H + -Dependent ATPase and K+ channel activities in the rat thyroid cell strain FRTL-5 D. J. Woods, J. Soden and S. P. Bidey Department of Medicine, University of Manchester, Stopford Building,

Oxford Road, Manchester M13 9PT (D. J. Woods is now at Animal Health Research and Development, Pfizer Central Research, Sandwich, Kent CT13 9NJ) (Requests for offprints should be addressed to S. P. Bidey)

received

23

May

1991

ABSTRACT

Using the fluorescent indicators 2\m='\,7\m='\-bis(2-carboxyethyl)-5\m='\-(6\m='\)-carboxyfluoresceinand Oxonol V to monitor intracellular pH (pHi) and cell membrane potential respectively, we have investigated the in-

volvement of H+-dependent ATPase and H+-dependent K+ channels in the recovery of the rat thyroid cell strain FRTL-5 from experimentally induced cytosolic acidification and membrane hyperpolarization events. Following exposure of cells to the weak acid sodium butyrate (24mmol/l) under bicarbonate-free incubation conditions, cytoplasmic acidification was maximal after 3 min, attaining a pHi of 6.42. The subsequent recovery of pHi was unimpaired by the absence of extracellular K+, but was reduced in the presence of the Na+ antagonist amiloride (1 mmol/l), recovering by 0.11\m=+-\0.003 units, compared with 0.27\m=+-\0.02units under amiloride-free conditions. In the presence of the H+-dependent ATPase antagonist N,N\m='\-dicyclohexylcarbodiimide (DCC), the pH, recovery observed in amiloride-containing, K+-free buffer was abolished. The recovery of pHi in Na+- and K+-containing

INTRODUCTION

The regulation of intracellular pH (pH,) in mam¬ malian epithelial cells is fundamental both to the maintenance of cellular integrity and to the control of diverse differentiated cellular events (Roos & Boron, 1981; Busa, 1986; Moolenar, 1986). The transmembrane movement of H+ is known to be predominantly controlled by two mechanisms which involve, respectively, the extrusion of intracellular H+ in exchange for extracellular Na+ through an electroneutral Na+/H+ antiport (Boron, 1983; Aronson, 1985), and the direct exchange of Cl~ for

buffer was accompanied by hyperpolarization of the cell membrane, the later stage of which was reduced after blockade of K+ channels with BaCl2, implying a major contribution of transmembrane K+ movement to such events. In contrast to its attenuating effect on pHi recovery, DCC was ineffective in reducing butyrate-dependent membrane hyperpolarization, suggesting that H+-dependent ATPase may not be a major contributory factor to this event. However, when K+ channels were blocked by addition of BaCl2, addition of DCC abolished the butyrate-induced membrane depolarization. These findings are consistent with the presence of two independent hyperpolarizing transport processes in the FRTL-5 cell membrane which appear to involve (i) a H+-dependent ATPase, activated in response to cytosolic acidification, and allowing partial recovery of pHi in the absence of extracellular Na+ and HCO3\m=-\, and (ii) H+-dependent K+ channels which, while contributing to membrane hyperpolarization, may not play a major role in the normal maintenance of pHi.

Journal of Molecular Endocrinology (1992) 8, 79-86

HCO3- (Roos & Boron, 1981). In addition, an electrogenic Na+/HCC>3~ co-transporter has been

identified in certain non-mammalian cells (Boron & Boulpaep, 1983), while a Na+-dependent Cl-/ HC03~ exchanger has been demonstrated in a number of cells, including those of the mammalian kidney tubule (Boron, 1983). In many mammalian epithelial cells, Na+/H+ antiport activity is the predominant mechanism maintaining a constant pH¡ in an acidic extracellular environment (Grinstein & Rothstein, 1986; Moolenar, 1986). Several recent studies have provided evidence for Na+/H+ antiport activity in primary cultures of thyroid follicular

cells

(Gerard, Gabrion, Verrier et al. 1985; Takasu, Nagasawa, Komiya et al. 1989; Chow, Woodbury & Yen-Chow, 1990). In the rat thyroid cell strain FRTL-5, Na+/H+ antiport activity has been shown to be involved in both the short-term mitogenic response to insulin-like growth factor-I (Woods, Soden, Tomlinson & Bidey, 1990) and in the delayed proliferative response to thyrotrophin (TSH) (Marcocci & Grollman, 1988). Although, as in many other epithelial cells, the Na+/H+ antiport plays a major role in transmem¬

brane H+ movement in the FRTL-5 cell membrane, have previously obtained evidence for the reten¬ tion of a minor component of transmembrane H+ movement under experimental in-vitro conditions in which both the Na+/H+ antiport and HC03~dependent Cl~ channels are inoperative (Woods et al. 1990). In several epithelial cell types, a com¬ ponent of transmembrane H+ movement has been attributed to the activity of K+- or voltage-depen¬ dent transporters (Steinmetz & Andersen, 1982; Keller, Jentsch, Koch & Weiderholt, 1986), and a number of such cells also possess an electrogenic H+-dependent ATPase which is activated at low pH¡ values (Keller et al. 1986). In the process of extruding H+ from the cytosol, the activity of this ATPase leads to electrical hyperpolarization of the plasma membrane (Bichara, Paillard, Leviel et al.

we

1983). Using

the intracellularly trapped fluorescent pHsensitive indicator 2',7'-bis-(2-carboxyethyl)-5'(6')carboxyfluorescein (BCECF), the Na+/H+ antiport antagonist amiloride, and incubation buffers nomi¬ nally free of HC03~ to eliminate the contribution of CP/HC03^ exchange to pH¡-regulatory events, we have investigated for the presence of a functional K+-dependent ATPase in enzymatically dispersed FRTL-5 cells. In parallel studies, using the cell membrane potential-sensitive dye Oxonol V (Wag¬ goner, 1979), we have determined whether the

depolarization

events

occurring subsequent

to

cyto-

solic acidification may involve the activation of a K + -dependent ATPase. Simultaneously, and using the specific pharmacological antagonist N,N'-dicyclohexyl-carbodiimide (DCC), we have demon¬ strated a H+-dependent ATPase activity in the FRTL-5 cell membrane, and have determined the contribution afforded by such activity to membrane hyperpolarization and depolarization events and transmembrane H+ movement. Some of these studies were presented at the 10th Joint Meeting of British Endocrine Societies (Woods, Soden & Bidey,

1991).

