Mechanisms of the interaction of the leukemic cell with endogenous regulators of the proliferation of hematopoietic tissue

A substance inhibiting DNA synthesis in mouse leukemic cells was isolated from the regenerating calf spleen. When added to a suspension of leukaemic cells, this substance is adsorbed on their surface. The following changes in cell features being noticed: 1) a minute decrease in electrophoretic cell motility, 2) a decrease in esterase activity of the cells, 3) an increase in microviscosity of membrane lipids, 4) an increase in the intracellular pH values. With a longer contact with this substance, changes in nuclear chromatin structure were noticed, with special reference to weakened bonds between DNA and proteins. The data obtained are of significance for revealing molecular mechanisms of hematopoietic mediator action on target cells.     

Protein synthesis in cells infected with Semliki Forest virus is not controlled by intracellular cation changes

Treatment of BHK cells with 1 microM nigericin results in a 55% decrease in K+ and a 3.3-fold increase in intracellular Na+; protein synthesis under these conditions is depressed by 35%. In BHK cells infected with Semliki Forest virus (SFV), protein synthesis is depressed by 76% 6.5 h after infection; intracellular K+ is unchanged, and intracellular Na+ is increased 1.8-fold at this time. These results suggest that the increase in intracellular Na+ in SFV-infected BHK cells does not adequately account for the decrease in protein synthesis, and makes it likely that an increased Na+ concentration is a consequence, not a cause, of alterations in protein synthesis in virally-infected cells. No evidence was obtained for the purported [Alonso, M. A. and Carrasco, L. (1980) Eur. J. Biochem. 109, 535-540; (1981) Eur. J. Biochem. 118, 289-294; (1981) FEBS Lett. 127, 112-114] ability of 1 microM nigericin to permeabilize' cells.     

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.     

Treatment with phorbol esters leads to spermine depletion and inhibition of DNA synthesis in phytohemagglutinin-activated bovine lymphocytes

Phorbol 12-myristate-13-acetate (PMA) inhibited an increase in [3H]thymidine incorporation induced by phytohemagglutinin (PHA) in cultured bovine lymphocytes. Cellular levels of putrescine increased in the presence of PHA and PMA but the levels of spermidine and spermine had decreased to the control levels by 40 h. In cells treated with PHA and PMA, the activity of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme in polyamine biodegradation, was stimulated synergistically. Phorbol esters with tumor-promoting ability also stimulated the enzyme activity and a reciprocal correlation between the enzyme activity and DNA synthesis was observed. Addition of spermine reversed the PHA- and PMA-induced inhibition of DNA synthesis but putrescine and spermidine failed to restore it. These results suggest that the enhancement of spermidine/spermine N1-acetyltransferase activity results in the depletion of intracellular spermine and a concomitant decrease in DNA synthesis.     

Sodium butyrate is a potent modulator of smooth muscle cell proliferation and gene expression

Sodium butyrate is a small, naturally occurring molecule with demonstrated activity on cell growth and differentiation. However, its effect on smooth muscle cells had not been examined. We have found that sodium butyrate and its more stable in vivo analogue tributyrin are potent DNA synthesis and cell proliferation inhibitors. The inhibitory activity of sodium butyrate was not mediated by an elevation of endogenous cAMP levels, a known pathway involved in SMC growth-arrest and maintenance of the contractile phenotype. Consistent with the concept that its activity is mediated by a cAMP-independent pathway, butyrate was able to augment the maximum DNA synthesis inhibitory effect of various agents that elevated intracellular cAMP levels. Additionally, butyrate present for just the initial 8 h or present as late as 16 h after serum addition was able to inhibit DNA synthesis. By contrast, the cAMP analogue, 8Br-cAMP had to be present throughout the entire G₀ to S phase of the cell cycle to effectively inhibit DNA synthesis. These results indicated that sodium butyrate inhibited SMC growth through a cAMP-independent mechanism. We also found that sodium butyrate was unable to abrogate the expression of the serum-inducible genes c- fos , c- myc , Ki-Ras and PS4, but was able to directly stimulate the expression of PS4 and thrombospondin. These results indicate that a number of important early G₁ events initiated by serum growth factors are unaltered by sodium butyrate and that this compound is able to directly stimulate the expression of certain genes normally associated with SMC proliferation.     

