November 1989

FEB 07793

Volume 257, number 2, 361-364

The dependence on intracellular ATP concentration of ATP-sensitive K-channels and of Na,K-ATPase in intact HIT-T 1Sp-cells Ichiro Niki, Frances

M. Ashcroft+

and Stephen J.H. Ashcroft

Nufield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford and + University Laboratory of Physiology, Parks Road, Oxford, England

Received 12 September 1989 We have studied the effects of changes of intracellular ATP concentration ([ATP],) on the activity of ATP-sensitive K-channels (IK(ATP1) and of Na,K-ATPase in intact cells of the insulin-secreting cell-line HIT-Tl5. Pre-exposure of HIT /p-cells to oligomycin caused a dose-dependent reduction in [ATP],. Marked activation of ZK(.,rpjactivity was found when ATP was lowered below 3 mM. Na,K-ATPase was progressively inhibited as ATP was lowered to 1.5 mM. These data demonstrate that changes in intracellular ATP in the millimolar range markedly influence the activity of two b-cell membrane proteins having affinities for ATP in the micromolar range. This suggests that submembrane [ATP] may be considerably below the measured bulk cytosolic concentration. The findings also support the proposed role of intracellular ATP in mediating effects of changes in glucose concentration on the activity of @ell IKcATr.) and insulin secretion. ATP-sensitive-K-channel;

Potassium channel; Na,K-ATPase; Insulin secretion; (HIT-T1 5 ,%cells, Pancreatic /?-cells)

1. INTRODUCTION

Recent studies have identified a key role for ATPsensitive K-channels (ZK(ATP))in control of insulin release from the pancreatic P-cell [l-B]. It is currently envisaged that an increase in extracellular glucose concentration elicits an increased rate of glucose metabolism within the P-cell and that the subsequent increase in intracellular ATP/ADP ratio inhibits ZK(ATP) activity. The resulting membrane depolarization opens voltage-dependent Ca-channels and leads to increases in Ca2+ influx, intracellular free Ca’+ concentration ([Ca*‘]i) and insulin secretion. Despite the considerable evidence that [ATP]i acts as the primary physiological regulator of Zx(~rp) activity [ 11, there remains a disparity between measured [ATP]i in the pancreatic ,&cell and the very much lower concentration of ATP that maximally inhibits the channel activity in excised membrane patches [9, lo]. A number of explanations have been suggested for this discrepancy, in particular the ability of ADP to oppose the effect of ATP on Z~(ATP)[I 1,121 and the fact that even in the unstimulated &cell the majority of Zx(~rp) channels are closed [13]. In the present study we demonstrate that

Correspondence address: S.J.H. Ashcroft, Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, England Abbreviations: IK(ATP), ATP-sensitive K-channel; [ATP]i, intracellular ATP concentration; [Cal,, intracellular free Ca2+ concentration; DMSO, dimethylsulfoxide Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/89/$3.50 0 1989 Federation of European Biochemical Societies

changes in &cell ATP content in the millimolar range can indeed cause changes in ZK(ATP) activity in intact ,& cells. We additionally show that another plasma membrane protein, Na,K-ATPase, is also markedly affected in intact P-cells by similar changes in [ATP]i despite the fact that much lower concentrations of ATP saturate the enzyme in isolated membrane preparations [ 141. These data can be explained by postulating that in the P-cell the sub-membrane concentration of ATP is lower than the bulk cytosolic concentration. 2. MATERIALS

AND METHODS

2.1. Cell culture HIT-T15 @cells (passage numbers 75-90) were cultured in RPM1 1640 tissue culture medium containing penicillin (100 U/ml), streptomycin (0.1 mg/ml), fungizone (0.25 /g/ml) and fetal calf serum (10%) at 37°C in an atmosphere of humidified air (95%) and CO2 (5%) as previously described in detail [ 151. Cells were passaged weekly and harvested using trypsin-EDTA. HIT cells were seeded in Multiwell plates at a density of 5 x lo5 cells per well (for s6Rb-efflux and 86Rb-uptake studies) and in culture flasks at 3 x 10’ cells per flask (for Quin 2 studies). The cells were cultured for 3-6 days before the experiments. All experiments were performed at 37°C. 2.2. 86Rb-efflux measurements for assay of IK(ATp) IK(~TP)activity was assayed by measuring glibenclamide-sensitive 86Rb-efflux [16,17]. The day before an experiment, 86RbCl (from Amersham, spec. act. 86-682 Ci/mol) was added to wells (0.1 ,&i/well) and cells were loaded with the isotope overnight. On the day of the experiment, the culture medium was removed and replaced with solution A containing (mM): NaCl 124, CaCla 1.8, MgClr 0.8, KC1 10, Hepes 20 (pH 7.5 with NaOH) to which was added *"RbCl (0.1 ,&/ml). For Ca-free solution (B), Ca was replaced by Mg and 1 mM EGTA was added. To deplete the cells of ATP, prein-

