©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Metabolism, Cytoplasmic Ca, and pH-regulating Exchangers in Glucose-induced Rise of Cytoplasmic pH in Normal Mouse Pancreatic Islets (*)

(Received for publication, August 22, 1994; and in revised form, December 21, 1994)

Ruth M. Shepherd Jean-Claude Henquin (§)

From the Unité d'Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, UCL 55.30, Avenue Hippocrate 55, B-1200 Brussels, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Intact mouse islets were loaded with 2`,7`-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to study the effects of glucose on cytoplasmic pH (pH) in pancreatic B-cells. In HCO(3) buffer, glucose produced a steady-state increase in pH that required metabolism of the sugar and was concentration-dependent between 0 and 10 mM (K 5 mM) before plateauing at a maximum value of 0.2 pH units. In HEPES buffer, glucose concentrations above 7 mM caused an initial rise followed by a secondary decrease and an eventual return to about initial values. Inhibition of Ca influx had little effect on the pH changes produced by glucose in HCO(3) medium, but unmasked an alkalinizing effect in HEPES buffer. Raising cytoplasmic Ca by 30 mM potassium caused a larger acidification in HEPES than in HCO(3) buffer, but a subsequent rise in glucose now increased pH in both types of buffer. In the presence of 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid (DIDS; inhibitor of HCO(3)/Cl exchange), the effect of glucose on pH in HCO(3) buffer became similar to that in HEPES buffer. After inhibition of the Na/H exchanger by dimethylamiloride, glucose produced a marked and sustained fall in pH in HEPES buffer. A similar fall was seen in HCO(3) buffer only when DIDS and dimethylamiloride were present together. However, if Ca influx was prevented when both exchangers were blocked, glucose increased pH. In conclusion, the metabolism of glucose tends to increase pH in B-cells, whereas the concomitant rise in [Ca] exerts an acidifying action. In HEPES buffer, this acidifying effect of Ca is offset by the operation of the Na/H exchanger. In physiological HCO(3) buffer, the activity of the HCO(3)/Cl exchanger overcompensates and leads to an increase in pH.


INTRODUCTION

Glucose plays a pre-eminent role in the control of pancreatic B-cell function(1) . The mechanisms by which it stimulates insulin release involve regulation of a number of ionic events through changes in B-cell metabolism(1, 2, 3, 4, 5, 6) . The major events can be summarized as follows. Glucose entry in B-cells is followed by an acceleration of glycolysis and glucose oxidation, which generates signals that close ATP-sensitive K channels in the plasma membrane. The resulting decrease in K conductance leads to depolarization with subsequent opening of voltage-dependent Ca channels. Ca influx through these channels increases, causing a rise in free cytoplasmic calcium [Ca], (^1)which serves as the triggering signal for the exocytosis of insulin granules. The metabolism of glucose also augments insulin release by amplifying the effectiveness of [Ca] on the secretory machinery(7) .

It has been speculated that protons might be one of the signals produced by glucose metabolism and that changes in cytoplasmic pH (pH) in B-cells might influence certain steps of stimulus-secretion coupling(8, 9, 10, 11) . Therefore, several studies have examined the effect of glucose on B-cell pH, but rather contradictory results have been obtained probably because of the use of different preparations (whole islets, dispersed islet cells, tumoral cell lines), of different methods of pH measurement, and of different buffers. The weight of the evidence, however, indicates that glucose produces a slight alkalinization of B-cells(12, 13, 14, 15, 16) . On the other hand, the mechanisms by which this alkalinization might occur are not established(11) . The current hypothesis that it is brought about by an activation of the Na/H exchanger with overcorrection of the acidifying action of glucose metabolism (16) has not taken into consideration the possible contributions of [Ca] and of the HCO(3)/Cl exchanger.

In this study, intact pancreatic islets from normal mice were loaded with the pH-sensitive dye BCECF and examined by microspectrofluorometry. Our aim was to monitor pH in islet cells over longer periods of time than in previous studies, during stimulation by various concentrations of glucose in the presence and absence of HCO(3), Ca, and inhibitors of the Na/H and HCO(3)/Cl exchangers.


EXPERIMENTAL PROCEDURES

Solutions

A bicarbonate-buffered medium (HCO(3) medium) and a HEPES-buffered medium (HEPES medium) were used. The HCO(3) medium contained 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl(2), 1.2 mM MgCl(2), and 24 mM NaHCO(3) and was gassed with O(2)/CO(2) (94:6) to maintain pH at 7.4. The HEPES medium contained 135 mM NaCl, 4.8 mM KOH, 2.5 mM CaCl(2), 1.2 mM MgCl(2), and 10 mM HEPES and was gassed with O(2). Its pH was adjusted to 7.4 at 37 °C with NaOH. When the KCl concentration was raised to 30 mM, the concentration of NaCl was decreased accordingly. In Ca-free solutions, MgCl(2) was substituted for CaCl(2), and 50 µM EGTA was added. All solutions contained 1 mg/ml bovine serum albumin (fraction V; Boehringer Mannheim). DIDS and DMA (Sigma) were added from 133 and 100 mM stock solutions in Me(2)SO, respectively.

