00l3-7227/91/1292-0656$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 2 Printed in U.S.A.
Epidermal Growth Factor Elevates Intracellular pH in Chicken Granulosa Cells* MING Lit, PAUL MORLEYt ELIKPLIMI K. ASEM, AND BENJAMIN K. TSANG Reproductive Biology Unit, Department of Obstetrics and Gynecology, University of Ottawa, Loeb Medical Research Institute, Ottawa Civic Hospital, Ottawa, Ontario, Canada Kl Y 4E9; and Department of Physiology and Pharmacology (E.K.A.), School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907
ABSTRACT. 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+0) 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+0, since the EGF effect was attenuated when Na+0 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-a but not by insulin, insulin-like growth factor-I, or transforming growth factor-/?. 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. {Endocrinology 129: 656-662,1991)
E
cells of many species (10-12). Studies have shown that interaction of growth factors with membrane receptors leads to protein phosphorylation (1), the breakdown of inositol phospholipids leading to the production of diacylglycerol (13-15), a rise in cytoplasmic free calcium (16, 17), and an increase in ion transport across the plasma membrane (18). Recent evidence suggests that mitogens rapidly activate a membrane-bound Na + /H + antiporter which extrudes protons from the cell using the inward Na+ gradient as a driving force to cause intracellular alkalinization (19-21). The antiporter is present in most, if not all, vertebrate cells and is thought to play a role in the regulation of cell volume, cytoplasmic pH regulation, and the initiation of growth and proliferation (22). The antiporter is inhibited by amiloride, which binds competitively to the sodium binding site (23-25) and is dependent on the concentration of extracellular Na+ (23). These observations implicate a role for pHi changes as a potential messenger for the actions of EGF (26-28). Previous studies from our laboratory have shown in chicken granulosa cells the presence of a Na + /H + exchange system which is dependent on extracellular Na+ and sensitive to amiloride (29). The present studies show for the first time that EGF can induce intracellular
PIDERMAL growth factor (EGF) enhances growth in several epidermal and nonepidermal tissues (1). In granulosa cells, EGF stimulates mitosis (2) and plays a significant role, primarily inhibitory, in regulating differentiation (3-5). In chicken granulosa cells, EGF stimulates proliferation (6) and inhibits LH-stimulated cAMP and progesterone production (7). In addition, this growth factor enhances both cell-associated and secreted plasminogen activator activity in hen granulosa cells (8, 9). The enhanced plasminogen activator activity by EGF is mediated via the activation of protein kinase C and is independent of cAMP (9). The molecular mechanisms by which growth factors influence metabolism and cell function are not fully understood. The effects of EGF are presumably mediated via EGF receptors known to be present on the granulosa
Received March 25, 1991. Address all correspondence and requests for reprints to: Dr. Benjamin K. Tsang, Reproductive Biology Unit, Loeb Medical Research Institute, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario, Canada KlY 4E9. * This study was supported by Medical Research Council of Canada Grant MT-10369 (to B.K.T.) and NIH Grant HD-27354-01 (to E.K.A.). t Visiting scholar from Fushan Veterinary College, Fushan, Peoples Republic of China. X Medical Research Council of Canada Postdoctoral Fellow.
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The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 01:49 For personal use only. No other uses without permission. . All rights reserved.
EGF INCREASES GRANULOSA CELL pH alkalinization by activating the Na + /H + antiporter in chicken granulosa cells.
