epidermal-growth-factor and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

The modulation by epidermal growth factor (EGF) of the Na+/H+ antiport in fetal and adult rat hepatocytes was studied in nominally HCO3- free solution. EGF (10 nM) activated the antiport in adult rat hepatocytes by 0.22 +/- 0.03 (mean +/- SD;n=10) pH units over basal value, measured with the fluorescent pH-sensitive intracellular probe, 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein (BCECF). The effect of EGF was inhibited by amiloride analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA), by ouabain, inhibitor of the Na+ pump, and by erbstatin analogue, an inhibitor of the tyrosine kinase activity of the EGF receptor. The effect of EGF on Na+/H+ antiport in adult rat hepatocytes appeared to be mediated by both protein kinase C (PKC) and G protein system. No effect of EGF and phorbol 12-myristate 13-acetate, an activator of PKC, on the Na+/H+ antiport was observed in fetal hepatocytes of 20 and 22 days. A different sensitivity of the antiport to high concentrations of amiloride and EIPA suggests that altered amount of the Na+/H+ antiport units or different isoforms could be expressed in fetal compared with adult cells.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Cells, Cultured; Enzyme Inhibitors; Epidermal Growth Factor; Fetus; Fluoresceins; Fluorescent Dyes; GTP-Binding Proteins; Hydroquinones; Kinetics; Liver; Ouabain; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate

Transforming growth factor beta 1 (TGF beta 1) is involved in promoting cell death by apoptosis in the liver, whereas the activation of Na+/H+ exchanger has been related to cell proliferation. The aim of this study was to gain information on the effects of TGF beta 1 on intracellular pH and Na+/H+ exchange activity in isolated periportal (PP) and perivenular (PV) rat hepatocytes using the pH-sensitive dye BCECF in a perfused subconfluent hepatocyte monolayer. Steady-state intracellular pH (pHi) in a bicarbonate-free solution (HEPES) were 7.17 +/- 0.031 in PP and 7.15 +/- 0.041 in PV cells. Treatment with TGF beta 1 (120 pmol/L) for 7 hours increased the number of apoptotic bodies by 25% and 38%, and decreased steady-state pHi to 7.11 +/- 0.018 (P = .05) and to 7.07 +/- 0.021 (P < .02), respectively, in PP and PV hepatocytes. In HEPES, cells recovered from an acid load, extruding protons at a rate (JH) of 4.85 +/- 1.01 mmol/L/min in PP cells and of 4.91 +/- 0.99 mmol/L/min in PV hepatocytes. This recovery appeared amiloride inhibitable (1 mmol/L). Culture with TGF beta 1 for 7 hours induced (in HEPES) a decrease of pHi recovery rate from an acid load more in PV (by 46%) than in PP hepatocytes (by 35%, P < .05). Acute administration of epidermal growth factor (EGF) (10 to 100/mL) induced an increase in Na+/H+ exchange activity by 32% and 27%, respectively, in PP and PV cells compared with controls. In contrast, in cells cultured for 7 hours with 120 pmol/L TGF beta 1, the acute administration of EGF slightly increased Na+/H+ exchange activity (by 18%, P < .05) only in PP cells. This study demonstrates that pHi and Na+/H+ exchange activity are decreased by TGF beta 1, which increases the number of apoptotic bodies in periportal and perivenular rat hepatocyte primary cultures.

Topics: Animals; Apoptosis; Cells, Cultured; Epidermal Growth Factor; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Ion Transport; Liver; Male; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Intracellular pH (pHi) plays an important role in the metabolic activation of quiescent cells after a proliferative stimulus, and Na+/H+ exchange activity is required for growth in some extrahepatic tumors. To investigate intracellular acid/base homeostasis in hepatoma cells and the effects of putative liver growth factors on Na+/h+ exchange activity, we have studied intracellular pH (pHi) regulation in Hep G2 cells, a well-differentiated hepatoma cell line, both in resting conditions and after administration of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulinlike growth factor-II (IGF-II). The effects of fetal calf serum, TGF alpha, and amiloride on 3H-Thymidine incorporation were also studied. Amiloride (1 mmol/L) and external Na+ removal decreased baseline pHi in both HEPES and KRB. In HEPES, cells recovered from an acid load (20 mmol/L NH4Cl) by an amiloride inhibitable Na+/H+ exchange. In KRB, an additional, DIDS-inhibitable, Na(+)- and HCO3- dependent, but Cl(-)-independent acid extruder (Na:HCO3 cotransport) was activated. No evidence was found for a Cl/HCO3 exchange acting as acid loader. Administration of EGF and TGF alpha, but not of IGF-II, induced a dose-dependent, amiloride-inhibitable increase in baseline pHi, together with an increase in Na+/H+ exchange activity, shifting to the right the JH/pHi curve. Finally, 3H-thymidine incorporation in Hep G2 cells, in the presence of FCS or TGF alpha, was strongly inhibited by amiloride. In conclusion, in Hep G2 cells, pHi is mainly regulated by Na+/H+ exchange, which activity can be stimulated by EGF and TGF alpha, but not by IGF-II. Administration of TGF alpha stimulates DNA synthesis, an effect that is blocked by amiloride, an inhibitor of Na+/H+ exchanger. These data suggest that Na+/H+ exchange activation may play a critical role in the growth of some hepatic tumors.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonium Chloride; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cattle; Cell Division; DNA; Epidermal Growth Factor; Fetal Blood; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Insulin-Like Growth Factor II; Liver Neoplasms; Sodium-Bicarbonate Symporters; Sodium-Hydrogen Exchangers; Transforming Growth Factor alpha; Tumor Cells, Cultured

