0013-7227/90/1271-0236$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 1 Printed in U.S.A.
Na+-H+ Exchanger Kinetics in Adrenal Glomerulosa Cells and Its Activation by Angiotensin II* PAUL R. CONLIN, SEONG Y. KIM, GORDON H. WILLIAMS, AND MITZY L. CANESSA Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 Na + /mg protein x min. By varying pH; (from pHj 7.1 to 6.2),
ABSTRACT. We have studied the kinetic properties of basal and angiotensin II (ANG II) stimulated Na+-H+ exchange in adrenal glomerulosa cells by measuring changes in cytosolic pH (pHO and initial rates of 22Na uptake in the presence or absence of dimethylamiloride (DMA). The cells were studied 1) under basal conditions, 2) at constant pH; (6.8) with varied external sodium (Na+0), and 3) at varied pH; with constant Na+0 (50 mM). In 2,7-biscarboxyethyl-5(6)-carboxyfluorescein loaded cells under basal conditions, pH; rose from 7.09 ± 0.02 to 7.19 ± 0.02 (P < 0.05) with addition of ANG II (100 nM). Similarly, DMAsensitive Na influx was enhanced from 9.2 ± 1.3 to 14.8 ± 2.1 nmol Na+/mg protein X min (P < 0.01) by ANG II. In cells acidloaded by preincubation in Na+-free media (pH; 6.8), addition of varying Na+O resulted in a rapid H+ efflux that was markedly inhibited by DMA. DMA-sensitive Na+ influx into these acidified cells with varied Na+0 exhibited a Michaelis-Menten constant (Km) of 23 mM and a maximum velocity (VmaJ of 43 nmol
DMA-sensitive Na+ influx likewise snowed activation with cellular acidification with a pK at pH; 7.09. At pH; 6.8, ANG II decreased the Km for Na+0 from 23 to 17 mM and increased the Vmax from 43 to 53 nmol Na+/mg protein x min. The pHi dependence of DMA-sensitive Na+ influx was not affected by ANG II (pK at pH; 7.03). DMA also inhibited All-stimulated aldosterone secretion and Na+ influx similarly. These results indicate that Na+-H+ exchange in adrenal glomerulosa cells is 1) functioning under basal conditions, and 2) is modulated by ANG II with enhanced Na+0 affinity and Vma, but without a shift in pHi dependence (similar to ANG II effects on vascular smooth muscle cells). These effects suggest an important role for Na+-H+ exchange during ANG II stimulation of aldosterone production by glomerulosa cells. (Endocrinology 127: 236-244, 1990)
T
HE ROLE of Na + -H + exchange in regulating intracellular pH (pHi) has been well established in several cell types particularly in relation to the cellular response to various hormones and growth factors. Although the exchanger may be ubiquitous, its activation is usually linked to ligand-receptor interactions specific for individual cell types which produce phosphoinositide breakdown, calcium mobilization, and activation of protein kinase C, such as growth factors {e.g. epidermal growth factor, platelet-derived growth factor) (1, 2) and vasoconstrictor agonists {e.g. arginine vasopressin, «i adrenergic agonists) (3, 4). Angiotensin II (ANG II) modulation of the Na + -H + exchanger likewise has been studied in cultured vascular smooth muscle cells which have partially lost the contractile response (5, 6). In
adrenal glomerulosa cells, ANG II stimulation produces a prompt increase in steroidogenesis and secretion of aldosterone through the generation of inositol phosphates (7, 8) and may potentially modulate functioning of the Na + -H + exchanger (9). Unlike cultured vascular smooth muscle cells, glomerulosa cells provide the opportunity to study the role of Na + -H + exchange in ANG II stimulated aldosterone secretion while monitoring both membrane and secretion events. The presence of Na + -H + exchange in rat glomerulosa cells has been suggested by Hunyady et al. (10) through the ability of an amiloride analog to inhibit Na+ uptake into acid loaded cells. However, modulation of exchanger activity by ANG II could not be demonstrated. Horiuchi et al. (9) have extended these findings by showing inhibition of ANG II stimulated aldosterone secretion and cellular alkalinization by specific amiloride analogs. In this report the kinetic properties of the Na + -H + exchanger in adrenal glomerulosa cells are studied as a functional assessment of the antiporter under basal and agonist stimulated conditions. In addition, the ability of ANG II to activate Na + -H + exchange is demonstrated as is the importance of this pH; regulator in the mainte-
Received December 15,1989. Address requests for reprints to: Paul R. Conlin, M.D., EndocrineHypertension Division, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. * Results of this manuscript were presented in part at the meeting of the American Federation for Clinical Research, May 1989, Washington, D.C. This work was supported by NIH Grants HL-42120, HL35664, a Specialized Center of Research (SCOR) in Hypertension Grant HL-36568 and the Training Program in Hypertension Grant HL-07609.