Materials and Methods Chicken granulosa cell isolation White Leghorn hens in their first year of reproductive activity were caged individually in a windowless, air-conditioned room with a 14-h light and 10-h dark light cycle. The birds had free access to a pelleted laying ration and tap water. The bird colony was patrolled, and the time of egg laying was recorded to the nearest 30 min. Granulosa cells were isolated as described previously (30). Briefly, birds were killed by cervical dislocation 10-14 h before expected ovulation. The granulosa cell layer from the two largest preovulatory follicles was separated, and the cells were dispersed in 4 ml Medium 199 (GIBCO Laboratories, Grand Island, NY) containing 0.2% (wt/vol) collagenase (Type 1A; Sigma Chemical Co., St. Louis, MO) for 10 min in a shaking water bath at 37 C. The Medium 199 was supplemented with 6.0 g/liter HEPES and antibiotics (50 U/ml penicillin, 50 ^g/ml streptomycin, and 0.625 Mg/ml Fungizone). The cells were recovered by centrifugation at 200 X g for 10 min at room temperature. Cell viability, as determined by trypan blue exclusion, was greater than 95%. Reagents Amiloride hydrochloride (amiloride), bovine pancreatic insulin, and Nigericin were obtained from Sigma. Ethylisopropyl amiloride (EIPA) and dimethyl amiloride (DMA) were generously provided by Merck, Sharpe, and Dohme (Rahway, NJ). Choline chloride was purchased from BDH Chemicals (Toronto, Ontario, Canada). Mouse submaxillary gland EGF, synthetic insulin-like growth factor-I, synthetic transforming growth factor-« (TGF-cv), and porcine transforming growth factor-^ (TGF-jtf) were obtained from ICN Biochemicals (Cleveland, OH). All growth factors were dissolved in sodium solution (see below) and stored at 4 C until used. Triton X-100 was obtained from Aldrich Chemical Co. Inc. (Milwaukee, WI). Determination of pHi Granulosa cells were loaded with 2',7'-bis(carboxyethyl5(6)-carboxyfluorescein (BCECF) as described previously (29). Briefly, freshly dispersed cells were resuspended in Na+ solution (144 mM NaCl, 5 mM KC1, 1 DIM CaCl2, 1 mM MgCl2, 10 mM glucose, and 20 mM Tris-HCl, pH 7.3) and loaded with BCECF by incubation with BCECF-AM (5 nM; Molecular Probes Inc., Eugene, OR) in Na+ solution at 37 C for 30 min. The stock solution was dissolved in dimethylsulfoxide and stored at -20 C. BCECF-AM diffuses across the cell membrane and is hydrolyzed by internal esterases in the cytoplasm, releasing the poorly permeant BCECF which serves as a pH; indicator (31). After loading, the cells were washed twice with Na+ solution to remove extracellular dye, recovered by centrifugation at 200 X g and resuspended in Na+ solution to give a final concentration of 1 x 10(! cells/ml. The fluorescence of BCECF-loaded cells was monitored in a Perkin Elmer (Montreal, Quebec, Canada) LS-5 spectrofluorometer attached to a Perkin Elmer R-100A recorder and set
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at excitation and emission wavelengths of 500 and 525 nm, respectively, with slit widths of 2.5 nm. The measurements were made at 37 C in a 3-ml cuvette containing 2 ml of the granulosa cell suspension (1 x 106 cells/ml) with constant stirring. At the end of each experiment a standard curve was obtained by calibrating pH vs. fluorescence by lysing the cells with 0.125% (vol/vol) Triton X-100, followed by titration of the medium with HEPES (1 M) or Tris (1 M). At each titration the fluorescence was noted and the pH determined. Standard curves of pH us. fluorescence were constructed by regression analysis. To obtain a value for pHj, the fluorescence before cell disruption was multiplied by a correction factor, and the corresponding pH value read on the graph. Since the fluorescence of the intracellularly trapped fluorescein derivative is less than that of the free dye (32) and varies between batches of cells, a correction factor was determined for each batch of cells as described by Thomas et al. (33) using nigericin in high K+containing medium. Nigericin [a carboxylic ionophore that enhances the electroneutral exchange of intracellular K+ (K+0 for extracellular H + (H+o)] sets the ratio of K+i and H+i equal to that of extracellular potassium (K+o) and H+o, such that K V K+o = H+i/H+o. Therefore, if cells are suspended in media with approximately the same K+ concentration as the cytoplasm, H+i will approach H+o. To obtain the correction factor, granulosa cells were suspended in K+ solution and nigericin (5 /XM) was added. After a steady state was reached, the cells were disrupted with Triton X-100 (0.125%) and the fluorescence was allowed to reach another steady state. The correction factor was taken as the ratio of the fluorescence before and after cell lysis. Typically, the pH; of granulosa cells exposed to nigericin (suspended in K+ solution; pH 7.3) increased rapidly and equilibrated with that of the external medium within 2 min. After steady state had been reached, addition of Triton produced a small additional increase in pH; (results not shown). Statistical analyses Statistical analyses were made by analysis of variance. Where required, data were transformed logarithmically before statistical analyses to remove heterogeneity of variance as determined by Bartlett's test. When significant effects were observed, Scheffe's test was used for multiple comparisons. Data are the mean ± SEM of three determinations from three different experiments. All tests were performed as described by Steel and Torrie (34), and statistical significance was inferred at P < 0.05.
