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Endocrinology Vol. 145, No. 10 4540-4549
Copyright © 2004 by The Endocrine Society

Ca2+-Induced Ca2+ Release in Pancreatic Islet ß-Cells: Critical Evaluation of the Use of Endoplasmic Reticulum-Targeted "Cameleons"

Aniko Varadi and Guy A. Rutter

Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom

Address all correspondence and requests for reprints to: Dr. G. A. Rutter, Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. E-mail: g.a.rutter{at}bris.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Elevated glucose concentrations cause Ca2+ influx and the exocytotic release of insulin from pancreatic islet ß-cells. Whether increases in cytosolic free Ca2+ concentration also mobilize Ca2+ from intracellular stores (Ca2+-induced Ca2+ release) is unresolved. Endoplasmic reticulum-targeted cameleons have previously been used to explore the involvement of endoplasmic reticulum (ER) Ca2+ release in these cells, albeit with differing conclusions. Cameleons comprise two spectrally shifted green fluorescent proteins, enhanced cyan and yellow fluorescent protein, whose orientation is affected by Ca2+, changing intramolecular fluorescence resonance energy transfer. By measuring pH in the cytosol and ER lumen, we demonstrate that high K+ concentrations (>20 mM) acidify both compartments in clonal MIN6 ß-cells when external bicarbonate concentrations are low (<5 mM), interfering with measurements using Ycam-2 and Ycam-4ER. However, when intracellular pH is strongly buffered (24 mM HCO3–), glucose or cell depolarization increases ER [Ca2+] monitored with Ycam-4ER. KCl-induced increases in ER [Ca2+] were diminished when intracellular stores were sensitized with 1 mM caffeine and inhibited by pretreatment with ryanodine. Furthermore, preincubation with ryanodine tended to slow the falling phase of the ER Ca2+ transient after cell depolarization with KCl and reduced the peak cytosolic [Ca2+]. By contrast, stimulation with glucose increased ER [Ca2+] both in the absence and presence of caffeine or ryanodine. These observations suggest that Ca2+-induced ER Ca2+ release can occur in ß-cells under some conditions but may not be essential for glucose-stimulated insulin secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PANCREATIC ISLET ß-cells respond to high glucose concentrations with the exocytosis of stored insulin (1). The triggering of release involves enhanced oxidative metabolism, closure of ATP-sensitive K+ channels, plasma membrane depolarization, and Ca2+ influx through voltage-gated Ca2+ channels (1). ß-Cells are equipped with extensive intracellular Ca2+ stores that correspond to both the endoplasmic reticulum (ER)/Golgi apparatus (2, 3, 4) and secretory vesicles (5, 6). ER/Golgi stores are mobilized by neurotransmitters and other agents that generate intracellular inositol 1,4,5-Trisphosphate (7). Whereas both the ER/Golgi (8, 9) and vesicles (5, 6) also appear to possess ryanodine receptors, the role of Ca2+-induced Ca2+ release (CICR) in ß-cells is uncertain (10). Using targeted aequorin in ß-cell lines, we (5) and others (3) have reported increases in the concentration of Ca2+ within the ER ([Ca2+]ER) in response to elevated glucose concentrations or cell depolarization with KCl. Moreover, measurements with the fluorescent probe furaptra provided similar findings in primary mouse ß-cells (2). Finally, a recombinant fluorescence resonance energy transfer (FRET)-based cameleon, molecularly targeted to the ER (Ycam-4ER) (11), reported an increase in free [Ca2+] in this compartment in single MIN6 ß-cells (12) in parallel with increases in the concentration of free Ca2+ in the cytosol [Ca2+]c, reported with the analogous cytosolic construct (Ycam-2). Furthermore, transgenic expression of a FRET-based cameleon in primary mouse ß-cells also reported an increase in ER Ca2+ concentration after glucose stimulation (13).

In contrast to the above findings, a recent study (14) suggested that Ycam-4ER reports decreases in [Ca2+]ER in MIN6 cells depolarized with KCl. However, in this study, cytosolic Ca2+ concentrations were measured using only the relatively pH-insensitive Ca2+ probe, fura 2, and the potential confounding effects of changes in intracellular pH on cameleon fluorescence (11, 15) were not examined.

