Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation

The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1-oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.     

Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes

The Na+/H+ antiport of rat thymic lymphocytes is activated when protein kinase C is stimulated by phorbol esters. A similar activation of the antiport is obtained when the cells are treated with hypertonic solutions. We tested the possibility that protein kinase C also mediates the osmotic activation of Na+/H+ exchange. Protein kinase C was depleted by preincubation of thymocytes for 24 hr in the presence of high concentrations of phorbol ester. Disappearance of the enzyme was assessed by direct measurement of phosphotransferase activity, and by the loss of biological responses to phorbol esters. The Na+/H+ antiport in protein kinase C-depleted cells was not stimulated by addition of phorbol ester, but responded normally to hypertonic treatment. The results indicate that the osmotic activation of countertransport does not require stimulation of protein kinase C.     

Pertussis toxin inhibits thrombin-induced activation of phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts.

Prior treatment with pertussis toxin of G0-arrested hamster fibroblasts (CCL39) results in a dose-dependent inhibition of two early events of the mitogenic response elicited by alpha-thrombin: accumulation of inositol phosphates in Li+-treated cells, and activation of the Na+/H+ antiport, measured either by the amiloride-sensitive 22Na+ influx or by the increase in intracellular pH. At 10(-1) U/ml of alpha-thrombin, the maximal inhibition was approximately 50% for these two early cellular responses, but the pertussis toxin effect was more pronounced at lower thrombin concentrations. In contrast, pertussis toxin does not affect the Na+/H+ antiport activation induced by phorbol esters or EGF, the action of which is not mediated by the phosphoinositide-metabolizing pathway in CCL39 cells. Therefore, our data suggest the following. A GTP-binding regulatory protein is probably involved in signal transduction between thrombin receptors and the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. This regulation does not seem to be exerted via modulations of cyclic AMP levels. The thrombin-induced activation of Na+/H+ antiport is, at least in part, mediated by the protein kinase C, as a consequence of stimulation of phosphatidylinositol turnover.     

Cytoplasmic [Ca2+] and intracellular pH in lymphocytes. Role of membrane potential and volume-activated Na+/H+ exchange

The effect of elevating cytoplasmic Ca2+ [( Ca2+]i) on the intracellular pH (pHi) of thymic lymphocytes was investigated. In Na+-containing media, treatment of the cells with ionomycin, a divalent cation ionophore, induced a moderate cytoplasmic alkalinization. In the presence of amiloride or in Na+-free media, an acidification was observed. This acidification is at least partly due to H+ (equivalent) uptake in response to membrane hyperpolarization since: it was enhanced by pretreatment with conductive protonophores, it could be mimicked by valinomycin, and it was decreased by depolarization with K+ or gramicidin. In addition, activation of metabolic H+ production also contributes to the acidification. The alkalinization is due to Na+/H+ exchange inasmuch as it is Na+ dependent, amiloride sensitive, and accompanied by H+ efflux and net Na+ gain. A shift in the pHi dependence underlies the activation of the antiport. The effect of [Ca2+]i on Na+/H+ exchange was not associated with redistribution of protein kinase C and was also observed in cells previously depleted of this enzyme. Treatment with ionomycin induced significant cell shrinking. Prevention of shrinking largely eliminated the activation of the antiport. Moreover, a comparable shrinking produced by hypertonic media also activated the antiport. It is concluded that stimulation of Na+/H+ exchange by elevation of [Ca2+]i is due, at least in part, to cell shrinking and does not require stimulation of protein kinase C.     

Regulation of amino acid uptake by phorbol esters and hypertonic solutions in rat thymocytes

Growth factors, mitogens, and malignant transformation can alter the rate of amino acid uptake in mammalian cells. It has been suggested that the effects of these stimuli on proliferation are mediated by activation of Na+/H+ exchange. In lymphocytes, Na+/H+ exchange can also be activated by phorbol esters and by hypertonic media. To determine the relationship between the cation antiport and amino acid transport, we tested the effects of these agents on the uptake of alpha-aminoisobutyric acid (AIB), methyl-AIB, proline, and leucine in rat thymocytes. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and hypertonicity stimulated amino acid uptake through system A (AIB, proline, and methyl-AIB). In addition, TPA, but not hypertonicity, also elevated leucine uptake. The stimulation of the Na+ -dependent system A was not due to an increased inward electrochemical Na+ gradient. The effects of TPA and hypertonic treatment were not identical: Stimulation of AIB uptake by TPA was observed within minutes, whereas at least 1 hr was required for the effect of hypertonicity to become noticeable. Moreover, stimulation by hypertonicity but not that by TPA, was partially inhibited by cycloheximide, suggesting a role of protein synthesis. That stimulation of Na+/H+ exchange does not mediate the effects on amino acid transport is suggested by two findings: 1) the stimulation of AIB uptake was not prevented by concentrations of amiloride or of 5-(N,N-disubstituted) amiloride analogs that completely inhibit the Na+/H+ antiport and 2) conditions that mimic the effect of the antiport, namely, increasing [Na+]i or raising pHi failed to stimulate amino acid uptake. Thus, in lymphocytes, activation of Na+/H+ exchange and stimulation of amino acid transport are not casually related.     

