0013.7227/92/1302-0717$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. 130, No. 2 Society

Printed

pH Homeostasis in Pituitary Intracellular pH Is Regulated Concentration* KID

TGRNQUIST

AND

ARMEN

H. TASHJIAN,

GH4C1 Cells: by Cytosolic

in U.S.A.

Basal Free Ca2+

JR.

Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Helsinki, Finland; and the Department of Molecular and Cellular Toxicology, Harvard School of Public Health, and the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

15-20 min and observed a decrease in basal pH, to 6.75 + 0.03 (P < 0.05). No additional acidification was obtained when 2deoxy-D-glucose-treated cells were depolarized, and no TRHinduced activation of Na+/H+ exchange was observed. Addition of ionomycin or 12-O-tetradecanoyl-phorbol-13-acetate separately to acidified cells had only modest effects on pH,; however, addition of 12-O-tetradecanoyl-phorbol-13-acetate and ionomytin together increased pH, markedly. We conclude that in GH,Ci cells, increasing [Ca’+], reduces basal pH, through a mechanism dependent on influx of extracellular Ca” and independent of Na+/H+ exchange. In addition, elevation of [Ca’+], and activation of protein kinase C act synergistically to enhance Na+/H+ exchange and increase pH, in acidified cells. Finally, normal cellular ATP is necessary for the activation of Na+/H+ exchange. (Endocrinology 130: 717-725, 1992)

ABSTRACT. In GHICl cells, membrane depolarization induces a rapid and sustained increase in the cytosolic free calcium concentration ([Ca*+],). In the present study we have investigated the role of [Ca’+], in the regulation of basal intracellular pH (pH,). Depolarizing GH,C, cells in buffer containing 0.4 mM extracellular Ca” decreased basal pH, from 7.02 + 0.04 to 6.85 + 0.03 (I’ < 0.05). If the depolarization-induced influx of Ca2+ was inhibited by chelating extracellular Ca*+ or blocking influx with nimodipine, no through voltage-operated Ca’+ channels acidification was observed. Addition of TRH induced a rapid activation of Na+/H’ exchange in acidified cells, increasing pH, by 0.14 f 0.03 U. The action of TRH was blunted if extracellular Ca*+ was chelated; however, if influx of Ca’+ via voltage-operated channels was blocked by nimodipine, TRH still increased pH;. To deplete ATP, we incubated cells with 2-deoxy-D-glucose for

T

HE ROLE of Na+/H+ exchange in regulating intracellular pH (pHi) has been well established in several cell systems (1, 2). A number of agonists stimulate the hydrolysis of phosphatidylinositol-4-5-bisphosphate to produce inositol-1,4,&trisphosphate and 1,2-diacylglycerol (1,2-DG) (3); these second messenger molecules cause a rise in cytosolic free Ca’+ ([Ca’+]i) and activation of protein kinase C (PKC), respectively (3). Agonistinduced activation of PKC has generally been considered to be an important mechanism for enhancing Na’/H’ exchange (l), although evidence for Ca’+-induced activation of the antiporter also exists (4-6). Changes in pHi are of importance in regulating essential cellular func-

tions, including sequestration of intracellular Ca2+ (7), and the binding of inositol-1,4,&trisphosphate to its receptor (8, 9). We have recently shown that pHi in GHICl cells can be regulated by activation of Na+/H+ exchange through two pathways, one dependent on activation of PKC, and a second dependent on an increase in [Ca’+]; (6, 10). However, the possible mechanistic relationships between PKC and [Ca*+]i activation of Na+/H+ exchange are not well understood. Considering the importance of changes in [Ca*+]i in the regulation of pituitary cell function, we have continued our examination of the relationships between [Ca*+]i and pHi in GH4C1 cells. In the present study we investigated whether a persistent increase in [Ca*+]i can alter basal pHi or modify the TRH-induced activation of Na+/H+ exchange. Our results show that increasing [Ca*+]i by depolarizing the cells induces rapid acidification of the cytosol independent of Na+/H+ exchange, but dependent on influx of extracellular Ca*+. The acidification is reversed by activating Na+/H+ exchange by TRH. Both [Ca*+],-induced acidification and TRH-induced activation of Na+/H’ exchange were dependent

Received August 23, 1991. Address all correspondence and requests for reprints to: Armen H. Tashjian, Jr., M.D., Department of Molecular and Cellular Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115. *This investigation was supported in part ,by the Academy of Finland, a grant from the University of Helsinki, and a research grant from the NIH (DK-11011). Part of this study was performed while K.T. was a Visiting Fellow in the Laboratory of Toxicology at the Harvard School of Public Health.

111

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Ca’+-INDUCED

718

ACIDIFICATION

on cellular ATP. Furthermore, the actions of [Ca’+]i and PKC activation on Na’/H+ exchange were found to be synergistic.

Endo. 199‘2 Vol 130. No 2

IN GHC,_ . CELLS

fura-2.

