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• W2070278889 abstract "Intact mouse islets were loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to study the effects of glucose on cytoplasmic pH (pHi) in pancreatic B-cells. In HCO3− buffer, glucose produced a steady-state increase in pHi that required metabolism of the sugar and was concentration-dependent between 0 and 10 mM (Km∼ 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 Ca2+ influx had little effect on the pHi changes produced by glucose in HCO3− medium, but unmasked an alkalinizing effect in HEPES buffer. Raising cytoplasmic Ca2+ by 30 mM potassium caused a larger acidification in HEPES than in HCO3− buffer, but a subsequent rise in glucose now increased pHi in both types of buffer. In the presence of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS; inhibitor of HCO3−/Cl− exchange), the effect of glucose on pHi in HCO3− 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 pHi in HEPES buffer. A similar fall was seen in HCO3− buffer only when DIDS and dimethylamiloride were present together. However, if Ca2+ influx was prevented when both exchangers were blocked, glucose increased pHi. In conclusion, the metabolism of glucose tends to increase pHi in B-cells, whereas the concomitant rise in [Ca2+]i exerts an acidifying action. In HEPES buffer, this acidifying effect of Ca2+ is offset by the operation of the Na+/H+ exchanger. In physiological HCO3− buffer, the activity of the HCO3−/Cl− exchanger overcompensates and leads to an increase in pHi. Intact mouse islets were loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to study the effects of glucose on cytoplasmic pH (pHi) in pancreatic B-cells. In HCO3− buffer, glucose produced a steady-state increase in pHi that required metabolism of the sugar and was concentration-dependent between 0 and 10 mM (Km∼ 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 Ca2+ influx had little effect on the pHi changes produced by glucose in HCO3− medium, but unmasked an alkalinizing effect in HEPES buffer. Raising cytoplasmic Ca2+ by 30 mM potassium caused a larger acidification in HEPES than in HCO3− buffer, but a subsequent rise in glucose now increased pHi in both types of buffer. In the presence of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS; inhibitor of HCO3−/Cl− exchange), the effect of glucose on pHi in HCO3− 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 pHi in HEPES buffer. A similar fall was seen in HCO3− buffer only when DIDS and dimethylamiloride were present together. However, if Ca2+ influx was prevented when both exchangers were blocked, glucose increased pHi. In conclusion, the metabolism of glucose tends to increase pHi in B-cells, whereas the concomitant rise in [Ca2+]i exerts an acidifying action. In HEPES buffer, this acidifying effect of Ca2+ is offset by the operation of the Na+/H+ exchanger. In physiological HCO3− buffer, the activity of the HCO3−/Cl− exchanger overcompensates and leads to an increase in pHi. Glucose plays a pre-eminent role in the control of pancreatic B-cell function(1Henquin J.-C. Kahn C.R. Weir G.C. Joslin's Diabetes Mellitus. 13th Ed. Lea & Febiger, Philadelphia1994: 56-80Google Scholar). The mechanisms by which it stimulates insulin release involve regulation of a number of ionic events through changes in B-cell metabolism(1Henquin J.-C. Kahn C.R. Weir G.C. Joslin's Diabetes Mellitus. 13th Ed. Lea & Febiger, Philadelphia1994: 56-80Google Scholar, 2Prentki M. Matschinsky F.M. Physiol. Rev. 1987; 67: 1185-1248Crossref PubMed Google Scholar, 3Ashcroft F.M. Rorsman P. Prog. Biophys. Mol. Biol. 1989; 54: 87-143Crossref PubMed Scopus (955) Google Scholar, 4Cook D.L. Taborsky G.J. Rifkin H. Porte D. Diabetes Mellitus: Theory and Practice. 4th Ed. Elsevier Science Publishers B.V., Amsterdam1990: 89-103Google Scholar, 5Henquin J.-C. Debuyser A. Drews G. Plant T.D. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 173-191Google Scholar, 6Misler S. Barnett D.W. Gillis K.D. Pressel D.M. Diabetes. 1992; 41: 1221-1228Crossref PubMed Google Scholar). 