Using the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we show here that depolarization of MIN6 cells with KCl concentrations exceeding approximately 20 mM causes progressive acidification, especially in media containing low bicarbonate concentrations (2.5 or 5.0 mM). This leads to a marked suppression of enhanced yellow fluorescent protein (EYFP) fluorescence with little if any change in enhanced cyan fluorescent protein (ECFP) fluorescence (11). These changes in the relative fluorescence of EYFP and ECFP give the appearance of a reduction in intramolecular FRET when Ycam-2 or Ycam-4ER are used, thus falsely reporting a fall in [Ca2+]c or [Ca2+]ER, respectively (14). Correspondingly, under the same depolarizing conditions, the less pH-sensitive Ca2+ probe fura 2 reports the expected increase in cytosolic [Ca2+], whereas Ycam-2 reports an apparent decrease in [Ca2+]c. However, at higher bicarbonate concentrations (24 mM), KCl-induced acidification of the cytosol (16) and ER are greatly reduced, permitting faithful recordings of cytosolic and ER [Ca2+]. Whereas cell depolarization or treatment with elevated glucose concentrations are confirmed as usually producing net increases in [Ca2+]ER (12), evidence that cytosolic Ca2+ ions regulate the activity of ER Ca2+ release pathways under some circumstances is provided.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Optimem I serum-free medium and Lipofectamine were from Life Technologies, Inc. (Life Science Research, Paisley, Scotland, UK). Fura 2 and BCECF were from Molecular Probes (Eugene, OR). ER-ECFP and ER-EYFP were obtained from BD Biosciences Clontech UK (Oxford, UK). All other chemicals and tissue culture materials were from Sigma (Poole, Dorset, UK).

Cell culture
MIN6 pancreatic ß-cells (passages 19–35) were cultured in DMEM supplemented with 15% (vol/vol) fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 C in an atmosphere of humidified air (95%) and CO2 (5%) as described previously (17). Assay of the release of cotransfected human GH, used as an insulin surrogate (18), revealed a 3.2 ± 0.7-fold (n = three separate cultures) stimulation of hormone release when cells were incubated 20 min at 30 vs. 3 mM glucose.

Transient transfection of MIN6 cells with cameleon, EYFP, and ECFP constructs
MIN6 cells were seeded at a density of 0.4–0.6 x 106/ml on 24-mm-diameter poly-L-lysine-coated coverslips and cultured overnight. Cells were transfected with 1 µg plasmid/coverslip encoding the untargeted (Ycam-2) or ER-targeted (Ycam-4ER) cameleon (19) and 10 µg/ml–1 Lipofectamine in Optimem I medium for 4 h. Cells were then cultured for 2–4 d in growth medium containing 25 mM glucose, which was then replaced with DMEM containing 3 mM glucose 12 h before Ca2+ imaging.

ECFP, EYFP, ER-targeted ECFP, and EYFP were expressed from separate plasmids as described (20) and imaged using the excitation and emission wavelengths given elsewhere (see Fig. 2Go).



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  		FIG. 2. Effect of KCl on pHi reported with BCECF (A) and fluorescence of EYFP (B) or ECFP (C). A, Cells were loaded with BCECF and measurements of fluorescence performed as described in Materials and Methods. Calibration experiments (40 ) revealed that an increase in fluorescence emission (440/490) of 1% was equivalent to a decrease in pHi of approximately 0.015. BA, Butyric acid. B and C, Effects of KCl on the fluorescence of EYFP (B) and ECFP (C) during excitation at 440 nm (emission 480 nM) and excitation at 505 (emission 535), respectively. Data are the means ± SEM of measurements on 12–21 separate cells in each case. **, P < 0.01 for the effects of KCl.