Gene replication in the presence of aphidicolin

DNA replication in the nucleus of eukaryotic cells is restricted to the S phase of the cell cycle, and different genes are duplicated at specific times, according to a well-defined temporal order. We have investigated whether activation of initiation sites, in proximity to genes that are replicated in different portions of the S phase, could be detected when synchronized 10T1/2 cells were maintained in aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta. Cells released from confluence arrest into medium containing 2 micrograms/mL APC progressed into the S phase, and nascent DNA accumulated during incubations of 24 and 32 h. Exposure to APC for 40 or 48 h resulted in growth of the radiolabeled DNA into larger molecules. Replicating DNA was isolated in CsCl gradients and probed with 32P-labeled gene probes for early-replicating genes (e.g., Ha-ras, mos, and myc) and a late-replicating gene (VH Ig). DNA replicated during the 24-h incubation in APC was enriched in Ha-ras gene sequences. The VH Ig gene did not replicate in cells incubated for as long as 56 h with APC. The myc and the mos genes were detected after 32 and 40 h in APC, respectively. The myc gene is replicated in 10T1/2 cells after Ha-ras but before mos. Therefore, the order of activation of these genes was conserved in the presence of APC. The delay in replication of myc and mos correlated well with the slowing of DNA replication by APC.     

Stimulation of epithelial cell growth by the neuropeptide substance P

The neuropeptide substance P (SP) was found to stimulate DNA synthesis and cell growth for epithelial cells (cornea and lens) in a serum-free environment. The length of treatment time was shown to be important since longer times shifted the dose-response curve to the left. In short-term DNA synthesis studies (40 h) the stimulation with SP (or synergism with insulin) was not apparent until close to 10 μM, however, when DNA synthesis assays were carried out over a long period of time (5 days) stimulation with SP was seen at 1 pM. The stimulation of DNA synthesis by SP was synergistic with insulin for lens epithelial cells, but little synergism was seen with corneal epithelial cells. It cell growth studies on lens epithelial cells SP also showed growth stimulation by itself and synergism with insulin at concentrations of 1–2 pM. The neuropeptide calcitonin gene related peptide (CGRP) showed no DNA synthesis stimulating ability on epithelial cells by itself at concentrations as high as 2.5 μM; however, it was synergistic with SP at a concentration of 0.025 μM. SP pretreatment of epithelial cells for 2 h causes an increase in cellular sensitivity to subsequent addition of either SP or insulin. This increase is consistent with the hypothesis that either the signal from SP persists after its removal from the cell or the dissociation time for SP from its receptor is longer than the wash time.     

碱性成纤维细胞生长因子对大鼠肾小球系膜细胞原癌基因c-fos和c-myc表达的影响

在建立大鼠肾小球系膜细胞（ＭＣ）体外培养方法的基础上,通过３Ｈ－ＴｄＲ参入实验,ＲＮＡ印迹分析和斑点杂交观察ｂＦＧＦ对ＭＣＤＮＡ合成及原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达的影响．结果表明,ｂＦＧＦ作用于ＭＣ１８ｈ,ＭＣ的３Ｈ－ＴｄＲ参入率明显增加（Ｐ＜０．０５）,２４ｈ达到高峰（Ｐ＜０．０１）;ｂＦＧＦ显著诱导原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达,其表达活性分别于３０ｍｉｎ和１ｈ达到高峰．提示ｂＦＧＦ是ＭＣ的强效丝裂原,其对ＭＣＤＮＡ合成的促进作用与诱导原癌基因ｃ－ｆｏｓ和ｃ－ｍｙｃ表达有关．     

Cd++ inhibits EGF-induced DNA synthesis but not EGF induced myc mRNA accumulation in serum starved NRK-49F cells