Preparation

All experiments were performed with islets isolated by collagenase digestion of the pancreata of fed female NMRI mice. The isolation procedure was carried out in HCO(3) buffer containing 10 mM glucose and supplemented with 5 mM HEPES. After isolation, the islets were cultured for 18-48 h in RPMI 1640 medium containing 10 mM glucose(17) .

Measurements of pH(i)

Cultured islets were first loaded with BCECF during 40 min of incubation at 37 °C in 2 ml of medium supplemented with 0.5 µM BCECF acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR) added from a 0.5 mM stock solution in Me(2)SO. The medium had the same composition (HCO(3) or HEPES) as that to be used during the experiment and always contained 3 mM glucose. Loaded islets were then transferred into a temperature-controlled perifusion chamber (Applied Imaging, Sunderland, United Kingdom) with a bottom made of a glass coverslip. They were held in place by gentle suction with a glass micropipette and perifused at a flow rate of 1.4 ml/min. The dead space of the system corresponded to 2 min and has been corrected for in the figures. Perifusion solutions were kept at 37 °C in a water bath, and the temperature controller ensured a temperature of 37.2 ± 0.3 °C close to the islet as monitored by a thermistor placed near the tissue.

The perifusion chamber was mounted on the stage of an inverted microscope (Nikon Diaphot) used in the epifluorescence mode with a times20 objective. BCECF was successively excited at 440 and 490 nm by means of two narrow band-pass filters mounted on a computer-controlled motorized filter wheel placed in front of a 75-watt xenon lamp. A dichroic mirror centered at 510 nm reflected the UV light to the perifusion chamber and transmitted the emitted fluorescence, which then passed through another filter of 535 nm. Fluorescent images were obtained with a CCD video camera (Photonic Science Ltd., Tunbridge Wells, UK) at a resolution of 256 times 256 pixels. They were then digitized into 256 gray levels and analyzed with the MagiCal system (Applied Imaging). To improve the signal-to-noise ratio, eight consecutive 40-ms frames were averaged at each wavelength before ratioing. The time interval between successive series of 440-490 images varied according to the length of the experiment, being 3.5 s over 15 min and 7.0 s over 30 min. Hence, the period of exposure to the excitatory light was the same irrespective of the duration of the experiment. The pH(i) was calculated from an in vitro calibration curve constructed from the ratio values obtained by perifusing solutions of different pH values (ranging between 5 and 9.5) containing 1.8 µM BCECF-free acid (the concentration giving a similar intensity of signal as that of BCECF-loaded islets). The medium had the following composition: 136 mM KCl, 4 mM NaCl, 5 mM MgCl(2), 5 mM glucose, and 20 mM HEPES. Bovine serum albumin was omitted to prevent its precipitation at high pH values.

Insulin Release

After preincubation, cultured islets were transferred, in batches of 20, into a previously described perifusion system(18) . Insulin was measured (19) in the effluent fractions collected every minute.

Presentation of Results

The results of pH(i) measurements are presented as traces that are the means ± S.E. for the indicated number of islets. Several islets from the same culture were tested with the same protocol, but each protocol was tested on at least three different cultures. For insulin release, each protocol was repeated four to five times with islets from different cultures. The statistical significance between means was assessed by analysis of variance followed by Dunnett's test or by unpaired t test when only two groups were compared.


RESULTS

Basal islet cell pH(i) values (in 3 mM glucose) averaged 6.98 ± 0.01 (n = 189) and 6.95 ± 0.01 (n = 134) in HCO(3) and HEPES buffers, respectively. This small, statistically significant (p < 0.05) difference was observed in all experimental series, as will be seen in the figures. It is, however, important to emphasize that these values correspond to steady-state pH(i). Sudden omission of HCO(3)/CO(2) by changing to HEPES buffer was followed by a rapid increase in pH(i) before the secondary decrease below initial values. Conversely, changing from HEPES buffer to HCO(3) buffer initially caused a transient acidification (data not shown). Qualitatively similar observations have been made with single mouse B-cells(20) .

Effects of Different Glucose Concentrations on Islet pH(i)

When the islets were perifused throughout with HCO(3) medium containing 3 mM glucose, pH(i) only marginally decreased with time. A larger fall occurred when glucose was omitted from the medium (Fig. 1A). In contrast, raising the concentration of glucose from 3 to 7 mM and above provoked an increase in pH(i) that consistently displayed a biphasic pattern (Fig. 1, B and C). A rapid increase was followed by a small transient decrease and then by a larger sustained increase. Because the small decrease varied in size and timing, the biphasicity of the pH(i) change is sometimes masked by pooling results obtained in different islets (e.g.Fig. 4).