236
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GLOMERULOSA CELL NA+-H+ EXCHANGE
nance of ANG II-stimulated aldosterone secretion. Materials and Methods Materials
237
wells, previously treated with fibronectin, at a density of 0.51.0 X 106 cells per well and incubated for 16-20 h before study. Cells cultured under these conditions retain the ability to secrete aldosterone for at least 7 days (data not shown). Aldosterone secretion studies
Dulbecco's modified Eagle's medium, essential and nonessential amino acids, trypsin-EDTA, insulin, a-tocopherol, transferrin, and antibiotics were from GIBCO (Grand Island, NY); nigericin and choline chloride were from Calbiochem (San Diego, CA). Ouabain, HEPES, Tris, ANG II, Percoll, metyrapone, L-glutamine, sodium selenite, and ammonium chloride were from Sigma (St. Louis, MO); 2, 7-biscarboxyethyl-5(6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) was from Molecular Probes (Eugene, OR). Ascorbic acid was from Fisher (Fairlawn, NJ), collagenase was from Worthington (Freehold, NJ), and BSA, fraction V, was from ICN ImmunoBiologicals (Lisle, IL). 22Na was from Amersham (Arlington Heights, IL); bumetanide was from Leo Lab (Vernouillet, France); and dimethylamiloride (DMA) was kindly supplied by Dr. E. Cragoe from Merck Sharpe and Dohme (Rahway, NJ). Cell isolation and culture Bovine adrenal glands were obtained from a local slaughterhouse and transported in ice cold 0.9% NaCl solution. The adrenals were bisected and the capsular portion was separated from the remainder of the gland by slicing the outer 0.3 mm with a microtome. These tissue slices were incubated in a modified Krebs-Ringer bicarbonate solution (MKRB) containing (in millimolars) 140 Na+, 3.7 K+, 1.25 Ca2+, 1.0 Mg>+, 1.0 SO42", 1.0 PO43-, 120 Cr, 25 HCO3-, 1 mg/ml BSA, 10 mM glucose, 1 mM L-glutamine, and an essential and nonessential amino acid mixture. The solution was equilibrated with 95% O2-5% CO2 before use. Crude collagenase (3.5 mg/ml) was added to the MKRB which contained the capsular portions and the mixture was allowed to incubate at 37 C for 30 min in a shaking water bath under a 95% O2-5% CO2 atmosphere. Dispersed cells were separated from tissue fragments by filtration through nylon mesh with 50-jtm pore size. After washing twice with MKRB, the cell suspension was loaded onto a discontinuous Percoll gradient consisting of layers containing 10, 30, 35, 60, and 80% Percoll isosmotically diluted with concentrated MKRB. After centrifugation at 400 X g for 30 min at 4 C, the cells separated into bands with glomerulosa cells appearing predominantly at the 35-60% interface. This band was collected and washed twice with MKRB. The cells were then used either for acute incubation studies or prepared for overnight culture. Cells prepared by this method routinely exclude Trypan blue greater than 95% and contain approximately 5% contamination with fasciculata-reticularis cells (as determined by visual inspection of the size difference between the two cell types). Those cells that were to be readied for culture were resuspended in Dulbecco's modified Eagle's medium supplemented with 2 nM L-insulin, 1 mM ascorbic acid, 1 /iM a-tocopherol, 1 mM L-glutamine, 50 nM sodium selenite, 50 nM metyrapone, 1.2 JUM transferrin, 11 mM glucose, 50 mg/liter gentamicin, and 3.7 mM potassium. The cells were plated into 16-mm culture