Cd⁺⁺ inhibits EGF-induced ³H-thymidine incorporation in serum deprived NRK-49F cells in a dose dependent pattern. The underlying mechanisms for this inhibition are largely unknown. EGF-induced myc mRNA accumulation in NRK-49F cells and the effects of Cd⁺⁺ on this response were examined under conditions that result in partial or complete inhibition of EGF-induced DNA synthesis. It was found that doses of Cd⁺⁺ that inhibit EGF-induced DNA synthesis do not inhibit EGF-induced protein synthesis and myc mRNA accumulation. Cd⁺⁺ doses of 0.5 µM and 1 µM were found actually to increase EGF-induced myc mRNA accumulation and amino acid incorporation. These results show that the effect of Cd⁺⁺ on EGF-induced DNA synthesis is not due to inhibition of entrance into G₁, but rather that Cd⁺⁺ acts on events subsequent to myc accumulation; that is, events associated with either G₁ progression, entry into S or DNA synthesis.Abbreviations EGF Epidermal Growth Factor - ³HTdr Tritium thymidine - MeAIB Methylaminoisobutyrate     

Alteration of intracellular GSH levels and its role in microcystin-LR-induced DNA damage in human hepatoma HepG2 cells

Microcystin-LR (MCLR) is a liver-specific toxin known as a tumour promoter in experimental animals. Its mechanisms of hepatotoxicity have been well documented; however, the mechanisms of other effects, in particular those related to its genotoxicity, are not well understood. In our previous studies, we showed that MCLR-induced DNA strand breaks are transiently present and that the damage is mediated by reactive oxygen species (ROS). In this study, we show that exposure of HepG2 cells to non-cytotoxic doses of MCLR-induced time-dependent alterations in the level of intracellular reduced glutathione (GSH). These comprised a rapid initial decrease followed by a gradual increase, reaching a maximum after 6h of exposure, before returning to the control level after 8h. During the first 4h, expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, increased, indicating an increased rate of de novo synthesis of GSH. The most important observation of this study, combined with the results of our previous studies is the correlation between the time course of alterations of intracellular GSH content and the formation and disappearance of MCLR-induced DNA damage. When the intracellular GSH level was reduced, MCLR-induced DNA damage was observed to increase. Later, when the level of intracellular GSH was normal or elevated, new DNA damage was not induced and existing damage was repaired. To confirm the role of GSH system in MCLR-induced genotoxicity, the intracellular GSH level was moderated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor. Pre-treatment with BSO dramatically increased the susceptibility of HepG2 cells to MCLR-induced DNA damage, while pre-treatment with NAC almost completely prevented MCLR-induced DNA damage. Thus, intracellular GSH is shown to play a critical role in the cellular defence against MCLR-induced DNA damage in HepG2 cells.     

Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells

Lead at slightly subtoxic concentrations markedly stimulated the rate of DNA synthesis in cultured animal cells. This stimulation was closely correlated with formation of a precipitate that was adsorbed and taken up by the cells under certain medium conditions. Data suggest that a precipitate-induced perturbation of the surface membrane leads to intracellular changes responsible for stimulation of DNA synthesis. Maximum stimulation of³H-thymidine incorporation by optimum concentrations of lead is delayed about 8 h compared to that in serum stimulation. In cells stimulated significantly by lead, but not in unstimu-lated cells, a reproducible rise of about 13% in intracellular magnesium occurred over a 24 h period, with an 8 h lag in the increase compared to that observed in serum stimulation. In view of the increases in intracellular magnesium consistently associated with and preceding stimulation of DNA synthesis by several different mitogens including serum and insulin, the present time-coordinated positive correlation between magnesium and DNA synthesis provides evidence for the primary involvement of this divalent cation in growth stimulation produced by lead.     

Changes in intracellular cations during the cell cycle in HeLa cells

Intracellular Na+, K+, and Mg2+ concentrations have been measured during the HeLa cell cycle and compared with changes in oxygen utilization and macromolecular synthesis. Cell water content remains relatively constant at 79 +/- 1% during the cell cycle. A biphasic change in intracellular Na+ occurs with low values as cells reach peak S phase and again in early G1. The decrease in S coincides with an increase in cell volume during increased macromolecular synthesis. The fall in intracellular Na+ during mitosis/early G1 coincides with decreased energy utilization as macromolecular synthesis decreases with a continued decrease in [Na+]i in G1 corresponding to a period of increasing cell volume and an increase in protein synthesis. Intracellular Na+ is relatively high during late S/G2 when phosphate incorporation into protein and phospholipid is maximal. Intracellular K+ concentrations largely parallel intracellular Na+ levels although the intracellular K+:Na+ ratio is significantly lower as the cell volume increases during late G2/mitosis. Additions of a Na+-pump inhibitor (strophanthidin) not only caused a rise in [Na+]i and fall in [K+]i but also inhibited protein synthesis. Conversely, addition of a protein synthesis inhibitor (cycloheximide) blocked amino acid incorporation and produces a fall in intracellular Na+ levels. These findings indicate that intracellular Na+ and K+ play an important role in regulating cell hydration during the cell cycle and that changes in Na+, K+-ATPase activity, synthesis and/or utilization of high energy phosphate compounds, fluid phase turnover (endocytosis), Na+:H+ exchange (pHi), Donnan forces, and ionic adsorption may all be involved.     