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Volume 257, number 2

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November 1989

cubation was carried out for 20 min at 37°C in 1 ml of solution A or B containing 86RbC1 (0.1 pCi/ml) in the absence or presence of 0.12-2.4 pg/ml oligomycin and 1 mM 2-deoxy-D-glucose. After the preincubation, the medium was replaced with solution A or B without 86RbCl and the cells were incubated for 10 min in the absence or presence of 1 PM glibenclamide and/or I mM ouabain. To determine the cellular content of s6Rb, cells were washed twice with solution A supplemented with 1 PM glibenclamide (to prevent loss of 86Rb via ZK(ATP)) and then extracted with 1 ml of 0.1 M boric acid (pH 8 with NaOH) and 0.5 M NaCl. The extract was counted using Cerenkov counting. Stock solutions of oligomycin were prepared in dimethylsulphoxide (DMSO) at 2.4 mg/ml and of glibenclamide (Hoechst) at 1 mM in DMSO. 2.3. 86Rb-uptake measurements for assay of Na,K-ATPase Na,K-ATPase activity was measured as ouabain-sensitive 86Rbuptake. HIT cells were preincubated for 20 min in buffer containing (mM): NaCl 134, CaC12 1.8, MgCl2 0.8, Hepes 20 (pH 7.5 with NaOH) in the presence or absence of the ATP-depleting agents described above. K+ was omitted from the preincubation medium in order to increase the intracellular Na+ concentration. After preincubation, the cells were further incubated for 5 min in 1 ml of solution C containing (mM): NaCl 129, CaCls 1.8, MgCls 0.8, KC1 5; Hepes 20 (pH 7.5 with NaOH) with 0.5 pCi 86RbCl in the presence or absence of 1 mM ouabain or 1 pM glibenclamide. After washing twice with 1 ml of solution C, supplemented with 1 pM glibenclamide to prevent Rb loss, 86Rb was extracted and counted as described above. 2.4. ATP measurements To measure [ATP],, cells incubated under parallel conditions to those used for the 86Rb-flux measurements were extracted in 0.5 ml of 4% perchloric acid. ATP in the extracts was quantitated using a luciferase assay and a commercial luminometer (LKB 1250). The intracellular concentration of ATP was calculated assuming a volume of 1 pl per cell [18]. 2.5. Measurement of intracellular Cd’ HIT cells (approximately 5 x 10’) were detached with trypsinEDTA, washed in 20 ml of RPM1 medium and resuspended in 4 ml of solution A. Cells were loaded with Quin 2 as previously descdribed (191 and incubated for 20 min. After loading, cells were diluted 4-fold in the medium and incubated for a further 20 min. They were then collected by centrifugation (190 x g for 5 min), resuspended in a Ca-free solution (B) or Ca2+-containing solution (A) at a density of 25 x lo6 cells/ml and stored on ice before use. Quin 2-loaded HIT cells (5 x 106) were transferred to a cuvette (final vol. 2 ml) in a Perkin-Elmer LS5 luminescence spectrometer and preincubated for 10 min with continuous stirring before addition of ATP-depleting agents. The intracellular free Ca2+ concentration was calculated as described in [20]. All data are expressed as mean + SE for n observations.

Oi

I//

0.1

1

Oligomycin

5

(pg/ml)

Fig. I. Effect of oligomycin on the intracellular concentration of ATP in HIT cells. ATP was extracted from HIT cells incubated for 20 min in the absence or presence of oligomycin (0.12-2.4pg/ml) plus 2-deoxyglucose (1 mM) and measured by a luciferase assay. The data are given as mean & SE for 3-8 observations.