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 Ca2+ channels. Ca2+ influx through these channels increases, causing a rise in free cytoplasmic calcium [Ca2+]i, 1The abbreviations used are: [Ca2+]ifree cytoplasmic calciumpHicytoplasmic pHBCECF2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluoresceinDIDS4,4′-diisothiocyanostilbene-2,2′-disulfonic acidDMA5-N,N-dimethylamiloride. 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 [Ca2+]i on the secretory machinery(7Gembal M. Gilon P. Henquin J.-C. J. Clin. Invest. 1992; 89: 1288-1295Crossref PubMed Scopus (426) Google Scholar). free cytoplasmic calcium cytoplasmic pH 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid 5-N,N-dimethylamiloride. It has been speculated that protons might be one of the signals produced by glucose metabolism and that changes in cytoplasmic pH (pHi) in B-cells might influence certain steps of stimulus-secretion coupling(8Pace C.S. Tarvin J.T. Smith J.S. Am. J. Physiol. 1983; 244: E3-E18Crossref PubMed Google Scholar, 9Malaisse W.J. Malaisse-Lagae F. Sener A. Experientia (Basel). 1984; 40: 1035-1043Crossref PubMed Scopus (48) Google Scholar, 10Sugimoto Y. Biomed. Res. 1988; 9: 383-394Crossref Scopus (2) Google Scholar, 11Best L. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 157-171Google Scholar). Therefore, several studies have examined the effect of glucose on B-cell pHi, 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 pHi measurement, and of different buffers. The weight of the evidence, however, indicates that glucose produces a slight alkalinization of B-cells(12Lindström P. Sehlin J. Biochem. J. 1984; 218: 887-892Crossref PubMed Scopus (43) Google Scholar, 13Deleers M. Lebrun P. Malaisse W.J. Horm. Metab. Res. 1985; 17: 391-395Crossref PubMed Scopus (21) Google Scholar, 14Arkhammar P. Berggren P.-O. Rorsman P. Biosci. Rep. 1986; 6: 355-361Crossref PubMed Scopus (18) Google Scholar, 15Best L. Bone E.A. Meats J.E. Tomlinson S. J. Mol. Endocrinol. 1988; 1: 33-38Crossref PubMed Scopus (27) Google Scholar, 16Juntti-Berggren L. Arkhammar P. Nilsson T. Rorsman P. Berggren P.-O. J. Biol. Chem. 1991; 266: 23537-23541Abstract Full Text PDF PubMed Google Scholar). On the other hand, the mechanisms by which this alkalinization might occur are not established(11Best L. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 157-171Google Scholar). 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 (16Juntti-Berggren L. Arkhammar P. Nilsson T. Rorsman P. Berggren P.-O. J. Biol. Chem. 1991; 266: 23537-23541Abstract Full Text PDF PubMed Google Scholar) has not taken into consideration the possible contributions of [Ca2+]i and of the HCO3−/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 pHi 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 HCO3−, Ca2+, and inhibitors of the Na+/H+ and HCO3−/Cl− exchangers. A bicarbonate-buffered medium (HCO3− medium) and a HEPES-buffered medium (HEPES medium) were used. The HCO3− medium contained 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, and 24 mM NaHCO3 and was gassed with O2/CO2 (94:6) to maintain pH at 7.4. The HEPES medium contained 135 mM NaCl, 4.8 mM KOH, 2.5 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES and was gassed with O2. 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 Ca2+-free solutions, MgCl2 was substituted for CaCl2, 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 Me2SO, respectively. 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 HCO3− 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(17Gilon P. Henquin J.-C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar). 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 Me2SO. The medium had the same composition (HCO3− 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 ×20 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 × 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 pHi 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 MgCl2, 5 mM glucose, and 20 mM HEPES. Bovine serum albumin was omitted to prevent its precipitation at high pH values. After preincubation, cultured islets were transferred, in batches of 20, into a previously described perifusion system(18Henquin J.