Figure 1: Influence of various glucose concentrations, of mannoheptulose, and of dihydroxyacetone on pH in mouse islets. BCECF-loaded islets were perifused with HCO(3) medium (solidlines) or HEPES medium (brokenlines). A-C, the concentration of glucose (G) was 3 mM at the start of the experiments and was then changed as indicated on top of each panel. D, the concentration of glucose was raised from 3 to 15 mM in a medium containing 20 mM mannoheptulose (MH) throughout. E, dihydroxyacetone (DHA) was added to a medium containing 3 mM glucose. Each trace is the mean ± S.E. for nine islets.




Figure 4: Influence of the omission of extracellular Ca on pH in mouse islets. BCECF-loaded islets were perifused with HCO(3) medium (solidline) or HEPES medium (brokenline). The concentration of glucose (G) was increased from 3 to 15 mM after 5 min. Ca was omitted, and 50 µM EGTA added between 15 and 25 min. The traces are the means ± S.E. for 9 and 10 islets, respectively.



In the absence of HCO(3), the decrease in pH(i) that followed the omission of glucose from the perifusion medium (Fig. 1A) was again larger (p < 0.01) than that occurring spontaneously when the islets were perifused with a medium containing 3 mM glucose throughout. A small monophasic rise in pH(i) was observed on the change from 3 to 7 mM glucose (Fig. 1B). When the glucose concentration was raised to 10 mM and above, islet pH(i) changed in three phases: a transient rise was followed by a decrease and an eventual return to about initial values (Fig. 1C).

Fig. 2illustrates the concentration dependence of the glucose-induced rise in islet pH(i). The measurements were made at steady state, 15 min after the change from 3 mM glucose to a medium containing another glucose concentration. They are presented as absolute pH(i) values (Fig. 2A) or as the change in pH(i) within the same islet (Fig. 2B). In a HCO(3) buffer, the relationship was hyperbolic with a K(m) of 5 mM and a maximum at 15 mM glucose. In the absence of HCO(3), the relationship was more complex. pH(i) increased between 0 and 7 mM glucose, while the size of the pH(i) rise became smaller at higher glucose concentrations (Fig. 2B).


Figure 2: Concentration dependence of the effects of glucose on pH in mouse islets. pH was measured in experiments similar to those shown in Fig. 1. A, pH at 19 min, i.e. 15 min after changing the glucose concentration; B, change in pH relative to the control value in the same islet at the start of the experiment (in glucose at 3 mM). Values are the means ± S.E. for 7-13 islets perifused with HCO(3) or HEPES medium as indicated.



Because of the distinct effects of high glucose concentrations on pH(i) in islets perifused with HCO(3) or HEPES medium, we checked that the discrepancy was not due to local alkalinization of the HCO(3) medium by CO(2) loss in the open perifusion chamber. In the first series, the flow rate of the HCO(3) solution was doubled, but this did not prevent 15 mM glucose from increasing pH(i) by 0.10 ± 0.02 units compared with 0.11 ± 0.01 units at the usual flow rate of 1.4 ml/min. In the second series, 5 mM HEPES was added to the HCO(3) buffer. Under these conditions, 15 mM glucose still increased pH(i) by 0.10 ± 0.01 units. These values are not statistically different.

Role of Metabolism in Glucose-induced pH(i) Changes

These experiments were done in a HCO(3) medium only. 3-O-Methylglucose is transported in B-cells like glucose, but is not metabolized(21) . The addition of a 12 mM concentration of this analogue to a medium containing 3 mM glucose did not influence pH(i) (data not shown). When the concentration of glucose was raised from 3 to 15 mM in a medium supplemented with 20 mM mannoheptulose, an inhibitor of glucose phosphorylation by glucokinase(21) , practically no increase in pH(i) occurred (Fig. 1D). On the other hand, the effect of glucose was reproduced by the glycolytic intermediate dihydroxyacetone (Fig. 1E). Ketoisocaproic acid, a leucine derivative that is metabolized only in the mitochondria, produced a biphasic increase in pH(i) similar to that caused by glucose (data not shown). Finally, the rise in pH(i) brought about by glucose was reversed by 2 mM azide, a mitochondrial poison (data not shown).