Alteration of intracellular GSH levels and its role in microcystin-LR-induced DNA damage in human hepatoma HepG2 cells

Microcystin-LR (MCLR) is a liver-specific toxin known as a tumour promoter in experimental animals. Its mechanisms of hepatotoxicity have been well documented; however, the mechanisms of other effects, in particular those related to its genotoxicity, are not well understood. In our previous studies, we showed that MCLR-induced DNA strand breaks are transiently present and that the damage is mediated by reactive oxygen species (ROS). In this study, we show that exposure of HepG2 cells to non-cytotoxic doses of MCLR-induced time-dependent alterations in the level of intracellular reduced glutathione (GSH). These comprised a rapid initial decrease followed by a gradual increase, reaching a maximum after 6 h of exposure, before returning to the control level after 8 h. During the first 4 h, expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, increased, indicating an increased rate of de novo synthesis of GSH. The most important observation of this study, combined with the results of our previous studies is the correlation between the time course of alterations of intracellular GSH content and the formation and disappearance of MCLR-induced DNA damage. When the intracellular GSH level was reduced, MCLR-induced DNA damage was observed to increase. Later, when the level of intracellular GSH was normal or elevated, new DNA damage was not induced and existing damage was repaired. To confirm the role of GSH system in MCLR-induced genotoxicity, the intracellular GSH level was moderated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor. Pre-treatment with BSO dramatically increased the susceptibility of HepG2 cells to MCLR-induced DNA damage, while pre-treatment with NAC almost completely prevented MCLR-induced DNA damage. Thus, intracellular GSH is shown to play a critical role in the cellular defence against MCLR-induced DNA damage in HepG2 cells.     

Alteration of the Intracellular Energetic and Ionic Conditions by Mengovirus Infection of Ehrlich Ascites Tumor Cells and Its Influence on Protein Synthesis in the Midphase of Infection

Mengovirus infection of Ehrlich ascites tumor cells caused a change of the intracellular ATP concentration. It increased by 35% within the first 3 h postinfection and then declined to zero within the next 5 h. The decrease in the ATP concentration was due, at least in part, to leakage of ATP into the medium, where it could be demonstrated by the luciferin-luciferase assay. Gross leakage of ATP was observed at 4.5 h postinfection, concomitant with the production of the first intracellular, infectious virus particles. A similar concentration decrease was detected for Mg(2+), the polyamines, and K(+), whereas an increase in the Na(+) concentration was observed. The intracellular Mg(2+) concentration varied synchronously with the ATP level, rising by 16% during the first 3 h postinfection and then progressively falling to lower values in the late period of the infectious cycle. After an initial slight enhancement, the putrescine, spermidine, and spermine concentrations declined at about 1.5 h postinfection. Wherease the intracellular K(+) concentration increased by 17% during the first hour postinfection, the Na(+) concentration diminished by the same value within the same time period, leaving the internal ionic strength unchanged early in infection. Three hours after the beginning of virus infection, there was a rapid decline of K(+) and enhancement of Na(+) within the cell. These alterations of the intracellular energetic and ionic conditions seem to be, at least in part, responsible for the cessation of virus-specific protein synthesis in mengovirus-infected Ehrlich ascites tumor cells commencing 3 to 3.5 h postinfection.     

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I)-stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I)-stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.     

Down-regulation of cellular proto-oncogenes during inhibition of rat parotid acinar cell proliferation.