Results EGF-stimulated granulosa cell alkalinization The resting pHj of chicken granulosa cells in Na + solution at 37 C was 6.81 ± 0.006 (n = 30). To determine if EGF affected chicken granulosa cell pH;, increasing concentrations of EGF (5-100 ng/ml) were added to BCECF-loaded granulosa cells in 144 mM Na+ solution
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 01:49 For personal use only. No other uses without permission. . All rights reserved.
EGF INCREASES GRANULOSA CELL pH
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pH 7.3, and the fluorescence was monitored for 60 min. In control suspensions, with either vehicle alone or no additions, small increases (0.040 ± 0.005 pH units; n = 14) in granulosa cell pH; were routinely observed over the 1-h observation period (data not shown). Addition of EGF induced a concentration-dependent cytosolic alkalinization. A maximum increase of 0.217 ± 0.009 pH units, compared to controls, was observed 1 h after the addition of 100 ng/ml EGF (Fig. 1). The half-maximal effective concentration of EGF (EC50) was 15 ng/ml. A significant (P < 0.05) cytosolic alkalinization was observed within 10 min of the addition of EGF (10 and 100 ng/ml) to the cell suspension (the earliest time examined) and progressively increased during the 60-min observation period (Fig. 2). Analysis of variance revealed a significant (P < 0.0001) interaction between EGF concentration and time. 0.300 0.250 0.200 0.150 0.100
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EGF (ng/ml) FIG. 1. EGF-induced alkalinization of cytoplasmic pH. Cells were incubated with vehicle alone (control) or EGF (5-100 ng/ml) and the change in pH; was determined after 60 min. Values represent the mean ± SEM of three determinations from three different experiments. Values with different superscripts are significantly (P < 0.0001) different. 0.3001 0.250-
Role of Na+/H+ exchange in the EGF-induced alkalinization of granulosa cell pHi The possible involvement of the Na + /H + antiporter in the EGF-induced increase in pHj was examined in BCECF-loaded granulosa cells exposed to a maximally stimulating concentration of EGF (100 ng/ml) in media containing 144, 72, 36, and 0 mM sodium (Na+0), pH 7.3. In these experiments, extracellular sodium was substituted with equimolar nonpermeant choline chloride which prevents H + extrusion via the Na + /H + antiport. In the absence of EGF, the pH; of granulosa cells was independent of Na+ concentration in the media (Na+0 = 0 mM, pH; = 6.82; Na+0 = 36 mM, pH; = 6.81; Na+0 = 72 mM, pHi = 6.81; Na+0 = 144 mM, pH; = 6.80). However, reduction of the extracellular sodium concentration from 144 mM to 0 mM caused a significant (P < 0.0001) concentration-dependent decrease in the ability of EGF to increase granulosa cell pH ; (Fig. 3). Analysis of variance showed a significant (P < 0.0001) interaction between Na+0 concentration and time. To further determine the involvement of Na + /H + exchange in the regulation of granulosa cell pHi by EGF, the influence of inhibitors of the Na + /H + antiporter (amiloride, DMA, and EIPA) was examined in BCECFloaded granulosa cell suspensions in the presence of 144 mM Na+0, pH 7.3. Amiloride (0.001-0.01 mM) had no effect on basal pH; (Control - pH; = 6.79 ± 0.015; 0.001 mM amiloride - pHj = 6.79 ± 0.022; 0.005 mM amiloride - pHi = 6.79 ± 0.003; 0.01 mM amiloride - pH; = 6.76 ± 0.015) measured 10 min after the addition to the medium; although high concentrations (0.5 and 5 mM) were inhibitory (data not shown). However, when amiloride (0.001-0.01 mM) was added to granulosa cell suspensions 10 min before EGF (100 ng/ml) amiloride sig0.300
• — • Control • — • EGF 10 ng/ml • — • EGF 100 ng/ml
Endo • 1991 Vol 129 • No 2
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Vol. 129, No. 2 Printed in U.S.A.