-C. Nature. 1978; 271: 271-273Crossref PubMed Scopus (153) Google Scholar). Insulin was measured (19Henquin J.-C. Lambert A.E. Am. J. Physiol. 1976; 231: 713-721Crossref PubMed Google Scholar) in the effluent fractions collected every minute. The results of pHi 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. Basal islet cell pHi values (in 3 mM glucose) averaged 6.98 ± 0.01 (n = 189) and 6.95 ± 0.01 (n = 134) in HCO3− 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 pHi. Sudden omission of HCO3−/CO2 by changing to HEPES buffer was followed by a rapid increase in pHi before the secondary decrease below initial values. Conversely, changing from HEPES buffer to HCO3− buffer initially caused a transient acidification (data not shown). Qualitatively similar observations have been made with single mouse B-cells(20Grapengiesser E. Gylfe E. Hellman B. Biochim. Biophys. Acta. 1989; 1014: 219-224Crossref PubMed Scopus (23) Google Scholar). When the islets were perifused throughout with HCO3− medium containing 3 mM glucose, pHi 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 pHi 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 pHi change is sometimes masked by pooling results obtained in different islets (e.g.Fig. 4).Figure 4:Influence of the omission of extracellular Ca2+ on pHi in mouse islets. BCECF-loaded islets were perifused with HCO3− medium (solidline) or HEPES medium (brokenline). The concentration of glucose (G) was increased from 3 to 15 mM after 5 min. Ca2+ 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the absence of HCO3−, the decrease in pHi 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 pHi 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 pHi changed in three phases: a transient rise was followed by a decrease and an eventual return to about initial values (Fig. 1C). Fig. 2 illustrates the concentration dependence of the glucose-induced rise in islet pHi. 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 pHi values (Fig. 2A) or as the change in pHi within the same islet (Fig. 2B). In a HCO3− buffer, the relationship was hyperbolic with a Km of ∼5 mM and a maximum at ∼15 mM glucose. In the absence of HCO3−, the relationship was more complex. pHi increased between 0 and 7 mM glucose, while the size of the pHi rise became smaller at higher glucose concentrations (Fig. 2B). Because of the distinct effects of high glucose concentrations on pHi in islets perifused with HCO3− or HEPES medium, we checked that the discrepancy was not due to local alkalinization of the HCO3− medium by CO2 loss in the open perifusion chamber. In the first series, the flow rate of the HCO3− solution was doubled, but this did not prevent 15 mM glucose from increasing pHi 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 HCO3− buffer. Under these conditions, 15 mM glucose still increased pHi by 0.10 ± 0.01 units. These values are not statistically different. These experiments were done in a HCO3− medium only. 3-O-Methylglucose is transported in B-cells like glucose, but is not metabolized(21Zawalich W.S. Diabetes. 1979; 28: 252-260Crossref PubMed Scopus (34) Google Scholar). The addition of a 12 mM concentration of this analogue to a medium containing 3 mM glucose did not influence pHi (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(21Zawalich W.S. Diabetes. 1979; 28: 252-260Crossref PubMed Scopus (34) Google Scholar), practically no increase in pHi 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 pHi similar to that caused by glucose (data not shown). Finally, the rise in pHi brought about by glucose was reversed by 2 mM azide, a mitochondrial poison (data not shown). Glucose metabolism in B-cells leads to closure of ATP-sensitive K+ channels in the plasma membrane, depolarization, stimulation of Ca2+ influx through voltage-dependent Ca2+ channels, and a rise in cytoplasmic [Ca2+]i. Hypoglycemic sulfonylureas, like tolbutamide, trigger the same sequence of events without changing B-cell metabolism(3Ashcroft F.M. Rorsman P. Prog. Biophys. Mol. Biol. 1989; 54: 87-143Crossref PubMed Scopus (955) Google Scholar, 4Cook D.L. Taborsky G.J. Rifkin H. Porte D. Diabetes Mellitus: Theory and Practice. 