Influence of Ca and Membrane Depolarization on pH(i)

Glucose metabolism in B-cells leads to closure of ATP-sensitive K channels in the plasma membrane, depolarization, stimulation of Ca influx through voltage-dependent Ca channels, and a rise in cytoplasmic [Ca](i). Hypoglycemic sulfonylureas, like tolbutamide, trigger the same sequence of events without changing B-cell metabolism(3, 4, 5, 6) . In contrast to a rise in glucose concentration, the addition of 200 µM tolbutamide to a HCO(3) medium containing 3 mM glucose decreased islet pH(i) by 0.05 ± 0.004 units (n = 9). Tolbutamide (1 mM) has previously been reported to lower B-cell pH(i) in HEPES buffer(20) .

Diazoxide opens ATP-sensitive K channels without interfering with metabolism and thereby prevents glucose from depolarizing B-cells and from increasing [Ca](i)(17, 22) . When the concentration of glucose was increased from 3 to 15 mM in the presence of diazoxide, a sustained rise in pH(i) was observed not only in HCO(3) buffer, but also in HEPES buffer (Fig. 3A).


Figure 3: Influence of B-cell membrane potential on pH in mouse islets. BCECF-loaded islets were perifused with HCO(3) medium (solidlines) or HEPES medium (brokenlines). A, diazoxide (Dz) was added to the medium before the concentration of glucose (G) was raised from 3 to 15 mM to prevent the depolarization of B-cells. B, extracellular potassium was raised to 30 mM at the time of diazoxide addition to depolarize B-cells. The concentration of glucose was then raised from 3 to 15 mM in the presence of high potassium and diazoxide. The traces are the means ± S.E. for 9-12 islets.



When the B-cell membrane was depolarized and cytoplasmic [Ca](i) was increased by 30 mM potassium in the presence of 3 mM glucose and diazoxide (7) , a fall in pH(i) occurred (Fig. 3B). This fall was much larger in HEPES than in HCO(3) buffer. A subsequent rise in the glucose concentration to 15 mM was followed by a sustained rise in pH(i) in both types of solutions (Fig. 3B). Under these conditions, the rise in glucose does not affect steady-state [Ca](i)(7) .

Omission of extracellular Ca during stimulation with 15 mM glucose markedly lowers [Ca](i) in islet cells(17, 23) . This did not result in any major change in pH(i) when the islets were perifused with HCO(3) buffer, but caused a marked alkalinization in HEPES buffer (Fig. 4). Ca reintroduction into the medium was followed by an acidification only in HEPES buffer.

Role of Na/H and HCO(3)/Cl Exchangers in Glucose Effects on pH(i)

Inhibiting the Na/H exchanger with 40 µM DMA in HCO(3) buffer had no significant effect on pH(i) in the presence of 3 mM glucose and did not interfere with the alkalinizing effect of 15 mM glucose (Fig. 5A). Qualitatively similar results were obtained with 100 µM DMA. The rise in pH(i) brought about by 15 mM glucose amounted to 0.12 ± 0.02 and 0.09 ± 0.02 in the presence of 40 and 100 µM DMA, respectively. These values are not significantly different (p = 0.3) from those obtained in the absence of DMA (0.11 ± 0.01). In HEPES medium, when no HCO(3)/Cl exchanger is operative, DMA caused a decrease in pH(i) in the presence of 3 mM glucose (Fig. 5A). On raising the concentration of glucose to 15 mM, there occurred a transient rise in pH(i), as that seen under control conditions, but the subsequent fall in pH(i) was of a greater magnitude and showed no sign of reversing as observed over the same time course in the absence of DMA (compare with Fig. 1C).


Figure 5: Influence of blockers of the HCO(3)/Cl and Na/H exchangers on the pH changes induced by glucose in mouse islets. BCECF-loaded islets were perifused with HCO(3) medium (solid lines) or HEPES medium (broken lines). Ten min before the concentration of glucose (G) was raised from 3 to 15 mM, the following substances were added to the medium: 40 µM DMA (A), 200 µM DIDS (B), and 40 µM DMA and 200 µM DIDS (C). These substances remained present until the end of the experiment. The traces are the means ± S.E. for 9-12 islets.



As expected, DIDS did not affect pH(i) in HEPES buffer, when no HCO(3)/Cl exchanger is operative. DIDS was also without effect in HCO(3) buffer containing 3 mM glucose, but profoundly modified the changes in islet pH(i) induced by 15 mM glucose (Fig. 5B). These became similar to those observed in HEPES medium without DIDS (compare with Fig. 1C).

The combination of DMA and DIDS produced essentially similar effects in HCO(3) and HEPES buffers (Fig. 5C). There occurred a decrease in pH(i) in 3 mM glucose, and the rise in glucose concentration to 15 mM produced a small initial increase in pH(i) followed by a marked decrease.