Glomerulosa cells obtained by the above isolation procedure were allowed to preincubate in MKRB solution for 60 min under a 95% O2-5% CO2 atmosphere. The cells were then washed once and resuspended in a modified Krebs-Ringer solution similar to that described above except bicarbonate was replaced by 20 mM HEPES, Na+ and Cl" concentrations were adjusted to 130 and 136 mM respectively, and pH was titrated to 7.4 at 37 C by addition of Tris base (MKRH). In experiments studying the effect of Na+-H+ exchange inhibition on ANG IIstimulated aldosterone production, the amiloride analog, DMA in dimethyl sulfoxide, was added at varying doses in 5-^1 aliquots to a cell suspension of 106 cells/0.5 ml and allowed to incubate for 5 min. DMA has previously been shown to be a specific, high affinity probe for the Na+-H+ exchanger (IC50, 0.3 MM) (11) with little affinity (IC50 > 300 ^M) for Na+-Ca2+ exchange (12). ANG II (100 nM) was subsequently added and allowed to incubate further at 37 C for 60 min. Aldosterone measurements were performed in duplicate using a RIA kit (Coat-A-Count RIA kit, Diagnostic Products Corp, Los Angeles, CA). Measurement of intracellular pH Cultured glomerulosa cells were harvested from the culture plates by exposure to a trypsin (0.05%)-EDTA (0.53 mM) solution for 10 min at 37 C. The cells were washed twice with HEPES-buffered saline (HBS) which contained (in millimolars) 130 N a \ 136 Cl~, 3.7 K+, 1.25 Ca2+, 1.0 PO43-, 1.0 Mg2+, 1.0 SO42", 20 HEPES, and 1 mg/ml BSA with pH adjusted to 7.4 at 37 C with Tris base. The cells were then resuspended in the presence of 2 /xM BCECF-AM and allowed to incubate at 37 C for 30 min. Extracellular dye was removed by washing twice with HBS and the cells were divided into 1-ml portions and allowed to stand at room temperature until the time of study. Cell acidification when performed was accomplished by incubation of the cells for 15-30 min in a solution in which choline chloride had replaced the sodium chloride at pH 7.0 (Na+-free HBS). Before measurement of fluorescence, the cells were centrifuged, resuspended in fresh buffer, and placed in a quartz cuvette at a density of 2 X 106 cells/2 ml. Cell fluorescence was measured in a Perkin-Elmer spectrofluorimeter (model 65-10S) equipped with a thermostated cuvette holder and a mechanical stirring bar. The BCECF-loaded cells were alternately excited at wavelengths of 500 and 440 nm with emission recorded at 530 nm with band widths of 5 and 7 nm, respectively. The ratio of emitted fluorescence at the two excitation wavelengths permitted calculation of pH; without interference from differences in dye loading, dye bleaching, or cell number. Because autofluorescence of unloaded cells was less than 2% of total fluorescence, it was not routinely subtracted.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:34 For personal use only. No other uses without permission. . All rights reserved.
GLOMERULOSA CELL NA+-H+ EXCHANGE
238
Calibration of pH ; was accomplished by measuring fluorescence of BCECF-loaded cells in HBS in which sodium had been substituted with potassium (130 mM) also with the addition of 10 nM nigericin. In the presence of this K + -H + ionophore, pH; equilibrates with extracellular pH and a calibration curve equating fluorescence with pH ; can be generated by titration of the cell suspension with 0.1 M HC1 over the pH range 7.4-6.5 (13). Over this pH range there was a linear relationship of fluorescence and pH;. Intracellular buffering power was performed by the pH; titration of acidified cells with 5 mM NH4C1 using methods described by Roos and Boron (14).