3.2.

IK(ATP)

A decrease in intracellular ATP concentration increased 86Rb-efflux from HIT cells (fig.2). In nonATP-depleted cells (data points at >5 mM [ATPIt) 86Rb-efflux amounted to 8% of the initial 86Rb content at the start of the incubation and glibenclamide had little effect. Lowering [ATP]i below 3 mM progressively increased 86Rb-efflux, up to 6-fold for [ATP]i less than 1 mM. This augmented 86Rb-efflux was abolished by 1 PM glibenclamide indicating that Rb efflux stimulated by ATP-depletion was through IK(ATP). 86Rb-efflux from ATP-depleted&cells was not affected by 1 mM ouabain either in the presence or in the absence of glibenclamide (data not shown). Thus under the conditions of these experiments the contribution of Na,K-ATPase to the measured Rb fluxes is negligible. The effects of ATP-depletion and glibenclamide on 86Rb-efflux during a 10 min incubation were not changed when similar experiments were carried out in the

3. RESULTS 01.

3.1. ATP content Fig. 1 shows the effect of increasing concentrations of oligomycin on [ATP]i in the presence of 1 mM 2-deoxy-D-glucose. Oligomycin (0.12-2.4 pg/ml) caused a dose-dependent decrease in intracellular concentration & ATP from 5.2 mM in the absence of oligomycin to less than 1 mM at the highest concentration of oligomycin. Neither [ATP]i nor 86Rb-efflux was influenced by 1 mM 2-deoxyglucose alone (data not shown). 362

0

,*. 1

, . 2

lnlracellular

I

3

,

.:.

,

4

5

ATP

(mM)

6

Fig.2. Effect of ATP depletion on 86Rb-efflux from HIT cells. HIT cells were loaded overnight with 86Rb and then pre-incubated for 20 min in the absence or presence of varying concentrations of oligomycin plus 2-deoxyglucose. The 86Rb-efflux from HIT cells during a subsequent 10 min incubation in Rb-free medium with (0) or without (0) 1 pM glibenclamide is expressed as a percentage of the 86Rb content at the start of the incubation. The intracellular ATP concentrations of HIT cells are expressed as an average of the values at the start and the end of the incubation. Each point is the mean f SE of 4-6 observations.

FEBS LETTERS

Volume 257, number 2

November 1989

with no detectable inhibition for [ATP]i less than 1.5 mM. The addition of 1 PM glibenclamide to the incubation media did not affect the inhibition of 86Rbuptake by ATP-depletion indicating lack of significant flux through ZK(ATP)under these conditions.

01-.

0

I

1

,

I

2

.

I

3

I

I

4

5

.

I

6

Intracellular ATP (mM)

Fig.3. Effect of ATP depletion on s6Rb-uptake by HIT cells. The a6Rb-uptake by HIT cells during a 5 min incubation in Rb-free medium without additions (0) or in the presence of either 1 mM ouabain (0) or 1 PM glibenclamide (A) is expressed as a percentage of the maximal s6Rb-uptake. The intracellular ATP concentration of HIT cells is expressed as an average of the values at the start and the end of the incubation. Each point is the mean of 4 observations (SEs were less than the size of the symbols).

absence of extracellular Ca’+. Thus 86Rb-efflux from non-ATP-depleted cells ([ATP]i = 5.54 + 0.43 mM, n = 3) was 14.4 f 1.5% (n = 5) in Ca-free buffer and 18.3 + 1.8% (n = 4) in solution A. In cells depleted with 1 mM 2-deoxyglucose plus 1.2 fig/ml oligomycin ([ATP]i = 0.44 f 0.08 mM, n = 3) the increase in 86Rbefflux observed in Ca-free buffer (to 35.1 f 3.5%, n = 4) was similar to that in solution A (to 34.2 + 3.1070, n = 4). The increased 86Rb-efflux elicited by ATP depletion in Ca-free buffer was also inhibited by 1 PM glibenclamide (to 16.2 + 1.9%, n = 4). 3.3. Na, K-A TPase 86Rb-uptake via Na,K-ATPase in HIT cells was also modified by changes in [ATP]i in the millimolar range (fig.3). 86Rb-uptake in the absence of ATP-depletion (data points at 6 mM [ATP]i) was inhibited by 60% by addition of 1 mM ouabain. As [ATP]i was lowered, ouabain had progressively less effect on 86Rb-uptake ohgomycm +