Finally, we evaluated the contribution of [Ca](i) to the fall in pH(i) caused by glucose when both Na/H and HCO(3)/Cl exchangers are inhibited. The [Ca](i) rise normally produced by glucose was prevented by diazoxide (17) or by blocking Ca channels with nimodipine(24) . When diazoxide was present in HCO(3) medium supplemented with DIDS and DMA or in HEPES medium containing DMA, pH(i) no longer decreased, but increased following the rise in the glucose concentration from 3 to 15 mM (Fig. 6). An increase in pH(i) was also produced by 15 mM glucose in HEPES medium supplemented with DMA and nimodipine (data not shown). The acidification of islet cells, produced by glucose when the Na/H and HCO(3)/Cl exchangers are inhibited, can thus be ascribed to the rise in [Ca](i) that glucose causes.


Figure 6: Role of Ca in the pH changes induced by glucose in mouse islets after blockade of the HCO(3)/Cl and Na/H exchangers. BCECF-loaded islets were perifused with HCO(3) medium (solidline) or HEPES medium (brokenline). Ten min before the concentration of glucose (G) was raised from 3 to 15 mM, the following substances were added to the medium: 40 µM DMA, 200 µM DIDS, and 250 µM diazoxide (Dz) in HCO(3) buffer and 40 µM DMA and 250 µM diazoxide in HEPES buffer. These substances remained present until the end of the experiment. The traces are the means ± S.E. for 7-10 islets.



Effects of Glucose on Insulin Release in HCO(3) and HEPES Buffers

Raising the glucose concentration from 3 to 15 mM in HCO(3) medium triggered a rapid increase in insulin release followed by a sustained second phase (Fig. 7A). In HEPES buffer, the increase in release was delayed and displayed a monophasic pattern. At steady state, however, the rate of insulin release was similar in both types of buffer. These results differ somewhat from those obtained previously with freshly isolated rat islets, from which insulin release was decreased during both phases when the glucose stimulation was applied in the absence of HCO(3)(19) . Amiloride has been reported to decrease or increase insulin release from rat islets(8, 25, 26) , and DIDS has been reported to decrease it(8) . Under our experimental conditions, DMA potentiated and DIDS decreased glucose-induced insulin release in a similar way in HCO(3) and HEPES buffers (Fig. 7, B and C). Although unknown effects of the pharmacological agents on the releasing process are possible, no correlation can thus be found between the changes in pH(i) and those of insulin release.


Figure 7: Effects of glucose on insulin release from mouse islets. Batches of 20 islets were perifused with HCO(3) medium (bullet) or HEPES medium (circle). The concentration of glucose (G) was raised from 3 to 15 mM as indicated. In B and C, the medium was supplemented with 40 µM DMA and 200 µM DIDS, respectively. Note that the perifusion system is different from that used for pH measurements. Values are means ± S.E. for four to five paired experiments.




DISCUSSION

Intact pancreatic islets, which contain 80% insulin-secreting B-cells, were loaded with BCECF and perifused for 25-30 min with both HCO(3) and HEPES buffers to study the transient and sustained changes in pH(i) brought about by glucose. In previous studies, static systems, a single type of buffer, and/or dispersed pancreatic cells have generally been used for relatively short periods of time(11) .

The existence of Na/H exchangers in islet cells is established, but the presence and possible function of HCO(3)/Cl exchangers are less clear (review in (11) ). Experiments using the ammonium prepulse technique (27) showed that both types of exchangers contribute to the recovery from an imposed acid load in mouse B-cells. (^2)This study further shows that both the Na/H and HCO(3)/Cl exchangers also participate in the control of basal pH(i). When the islets were perifused with HCO(3) medium containing 3 mM glucose, pH(i) was little affected by separate blockade of the exchangers. A stronger acidification occurred when DMA and DIDS were combined in HCO(3) buffer or when DMA was added to HEPES buffer, in which DIDS alone was without effect, as expected. We therefore felt it important to compare the effects of high glucose concentrations in HCO(3) buffer (when both exchangers are operative) and in HEPES buffer (when the HCO(3)/Cl exchanger is not operative).

Effects of Glucose on Islet Cell pH(i) in HCO(3) Buffer

In physiological HCO(3) buffer, glucose produced a biphasic increase in islet cell pH(i). The dose-response relationship was hyperbolic with a K(m) of 5 mM. A similar relationship has been observed when pH(i) was measured using the 5,5-dimethyl[2-^14C]oxazolidine-2,4-dione technique(12) . Several lines of evidence support the contention that an acceleration of islet cell metabolism is essential for the alkalinizing effect of glucose. First, the nonmetabolized 3-O-methylglucose (21) did not affect pH(i). Second, inhibition of glucose phosphorylation with mannoheptulose (21) prevented the effect of glucose on pH(i), as already shown by others(12) . Third, inhibition of ATP production by mitochondria reversed the effect of glucose. Fourth, the effect of glucose was mimicked by dihydroxyacetone and by ketoisocaproic acid.