 
Single-cell Ca2+ imaging with targeted cameleons
MIN6 cells were perifused at a constant rate of 1–2 ml/min–1 with one of the following: 1) modified Krebs-Ringer-HEPES-bicarbonate (KRH buffer) (14) [140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 2.0 mM NaHCO3, 3 mM glucose, 10 mM HEPES (pH 7.4), and 1.0 mM CaCl2 equilibrated with O2/CO2 (95:5, vol/vol)]; 2) Krebs buffered Ringer’s solution (KBRS) (13) [117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5.0 mM NaHCO3, 20 mM HEPES (pH 7.4), 0.1% (wt/vol) BSA, and 3 mM glucose equilibrated with O2/CO2 (95:5, vol/vol)]; or 3) bicarbonate-buffered medium (BBM) (16) [120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 24 mM NaHCO3, and 3 mM glucose, gassed with O2/CO2 (95:5, vol/vol) to maintain pH at 7.4]. When the KCl concentration was raised to 25 mM, the concentration of NaCl was decreased accordingly. Cells were imaged at 37 C on an inverted optics Leica (Heidelberg, Germany) DM/IRBE epifluorescence microscope by using a x63 PL Apo 1.4 oil immersion objective. Cells were illuminated with a 100-W mercury arc lamp, and the excitatory light attenuated with a neutral density filter plus a 440 ± 21 nm excitation filter. The microscope was fitted with a 455-nm long pass dichroic mirror, and two emission filters (480DF30 for ECFP and 535DF25 for EYFP, all from Omega Optical, Glen Spectra, Middlesex, UK), alternated with a mechanical filter wheel (Ludl Electronic Products, Hawthorn, NY). Images were acquired on a 12-bit cooled charge- coupled device camera (C4742–95, Hamamatsu, Hamamatsu City, Japan) every 10–20 sec, and controlled by Openlab software (Improvision, Coventry, UK) running on an Apple Macintosh G3 Powerbook. Bleaching was corrected by fitting a simple exponential equation (Microsoft Excel, Microsoft Corp., Redmond, CA) to the ratio values preceding the first addition of stimulants and thus generate the predicted bleaching in the absence of further additions (12). Control experiments (without additions) showed a good approximation of the actual bleaching observed when stimulant was not added. Corrected traces were then generated by division of the observed ratio values by those predicted by the fitted exponential (12).

Measurement of [Ca2+]c with fura 2
Changes in [Ca2+]c were measured at 37 C with entrapped fura 2 (12) using a Leica DM-IRBI inverted microscope (x40 objective) and a Hamamatsu C4742–995 charge-coupled device camera driven by Openlab software (Improvision) (12). Cells were loaded with 5 µM fura 2/AM in the presence of 0.1% (wt/vol) Pluronic F-127 (BASF, Mount Olive, NJ) for 40 min in KRH buffer containing 3 mM glucose.

Measurement of changes in intracellular pH (pHi)
Intracellular pH changes were measured by monitoring fluorescence changes at the single-cell level using BCECF, a pHi indicator localized largely to the cell cytosol (21). Suspensions of MIN6 cells were loaded with 10 µM BCECF for 30 min at room temperature under continuous mixing and then washed with KRH buffer and seeded onto poly-L-lysine-coated coverslips. Cells were perifused continuously at a flow rate of 2 ml/min–1 on the stage of a Diaphot microscope (Nikon, Tokyo, Japan) equipped with a x40 oil immersion objective. The ratio of the emitted light (>510 nm) at two excitation wavelengths (440/490 nm) was used to monitor pHi, using commercially available software (Cairn Instruments, Faversham, UK) for data acquisition. The effects of KCl on pHi were measured in all three buffers listed above. KCl was added isotonically by decreasing NaCl concentrations in all experiments.

