Effects of amiloride on the induction of DNA synthesis and casein gene expression in rabbit mammary explants

Amiloride, an inhibitor of Na+/H+ exchange, was added at various concentrations to the culture medium of rabbit mammary explants. In the concentration range 100-250 microM, amiloride progessively inhibited 14C-thymidine incorporation induced by insulin, EGF or prolactin. Up to 250 microM, amiloride, which did not inhibit basal protein synthesis, was not cytotoxic, but it reduced basal DNA synthesis at the highest concentration. Addition of amiloride to the culture medium of mammary explants also strongly inhibited the induction of casein synthesis and casein mRNA accumulation by prolactin. The inhibition by amiloride is therefore not specific of the mitogenic action of prolactin since this drug also prevented its lactogenic action. The data reported here describe a new inhibitory action of amiloride on the transmission of the lactogenic signals.     

Stimulation by serum of the Na+/H+ antiporter in quiescent pig kidney epithelial (LLC-PK1) cells and role of the antiporter in the reinitiation of DNA synthesis

LLC-PK1 cells can be brought into a classical quiescent state by depriving them of serum for 6 days. At this time, pulse-labeling with [3H]-thymidine shows that only 3% of the cells are synthesizing DNA, but the quiescent cells can be stimulated with serum to re-enter the cell cycle at a point early in G1. The rate of amiloride-sensitive 22Na+ uptake (as a measure of the Na+/H+ antiporter) is relatively low during quiescence; it rises 2- to 3-fold within 4 h after serum addition. This increase in antiporter activity appears to be required for the resumption of DNA synthesis in the absence of bicarbonate, because ethylisopropylamiloride (EIPA) blocks [3H]-thymidine incorporation when serum is added to cells in bicarbonate-free medium. In the presence of bicarbonate, however, EIPA has no effect on [3H]-thymidine incorporation, indicating that another (bicarbonate-dependent) transport system can substitute for the antiporter under these conditions.     

Na+/H+ antiporter activity in hamster embryos is activated during fertilization

This study characterized the activation of the regulatory activity of the Na+/H+ antiporter during fertilization of hamster embryos. Hamster oocytes appeared to lack any mechanism for the regulation of intracellular pH in the acid range. Similarly, no Na+/H+ antiporter activity could be detected in embryos that were collected from the reproductive tract between 1 and 5 h post-egg activation (PEA). Activity of the Na+/H+ antiporter was first detected in embryos collected at 5.5 h PEA and gradually increased to reach maximal activity in embryos collected at 7 h PEA. Parthenogenetically activated one-cell and two-cell embryos demonstrate Na+/H+ antiporter activity, indicating that antiporter activity is maternally derived and initiated by activation of the egg. The inability of cycloheximide, colchicine, or cytochalasin D to affect initiation of antiporter activity indicates that antiporter appearance is not dependent on the synthesis of new protein or recruitment of existing protein to the cell membrane. In contrast, incubation of one-cell embryos with sphingosine did inhibit the appearance of Na+/H+ antiporter activity, showing that inhibition of normal protein kinase C activity is detrimental to antiporter function. Furthermore, incubation of oocytes with a phorbol ester which stimulates protein kinase C activity induced Na+/H+ antiporter activity in oocytes in which the activity was previously absent. Incubation with an intracellular calcium chelator also reduced the appearance of antiporter activity. Taken together, these data indicate that the appearance of Na+/H+ antiporter activity following egg activation may be due, at least in part, to regulation by protein kinase C and intracellular calcium levels.     

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.     

Second messenger signaling in the regulation of cytosolic pH and DNA synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblastic osteosarcoma cells: role of Na+/H+ exchange.

The present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH-related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR-106 which possessed PTH-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amiloride-inhibitable Na+/H+ exchange system. Both human (h)PTH-(1-34) and hPTHrP-(1-34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose-dependent manner with a minimal effective dose of 10(-10) M. Dibutyryl cAMP (10(-4) M and Sp-cAMPS (10(-4) M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10(-6) M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP- and Sp-cAMPS-induced decrease in pHi but did not affect calcium ionophore-induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore-induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA-inhibited Na+/H+ exchange and increased cytosolic calcium-induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/H+ exchange system.     