1,2-Diacylglycerols mimic phorbol 12-myristate 13-acetate activation of the sea urchin egg

Phorbol diesters have been reported to stimulate the Na+/H+ antiport of a variety of cells including sea urchin eggs. Since stimulation of the Na+/H+ antiport is necessary for metabolic derepression during fertilization and protein kinase C is a target of phorbol diesters, enhanced Na+/H+ exchange during fertilization may be a result of protein kinase C activity. Protein kinase C is probably physiologically activated by diacylglycerols, which are derived from hydrolysis of phosphatidylinositol. Treatment of sea urchin eggs with 1,2-diacylglycerols was found to stimulate the Na+/H+ antiport. The 1,3-isomers were without effect. Further, the effects of 1,2-diacylglycerol and phorbol diester are not additive with respect to Na+/H+ exchange. While a direct participation of protein kinase C activity during fertilization remains to be demonstrated, these data support the hypothesis that protein kinase C activity plays a role in fertilization. However, the cytotoxic effect of protein kinase C activators suggests effects associated with their pleiotropic nature.     

Stimulation of human platelets by collagen occurs by a Na+/H+ exchanger independent mechanism

In stimulated human platelets dense-granule secretion in response to the 'weak agonists' ADP, adrenaline, platelet activating factor and low concentrations of thrombin as well as Ca2+ mobilisation in response to thrombin are enhanced by a Na+/H+ exchanger. In the present study the role of this antiport in collagen stimulated human platelets was examined. While stimulation of platelets loaded with the fluorescent intracellular pH-sensitive dye, bis-carboxyethyl-5-(6)-carboxyfluorescein (BCECF) with thrombin resulted in the activation of the Na+/H+ exchanger, activation of this antiport did not occur in collagen-stimulated platelets. The lack of antiport activity in response to collagen using BCECF-loaded platelets correlated with the lack of any functional role of the antiport in collagen stimulated platelets. In the presence of a Na+/H+ exchange inhibitor, ethylisopropylamiloride, neither collagen-induced platelet aggregation or dense-granule secretion was affected. Furthermore, while the removal of extracellular Na+ (Na+ext), a condition that also prevents activation of the antiport, inhibited dense-granule secretion in response to a low concentration of thrombin, collagen-induced secretion was potentiated. This potentiatory effect could not be attributed to changes in either the membrane potential or in collagen-induced phospholipase C or protein kinase C activity. The present results indicate that in contrast to the 'weak agonists' (1) collagen-induced platelet activation does not require activation of the Na+/H+ exchanger and (2) Na+ext per se is an inhibitor of collagen-induced secretion.     

Mechanism of activation of lymphocyte Na+/H+ exchange by concanavalin A. A calcium- and protein kinase C-independent pathway

Treatment of thymic lymphocytes with the mitogenic lectin concanavalin A (ConA) increases the intracellular free Ca2+ concentration and stimulates phosphoinositide turnover. ConA also induced a rapid, amiloride-sensitive, Na+-dependent increase in cytosolic pH of 0.13 +/- 0.01, indicative of stimulation of the Na+/H+ antiport. To investigate the mechanism underlying activation of Na+/H+ exchange by ConA, the intracellular free Ca2+ concentration changes induced by this lectin were precluded by loading the cells with Ca2+-buffering agents and suspension in Ca2+-free media. Under these conditions, the ConA-induced cytoplasmic alkalinization proceeded normally. Two approaches were used to assess the role of protein kinase C. First, this enzyme was inhibited by the addition of 1-(5-isoquinolinysulfonyl)-2-methylpiperazine. In the presence of this potent antagonist, stimulation of the antiport by 12-O-tetradecanoylphorbol-13-acetate was greatly inhibited. In contrast, stimulation by ConA was unaffected. Second, protein kinase C was depleted by overnight incubation with phorbol esters. Following this treatment, Na+/H+ exchange was no longer activated by 12-O-tetradecanoyl-13-acetate, but was still stimulated by ConA. These data suggest that a Ca2+- and protein kinase C-independent mechanisms mediates the activation of Na+/H+ exchange by ConA. The possible role of GTP-binding proteins in the activation was also studied. The antiport was not stimulated by either fluoroaluminate or vanadate. Moreover, pretreatment with pertussis toxin failed to inhibit the ConA-induced cytoplasmic alkalinization. In contrast, preincubation with cholera toxin partially inhibited activation. Under these conditions, cholera toxin significantly elevated intracellular cAMP levels. Inhibition was also observed in cells treated with forskolin at concentrations that increased [cAMP]. The data suggest that a novel cAMP-sensitive signaling mechanism not involving Ca2+ and protein kinase C is involved in the stimulation of Na+/H+ exchange by mitogens in T lymphocytes.     

Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes

The activity of the Na+/H+ exchange system of rat thymic lymphocytes was determined by means of intracellular (pHi) and extracellular pH (pH0) measurements. In isotonic media, the antiport is virtually quiescent at physiological pHi (7.0-7.1), but is greatly activated by cytoplasmic acidification. At normal pHi, the antiport can also be activated by osmotic shrinking. Osmotic activation occurs after a delay of 20-30 s and is reversed several minutes after iso-osmolarity is restored. The mechanism of activation was analyzed by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. The affinities of the external substrate site for Na+ and H+ are not altered in shrunken cells. In contrast, the Hi+ sensitivity of the antiport (which is largely dictated by an allosteric modifier site) was increased, which accounted for the activation. The concentration of free cytoplasmic Ca2+ [( Ca2+]i) increased after osmotic shrinking. This increase was dependent on the presence of extracellular Ca2+ and Na+ and was blocked by inhibitors of Na+/H+ exchange, which suggests that it is a consequence, rather than the cause, of the activation of the antiport. It is concluded that the shift in the pHi dependence of the modifier site of the Na+/H+ antiport is the primary event underlying the regulatory volume increase that follows osmotic shrinkage.     

A phorbol ester and 1-oleoyl-2-acetylglycerol induce Na+/H+ exchange in human platelets

This study aimed at investigating the mechanisms by which stimulation of human platelets results in activation of Na+/H+ exchange. Platelets were suspended in a slightly buffered medium and the stimulus-induced, amiloride-sensitive H+ release, reflecting Na+/H+ exchange, was estimated from changes in the medium pH. H+ release could be evoked by thrombin and by activators of protein kinase C such as 1-oleoyl-2-acetylglycerol (OAG) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the thrombin-and the OAG-induced Na+/H+ exchange could be blocked by trifluoperazine, a protein kinase C inhibitor. The thrombin-induced H+ release was also sensitive to increased intracellular cAMP levels, probably due to inhibition of phospholipase C activation, whereas the OAG-induced activation of Na+/H+ exchange was unaffected. Our data suggest that activation of Na+/H+ exchange is mediated by protein kinase C.     

Interleukin-3-stimulated haemopoietic stem cell proliferation. Evidence for activation of protein kinase C and Na+/H+ exchange without inositol lipid hydrolysis.

Interleukin 3 (IL-3) is an important regulator of haemopoietic stem cell proliferation both in vivo and in vitro. Little is known about the possible mechanisms whereby this growth factor acts on stem cells to stimulate cell survival and proliferation. Here we have investigated the role of intracellular pH and the Na+/H+ antiport in stem cell proliferation using the multipotential IL-3-dependent stem cell line, FDCP-Mix 1. Evidence is presented that IL-3 can stimulate the activation of an amiloride-sensitive Na+/H+ exchange via protein kinase C activation. IL-3-mediated activation of the Na+/H+ exchange is not observed in FDCP-Mix 1 cells where protein kinase C levels have been down-modulated by treatment with phorbol esters. Also the protein kinase C inhibitor H7 can inhibit IL-3-mediated increases in intracellular pH. This activation of Na+/H+ exchange via protein kinase C has been shown to occur with no measurable effects of IL-3 on inositol lipid hydrolysis or on cytosolic Ca2+ levels. Evidence is also presented that this IL-3-stimulated alkalinization acts as a signal for cellular proliferation in stem cells.     