Statistics
Data are the means ± SEM for the number of experiments indicated. Statistical significance was calculated by one-tailed Student’s t test, using Microsoft Excel software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Monitoring FRET changes of Ycam-2 and Ycam-4ER in response to various KCl concentrations in KRH buffer
As previously reported, in media containing a low (2.5 mM) HCO3– concentration (12), exposure of MIN6 cells expressing Ycam-2 to 15 mM KCl increased both the apparent intramolecular FRET of Ycam-2 (Fig. 1AGo) and the fluorescence ratio (340/380 nm) of fura 2 (Fig. 1FGo), consistent with the opening of voltage-gated Ca2+ channels and increases in [Ca2+]c. By contrast, a higher KCl concentration (30 mM) decreased the FRET ratio of both Ycam-2 (Fig. 1CGo) and the ER-targeted cameleon, Ycam-4ER (Fig. 1DGo) but still provoked the expected marked increase in cytosolic [Ca2+] as reported with fura 2 (Fig. 1FGo). The effects of KCl were concentration dependent (Fig. 1EGo) with no significant change in FRET ratio reported by either Ycam-2 or Ycam-4ER at 20 mM KCl but marked decreases at 25, 30, or 70 mM KCl (Fig. 1EGo), in line with previous findings (14) using 25 mM KCl.



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  		FIG. 1. Apparent [Ca2+]c and [Ca2+]ER changes induced by KCl. MIN6 cells were transfected with plasmids encoding Ycam-2 (A and C) or Ycam-4ER (B and D) and 2–4 d later used for Ca2+ imaging. Cells were cultured in medium containing 3 mM glucose 12 h before imaging. The effects of 15 (A and B) or 30 mM KCl (C and D) on ratio values in KRH buffer (containing 10 mM HEPES and 2.5 mM HCO3–; see Materials and Methods) are shown. Ratio values were obtained from 72 (A), 115 (B), 56 (C), and 47 (D) cells from three to six independent experiments. When added (C), ionomycin was present at 10 µM. E, Effects of KCl on the apparent ratio changes of Ycam-2 and Ycam-4ER; data are means ± SEM from three to seven independent experiments involving at least 13 cells in total. F, KCl-induced increases in cytosolic Ca2+ concentration as reported with fura 2 (see Materials and Methods). Data are the means ± SEM of experiments on 151 separate cells (six independent experiments).

 
This discrepancy between the apparent decrease in [Ca2+]c in response to 30 mM KCl reported by Ycam-2 on the one hand (Fig. 1CGo), and the increase in [Ca2+]c reported with fura 2 (Fig. 1FGo), led us determine whether other factors might influence the FRET ratio of Ycam-2. In addition to increases in [Ca2+]c, both decreases in pH (16, 22) and increases in free [ATP] (23, 24) have been reported to accompany cell depolarization. Whereas changes in ATP concentration seemed unlikely to affect the fluorescence of either of the green fluorescent protein (GFP) mutants in Ycam-2 or the calmodulin-M13 Ca2+ sensing unit, EYFP is known to be highly sensitive to changes in pH (25). As shown in Fig. 2Go, whereas 15 mM KCl had no detectable effect on intracellular pH reported with the fluorescent probe BCECF, under these conditions (2.5 mM HCO3–; KRH), KCl concentrations of 30 mM or above caused a profound acidification of the cell cytosol (by ~0.3 or 0.6 pH units at 30 and 70 mM, respectively). These changes occurred from a resting intracellular pH value of 7.041 ± 0.024 (mean ± SEM for three separate experiments) in KRH buffer, as determined by a null point method (26) (Fig. 2AGo). This value lies within the range (6.95–7.1) previously reported for both primary mouse and clonal (HIT-T15) ß-cells at substimulatory glucose concentrations (16, 22, 27, 28).

Changes in EYFP and ECFP fluorescence in response to KCl in KRH buffer
To determine whether KCl-dependent decreases in pH led to the predicted decreases in EYFP and ECFP fluorescence, these proteins were expressed separately in MIN6 cells. Whereas the addition of 15 mM KCl had no effect on EYFP (Fig. 2BGo) or ECFP (not shown) fluorescence, 30 or 70 mM KCl caused marked (21 and 44%) falls in the fluorescence of EYFP, but no significant change in the fluorescence of ECFP, measured at the respective excitation and emission maximum of each fluorophore (Fig. 2Go, B and C). These changes are exactly in line with the expected behavior of these two GFP mutants reported previously in vitro and in living cells (pKa of EYFP = 7.1, Hill coefficient, h = 1.1; pKa of ECFP = 6.4, h = 0.6) (25), given a resting pHi value of approximately 7.05 (see above).