7.1
Endo • 1990 Voll27«Nol
A From: Na 130mM pH 7.4 To: Choine Cl 130mM pH 7.0
I
=5 6.9 O 63
4
10
6 TME, minutes
NaCl pH 7.4
B 7.2 7.0
+
Na influx measurement
130mM Choline Cl pH 7.0
25mM 10mM OmM+DMA
Glomerulosa cells on the culture plates were placed in a 37 C water bath and the culture media were replaced by HBS. Cell acidification, when performed, was accomplished by preincubation with Na+-free HBS with pH 6.0, 6.5, 7.0, and 7.4 for 30 min. When determining external sodium dependence of Na+ influx, Na+-free HBS at pH 7.0 was used. Five minutes before influx measurement, 1 mM ouabain and 0.1 mM bumetanide were added. To initiate the measurement of influx, the preincubation solution was replaced by a solution containing 5-100 mM NaCl with isosmotic substitution of sodium with choline, 1-2 /iCi/ml 22Na, 1 mM ouabain, 0.1 mM bumetanide (pH 7.4) with or without the addition of DMA (20 fiM) and/or ANG II (100 nM). Influx was terminated at 2 min by rapid aspiration of the influx media and washing with ice-cold 0.1 M MgCl2 (pH 7.2). The culture plates were extracted with 0.1% sodium dodecyl sulfate and total radioactivity was counted for each well. Protein was determined by the Lowry method (15). DMAsensitive Na+ influx was determined as the difference between 22 Na uptake in the absence and presence of DMA.
4
6
8
12
10
Time, minutes
FIG. 1. Evidence for Na+-H+ exchange in adrenal glomerulosa cells. A, BCECF-loaded cells, initially suspended in 130 mM Na+, pH 7.4, were centrifuged and resuspended in Na+-free solution (isosmotic replacement of sodium by choline), pH 7.0. Cellular acidification occurred over 5-10 min. B, Cells acid loaded as described in A were centrifuged and resuspended in solutions containing 10, 25, 50, and 130 mM Na+ (isosmotically replaced with choline), pH 7.4, with recording begun at the arrow. Note that in the presence of 130 mM Na+ and 20 /xM DMA there was no cellular alkalinization observed. pHj-6.8
12
PHQ-7.4
i^4-
10 UJ
Vol. 127, No. 1 Printed in U.S.A.
Na+-H+ Exchanger Kinetics in Adrenal Glomerulosa Cells and Its Activation by Angiotensin II* PAUL R. CONLIN, SEONG Y. KIM, GORDON H. WILLIAMS, AND MITZY L. CANESSA Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 Na + /mg protein x min. By varying pH; (from pHj 7.1 to 6.2),
ABSTRACT. We have studied the kinetic properties of basal and angiotensin II (ANG II) stimulated Na+-H+ exchange in adrenal glomerulosa cells by measuring changes in cytosolic pH (pHO and initial rates of 22Na uptake in the presence or absence of dimethylamiloride (DMA). The cells were studied 1) under basal conditions, 2) at constant pH; (6.8) with varied external sodium (Na+0), and 3) at varied pH; with constant Na+0 (50 mM). In 2,7-biscarboxyethyl-5(6)-carboxyfluorescein loaded cells under basal conditions, pH; rose from 7.09 ± 0.02 to 7.19 ± 0.02 (P < 0.05) with addition of ANG II (100 nM). Similarly, DMAsensitive Na influx was enhanced from 9.2 ± 1.3 to 14.8 ± 2.1 nmol Na+/mg protein X min (P < 0.01) by ANG II. In cells acidloaded by preincubation in Na+-free media (pH; 6.8), addition of varying Na+O resulted in a rapid H+ efflux that was markedly inhibited by DMA. DMA-sensitive Na+ influx into these acidified cells with varied Na+0 exhibited a Michaelis-Menten constant (Km) of 23 mM and a maximum velocity (VmaJ of 43 nmol
DMA-sensitive Na+ influx likewise snowed activation with cellular acidification with a pK at pH; 7.09. At pH; 6.8, ANG II decreased the Km for Na+0 from 23 to 17 mM and increased the Vmax from 43 to 53 nmol Na+/mg protein x min. The pHi dependence of DMA-sensitive Na+ influx was not affected by ANG II (pK at pH; 7.03). DMA also inhibited All-stimulated aldosterone secretion and Na+ influx similarly. These results indicate that Na+-H+ exchange in adrenal glomerulosa cells is 1) functioning under basal conditions, and 2) is modulated by ANG II with enhanced Na+0 affinity and Vma, but without a shift in pHi dependence (similar to ANG II effects on vascular smooth muscle cells). These effects suggest an important role for Na+-H+ exchange during ANG II stimulation of aldosterone production by glomerulosa cells. (Endocrinology 127: 236-244, 1990)
T