Epidermal Growth Factor Elevates Intracellular pH in Chicken Granulosa Cells* MING Lit, PAUL MORLEYt ELIKPLIMI K. ASEM, AND BENJAMIN K. TSANG Reproductive Biology Unit, Department of Obstetrics and Gynecology, University of Ottawa, Loeb Medical Research Institute, Ottawa Civic Hospital, Ottawa, Ontario, Canada Kl Y 4E9; and Department of Physiology and Pharmacology (E.K.A.), School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907
ABSTRACT. 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+0) 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+0, since the EGF effect was attenuated when Na+0 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-a but not by insulin, insulin-like growth factor-I, or transforming growth factor-/?. 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. {Endocrinology 129: 656-662,1991)
E
cells of many species (10-12). Studies have shown that interaction of growth factors with membrane receptors leads to protein phosphorylation (1), the breakdown of inositol phospholipids leading to the production of diacylglycerol (13-15), a rise in cytoplasmic free calcium (16, 17), and an increase in ion transport across the plasma membrane (18). Recent evidence suggests that mitogens rapidly activate a membrane-bound Na + /H + antiporter which extrudes protons from the cell using the inward Na+ gradient as a driving force to cause intracellular alkalinization (19-21). The antiporter is present in most, if not all, vertebrate cells and is thought to play a role in the regulation of cell volume, cytoplasmic pH regulation, and the initiation of growth and proliferation (22). The antiporter is inhibited by amiloride, which binds competitively to the sodium binding site (23-25) and is dependent on the concentration of extracellular Na+ (23). These observations implicate a role for pHi changes as a potential messenger for the actions of EGF (26-28). Previous studies from our laboratory have shown in chicken granulosa cells the presence of a Na + /H + exchange system which is dependent on extracellular Na+ and sensitive to amiloride (29). The present studies show for the first time that EGF can induce intracellular
PIDERMAL growth factor (EGF) enhances growth in several epidermal and nonepidermal tissues (1). In granulosa cells, EGF stimulates mitosis (2) and plays a significant role, primarily inhibitory, in regulating differentiation (3-5). In chicken granulosa cells, EGF stimulates proliferation (6) and inhibits LH-stimulated cAMP and progesterone production (7). In addition, this growth factor enhances both cell-associated and secreted plasminogen activator activity in hen granulosa cells (8, 9). The enhanced plasminogen activator activity by EGF is mediated via the activation of protein kinase C and is independent of cAMP (9). The molecular mechanisms by which growth factors influence metabolism and cell function are not fully understood. The effects of EGF are presumably mediated via EGF receptors known to be present on the granulosa
Received March 25, 1991. Address all correspondence and requests for reprints to: Dr. Benjamin K. Tsang, Reproductive Biology Unit, Loeb Medical Research Institute, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario, Canada KlY 4E9. * This study was supported by Medical Research Council of Canada Grant MT-10369 (to B.K.T.) and NIH Grant HD-27354-01 (to E.K.A.). t Visiting scholar from Fushan Veterinary College, Fushan, Peoples Republic of China. X Medical Research Council of Canada Postdoctoral Fellow.
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The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 01:49 For personal use only. No other uses without permission. . All rights reserved.
EGF INCREASES GRANULOSA CELL pH alkalinization by activating the Na + /H + antiporter in chicken granulosa cells.