Glucose metabolism in B-cells leads to closure of ATP-sensitive K channels, membrane depolarization, and rise in [Ca](i). This study shows that the alkalinizing effect of glucose does not depend on closure of ATP-sensitive K channels and B-cell depolarization because it persisted when both events were prevented by diazoxide (5, 22) and was not reproduced by tolbutamide, which depolarizes B-cells by closing ATP-sensitive K channels(5, 22) . The alkalinization is also independent of the membrane potential itself because it occurred when glucose was added to both polarized B-cells (4.8 mM potassium + diazoxide) and depolarized B-cells (30 mM potassium + diazoxide). Finally, the rise in pH(i) produced by glucose is not secondary to the [Ca](i) rise because it occurred under conditions where glucose is known not to increase [Ca](i) (4.8 mM potassium + diazoxide) (17) or to only transiently decrease an already elevated [Ca](i) (30 mM potassium + diazoxide)(7) . Omission of extracellular Ca was also without significant effect.

It should be noted, however, that the rise in pH(i) brought about by 15 mM glucose did not display a biphasic pattern when glucose could not also raise [Ca](i). Recent experiments have shown that the rise in [Ca](i) lags slightly behind the rise in pH(i) in single B-cells(28) . Further experiments will be necessary to determine whether it is involved in this biphasic change.

We also wish to point out that, before starting the pH experiments, the quality of all islet preparations was assessed by measuring glucose-induced [Ca](i) changes. Oscillations in [Ca](i) similar to those previously reported (17) were consistently seen. On the other hand, no oscillations in pH(i) were detected even when the data acquisition rate was identical to that at which [Ca](i) oscillations could easily be identified.^2 We cannot exclude the possibility that pH(i) oscillations escaped detection with our technique or could not be seen in a multicellular preparation because they are asynchronous in different cells.

Effects of Glucose on Islet Cell pH(i) in HEPES Buffer

These effects were substantially different from those produced in physiological HCO(3) buffer. Glucose also dose-dependently increased pH(i), but the maximum effect was reached at 7 mM. The smaller effect of higher concentrations may be ascribed to an acidifying action of [Ca](i) for the following reasons.

Omission of extracellular Ca from a medium containing 15 mM glucose, which results in a fall in [Ca](i)(17, 23) , was followed by a prompt rise in pH(i), which attained values similar to those measured in HCO(3) buffer. Moreover, when the glucose-induced [Ca](i) rise was prevented with diazoxide, a rapid sustained alkalinization was caused by 15 mM glucose. This alkalinization also occurred in the presence of high potassium and diazoxide, when glucose does not increase the already elevated [Ca](i)(7) . Finally, the relationship between glucose concentration and pH(i) in HEPES and HCO(3) buffers started to diverge above 7 mM, i.e. above the threshold for membrane depolarization and stimulation of Ca influx(1, 4, 5) . The stronger acidifying effect of [Ca](i) in HEPES compared with HCO(3) buffer is also evidenced by the larger fall in pH(i) produced by 30 mM potassium in the former buffer.

Our results therefore show that the genuine effect of glucose is similar in the absence or presence of HCO(3). The difference in the pH(i) changes results from the masking of the alkalinization by a strong [Ca](i)-induced acidification that is normally prevented by the HCO(3)/Cl exchanger. Further support for this interpretation is provided by the experiments using blockers of the exchangers (see below). That the HCO(3)/Cl exchanger is somehow activated in the presence of high glucose is compatible with the observations that glucose stimulates HCO(3) uptake by (29) and Cl efflux from (30) islet cells and that a fraction of this stimulation is Ca-dependent, even though the interpretation of this dependence is not straightforward.

Is There a Role of the pH-regulating Exchangers in the Effects of Glucose?

DMA is a potent and relatively selective blocker of the Na/H exchanger. At a concentration of 20 µM, it blocks the exchanger completely in other systems (31, 32) . However, 40 µM DMA did not prevent 15 mM glucose from increasing pH(i) in islets perifused with physiological HCO(3) buffer. Even at 100 µM, DMA reduced the effect of glucose by no more than 20%, yet 40 µM DMA was very effective in HEPES buffer or when combined with DIDS in HCO(3) buffer. Under these conditions, glucose provoked a small rise in pH(i) followed by a marked acidification. The effectiveness of DMA is also supported by its strong effects on recovery from an acid load in the presence of DIDS or in HEPES buffer.^2 We therefore cannot support the previous conclusion that the alkalinizing effect of glucose is caused by a stimulation of the Na/H exchanger(16) . This suggestion was primarily based on the observation that glucose decreased mouse islet cell pH(i) in HEPES buffer containing ethylisopropylamiloride. This observation is confirmed here, but we also show that the acidifying effect of glucose does not occur in physiological HCO(3) buffer and, as discussed below, can be explained differently. On the other hand, we have no explanation for the observation that glucose provoked a small decrease in rat islet pH(i) when 100 µM amiloride, a much weaker inhibitor of the Na/H exchanger(31, 32) , was present in HCO(3) buffer(13) . Whether this reflects a species difference or a limitation of the technique used in these early measurements is unclear.