HE ROLE of Na + -H + exchange in regulating intracellular pH (pHi) has been well established in several cell types particularly in relation to the cellular response to various hormones and growth factors. Although the exchanger may be ubiquitous, its activation is usually linked to ligand-receptor interactions specific for individual cell types which produce phosphoinositide breakdown, calcium mobilization, and activation of protein kinase C, such as growth factors {e.g. epidermal growth factor, platelet-derived growth factor) (1, 2) and vasoconstrictor agonists {e.g. arginine vasopressin, «i adrenergic agonists) (3, 4). Angiotensin II (ANG II) modulation of the Na + -H + exchanger likewise has been studied in cultured vascular smooth muscle cells which have partially lost the contractile response (5, 6). In
adrenal glomerulosa cells, ANG II stimulation produces a prompt increase in steroidogenesis and secretion of aldosterone through the generation of inositol phosphates (7, 8) and may potentially modulate functioning of the Na + -H + exchanger (9). Unlike cultured vascular smooth muscle cells, glomerulosa cells provide the opportunity to study the role of Na + -H + exchange in ANG II stimulated aldosterone secretion while monitoring both membrane and secretion events. The presence of Na + -H + exchange in rat glomerulosa cells has been suggested by Hunyady et al. (10) through the ability of an amiloride analog to inhibit Na+ uptake into acid loaded cells. However, modulation of exchanger activity by ANG II could not be demonstrated. Horiuchi et al. (9) have extended these findings by showing inhibition of ANG II stimulated aldosterone secretion and cellular alkalinization by specific amiloride analogs. In this report the kinetic properties of the Na + -H + exchanger in adrenal glomerulosa cells are studied as a functional assessment of the antiporter under basal and agonist stimulated conditions. In addition, the ability of ANG II to activate Na + -H + exchange is demonstrated as is the importance of this pH; regulator in the mainte-
Received December 15,1989. Address requests for reprints to: Paul R. Conlin, M.D., EndocrineHypertension Division, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. * Results of this manuscript were presented in part at the meeting of the American Federation for Clinical Research, May 1989, Washington, D.C. This work was supported by NIH Grants HL-42120, HL35664, a Specialized Center of Research (SCOR) in Hypertension Grant HL-36568 and the Training Program in Hypertension Grant HL-07609.
236
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GLOMERULOSA CELL NA+-H+ EXCHANGE
nance of ANG II-stimulated aldosterone secretion. Materials and Methods Materials
237
wells, previously treated with fibronectin, at a density of 0.51.0 X 106 cells per well and incubated for 16-20 h before study. Cells cultured under these conditions retain the ability to secrete aldosterone for at least 7 days (data not shown). Aldosterone secretion studies
Dulbecco's modified Eagle's medium, essential and nonessential amino acids, trypsin-EDTA, insulin, a-tocopherol, transferrin, and antibiotics were from GIBCO (Grand Island, NY); nigericin and choline chloride were from Calbiochem (San Diego, CA). Ouabain, HEPES, Tris, ANG II, Percoll, metyrapone, L-glutamine, sodium selenite, and ammonium chloride were from Sigma (St. Louis, MO); 2, 7-biscarboxyethyl-5(6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) was from Molecular Probes (Eugene, OR). Ascorbic acid was from Fisher (Fairlawn, NJ), collagenase was from Worthington (Freehold, NJ), and BSA, fraction V, was from ICN ImmunoBiologicals (Lisle, IL). 22Na was from Amersham (Arlington Heights, IL); bumetanide was from Leo Lab (Vernouillet, France); and dimethylamiloride (DMA) was kindly supplied by Dr. E. Cragoe from Merck Sharpe and Dohme (Rahway, NJ). Cell isolation and culture Bovine adrenal glands were obtained from a local slaughterhouse and transported in ice cold 0.9% NaCl solution. The adrenals were bisected and the capsular portion was separated from the remainder of the gland by slicing the outer 0.3 mm with a microtome. These tissue slices were incubated in a modified Krebs-Ringer bicarbonate solution (MKRB) containing (in millimolars) 140 Na+, 3.7 K+, 1.25 Ca2+, 1.0 Mg>+, 1.0 SO42", 1.0 PO43-, 120 Cr, 25 HCO3-, 1 mg/ml BSA, 10 mM glucose, 1 mM L-glutamine, and an essential and nonessential amino acid mixture. The solution was equilibrated with 95% O2-5% CO2 before use. Crude