Materials and Methods Chicken granulosa cell isolation White Leghorn hens in their first year of reproductive activity were caged individually in a windowless, air-conditioned room with a 14-h light and 10-h dark light cycle. The birds had free access to a pelleted laying ration and tap water. The bird colony was patrolled, and the time of egg laying was recorded to the nearest 30 min. Granulosa cells were isolated as described previously (30). Briefly, birds were killed by cervical dislocation 10-14 h before expected ovulation. The granulosa cell layer from the two largest preovulatory follicles was separated, and the cells were dispersed in 4 ml Medium 199 (GIBCO Laboratories, Grand Island, NY) containing 0.2% (wt/vol) collagenase (Type 1A; Sigma Chemical Co., St. Louis, MO) for 10 min in a shaking water bath at 37 C. The Medium 199 was supplemented with 6.0 g/liter HEPES and antibiotics (50 U/ml penicillin, 50 ^g/ml streptomycin, and 0.625 Mg/ml Fungizone). The cells were recovered by centrifugation at 200 X g for 10 min at room temperature. Cell viability, as determined by trypan blue exclusion, was greater than 95%. Reagents Amiloride hydrochloride (amiloride), bovine pancreatic insulin, and Nigericin were obtained from Sigma. Ethylisopropyl amiloride (EIPA) and dimethyl amiloride (DMA) were generously provided by Merck, Sharpe, and Dohme (Rahway, NJ). Choline chloride was purchased from BDH Chemicals (Toronto, Ontario, Canada). Mouse submaxillary gland EGF, synthetic insulin-like growth factor-I, synthetic transforming growth factor-« (TGF-cv), and porcine transforming growth factor-^ (TGF-jtf) were obtained from ICN Biochemicals (Cleveland, OH). All growth factors were dissolved in sodium solution (see below) and stored at 4 C until used. Triton X-100 was obtained from Aldrich Chemical Co. Inc. (Milwaukee, WI). Determination of pHi Granulosa cells were loaded with 2',7'-bis(carboxyethyl5(6)-carboxyfluorescein (BCECF) as described previously (29). Briefly, freshly dispersed cells were resuspended in Na+ solution (144 mM NaCl, 5 mM KC1, 1 DIM CaCl2, 1 mM MgCl2, 10 mM glucose, and 20 mM Tris-HCl, pH 7.3) and loaded with BCECF by incubation with BCECF-AM (5 nM; Molecular Probes Inc., Eugene, OR) in Na+ solution at 37 C for 30 min. The stock solution was dissolved in dimethylsulfoxide and stored at -20 C. BCECF-AM diffuses across the cell membrane and is hydrolyzed by internal esterases in the cytoplasm, releasing the poorly permeant BCECF which serves as a pH; indicator (31). After loading, the cells were washed twice with Na+ solution to remove extracellular dye, recovered by centrifugation at 200 X g and resuspended in Na+ solution to give a final concentration of 1 x 10(! cells/ml. The fluorescence of BCECF-loaded cells was monitored in a Perkin Elmer (Montreal, Quebec, Canada) LS-5 spectrofluorometer attached to a Perkin Elmer R-100A recorder and set
657
at excitation and emission wavelengths of 500 and 525 nm, respectively, with slit widths of 2.5 nm. The measurements were made at 37 C in a 3-ml cuvette containing 2 ml of the granulosa cell suspension (1 x 106 cells/ml) with constant stirring. At the end of each experiment a standard curve was obtained by calibrating pH vs. fluorescence by lysing the cells with 0.125% (vol/vol) Triton X-100, followed by titration of the medium with HEPES (1 M) or Tris (1 M). At each titration the fluorescence was noted and the pH determined. Standard curves of pH us. fluorescence were constructed by regression analysis. To obtain a value for pHj, the fluorescence before cell disruption was multiplied by a correction factor, and the corresponding pH value read on the graph. Since the fluorescence of the intracellularly trapped fluorescein derivative is less than that of the free dye (32) and varies between batches of cells, a correction factor was determined for each batch of cells as described by Thomas et al. (33) using nigericin in high K+containing medium. Nigericin [a carboxylic ionophore that enhances the electroneutral exchange of intracellular K+ (K+0 for extracellular H + (H+o)] sets the ratio of K+i and H+i equal to that of extracellular potassium (K+o) and H+o, such that K V K+o = H+i/H+o. Therefore, if cells are suspended in media with approximately the same K+ concentration as the cytoplasm, H+i will approach H+o. To obtain the correction factor, granulosa cells were suspended in K+ solution and nigericin (5 /XM) was added. After a steady state was reached, the cells were disrupted with Triton X-100 (0.125%) and the fluorescence was allowed to reach another steady state. The correction factor was taken as the ratio of the fluorescence before and after cell lysis. Typically, the pH; of granulosa cells exposed to nigericin (suspended in K+ solution; pH 7.3) increased rapidly and equilibrated with that of the external medium within 2 min. After steady state had been reached, addition of Triton produced a small additional increase in pH; (results not shown). Statistical analyses Statistical analyses were made by analysis of variance. Where required, data were transformed logarithmically before statistical analyses to remove heterogeneity of variance as determined by Bartlett's test. When significant effects were observed, Scheffe's test was used for multiple comparisons. Data are the mean ± SEM of three determinations from three different experiments. All tests were performed as described by Steel and Torrie (34), and statistical significance was inferred at P < 0.05.