Mitogen-stimulated activation of the Na+/H+ antiporter does not regulate S6 phosphorylation or protein synthesis in murine thymocytes or Swiss 3T3 fibroblasts

The activation of protein synthesis by mitogens in quiescent (G0) mammalian cells is obligatory for progression from G0 through G1 to DNA synthesis in S phase. When the activation of the Na+/H+ antiporter which occurs in mitogen-stimulated Swiss 3T3 fibroblasts or murine fibroblasts is completely blocked by dimethylamiloride, there is little or no effect on the phosphorylation of the ribosomal protein S6 or the activation of protein synthesis assayed by [35S]methionine incorporation. Furthermore, the accumulation of the protein product of the activated c-myc gene is unaffected by dimethylamiloride in 3T3 fibroblasts. The data show that there is no requirement for activation of the Na+/H+ antiporter for the activation of S6 phosphorylation or protein synthesis by mitogens but do not preclude the possibility that activation of the antiporter is necessary for some other response(s) obligatory for DNA synthesis. These data are contrasted with previous reports for Chinese hamster lung fibroblasts that the increase in intracellular pH which results from activation of the Na+/H+ antiporter in bicarbonate-free media is necessary for S6 phosphorylation, protein synthesis, and hence, for subsequent DNA synthesis (Pouyssegur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3935-3939; Chambard, J.C., and Pouyssegur, J. (1986) Exp. Cell Res. 164, 282-294).     

Inhibition and labeling of the rat renal Na+/H+-exchanger by an antagonist of muscarinic acetylcholine receptors

A covalently binding label for muscarinic acetylcholine receptors, propylbenzilylcholine mustard (PrBCM), irreversibly inhibits the Na+/H+ exchanger in rat renal brush-border membrane vesicles. Substrates of the antiporter, Na+ and Li+, as well as inhibitors, amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and propranolol, protect the antiporter from inactivation by PrBCM. With [3H]PrBCM a band with an app. Mr of 65 kDa is predominantly labeled. Amiloride protects this band from labeling with [3H]PrBCM and [14C]-N,N'-dicyclohexylcarbodiimide (DCCD) proving its identity with the renal Na+/H+ exchanger. Our data reveal a specific interaction of PrBCM with the Na+/H+ exchanger and suggest structural relations between antiporter and receptors.     

Characterization of potent Na+/H+ exchange inhibitors from the amiloride series in A431 cells

Na+/H+ exchange is stimulated in a variety of cell types by addition of mitogenic polypeptides such as epidermal growth factor or platelet-derived growth factor. In order to assess the importance of Na+/H+ exchange in the mitogenic response, it is desirable to have available inhibitors of this process which exhibit high affinity and good specificity. We characterize in this report a number of 5-alkylamino-substituted derivatives of amiloride [3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide++ +] which show much higher affinity than the parent compound for the Na+/H+ antiporter in A431 cells. High affinity is conferred by substitution with two alkyl groups and is increased by introducing a branched alkyl chain. An analogue bearing a 5-anilino group is also very potent. These analogues effectively inhibit the elevation of intracellular pH upon stimulation of Na+/H+ exchange by growth factors. We have assessed other potential inhibitory effects of these compounds on cellular metabolism. In agreement with previous reports, we find that amiloride inhibits protein synthesis both in cells and in cell-free translation systems. While amiloride and its analogues show similar inhibition of protein synthesis in a cell-free system, most analogues inhibit cellular protein synthesis at much lower concentrations than does amiloride. These analogues are also potent inhibitors of purified Na,K-ATPase and cause a profound decrease in intracellular K+ as well as ATP content. These latter effects, however, require analogue concentrations which are 5-7 times higher than those inhibiting cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line

The actions of insulin, hydrocortisone, prolactin and growth hormone on the synthesis of macromolecules in MCF-7 cells was determined in a serum-free defined medium. The inclusion of the polyamine spermidine in the medium was shown to enhance the insulin stimulation of the rate of [3H]uridine incorporation into RNA in a manner similar to that demonstrated for hydrocortisone. Spermidine, in addition to insulin and hydrocortisone, was also essential for prolactin to manifest a stimulation of the rate of [3H]uridine incorporation; this effect of spermidine was optimal with spermidine concentrations between 1 and 5 mM. Prolactin also stimulated the rate of [3H]leucine incorporation into total cellular protein and into an isoelectrically precipitable (pH 4.6) phosphoprotein fraction. The actions of prolactin on total protein and phosphoprotein synthesis were only expressed if spermidine, in addition to insulin and hydrocortisone, was contained in the culture medium. All of the prolactin responses were observed employing physiological concentrations of prolactin. Specificity of the prolactin responses was established by demonstrating that porcine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells.     

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.     