4th Ed. Elsevier Science Publishers B.V., Amsterdam1990: 89-103Google Scholar, 5Henquin J.-C. Debuyser A. Drews G. Plant T.D. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 173-191Google Scholar, 6Misler S. Barnett D.W. Gillis K.D. Pressel D.M. Diabetes. 1992; 41: 1221-1228Crossref PubMed Google Scholar). In contrast to a rise in glucose concentration, the addition of 200 μM tolbutamide to a HCO3− medium containing 3 mM glucose decreased islet pHi by 0.05 ± 0.004 units (n = 9). Tolbutamide (1 mM) has previously been reported to lower B-cell pHi in HEPES buffer(20Grapengiesser E. Gylfe E. Hellman B. Biochim. Biophys. Acta. 1989; 1014: 219-224Crossref PubMed Scopus (23) Google Scholar). Diazoxide opens ATP-sensitive K+ channels without interfering with metabolism and thereby prevents glucose from depolarizing B-cells and from increasing [Ca2+]i(17Gilon P. Henquin J.-C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar, 22Trube G. Rorsman P. Ohno-Shosaku T. Pfluegers Arch. Eur. J. Physiol. 1986; 407: 493-499Crossref PubMed Scopus (527) Google Scholar). When the concentration of glucose was increased from 3 to 15 mM in the presence of diazoxide, a sustained rise in pHi was observed not only in HCO3− buffer, but also in HEPES buffer (Fig. 3A). When the B-cell membrane was depolarized and cytoplasmic [Ca2+]i was increased by 30 mM potassium in the presence of 3 mM glucose and diazoxide (7Gembal M. Gilon P. Henquin J.-C. J. Clin. Invest. 1992; 89: 1288-1295Crossref PubMed Scopus (426) Google Scholar), a fall in pHi occurred (Fig. 3B). This fall was much larger in HEPES than in HCO3− buffer. A subsequent rise in the glucose concentration to 15 mM was followed by a sustained rise in pHi in both types of solutions (Fig. 3B). Under these conditions, the rise in glucose does not affect steady-state [Ca2+]i(7Gembal M. Gilon P. Henquin J.-C. J. Clin. Invest. 1992; 89: 1288-1295Crossref PubMed Scopus (426) Google Scholar). Omission of extracellular Ca2+ during stimulation with 15 mM glucose markedly lowers [Ca2+]i in islet cells(17Gilon P. Henquin J.-C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar, 23Theler J.-M. Mollard P. Guérineau N. Vacher P. Pralong W.F. Schlegel W. Wollheim C.B. J. Biol. Chem. 1992; 267: 18110-18117Abstract Full Text PDF PubMed Google Scholar). This did not result in any major change in pHi when the islets were perifused with HCO3− buffer, but caused a marked alkalinization in HEPES buffer (Fig. 4). Ca2+ reintroduction into the medium was followed by an acidification only in HEPES buffer. Inhibiting the Na+/H+ exchanger with 40 μM DMA in HCO3− buffer had no significant effect on pHi 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 pHi 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 HCO3−/Cl− exchanger is operative, DMA caused a decrease in pHi in the presence of 3 mM glucose (Fig. 5A). On raising the concentration of glucose to 15 mM, there occurred a transient rise in pHi, as that seen under control conditions, but the subsequent fall in pHi 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). As expected, DIDS did not affect pHi in HEPES buffer, when no HCO3−/Cl− exchanger is operative. DIDS was also without effect in HCO3− buffer containing 3 mM glucose, but profoundly modified the changes in islet pHi 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 HCO3− and HEPES buffers (Fig. 5C). There occurred a decrease in pHi in 3 mM glucose, and the rise in glucose concentration to 15 mM produced a small initial increase in pHi followed by a marked decrease. Finally, we evaluated the contribution of [Ca2+]i to the fall in pHi caused by glucose when both Na+/H+ and HCO3−/Cl− exchangers are inhibited. The [Ca2+]i rise normally produced by glucose was prevented by diazoxide (17Gilon P. Henquin J.-C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar) or by blocking Ca2+ channels with nimodipine(24Warnotte C. Gilon P. Nenquin M. Henquin J.-C. Diabetes. 