collagenase (3.5 mg/ml) was added to the MKRB which contained the capsular portions and the mixture was allowed to incubate at 37 C for 30 min in a shaking water bath under a 95% O2-5% CO2 atmosphere. Dispersed cells were separated from tissue fragments by filtration through nylon mesh with 50-jtm pore size. After washing twice with MKRB, the cell suspension was loaded onto a discontinuous Percoll gradient consisting of layers containing 10, 30, 35, 60, and 80% Percoll isosmotically diluted with concentrated MKRB. After centrifugation at 400 X g for 30 min at 4 C, the cells separated into bands with glomerulosa cells appearing predominantly at the 35-60% interface. This band was collected and washed twice with MKRB. The cells were then used either for acute incubation studies or prepared for overnight culture. Cells prepared by this method routinely exclude Trypan blue greater than 95% and contain approximately 5% contamination with fasciculata-reticularis cells (as determined by visual inspection of the size difference between the two cell types). Those cells that were to be readied for culture were resuspended in Dulbecco's modified Eagle's medium supplemented with 2 nM L-insulin, 1 mM ascorbic acid, 1 /iM a-tocopherol, 1 mM L-glutamine, 50 nM sodium selenite, 50 nM metyrapone, 1.2 JUM transferrin, 11 mM glucose, 50 mg/liter gentamicin, and 3.7 mM potassium. The cells were plated into 16-mm culture
Glomerulosa cells obtained by the above isolation procedure were allowed to preincubate in MKRB solution for 60 min under a 95% O2-5% CO2 atmosphere. The cells were then washed once and resuspended in a modified Krebs-Ringer solution similar to that described above except bicarbonate was replaced by 20 mM HEPES, Na+ and Cl" concentrations were adjusted to 130 and 136 mM respectively, and pH was titrated to 7.4 at 37 C by addition of Tris base (MKRH). In experiments studying the effect of Na+-H+ exchange inhibition on ANG IIstimulated aldosterone production, the amiloride analog, DMA in dimethyl sulfoxide, was added at varying doses in 5-^1 aliquots to a cell suspension of 106 cells/0.5 ml and allowed to incubate for 5 min. DMA has previously been shown to be a specific, high affinity probe for the Na+-H+ exchanger (IC50, 0.3 MM) (11) with little affinity (IC50 > 300 ^M) for Na+-Ca2+ exchange (12). ANG II (100 nM) was subsequently added and allowed to incubate further at 37 C for 60 min. Aldosterone measurements were performed in duplicate using a RIA kit (Coat-A-Count RIA kit, Diagnostic Products Corp, Los Angeles, CA). Measurement of intracellular pH Cultured glomerulosa cells were harvested from the culture plates by exposure to a trypsin (0.05%)-EDTA (0.53 mM) solution for 10 min at 37 C. The cells were washed twice with HEPES-buffered saline (HBS) which contained (in millimolars) 130 N a \ 136 Cl~, 3.7 K+, 1.25 Ca2+, 1.0 PO43-, 1.0 Mg2+, 1.0 SO42", 20 HEPES, and 1 mg/ml BSA with pH adjusted to 7.4 at 37 C with Tris base. The cells were then resuspended in the presence of 2 /xM BCECF-AM and allowed to incubate at 37 C for 30 min. Extracellular dye was removed by washing twice with HBS and the cells were divided into 1-ml portions and allowed to stand at room temperature until the time of study. Cell acidification when performed was accomplished by incubation of the cells for 15-30 min in a solution in which choline chloride had replaced the sodium chloride at pH 7.0 (Na+-free HBS). Before measurement of fluorescence, the cells were centrifuged, resuspended in fresh buffer, and placed in a quartz cuvette at a density of 2 X 106 cells/2 ml. Cell fluorescence was measured in a Perkin-Elmer spectrofluorimeter (model 65-10S) equipped with a thermostated cuvette holder and a mechanical stirring bar. The BCECF-loaded cells were alternately excited at wavelengths of 500 and 440 nm with emission recorded at 530 nm with band widths of 5 and 7 nm, respectively. The ratio of emitted fluorescence at the two excitation wavelengths permitted calculation of pH; without interference from differences in dye loading, dye bleaching, or cell number. Because autofluorescence of unloaded cells was less than 2% of total fluorescence, it was not routinely subtracted.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:34 For personal use only. No other uses without permission. . All rights reserved.