Regulation of Na+/H+ and Cl-/HCO3- antiports in Vero cells

The effect of serum, phorbol-12-myristate-13-acetate (TPA), and forskolin on the activity Na+/H+ antiport and the Na(+)-coupled and Na(+)-independent Cl-/HCO3- antiport was studied in Vero cells by measuring 22Na+ and 36Cl- fluxes and changes in cytosolic pH (pHi). The Na(+)-independent Cl-/HCO3- antiport, which acts as an acidifying mechanism, is strongly pH-sensitive. In serum-starved cells it is activated at alkaline cytosolic pH, with a half-maximal activity at pHi approximately 7.20. Incubation with serum increased the activity of the Na(+)-independent Cl-/HCO3- antiport at pHi values from 6.8 to 7.2. Thus serum appeared to alter the pHi sensitivity of this antiporter such that the threshold value for activation of the antiport was shifted to a more acidic value. Na+/H+ antiport was somewhat stimulated initially by addition of serum, but further incubation with serum (greater than 45 min) decreased its activity. The activity of the Na(+)-coupled Cl-/HCO3- antiport, which is the major alkalinizing antiport in Vero cells, was not altered by short-term incubation with serum (less than 10 min) but decreased after prolonged incubation (greater than 45 min). Our findings with TPA and forskolin indicate that the effect of serum is partly mediated by the protein kinase C pathway, whereas the cyclic adenosine monophosphate pathway does not appear to play an important role. The net effect of serum on the pHi-regulating antiports was a slight decrease in intracellular pH.     

Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism.

A variety of cell types regulate their volume in anisotonic media by stimulating Na+/H+ exchange. Like growth factors, osmotic challenge activates the Na+/H+ antiport by increasing its sensitivity to intracellular [H+]. To investigate the molecular mechanism underlying this shift in pH sensitivity, the antiporter of 32P-labeled human bladder carcinoma cells and of Chinese hamster ovary cells was immunoprecipitated using antibodies raised against the cytosolic domain of the NHE-1 isoform of the Na+/H+ exchanger. Unlike the effects of growth promoters, activation of the antiport during volume regulation was not associated with increased phosphorylation. The possible coexistence of multiple antiporter isoforms was considered. The cytosolic alkalosis normally elicited by hypertonic media was found to be absent in Na+/H+ exchange-deficient fibroblasts. Responsiveness to osmotic challenge was restored by stable transfection of these cells with the cDNA encoding NHE-1. In these transfectants, phosphorylation of the antiporter was also unaffected during osmotic activation. The unchanged phosphate content of the antiporter might be explained by dephosphorylation of one site with concomitant phosphorylation at a different site. However, this possibility appears unlikely since phosphoamino acid analysis revealed that serine was the only residue phosphorylated in immunoprecipitated antiports of both control and osmotically stimulated cells. Moreover, phosphopeptide maps of control and hypertonically activated antiports were identical. These findings reveal a novel mode of activation of Na+/H+ exchange not requiring direct phosphorylation of the antiporter. We propose the existence of dual control of Na+/H+ exchange by phosphorylation-dependent and -independent mechanisms.     

Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways

Angiotensin II, a potent vasoconstrictor, is known to stimulate Ca2+ mobilization and Na+ influx in vascular smooth muscle cells (VSMC). The fact that the Na+/H+ exchange inhibitor, amiloride, blocks angiotensin II-stimulated Na+ influx and is itself a vasodilator suggests that Na+/H+ exchange may play a role in the angiotensin II-mediated effects on VSMC. We have used a pH-sensitive fluorescent dye to study Na+/H+ exchange in cultured rat aortic VSMC. Basal intracellular pH was 7.08 in physiological saline buffer. Angiotensin II stimulation caused an initial transient acidification, followed by a Na+-dependent alkalinization. Angiotensin II increased the rate of alkalinization with apparent threshold, half-maximal, and maximal effect of 0.01, 3, and 100 nM, respectively. Angiotensin II stimulation appeared to be mediated by a shift in the Km of the Na+/H+ exchanger for extracellular Na+. Since angiotensin II activates phospholipase C in VSMC, we tested the possibility that angiotensin II increased Na+/H+ exchange by activation of protein kinase C via stimulation of diacylglycerol formation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated Na+/H+ exchange in VSMC cultured for 24 h in serum-free medium, and the subsequent angiotensin II response was inhibited. However, VSMC grown in serum and treated for 24 h with TPA to decrease protein kinase C activity showed no inhibition of angiotensin II-stimulated Na+/H+ exchange. TPA caused no intracellular alkalinization of VSMC grown in serum, while the angiotensin II response was actually enhanced compared to VSMC deprived of serum for 24 h. We conclude that angiotensin II stimulates an amiloride-sensitive Na+/H+ exchange system in cultured VSMC which is mediated by protein kinase C-dependent and -independent mechanisms. Angiotensin II-mediated Na+ influx and intracellular alkalinization may play a role in excitation-response coupling in vascular smooth muscle.     

Thyrotropin-releasing hormone activates Na+/H+ exchange in rat pituitary cells.