P-deoxyglucose

Time (min)

Fig.4. Effect of ATP depletion on the intracellular Ca2+ concentration in HIT cells. Intracellular Ca2+ concentrations were estimated with Quin 2. After Quin 2/AM loading, the cells were resuspended and incubated in buffer containing 1.8 mM Ca (0) or in Ca-free buffer (0). Oligomycin (1.2 pg/ml) and 2-deoxyglucose (1 mM) were added to the media after 10 min and the incubation was continued for a further 20 min. Each point is the mean + SE of 3 observations.

3.4. Zntracellular free Cd’ Fig.4 shows that [Cali in HIT cells incubated in the presence of extracellular Ca2+ was 180 nM; a gradual increase in [Cali was observed on ATP depletion which reached 516 f 100 nM (n = 3) at the end of 20 min incubation with oligomycin plus 2-deoxyglucose. In Cafree buffer, however, the basal [Cali (50 nM) was not increased by the addition of the ATP-depleting agents. 4. DISCUSSION The cloned &cell line HIT-T1 5 provides an attractive model for studying the regulatory mechanisms of the pancreatic P-cell because it retains good secretory responses to agents modifying insulin release from normal P-cells including the main physiological regulator glucose and the sulfonylureas used in the treatment of non-insulin-dependent diabetes mellitus [ 15,18,19]. As in normal &cells, this cell line possesses an ZK(ATP) which is markedly inhibited by micromolar concentrations of ATP in excised membrane patches [21]. Using this P-cell line, we have now established the ATPdependence for the activities of ZK(ATP)and of Na,KATPase in intact cells. We have used glibenclamide-sensitive “jRb-efflux as a measurement of whole cell ZK(ATP) activity [ 16,171. We can discount the possibility that significant glibenclamide-sensitive 86Rb-efflux flows through Caactivated K-channels for two reasons. First, patchclamp studies have shown that these channels are insensitive to sulfonylureas [4,22]. Secondly, we show here that although [Cali rises on ATP-depletion this effect is only seen in the presence of extracellular Ca’+, whereas omission of external Ca2+ did not modify the glibenclamide-sensitive 86Rb-efflux evoked by ATPdepletion. The fact that insulin secretion is inhibited by ATP depletion [23] despite the increase in intracellular Ca2+ we report here suggests that a certain minimum concentration of ATP is necessary for the secretory process itself. The present Rb-efflux study is in agreement with an earlier study on RINmSF cells [17] in showing that the ATP concentration which is required for inhibition of ZK(ATP) in intact P-cells is about two orders of magnitude higher than that reported for isolated membrane systems [9,10]. One possible explanation for the discrepancy is that ADP modulates the ATP sensitivity of the channel [ 11,121. An alternative possibility is that the sub-membrane ATP concentration may be significantly lower than the value derived from whole cell measurements as previously shown for hepatocytes 363

Volume 257, number 2

FEBS LETTERS

[24] and oocytes [25]. Were such an ATP gradient to exist, other membrane proteins would also be exposed to an [ATP]i lower than the average intracellular concentration. To test this, we examined the effect of ATP-depletion on Na,K-ATPase activity in intact ,& cells. This plasma membrane enzyme is known to be activated by micromolar concentrations of ATP when studied in isolated membrane systems [ 141. Our data demonstrate that the activity of the Na,K-ATPase in intact P-cells was also sensitive to changes in [ATP]i within the millimolar range. The &cell Na,K-ATPase showed a progressive and non-saturating dependence on [ATP]i within the range 2-6 mM. Below about 2 mM [ATP]i there was no effect of ouabain on 86Rbuptake indicating that the &cell Na,K-ATPase was essentially inactive. Similar findings have been reported for hepatocytes [24]. These data are thus consistent with the concept that the actual concentration of ATP beneath the &cell plasma membrane is considerably lower than that expressed as an averaged intracellular concentration. Such an intracellular gradient of ATP might arise from unequal rates of ATP supply by mitochondria and utilization by membrane ATPases. Our findings thus support the view that a major physiological determinant of Zk(~rr) activity is the submembrane ATP concentration; however, additional regulatory mechanisms cannot be ruled out. Acknowledgements: We thank the Medical Research Council, the British Diabetic Association, the EP Abraham Trust and the Nordisk UK Fund for financial support. The technical assistance of Jeremy Chalk for the culture of HIT cells is gratefully acknowledged. HITT15 cells were generously provided by Professor A.E. Boyd iii, Houston, TX and glibenclamide was a gift from Hoechst. We are grateful to Dr T. Sasaguri, University of Kyushu, Japan and Dr Barbara Corkey, Boston, USA for helpful discussions.