Many bioregulators, such as epidermal growth factor (EGF), induce intracellular alkalinization by activating a membrane bound Na+/H+ antiporter. The present studies were designed to examine the influence of EGF on intracellular pH (pHi) in chicken granulosa cells. pHi in granulosa cells from the two largest preovulatory follicles of hens was determined spectrofluorometrically using the dye 2',7'-bis(carboxyethyl-5(6)-carboxyfluorescein. The resting pHi was 6.81 +/- 0.006 (n = 30) when the extracellular pH and sodium concentration (Na+o) were 7.3 and 144 mM, respectively. EGF (5-100 ng/ml) induced a concentration-dependent increase in pHi, which reached a maximum of 0.217 +/- 0.009 pH units at a concentration of 100 ng/ml EGF. Cytosolic alkalinization was observed within 10 min of the addition of EGF and lasted over the 60 min observation period. The increase in pHi was dependent upon the presence of Na+o, since the EGF effect was attenuated when Na+o was substituted with equimolar concentrations of nonpermeant choline chloride. The EGF-induced pHi change was also inhibited by amiloride, dimethyl amiloride, and ethylisopropyl amiloride, inhibitors of the Na+/H+ antiporter. The alkalinization effect of EGF was mimicked by transforming growth factor-alpha but not by insulin, insulin-like growth factor-I, or transforming growth factor-beta. These studies suggest for the first time that intracellular alkalinization resulting from activation of the Na+/H+ antiporter may be a part of the transmembrane signaling pathway in the action of EGF on chicken granulosa cells.

Topics: Animals; Carrier Proteins; Chickens; Epidermal Growth Factor; Female; Fluoresceins; Fluorescent Dyes; Granulosa Cells; Growth Substances; Hydrogen-Ion Concentration; Sodium; Sodium-Hydrogen Exchangers; Transforming Growth Factor alpha

We studied the effects of epidermal growth factor (EGF), thyroid-stimulating hormone (TSH) and amiloride on cytoplasmic pH (pHi) in cultured porcine thyroid cells. We used 2',7'-bis(2-carboxyethyl)-5- (and 6-)carboxyfluorescein (BCECF), an internalized fluorescent pH indicator, to measure pHi. EGF stimulated thyroid cell alkalinization and proliferation, which were blocked by amiloride. EGF-stimulated thyroid cell alkalinization depended on extracellular Na+ concentrations. EGF stimulation resulted in an activation of Na+/H+ exchange, which alkalinized the cells. The results indicated that Na+/H+ exchange or cell alkalinization might function as a transmembrane signal transducer in the action of EGF. In the present system, TSH did not stimulate alkalinization or proliferation.

Topics: Amiloride; Animals; Carrier Proteins; Cell Membrane; Cells, Cultured; Cytoplasm; Epidermal Growth Factor; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Protons; Signal Transduction; Sodium; Sodium-Hydrogen Exchangers; Swine; Thyroid Gland; Thyrotropin

An electroneutral Na+-H+ exchange system has been described in a variety of tissues and cell types, including those of renal origin, and has been proposed to play a role in the activation of growth. We have recently characterized the presence of this ubiquitous transporter in the apical domain of confluent epithelial LLC-PK1 cells (J. Biol. Chem. 261: 3252-3258, 1986). Because most apical membrane proteins appear late in cell growth, accompanying epithelial cell polarization, we determined whether the Na+-H+ exchanger is required for the growth of LLC-PK1 cells. The studies reported here show that there is no obligatory requirement for increased H+ efflux or Na+ entry via the Na+-H+ exchanger for the initiation of cell growth in this epithelial cell line. We used 22Na+ influx, acid extrusion, and intracellular pH determinations to show that onset of cell growth, as measured by DNA content, precedes the activity of the Na+-H+ exchanger in exponentially growing cells, whereas confluent monolayers express Na+-H+ exchanger activity. When confluent cells are replated at low density, Na+-H+ exchanger activity disappears within 8 h in contrast to high-density replated cells. The fact that Na+-H+ exchanger activity is only present in confluent monolayers suggests that the development of tight junctions and polar differentiation play a role in the expression of the Na+-H+ exchanger and that this exchanger is more important to the polar epithelial cell for transepithelial transport than for the maintenance of intracellular pH.

Topics: Amiloride; Ammonium Chloride; Biological Transport; Bombesin; Carrier Proteins; Cell Line; DNA; Epidermal Growth Factor; Epithelial Cells; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Kidney; Potassium Chloride; Protons; Sodium; Sodium-Hydrogen Exchangers