1994; 43: 703-711Crossref PubMed Scopus (207) Google Scholar). When diazoxide was present in HCO3− medium supplemented with DIDS and DMA or in HEPES medium containing DMA, pHi no longer decreased, but increased following the rise in the glucose concentration from 3 to 15 mM (Fig. 6). An increase in pHi 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 HCO3−/Cl− exchangers are inhibited, can thus be ascribed to the rise in [Ca2+]i that glucose causes. Raising the glucose concentration from 3 to 15 mM in HCO3− 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 HCO3−(19Henquin J.-C. Lambert A.E. Am. J. Physiol. 1976; 231: 713-721Crossref PubMed Google Scholar). Amiloride has been reported to decrease or increase insulin release from rat islets(8Pace C.S. Tarvin J.T. Smith J.S. Am. J. Physiol. 1983; 244: E3-E18Crossref PubMed Google Scholar, 25Biden T.J. Janjic D. Wollheim C.B. Am. J. Physiol. 1986; 250: C207-C213Crossref PubMed Google Scholar, 26Lebrun P. van Ganse E. Juvent M. Deleers M. Herchuelz A. Biochim. Biophys. Acta. 1986; 886: 448-456Crossref PubMed Scopus (19) Google Scholar), and DIDS has been reported to decrease it(8Pace C.S. Tarvin J.T. Smith J.S. Am. J. Physiol. 1983; 244: E3-E18Crossref PubMed Google Scholar). Under our experimental conditions, DMA potentiated and DIDS decreased glucose-induced insulin release in a similar way in HCO3− 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 pHi and those of insulin release. Intact pancreatic islets, which contain ∼80% insulin-secreting B-cells, were loaded with BCECF and perifused for 25-30 min with both HCO3− and HEPES buffers to study the transient and sustained changes in pHi 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(11Best L. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 157-171Google Scholar). The existence of Na+/H+ exchangers in islet cells is established, but the presence and possible function of HCO3−/Cl− exchangers are less clear (review in (11Best L. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 157-171Google Scholar)). Experiments using the ammonium prepulse technique (27Boron W.F. De Weer P. J. Gen. Physiol. 1976; 67: 91-112Crossref PubMed Scopus (683) Google Scholar) showed that both types of exchangers contribute to the recovery from an imposed acid load in mouse B-cells. 2R. M. Shepherd and J.-C. Henquin, unpublished data. This study further shows that both the Na+/H+ and HCO3−/Cl− exchangers also participate in the control of basal pHi. When the islets were perifused with HCO3− medium containing 3 mM glucose, pHi was little affected by separate blockade of the exchangers. A stronger acidification occurred when DMA and DIDS were combined in HCO3− 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 HCO3− buffer (when both exchangers are operative) and in HEPES buffer (when the HCO3−/Cl− exchanger is not operative). In physiological HCO3− buffer, glucose produced a biphasic increase in islet cell pHi. The dose-response relationship was hyperbolic with a Km of ∼5 mM. A similar relationship has been observed when pHi was measured using the 5,5-dimethyl[2-14C]oxazolidine-2,4-dione technique(12Lindström P. Sehlin J. Biochem. J. 1984; 218: 887-892Crossref PubMed Scopus (43) Google Scholar). 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 (21Zawalich W.S. Diabetes. 1979; 28: 252-260Crossref PubMed Scopus (34) Google Scholar) did not affect pHi. Second, inhibition of glucose phosphorylation with mannoheptulose (21Zawalich W.S. Diabetes. 1979; 28: 252-260Crossref PubMed Scopus (34) Google Scholar) prevented the effect of glucose on pHi, as already shown by others(12Lindström P. Sehlin J. Biochem. J. 1984; 218: 887-892Crossref PubMed Scopus (43) Google Scholar). 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 [Ca2+]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 (5Henquin J.-C. Debuyser A. Drews G. Plant T.D. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 173-191Google Scholar, 22Trube G. Rorsman P. Ohno-Shosaku T. Pfluegers Arch. Eur. J. Physiol. 1986; 407: 493-499Crossref PubMed Scopus (527) Google Scholar) and was not reproduced by tolbutamide, which depolarizes B-cells by closing ATP-sensitive K+ channels(5Henquin J.-C. Debuyser A. Drews G. Plant T.D. