasmic alkalinization per se is insufficient to trigger S6 phosphorylation, polysome formation, and subsequent DNA synthesis. Transient exposure to a non-permissive pHo (6.5) inhibits both the rate of leucine incorporation into proteins and the progression through the G1 phase of the cell cycle. In contrast, cells committed to DNA synthesis are unaltered by the acidic pHo. These observations suggest that pHi by controlling the rate of protein synthesis play a determinant role in the control of cell division.     

Relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase induction, and DNA synthesis

The relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis was examined with bovine small lymphocytes stimulated by concanavalin A (Con A). The Na+/H+ antiport activity was activated immediately after addition of concanavalin A; the maximum was reached 1 h after Con A addition and the activation continued at least 6 h. With increasing concanavalin A concentrations, the activities of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis increased in a parallel manner. In the presence of HCO3- in the medium, the internal alkalinization of lymphocytes was not induced by Con A. Ornithine decarboxylase and DNA synthetic activities were not inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a specific inhibitor of the Na+/H+ antiporter. In contrast, in the absence of HCO3- in the medium, the internal pH was alkalinized approximately 0.06 pH units by Con A. EIPA did inhibit the alkalinization of the internal pH or DNA synthesis significantly. Ornithine decarboxylase activity was not inhibited by EIPA. These results indicate that the activation of a Na+/H+ antiporter is not a trigger for cell proliferation, but its activation is important probably through the maintenance of the internal pH optimum, especially in HCO3(-)-free medium.