The effects of thyrotropin-releasing hormone (TRH) and 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytosolic pH (pHi) were studied on GH4C1 pituitary cells loaded with the fluorescent pH indicator bis(carboxyethyl)carboxyfluorescein (BCECF) and the fluorescent Ca2+ indicator quin2. TRH, which was minimally effective at around 10(-9) M, and TPA, 100 nM, produced very small elevations in pHi of about 0.05 pH units from the normal basal resting pHi of GH4C1 cells of around 7.05. The effects were more marked after acid-loading the cells using 1 micrograms of nigericin/ml. Preincubation with amiloride or replacing the extracellular Na+ with choline+ completely blocked the elevations stimulated by TRH or TPA, consistent with an activation of the Na+/H+ antiport mechanism. The effects were completely independent of the cytoplasmic free calcium concentration ([Ca2+]i). The calcium ionophore ionomycin produced an elevation in [Ca2+]i with no concomitant effect on pHi, and amiloride, although completely inhibiting the pH change stimulated by TRH, failed to affect the initial stimulated [Ca2+]i transient. Although the data are consistent with an elevation in pHi by TRH which is caused by stimulation of a protein kinase C and subsequent activation of the antiporter, the rapidity of the onset of the pHi response to TRH could not be mimicked by a combination of TPA and ionomycin. These results, together with previous findings which show that secretion can be mimicked by TPA and ionomycin, suggest that TRH-stimulated Na+/H+ exchange plays no part in the acute stimulation of secretion, but that TRH increases the pH-sensitivity of the antiport system during increased synthesis of prolactin and growth hormone.     

Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+H+ exchange?

The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport.     

Effects of phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of epidermal growth factor-stimulated formation of inositol phosphates in A-431 cells by calcium and protein kinase C

Epidermal growth factor (EGF) treatment of A-431 cells induces a biphasic increase in the levels of inositol phosphates. The growth factor produces an initial, rapid increase in the level of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) due to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (Wahl, M., Sweatt, J. D., and Carpenter, G. (1987) Biochem. Biophys. Res. Commun. 142, 688-695). The level of inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) also rises rapidly in response to treatment with EGF. The initial formation (less than 1 min) of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 does not require Ca2+ present in the culture medium. However, the addition of Ca2+ to the medium at levels of 100 microM or greater potentiates the growth factor-stimulated increases in the levels of all inositol phosphates at later times after EGF addition (1-60 min). The data suggest that EGF-receptor complexes initially stimulate the enzyme phospholipase C in a manner that is independent of an influx of extracellular Ca2+. The presence of Ca2+ in the medium allows prolonged growth factor activation of phospholipase C. Treatment of A-431 cells with Ca2+ ionophores (A23187 and ionomycin) did not mimic the activity of EGF in producing a rapid increase in the formation of the Dowex column fraction containing Ins-1,4,5-P3, Ins-1,3,4,5-P4, and inositol 1,3,4-trisphosphate (InsP3). However, the initial EGF-stimulated formation of inositol phosphates was substantially diminished in cells loaded with the Ca2+ chelator Quin 2/AM. EGF receptor occupancy studies indicated that maximal stimulation of InsP3 accumulation by EGF requires nearly full (75%) occupancy of available EGF binding sites, while half-maximal stimulation requires 25% occupancy. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an exogenous activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), causes a dramatic, but transient, inhibition of the EGF-stimulated formation of inositol phosphates. Tamoxifen and sphingosine, reported pharmacologic inhibitors of protein kinase C activity, potentiate the capacity of EGF to induce formation of inositol phosphates. Neither TPA nor tamoxifen significantly affects the 125I-EGF binding capacity of A-431 cells; however, TPA appeared to enhance internalization of the ligand. Ligand occupation of the EGF receptor on the A-431 cell appears to initiate a complex signaling mechanism involving production of intracellular messengers for Ca2+ mobilization and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C

Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.     

Characterization of Na+/H+ exchange in FRTL-5 thyroid cells. Evidence for dependence on activation of protein kinase C.

Na+/H+ exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pHi) was 7.13 +/- 0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity beta i was determined using the NH4Cl-pulse method, yielding a beta i value of 85 +/- 12 mM/pH unit. The relationship between extracellular Na+ and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent Km value of 44 +/- 26 mM, and an Vmax value of 0.3 +/- 0.01 pH unit/min. The agonist-induced activation of Na+/H+ exchange was investigated in cells acidified with nigericin. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 microM amiloride, by substituting extracellular Na+ with equimolar concentrations of choline+, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca2+, or depleating intracellular Ca2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na+/H+ exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.