GLOMERULOSA CELL NA+-H+ EXCHANGE
238
Calibration of pH ; was accomplished by measuring fluorescence of BCECF-loaded cells in HBS in which sodium had been substituted with potassium (130 mM) also with the addition of 10 nM nigericin. In the presence of this K + -H + ionophore, pH; equilibrates with extracellular pH and a calibration curve equating fluorescence with pH ; can be generated by titration of the cell suspension with 0.1 M HC1 over the pH range 7.4-6.5 (13). Over this pH range there was a linear relationship of fluorescence and pH;. Intracellular buffering power was performed by the pH; titration of acidified cells with 5 mM NH4C1 using methods described by Roos and Boron (14).
7.1
Endo • 1990 Voll27«Nol
A From: Na 130mM pH 7.4 To: Choine Cl 130mM pH 7.0
I
=5 6.9 O 63
4
10
6 TME, minutes
NaCl pH 7.4
B 7.2 7.0
+
Na influx measurement
130mM Choline Cl pH 7.0
25mM 10mM OmM+DMA
Glomerulosa cells on the culture plates were placed in a 37 C water bath and the culture media were replaced by HBS. Cell acidification, when performed, was accomplished by preincubation with Na+-free HBS with pH 6.0, 6.5, 7.0, and 7.4 for 30 min. When determining external sodium dependence of Na+ influx, Na+-free HBS at pH 7.0 was used. Five minutes before influx measurement, 1 mM ouabain and 0.1 mM bumetanide were added. To initiate the measurement of influx, the preincubation solution was replaced by a solution containing 5-100 mM NaCl with isosmotic substitution of sodium with choline, 1-2 /iCi/ml 22Na, 1 mM ouabain, 0.1 mM bumetanide (pH 7.4) with or without the addition of DMA (20 fiM) and/or ANG II (100 nM). Influx was terminated at 2 min by rapid aspiration of the influx media and washing with ice-cold 0.1 M MgCl2 (pH 7.2). The culture plates were extracted with 0.1% sodium dodecyl sulfate and total radioactivity was counted for each well. Protein was determined by the Lowry method (15). DMAsensitive Na+ influx was determined as the difference between 22 Na uptake in the absence and presence of DMA.
4
6
8
12
10
Time, minutes
FIG. 1. Evidence for Na+-H+ exchange in adrenal glomerulosa cells. A, BCECF-loaded cells, initially suspended in 130 mM Na+, pH 7.4, were centrifuged and resuspended in Na+-free solution (isosmotic replacement of sodium by choline), pH 7.0. Cellular acidification occurred over 5-10 min. B, Cells acid loaded as described in A were centrifuged and resuspended in solutions containing 10, 25, 50, and 130 mM Na+ (isosmotically replaced with choline), pH 7.4, with recording begun at the arrow. Note that in the presence of 130 mM Na+ and 20 /xM DMA there was no cellular alkalinization observed. pHj-6.8
12
PHQ-7.4
i^4-
10 UJ