Results EGF-stimulated granulosa cell alkalinization The resting pHj of chicken granulosa cells in Na + solution at 37 C was 6.81 ± 0.006 (n = 30). To determine if EGF affected chicken granulosa cell pH;, increasing concentrations of EGF (5-100 ng/ml) were added to BCECF-loaded granulosa cells in 144 mM Na+ solution
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 01:49 For personal use only. No other uses without permission. . All rights reserved.
EGF INCREASES GRANULOSA CELL pH
658
pH 7.3, and the fluorescence was monitored for 60 min. In control suspensions, with either vehicle alone or no additions, small increases (0.040 ± 0.005 pH units; n = 14) in granulosa cell pH; were routinely observed over the 1-h observation period (data not shown). Addition of EGF induced a concentration-dependent cytosolic alkalinization. A maximum increase of 0.217 ± 0.009 pH units, compared to controls, was observed 1 h after the addition of 100 ng/ml EGF (Fig. 1). The half-maximal effective concentration of EGF (EC50) was 15 ng/ml. A significant (P < 0.05) cytosolic alkalinization was observed within 10 min of the addition of EGF (10 and 100 ng/ml) to the cell suspension (the earliest time examined) and progressively increased during the 60-min observation period (Fig. 2). Analysis of variance revealed a significant (P < 0.0001) interaction between EGF concentration and time. 0.300 0.250 0.200 0.150 0.100
ob
0.050 0.000 5
10
50
100
EGF (ng/ml) FIG. 1. EGF-induced alkalinization of cytoplasmic pH. Cells were incubated with vehicle alone (control) or EGF (5-100 ng/ml) and the change in pH; was determined after 60 min. Values represent the mean ± SEM of three determinations from three different experiments. Values with different superscripts are significantly (P < 0.0001) different. 0.3001 0.250-
Role of Na+/H+ exchange in the EGF-induced alkalinization of granulosa cell pHi The possible involvement of the Na + /H + antiporter in the EGF-induced increase in pHj was examined in BCECF-loaded granulosa cells exposed to a maximally stimulating concentration of EGF (100 ng/ml) in media containing 144, 72, 36, and 0 mM sodium (Na+0), pH 7.3. In these experiments, extracellular sodium was substituted with equimolar nonpermeant choline chloride which prevents H + extrusion via the Na + /H + antiport. In the absence of EGF, the pH; of granulosa cells was independent of Na+ concentration in the media (Na+0 = 0 mM, pH; = 6.82; Na+0 = 36 mM, pH; = 6.81; Na+0 = 72 mM, pHi = 6.81; Na+0 = 144 mM, pH; = 6.80). However, reduction of the extracellular sodium concentration from 144 mM to 0 mM caused a significant (P < 0.0001) concentration-dependent decrease in the ability of EGF to increase granulosa cell pH ; (Fig. 3). Analysis of variance showed a significant (P < 0.0001) interaction between Na+0 concentration and time. To further determine the involvement of Na + /H + exchange in the regulation of granulosa cell pHi by EGF, the influence of inhibitors of the Na + /H + antiporter (amiloride, DMA, and EIPA) was examined in BCECFloaded granulosa cell suspensions in the presence of 144 mM Na+0, pH 7.3. Amiloride (0.001-0.01 mM) had no effect on basal pH; (Control - pH; = 6.79 ± 0.015; 0.001 mM amiloride - pHj = 6.79 ± 0.022; 0.005 mM amiloride - pHi = 6.79 ± 0.003; 0.01 mM amiloride - pH; = 6.76 ± 0.015) measured 10 min after the addition to the medium; although high concentrations (0.5 and 5 mM) were inhibitory (data not shown). However, when amiloride (0.001-0.01 mM) was added to granulosa cell suspensions 10 min before EGF (100 ng/ml) amiloride sig0.300
• — • Control • — • EGF 10 ng/ml • — • EGF 100 ng/ml
Endo • 1991 Vol 129 • No 2
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