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 173-191Google Scholar, 22Trube G. Rorsman P. Ohno-Shosaku T. Pfluegers Arch. Eur. J. Physiol. 1986; 407: 493-499Crossref PubMed Scopus (527) Google Scholar). 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 pHi produced by glucose is not secondary to the [Ca2+]i rise because it occurred under conditions where glucose is known not to increase [Ca2+]i (4.8 mM potassium + diazoxide) (17Gilon P. Henquin J.-C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar) or to only transiently decrease an already elevated [Ca2+]i (30 mM potassium + diazoxide)(7Gembal M. Gilon P. Henquin J.-C. J. Clin. Invest. 1992; 89: 1288-1295Crossref PubMed Scopus (426) Google Scholar). Omission of extracellular Ca2+ was also without significant effect. It should be noted, however, that the rise in pHi brought about by 15 mM glucose did not display a biphasic pattern when glucose could not also raise [Ca2+]i. Recent experiments have shown that the rise in [Ca2+]i lags slightly behind the rise in pHi in single B-cells(28Juntti-Berggren L. Civelek V.N. Berggren P.-O. Schultz V. Corkey B.E. Tornheim K. J. Biol. Chem. 1994; 269: 14391-14395Abstract Full Text PDF PubMed Google Scholar). 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 [Ca2+]i changes. Oscillations in [Ca2+]i similar to those previously reported (17Gilon P. Henquin J.-C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar) were consistently seen. On the other hand, no oscillations in pHi were detected even when the data acquisition rate was identical to that at which [Ca2+]i oscillations could easily be identified.2 We cannot exclude the possibility that pHi oscillations escaped detection with our technique or could not be seen in a multicellular preparation because they are asynchronous in different cells. These effects were substantially different from those produced in physiological HCO3− buffer. Glucose also dose-dependently increased pHi, but the maximum effect was reached at ∼7 mM. The smaller effect of higher concentrations may be ascribed to an acidifying action of [Ca2+]i for the following reasons. Omission of extracellular Ca2+ from a medium containing 15 mM glucose, which results in a fall in [Ca2+]i(17Gilon P. Henquin J.-C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar, 23Theler J.-M. Mollard P. Guérineau N. Vacher P. Pralong W.F. Schlegel W. Wollheim C.B. J. Biol. Chem. 1992; 267: 18110-18117Abstract Full Text PDF PubMed Google Scholar), was followed by a prompt rise in pHi, which attained values similar to those measured in HCO3− buffer. Moreover, when the glucose-induced [Ca2+]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 [Ca2+]i(7Gembal M. Gilon P. Henquin J.-C. J. Clin. Invest. 1992; 89: 1288-1295Crossref PubMed Scopus (426) Google Scholar). Finally, the relationship between glucose concentration and pHi in HEPES and HCO3− buffers started to diverge above 7 mM, i.e. above the threshold for membrane depolarization and stimulation of Ca2+ influx(1Henquin J.-C. Kahn C.R. Weir G.C. Joslin's Diabetes Mellitus. 13th Ed. Lea & Febiger, Philadelphia1994: 56-80Google Scholar, 4Cook D.L. Taborsky G.J. Rifkin H. Porte D. Diabetes Mellitus: Theory and Practice. 4th Ed. Elsevier Science Publishers B.V., Amsterdam1990: 89-103Google Scholar, 5Henquin J.-C. Debuyser A. Drews G. Plant T.D. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 173-191Google Scholar). The stronger acidifying effect of [Ca2+]i in HEPES compared with HCO3− buffer is also evidenced by the larger fall in pHi 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 HCO3−. The difference in the pHi changes results from the masking of the alkalinization by a strong [Ca2+]i-induced acidification that is normally prevented by the HCO3−/Cl− exchanger. Further support for this interpretation is provided by the experiments using blockers of the exchangers (see below). That the HCO3−/Cl− exchanger is somehow activated in the presence of high glucose is compatible with the observations that glucose stimulates HCO3− uptake by (29Deleers M. Lebrun P. Malaisse W.J. FEBS Lett. 1983; 154: 97-100Crossref PubMed Scopus (29) Google Scholar) and Cl− efflux from (30Sehlin J. Am. J. Physiol. 