The important role of the HCO(3)/Cl exchanger is strikingly demonstrated by the effects of DIDS. This blocker of the exchanger (33, 34) was without effect in HEPES buffer, when the exchanger is not functioning because of the absence of HCO(3). On the other hand, in HCO(3) buffer supplemented with DIDS, the response to glucose became similar to that occurring in HEPES buffer. After a transient rise, pH(i) decreased slightly before returning to levels similar to those measured before the concentration of glucose was raised. This stabilization of pH(i) at steady state is obviously achieved by the Na/H exchanger. Thus, when both exchangers were inhibited, either by combination of the pharmacological blockers or by addition of DMA alone to HEPES buffer, the initial small increase in pH(i) produced by 15 mM glucose was followed by a marked acidification.

To determine whether this acidification is a proper effect of glucose or is the consequence of the rise in [Ca](i), the latter was prevented with diazoxide or nimodipine. Under these conditions, glucose increased pH(i). It is thus clear that the genuine effect of glucose is to increase pH(i), not to decrease it, when the Na/H and HCO(3)/Cl exchangers are not operative. The decrease in pH(i) is caused by the rise in [Ca](i) that glucose also produces.

Conclusions

It has been often stated that increased cellular metabolism generates acidic products, but it has generally been overlooked that ATP synthesis consumes protons, whereas ATP hydrolysis is the predominant source of intracellular acid load(35) . An acceleration of aerobic metabolism with an increase in the ATP/ADP ratio is usually accompanied by an alkalinization. We suggest that a similar mechanism explains the increase in islet cell pH(i) that occurs upon stimulation with glucose. The possibility that changes in B-cell pH(i) play a role in stimulus-secretion coupling has aroused much interest and prompted several, sometimes divergent hypotheses, which have been reviewed elsewhere(11) . Our experiments show that, during steady-state stimulation with glucose, similar rates of insulin secretion are measured in the face of sometimes substantial differences in pH(i). With the reservation that pH(i) might exert opposite effects on different steps of stimulus-secretion coupling, this study does not support the hypothesis that the changes in pH(i) play a significant role in the B-cell secretory response to glucose.

In conclusion, contrary to previous assumptions, the metabolism of glucose in islet cells tends to increase pH(i), whereas it is the rise in [Ca](i) occurring in glucose-stimulated B-cells that exerts an acidifying action. In HEPES buffer, the acidifying effect of [Ca](i) is offset by the operation of a Na/H exchanger, so that after transient fluctuations, pH(i) returns to approximately basal values at steady state. In physiological HCO(3) buffer, the activity of the HCO(3)/Cl exchanger overcompensates and leads to the increase in pH(i).


FOOTNOTES

*
This work was supported by Grant 3.4525.94 from the Fonds de la Recherche Scientifique Médicale, Brussels, Belgium, and by Grant SPPS-AC 89/95-135 from the Ministry of Scientific Policy, Brussels. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 32-2-7645532.

(^1)
The abbreviations used are: [Ca], free cytoplasmic calcium; pH, cytoplasmic pH; BCECF, 2`,7`-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid; DMA, 5-N,N-dimethylamiloride.

(^2)
R. M. Shepherd and J.-C. Henquin, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Dr. P. Gilon for advice on the use of the fluorometric system, to M. Nicaise for technical assistance, and to M. Nenquin for performing the experiments of insulin secretion and for editorial help.