MATERIALS AND METHODS

Chemicals

Amiloride hydrochloride (amiloride), DCC, Tris, choline chloride, valinomycin, nigericin, dimethylsulphoxide (DMSO) and Hepes were obtained from Sigma Chemical Co. Ltd, Poole, Dorset, U.K; Dispase was obtained from Boehringer-Mannheim Ltd, Eastbourne, E. Sussex, U.K; Oxonol V was purchased from Molecular Probes Inc., Eugene, OR, U.S.A., and the penta-acetomethvl ester of BCECF (BCECF-AM) from Calbiochem (Novabiochem, Nottingham, Notts, U.K.). TSH (Thytropar) was purchased from Armour Pharmaceuticals, Phoenix, AZ, U.S.A. Newborn calf serum (NCS; heat-inactivated), Hank's balanced salt solution

(HBSS; Ca2+/Mg2+-free), L-glutamine, penicillin streptomycin were obtained from Gibco Ltd, Paisley, Strathclyde, U.K. All other reagents were of the highest grades obtainable from commercial and

sources.

Solutions Stock solutions (lmmol/1) of BCECF-AM were made in DMSO and stored in 50 pi aliquots at 20°C. Amiloride and Oxonol V were dissolved in DMSO, and valinomycin was dissolved in ethanol. TSH was stored in aqueous stock solution at —70°C, while DCC was prepared in aqueous solution. Modified Krebs-Ringer buffer (mKRB) contained 120 mmol choline chloride/l, 5 mmol KCl/l, 1 mmol MgCl2/l, 1 mmol CaC^/l, 10 mmol glucose/l and —

20mmolTris-HCl/l.

FRTL-5 cell cultures The FRTL-5 cell strain (ATCC CRL8305) used in these studies was a continuous untransformed strain of rat thyroid follicular cells (Bidey, Lambert &

Robertson, 1988), having

a

population doubling

time of 36 h under the optimal growth conditions previously described (Ambesi-Impiombato, Parks & Coon, 1980; Ambesi-Impiombato, Picone & Tra¬ montano, 1982; Vitti, Rotella, Valente et al. 1983). Coon's modified F12 medium (mF12) used to support this cell strain was purchased from Imperial Laboratories Ltd, Andover, Hants, U.K. Studies of

pH¡

Preincubation FRTL-5 cells were maintained as stock monolayer cultures in Petri dishes in mF12 containing 5% (v/v) NCS, 100mU TSH/1 and 10mg insulin/l, under conditions previously described (Bidey, Chiovato, Day et al. 1984). Twenty-four hours before initiat¬ ing experiments, this medium was replaced by fresh medium without TSH or serum.

Cell

dispersion and test incubations of FRTL-5 cells

prepared from subconfluent monolayers using Dispase (2 g/l in HBSS). After washing, and recovery for 1 h in mF12, resuspended cells were loaded with BCECFAM (5 pmol/l) which, following hydrolysis by en¬ dogenous cytosolic esterases, yields the intracellularly trapped pH-sensitive fluorescent indicator BCECF. The dye-loading procedure was performed in mKRB, containing 0.5% (w/v) bovine serum albumin, for 20min at 37°C. After washing, cells were resuspended at a density of 106 cells in 2.5 ml

Suspensions

were

mKRB in a 3 ml quartz cuvette. The fluorescence emission of the BCECF-loaded cells was monitored in a Perkin-Elmer LS5B luminescence spectrometer, the cuvette holder of which had been modifed to contain a magnetic stirrer and a water jacket. The temperature of the latter was maintained at 37°C by means of an external water bath and pump. Exci¬ tation and emission wavelengths were 500 and 530nm respectively, each using a slit width of 5nm. Fluorescent emission was continuously monitored on a flat-bed, single-channel recorder (LKB Instru¬ ments, South Croydon, Surrey, U.K.), while the

machine-integrated values emission (4-s integration recorded for up to 16 min.

displayed for fluorescent periods) were manually

Manipulation of pH¡ Acidification of the cytosol of FRTL-5 cells in suspension was achieved using the weak acid sodium butyrate (24mmol/l), undissociated molecules of which are able to permeate the plasma membrane freely, giving rise to excess intracellular H+ after dissociation (Moolenar, 1986). In order to minimize the contribution to subsequent pH¡ recovery of transmembrane Na+-dependent and -independent 0~/HC03_ exchange, all incubations were per¬ formed in medium nominally free of HC03~ and buffered with Tris-HCl. Certain incubations were performed in K -free medium, in the presence or absence of the H+-dependent ATPase inhibitor DCC or the Na+/H+ antiporter blocker amiloride (lmmol/l) which, in each case, were added in a minimal volume (

H(+)-dependent ATPase and K+ channel activities in the rat thyroid cell strain FRTL-5.

Using the fluorescent indicators 2',7'-bis(2-carboxyethyl)-5'-(6')-carboxyfluorescein and Oxonol V to monitor intracellular pH (pHi) and cell membrane...
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