1978; 235: E501-E508PubMed Google Scholar) islet cells and that a fraction of this stimulation is Ca2+-dependent, even though the interpretation of this dependence is not straightforward. 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 (31Vigne P. Frelin C. Cragoe Jr., E.J. Lazdunski M. Mol. Pharmacol. 1984; 25: 131-136PubMed Google Scholar, 32L'Allemain G. Franchi A. Cragoe Jr., E. Pouysségur J. J. Biol. Chem. 1984; 259: 4313-4319Abstract Full Text PDF PubMed Google Scholar). However, 40 μM DMA did not prevent 15 mM glucose from increasing pHi in islets perifused with physiological HCO3− 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 HCO3− buffer. Under these conditions, glucose provoked a small rise in pHi 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(16Juntti-Berggren L. Arkhammar P. Nilsson T. Rorsman P. Berggren P.-O. J. Biol. Chem. 1991; 266: 23537-23541Abstract Full Text PDF PubMed Google Scholar). This suggestion was primarily based on the observation that glucose decreased mouse islet cell pHi 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 HCO3− 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 pHi when 100 μM amiloride, a much weaker inhibitor of the Na+/H+ exchanger(31Vigne P. Frelin C. Cragoe Jr., E.J. Lazdunski M. Mol. Pharmacol. 1984; 25: 131-136PubMed Google Scholar, 32L'Allemain G. Franchi A. Cragoe Jr., E. Pouysségur J. J. Biol. Chem. 1984; 259: 4313-4319Abstract Full Text PDF PubMed Google Scholar), was present in HCO3− buffer(13Deleers M. Lebrun P. Malaisse W.J. Horm. Metab. Res. 1985; 17: 391-395Crossref PubMed Scopus (21) Google Scholar). Whether this reflects a species difference or a limitation of the technique used in these early measurements is unclear. The important role of the HCO3−/Cl− exchanger is strikingly demonstrated by the effects of DIDS. This blocker of the exchanger (33Cabantchik Z.I. Knauf P.A. Rothstein A. Biochim. Biophys. Acta. 1978; 515: 289-302Google Scholar, 34Frelin C. Vigne P. Ladoux A. Lazdunski M. Eur. J. Biochem. 1988; 174: 3-14Crossref PubMed Scopus (282) Google Scholar) was without effect in HEPES buffer, when the exchanger is not functioning because of the absence of HCO3−. On the other hand, in HCO3− buffer supplemented with DIDS, the response to glucose became similar to that occurring in HEPES buffer. After a transient rise, pHi decreased slightly before returning to levels similar to those measured before the concentration of glucose was raised. This stabilization of pHi 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 pHi 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 [Ca2+]i, the latter was prevented with diazoxide or nimodipine. Under these conditions, glucose increased pHi. It is thus clear that the genuine effect of glucose is to increase pHi, not to decrease it, when the Na+/H+ and HCO3−/Cl− exchangers are not operative. The decrease in pHi is caused by the rise in [Ca2+]i that glucose also produces. 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(35Busa W.B. Nuccitelli R. Am. J. Physiol. 1984; 246: R409-R438PubMed Google Scholar). 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 pHi that occurs upon stimulation with glucose. The possibility that changes in B-cell pHi play a role in stimulus-secretion coupling has aroused much interest and prompted several, sometimes divergent hypotheses, which have been reviewed elsewhere(11Best L. Flatt P.R. Nutrient Regulation of Insulin Secretion. Portland Press Ltd., London1992: 157-171Google Scholar). 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 pHi. With the reservation that pHi might exert opposite effects on different steps of stimulus-secretion coupling, this study does not support the hypothesis that the changes in pHi play a significant role in the B-cell secretory response to glucose." @default.
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• W2070278889 title "The Role of Metabolism, Cytoplasmic Ca2+, and pH-regulating Exchangers in Glucose-induced Rise of Cytoplasmic pH in Normal Mouse Pancreatic Islets" @default.
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