The role of cell surface galactosyltransferase in mediating isoproterenol-induced parotid gland hypertrophy and hyperplasia was examined in rat parotid gland acinar cells. Introduction of the transferase modifier, alpha-lactalbumin, or galactosyltransferase-associated kinase inhibitor trifluoperazine, into beta-agonist-treated rats prevented acinar cell proliferation as determined by [3H]thymidine incorporation after 96 h of treatment. However, [3H]thymidine incorporation into DNA after 24 h of treatment, with injection of a combination of isoproterenol/alpha-lactalbumin or isoproterenol/trifluoperazine, was similar to injections of isoproterenol alone; suggesting that acinar cells could be stimulated to undergo a single round of DNA synthesis. Northern blot analysis of myc and fos expression followed a similar pattern of down-regulation to control levels after 96 h but not after 24 h. Hybridization with erb B showed little change with proliferation, confirming previous observations on protein levels of the EGF-receptor in acinar cells. Western blot analysis of nuclear protein expression of myc revealed that isoproterenol caused an increase in a 62-kDa protein which was again down-regulated with inhibition of cell proliferation. Analysis of protein levels of Rb110 protein showed no change in protein level in the nucleus with cell proliferation, but did show an associated increase in protein phosphorylation in response to growth stimulation.     

Phenotypic effects of overexpression of PKC beta 1 in rat liver epithelial cells

We have used a previously described retroviral expression vector pMV7-PKC beta 1 to develop derivatives of two rat liver epithelial cell lines, K16 and K22, that stably express about tenfold-higher PKC activity than control cells. Despite these high levels of PKC, these cells did not exhibit gross morphologic changes, anchorage-independent growth, or tumorigenicity. K16PKC-4 and K22PKC-2, two lines with the highest PKC enzyme activity, were studied further in terms of several responses to the phorbol ester tumor promoter TPA. When treated with 100 ng/ml of TPA, the control K16MV7 and K22MV7 cells displayed a slight change in morphology, whereas the K16PKC-4 and K22PKC-2 cells displayed a marked change in morphology. Northern blot analyses demonstrated that TPA induced increased levels of fos, myc, phorbin, and ODC RNAs in control K16MV7 and K22MV7 cells, with maximum induction occurring at about 0.5, 1, 8, and 8 h, respectively. In K16PKC-4 and K22PKC-2 cells, TPA induction of phorbin and ODC RNAs was markedly enhanced, but this was not the case for myc and fos RNAs. In addition, the levels of myc RNA were constitutively higher in both K16PKC-4 and K22PKC-2 cells than in the control cells. Taken together, these results provide direct evidence that PKC plays a critical role in modulating the expression of myc, phorbin, and ODC RNAs. On the other hand, overexpression of PKC beta 1 is not itself sufficient to cause cell transformation.     

Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.     

Extracellular Na+ and initiation of DNA synthesis: role of intracellular pH and K+

Initiation of DNA synthesis in confluent quiescent 3T3 cell cultures stimulated by epidermal growth factor (EGF), vasopressin, and insulin was abolished by removing extracellular Na+. The inhibition was reversible, time- and Na+-concentration-dependent, and not due to an effect on binding or internalization of 125I-EGF. Stimulation by combinations of other growth factors with different mechanisms of action was also affected by decreasing extracellular Na+, but with different half-maximal Na+ concentrations. When choline was used as an osmotic substitute for Na+, the decrease in DNA synthesis was correlated with the decrease in intracellular K+. In contrast, when sucrose was used there was stimulation of the Na+-K+ pump and maintenance of intracellular K+ that resulted in a somewhat higher rate of DNA synthesis at lowered extracellular Na+ compared to choline. Mitogenesis induced by epidermal growth factor, vasopressin, and insulin led to cytoplasmic alkalinization as determined by an increase in uptake of the weak acid 5,5-dimethyloxazolidine-2,4-dione. Experimental decrease in extracellular Na+ blocked this cellular alkalinization. Therefore, under some conditions the supply of extracellular Na+ may limit cellular proliferation because of a reduction in the provision of Na+ to the Na+/H+ antiport and resultant failure of alkalinization. We conclude that Na+ flux and its effect on intracellular K and pH has a major role in the complex system that regulates proliferation.