Changes

in [Ca’+]i

obtained

with

the Hitachi

F2000

fluorometer were calculated usinga computer program designed for use with this instrument. Statistics

Materials

and Methods

Results

Materials Bis-(carboxyethyl)carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2-AM, and BAPTA-AM were obtained from Molecular Probes (Eugene, OR). Digitonin was purchased from Fischer Scientific (Pittsburgh, PA). 2-Deoxy-D-glucose, TRH, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and nigericin were obtained from Sigma (St. Louis, MO), and ionomycin was obtained from Calbiochem (La Jolla, CA). All flasks and dishes used for cell culture were purchased from Nunc Plastics (Kamstrup, Denmark). Cell culture Clonal rat pituitary GHICl cells were grown in monolayer culture in Ham’s F-10 Nutrient Mixture with 15% (vol/vol) horse serum and 2.5% fetal bovine serum in a water-saturated atmosphere of 5% CO, and 95% air at 37 C, as described previously (11, 12). Before an experiment, cells from a single donor culture were harvested with 0.1% Viokase or 0.1% trypsin and subcultured in loo-mm dishes for 7-9 days. Cells were fed every 2-3 days and always on the day before an experiment. Measurement

of

pH,

The method for measuring pHi in GH,C, cells has been described previously (10). In brief, the cells were harvested in HEPES-buffered salt solution (HBSS; containing in millimolar concentrations: NaCl, 118; KCl, 4.6; glucose, 10; CaCl*, 0.4; HEPES, 20; pH 7.2) lacking CaCl, and containing 0.02% EDTA. The cells were then washed twice in HBSS (containing CaC12) and incubated for 35 min at 37 C with 5 pM BCECFAM. pH, was determined fluorometrically with a Spex Fluorolog F 1llA spectrophotometer (Spex Instruments, Edison, NJ) or a Hitachi F2000 fluorometer (Hitachi, Ltd., Tokyo, Japan), using an excitation wavelength of 500 nm and an emission wavelength of 530 nm. At the end of the experiment, the signal was calibrated by lysing the cells with digitonin and measuring the fluorescence at known pH values. To correct for the red shift in the spectrum of BCECF induced by calibrating in an extracellular solution, cells were incubated in a high K’ buffer, and known pH values were imposed across the plasma membrane using the K+/H+ ionophore nigericin. The cells were then lysed with digitonin, and a new calibration curve was constructed (13). The calibration curves were linear over the pH range between 6.4-7.2. Measuring

[Ca’+],

The method for determining [Ca’+li with fura- in GH4C1 cells has been previously

described

(14).

Fluorescence

was

measuredin a Spex Fluorolog F 1llA spectrophotometer (excitation wavelength, 342 nm; emissionwavelength, 492 nm) or a Hitachi

F2000

spectrophotometer

(excitation

wavelengths,

340 and 380 nm; emissionwavelength, 510 nm). [Ca’+li was calculated, as previously described(15), with Kd = 224 nM for

are given as the mean + SE, and each experiment

wasrepeatedat least five times using at least three independent cell preparations. Statistical analyses were made using Student’s t test for comparison between two means. Results [CP]i

and basal pHi

in GH4C1 cells

In many cell types, a transient or persistent increase in [Ca’+]; is associated with a decrease in pHi (16-18). In GHICl cells, addition of TRH induces a rapid transient increase in [Ca’+]i, followed by a prolonged plateau phase of rise in [Ca’+]i (19, 20). In the present experiments, TRH induced a transient spike in [Ca”]; to 548 f 108 nM’ and a plateau phase of 224 f 16 nM; however, no acute change in pHi was observed (data not shown). The basal pHi in GH4C1 cells was 7.02 f 0.02 (mean f SE). We then determined whether a more persistent increase in [Ca”];, induced by depolarizing the cells with high extracellular K+ and, thus, activating voltage-operated Ca*+ channels (VOCC) (21), could induce a change in pHi. In the present experiments, depolarization with 50 mM K’ increased [Ca*+]i from an average resting level of 107 + 14 nM to a plateau level of 512 f 106 nM. In Fig. 1A we show that addition of 50 mM K’ induced rapid acidification of the cytosol. The initial decrease in pHi measured during the first min was 0.09 f 0.01 pH U. Addition of vehicle alone produced no acute change in pHi (Fig. 1B). The immediate (first few seconds) decrease in fluorescence observed after the addition of K’ was probably due to the change in medium osmolarity. We have observed similar changes after the addition of either 50 mM Na+ or 50 mM choline+. To separate the action of depolarization per se (22) from that of the resultant increase in [Ca”];, we depolarized the cells with K+ in a Ca*+-free buffer. In Fig. 2A, we show that acidification in response to high K+ depends on influx of extracellular Ca’+, as little or no acidification was seen in a Ca’+-free buffer. Readdition of extracellular Ca*+ (final concentration, 1 mM) to the depolarized cell suspension led immediately to a decrease in pHi of 0.10 + 0.01 U (Fig. 2B), a value similar to the initial acidification observed after the addition of K’ to cells in Ca*+-containing buffer. Readdition of extracellular Ca2+ increased [Ca*+]i from 68 f 12 to 1019 f 325 1Numerical data given in the text for [Ca”], or pH, are the mean + for at least five separate experiments; therefore, they may not correspond exactly to the numerical value in a single representative experiment, as illustrated by the figures. SE

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Ca*+-INDUCED

A,

ACIDIFICATION

Action of TRH on pHi in cells previously acidified by a depolarization-induced increase in [Ca*+J,

v

FIG. 1. Action of 50 mM extracellular K’ on basal pH, and [Ca’+li in GH,C, cells. A, Effects of K’ on pHi and [Ca”],. B, Effect of vehicle alone.

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