Competitive inhibition of tritium-labeled platelet-activating factor binding to rabbit platelet membranes by amiloride and amiloride analogs

Amiloride and its structural analogs, ethylisopropyl amiloride, benzamil, and dichlorobenzamil, inhibit both the specific [3H]C18-PAF binding to rabbit platelet membranes and PAF-induced aggregation of gel-filtered rabbit platelets. Detailed analysis of binding inhibitions demonstrate that ethylisopropyl amiloride is a competitive inhibitor with an equilibrium dissociation constant (KB) of 23 microM. The concentration of amiloride and its analogs needed to inhibit the PAF-induced aggregation is high and there exists no correlation between their inhibitory activities of platelet aggregation and those of Na+/H+ antiporter. However, the inhibitory effects on the PAF-induced aggregation are parallel to those on the specific [3H]C18-PAF binding. The inhibitory effects of amiloride and its analogs on the activation of platelets are at the PAF-receptor binding step, rather than at the Na+/H+ antiporter.     

Effect of kinase C inhibitor, gossypol, on the actions of prolactin in cultured mouse mammary tissues

Gossypol, a drug which has been shown to be an inhibitor of kinase C activity in mouse mammary tissues, is shown to abolish several of the actions of prolactin in cultured mouse mammary gland explants. The prolactin effects that are abolished include its stimulatory effects on ornithine decarboxylase activity, the rate of [3H]uridine incorporation into RNA, the rate of [3H]leucine incorporation into a casein-rich phosphoprotein fraction, and the rate of [14C]acetate incorporation into lipids. Since the inhibitory concentrations of gossypol employed in these studies correspond well with the gossypol concentrations required to inhibit kinase C activity, we conclude that ongoing kinase C activity is essential for prolactin to express its differentiative actions in mammary tissues.     

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.     

T cell stimulation via CD2 molecules is regularly accompanied by an increase in cytoplasmic pH. Different effects of lectins and CD3 antibodies

The stimulation of different cell types with growth factors is often accompanied by a rapid intracellular alkalinization. By using mitogenic lectins, cluster of differentiation (CD)2 and CD3 mAb, as stimuli, we studied early changes of the intracellular pH in the activation process of resting human PBL. We found increases in free cytoplasmic Ca2+ levels and DNA synthesis but no intracellular alkalinization in the early activation phase upon stimulation with the mitogenic lectins, Con A, and PHA. Similarly stimulation with CD3 mAb led in most instances to no detectable pH shifts. Only in 7 out of 30 experiments was CD3 mAb-induced alkalinization observed. In contrast, stimulation with mitogenic combinations of anti-CD2 mAb led in all instances to rapid and clear-cut intracellular pH shifts very similar to those observed upon stimulation with PMA. In medium lacking sodium bicarbonate the intracellular alkalinization via the CD2 structure could be blocked by the amiloride analogue 5-(N-methyl-N-isobutyl)amiloride (MIA), which indicates that this increase in pH is mediated by the amiloride-sensitive Na+/H+ antiporter. Blockade of this antiporter had no negative effect, however, on T cell proliferation as measured by thymidine incorporation. In contrast, significantly enhanced proliferation rates were observed after stimulation with mitogenic combinations of anti-CD2 antibodies in the presence of MIA. No such effect of MIA could be observed in lectin induced T cell stimulation. These findings indicate that stimulation of the Na+/H+ antiporter via the CD2 structure is neither a prerequisite for T cell proliferation nor does it promote T cell growth. It rather seems to function in a regulatory role. In its absence, superinduction of proliferation can be achieved.     

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.     

Modulation of DNA synthesis in cultured muscle cells by 1,25-dihydroxyvitamin D-3

Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.     

Inhibition of T-lymphocyte activation by amiloride

The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occurring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H]leucine and [3H]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when T-lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75% decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 microM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.     

Mixed type inhibition of the renal Na+/H+ antiporter by Li+ and amiloride. Evidence for a modifier site

We studied the interactions of Na+, Li+, and amiloride on the Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pHin = 6.0; pHout = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (Km for Li+ = 1.2 +/- 0.1 mM; Km for Na+ = 14.3 +/- 0.8 mM; Vmax for Li+ = 2.40 +/- 0.07 fluorescence units/s/mg of protein; Vmax for Na+ = 7.10 +/- 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], [( Na+] = 90 mM; [Li+] less than 1 mM), we observed noncompetitive inhibition (Vmax for Na+/H+ exchange reduced by 25%). The apparent Ki for this noncompetitive inhibition was congruent to 50 microM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (Ki = 30 microM, Ki' = 90 microM) but was only a simple competitive inhibitor of Li+/H+ exchange (Ki = 10 microM). At [Li] = 1 mM and [amiloride] less than 100 microM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.     

Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos

We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.