REFERENCES

  1. Henquin, J.-C. (1994) in Joslin's Diabetes Mellitus (Kahn, C. R., and Weir, G. C., eds) 13th Ed., pp. 56-80, Lea & Febiger, Philadelphia
  2. Prentki, M., and Matschinsky, F. M. (1987) Physiol. Rev. 67, 1185-1248 [Free Full Text]
  3. Ashcroft, F. M., and Rorsman, P. (1989) Prog. Biophys. Mol. Biol. 54, 87-143 [CrossRef][Medline] [Order article via Infotrieve]
  4. Cook, D. L., and Taborsky, G. J. (1990) in Diabetes Mellitus: Theory and Practice (Rifkin, H., and Porte, D., eds) 4th Ed., pp. 89-103, Elsevier Science Publishers B.V., Amsterdam
  5. Henquin, J.-C., Debuyser, A., Drews, G., and Plant, T. D. (1992) in Nutrient Regulation of Insulin Secretion (Flatt, P. R., ed) pp. 173-191, Portland Press Ltd., London
  6. Misler, S., Barnett, D. W., Gillis, K. D., and Pressel, D. M. (1992) Diabetes 41, 1221-1228 [Abstract]
  7. Gembal, M., Gilon, P., and Henquin, J.-C. (1992) J. Clin. Invest. 89, 1288-1295 [Medline] [Order article via Infotrieve]
  8. Pace, C. S., Tarvin, J. T., and Smith, J. S. (1983) Am. J. Physiol. 244, E3-E18
  9. Malaisse, W. J., Malaisse-Lagae, F., and Sener, A. (1984) Experientia (Basel) 40, 1035-1043 [Medline] [Order article via Infotrieve]
  10. Sugimoto, Y. (1988) Biomed. Res. 9, 383-394
  11. Best, L. (1992) in Nutrient Regulation of Insulin Secretion (Flatt, P. R., ed) pp. 157-171, Portland Press Ltd., London
  12. Lindström, P., and Sehlin, J. (1984) Biochem. J. 218, 887-892 [Medline] [Order article via Infotrieve]
  13. Deleers, M., Lebrun, P., and Malaisse, W. J. (1985) Horm. Metab. Res. 17, 391-395 [Medline] [Order article via Infotrieve]
  14. Arkhammar, P., Berggren, P.-O., and Rorsman, P. (1986) Biosci. Rep. 6, 355-361 [Medline] [Order article via Infotrieve]
  15. Best, L., Bone, E. A., Meats, J. E., and Tomlinson, S. (1988) J. Mol. Endocrinol. 1, 33-38 [Abstract]
  16. Juntti-Berggren, L., Arkhammar, P., Nilsson, T., Rorsman, P., and Berggren P.-O. (1991) J. Biol. Chem. 266, 23537-23541 [Abstract/Free Full Text]
  17. Gilon, P., and Henquin, J.-C. (1992) J. Biol. Chem. 267, 20713-20720 [Abstract/Free Full Text]
  18. Henquin, J.-C. (1978) Nature 271, 271-273 [Medline] [Order article via Infotrieve]
  19. Henquin, J.-C., and Lambert, A. E. (1976) Am. J. Physiol. 231, 713-721 [Medline] [Order article via Infotrieve]
  20. Grapengiesser, E., Gylfe, E., and Hellman, B. (1989) Biochim. Biophys. Acta 1014, 219-224 [Medline] [Order article via Infotrieve]
  21. Zawalich, W. S. (1979) Diabetes 28, 252-260 [Medline] [Order article via Infotrieve]
  22. Trube, G., Rorsman, P., and Ohno-Shosaku, T. (1986) Pfluegers Arch. Eur. J. Physiol. 407, 493-499 [Medline] [Order article via Infotrieve]
  23. Theler, J.-M., Mollard, P., Guérineau, N., Vacher, P., Pralong, W. F., Schlegel, W., and Wollheim, C. B. (1992) J. Biol. Chem. 267, 18110-18117 [Abstract/Free Full Text]
  24. Warnotte, C., Gilon, P., Nenquin, M., and Henquin, J.-C. (1994) Diabetes 43, 703-711 [Abstract]
  25. Biden, T. J., Janjic, D., and Wollheim, C. B. (1986) Am. J. Physiol. 250, C207-C213
  26. Lebrun, P., van Ganse, E., Juvent, M., Deleers, M., and Herchuelz, A. (1986) Biochim. Biophys. Acta 886, 448-456 [Medline] [Order article via Infotrieve]
  27. Boron, W. F., and De Weer, P. (1976) J. Gen. Physiol. 67, 91-112 [Abstract]
  28. Juntti-Berggren, L., Civelek, V. N., Berggren, P.-O., Schultz, V., Corkey, B. E., and Tornheim, K. (1994) J. Biol. Chem. 269, 14391-14395 [Abstract/Free Full Text]
  29. Deleers, M., Lebrun, P., and Malaisse, W. J. (1983) FEBS Lett. 154, 97-100 [CrossRef][Medline] [Order article via Infotrieve]
  30. Sehlin, J. (1978) Am. J. Physiol. 235, E501-E508
  31. Vigne, P., Frelin, C., Cragoe, E. J., Jr., and Lazdunski, M. (1984) Mol. Pharmacol. 25, 131-136 [Abstract]
  32. L'Allemain, G., Franchi, A., Cragoe, E., Jr., and Pouysségur, J. (1984) J. Biol. Chem. 259, 4313-4319 [Abstract/Free Full Text]
  33. Cabantchik, Z. I., Knauf, P. A., and Rothstein, A. (1978) Biochim. Biophys. Acta 515, 289-302
  34. Frelin, C., Vigne, P., Ladoux, A., and Lazdunski, M. (1988) Eur. J. Biochem. 174, 3-14 [Abstract]
  35. Busa, W. B., and Nuccitelli, R. (1984) Am. J. Physiol. 246, R409-R438

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.