REFERENCES [l] Ashcroft, F.M. (1988) Annu. Rev. Neurosci. 11, 97-118.

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November 1989

121Ashcroft, F.M., Ashcroft, S. J.H. and Harrison, D.E. (1988) J. Physiol. 400, 501-527. ]31 Arkhammar, P., Nilsson, T., Rorsman, P. and Berggren, P.-O. (1987) J. Biol. Chem. 262, 5448-5454. ]41 Sturgess, N.C., Kozlowski, R.Z., Carrington, C.A., Hales, C.N. and Ashford, M.L.J. (1988) Br. J. Pharmacol. 95, 83-94. [51 Ribalet, B. and Ciani, S. (1987) Proc. Natl. Acad. Sci. USA 84, 1721-1725. 161 Ziinkler, B.J., Lenzen, S., Manner, K., Panten, U. and Trube, G. (1988) Naunyn-Schmiedeberg’s Arch. Pharmacol. 337, 225-230. [71 Misler, S., Falke, L.C., Gillis, K. and McDaniel, M.L. (1986) Proc. Natl. Acad. Sci. USA 83, 7119-7123. PI Dunne, M.J., Ilott, M.C. and Petersen, O.H. (1987) J. Membr. Biol. 99, 215-224. [91 Cook, D.L. and Hales, C.N. (1984) Nature 311, 271-273. [lOI Rorsman, P. and Trube, G. (1986) Pfliigers Arch. 405, 305-309. [Ill Kakei, M., Kelly, R.P., Ashcroft, S.J.H. and Ashcroft, F.M. (1986) FEBS Lett. 208, 63-66. I121 Dunne, M.J. and Petersen, O.H. (1986) FEBS Lett. 208, 59-62. 1131 Cook, D.L., Satin, L.S., Ashford, M.L.J. and Hales, C.N. (1988) Diabetes 37, 495-498. 1141 Neufeld, A.H. and Levy, H.M. (1969) J. Biol. Chem. 244, 6493-6497. I151 Ashcroft, S.J.H., Hammonds, P. and Harrison, D.E. (1986) Diabetologia 29, 727-733. [161 Niki, I., Kelly, R.P., Ashcroft, S.J.H. and Ashcroft, F.M. (1989) Pflugers Arch., in press. H., De Weille, J., Fosset, M. and P71 Schmid-Antomarchi, Lazdunski, M. (1987) J. Biol. Chem. 262, 15840-15844. H81 Ashcroft, S.J.H. and Stubbs, M. (1987) FEBS Lett. 219, 311-315. iI91 Hughes, S.J. and Ashcroft, S.J.H. (1988) J. Mol. Endocrinol. 1, 13-17. PO1 Rorsman, P. and Abrahamsson, H. (1985) Acta Physiol. Stand. 125, 639-647. Pll Eddlestone, G.T., Ribalet, B. and Ciani, S. (1989) Biophys. J. 55, 541A. WI Trube, G., Rorsman, P. and Ohno-Shosaku, T. (1986) Pfliigers Arch. 407, 493-499. 1231 Ashcroft, S.J.H., Weerasinghe, L.C.C. and Randle, P.J. (1973) Biochem. J. 132, 223-231. 1241 Aw, T.Y. and Jones, D.P. (1985) Am. J. Physiol. 249, C385-C392. P51 Miller, D.S. and Horowitz, S.B. (1986) J. Biol. Chem. 261, 13911-13915.

The dependence on intracellular ATP concentration of ...

increase in intracellular ATP/ADP ratio inhibits. ZK(ATP) activity. The resulting membrane depolarization opens voltage-dependent Ca-channels and leads to in-.

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