Monitoring FRET changes of Ycam-2 and Ycam-4ER in response to KCl in various extracellular buffers
To explore the impact of intracellular pH buffering on the reported [Ca2+] changes, we next monitored cameleon and BCECF fluorescence in three different extracellular buffers: 1) KRH buffer (containing 2.5 mM HCO3– and 10 mM HEPES), as used for our earlier experiments (12); 2) KBRS buffer (5 mM HCO3–, 20 mM HEPES) (14); and 3) a physiological BBM (24 mM HCO3–, zero HEPES) in which KCl has been reported to cause less cytosolic acidification than in HEPES buffer (16). Cells were stimulated by the addition of 25 mM KCl. Although the null point titration approach (26) could not be used to calibrate resting pHi values in the presence of these higher HCO3– concentrations, previous measurements in mouse ß-cells (16) indicated that resting pHi values are unlikely to differ significantly (i.e. by more than 0.05 pH units) in either KBRS or BBM, compared with those in KRH medium, consistent with resting pHi values close to 7.05. Correspondingly, we observed essentially identical resting values for the fluorescence excitation ratio of BCECF in each buffer (data not shown). In KRH and KBRS buffers, a drop in the emission ratio of both Ycam-2 and Ycam-4ER was observed (Fig. 3Go, A, B, D, E, and J). This was accompanied by a fall in pHi, as reported with BCECF (Fig. 3Go, C, F, and K). In contrast, a very small change in intracellular pH (Fig. 3Go, I and K) and an increase of both Ycam-2 and Ycam-4ER FRET (Fig. 3Go G, H, and J) were observed in physiological HCO3– buffer (BBM). These data suggest that at this KCl concentration, the cameleon constructs principally report pH-related changes rather than true [Ca2+] changes when measured in cells maintained in either KRH or KBRS buffers. However, in the physiological HCO3– buffer (BBM), 25 mM KCl prompted a much less pronounced decrease in pHi, (Fig. 3Go, I and K), and as expected the emission ratio of Ycam-2 and Ycam-4ER were increased (Fig. 3Go, G, H, and J).



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  		FIG. 3. Emission ratio changes of Ycam-2, Ycam-4ER, and BCECF induced by 25 mM KCl in the presence of KRH, KBRS, and BBM extracellular buffers. Experiments were performed as described in the legends to Figs. 1Go and 2Go. A, D, G; B, E, H; and C, F, I, Representative traces of Ycam-2, Ycam-4ER, and BCECF fluorescence ratio changes, respectively, in response to stimulation with 25 mM KCl. J, Effects of KCl on the apparent ratio changes of Ycam-2 and Ycam-4ER; data are means ± SEM from three to five independent experiments involving at least 20 cells in total. K, KCl-induced increases in cytosolic pH as reported with BCECF. Data are the means ± SEM of four to six separate measurements. For Ycam-2 and Ycam-4ER, images were taken every 10 sec, and time scales for BCECF are indicated.

 
To verify that changes in pHi measured with BCECF (largely reflecting cytosolic pH) were also reflected by changes in pH within the ER lumen, and thus likely to influence Ycam-4ER, we investigated the effect of KCl on the fluorescence of ER lumen-targeted EYFP and ECFP. The latter fluoroproteins were expressed separately in MIN6 cells. Addition of 25 mM KCl induced a significant drop in EYFP fluorescence, similar in magnitude to that observed in the cytosol (Fig. 2BGo vs. Fig. 4AGo) in both KRH and KBRS buffers. However, in the physiological HCO3– buffer (BBM), the fluorescence intensity of EYFP did not change significantly (Fig. 4AGo). By contrast, ECFP fluorescence was not affected by the addition of 25 mM KCl in any of the buffers used (Fig. 4BGo), consistent with the lower sensitivity to pH of this mutant (25). These data indicate that, in response to cell depolarization, qualitatively similar changes of pH occur in the lumen of the ER and the cytosol and are thus likely to affect recordings made with Ycam-4ER in the former case.



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  		FIG. 4. Effect of KCl on the fluorescence of ER-EYFP (A) or ER-ECFP (B). Cells were transfected with 1 µg of plasmid-encoding ER-EYFP or ER-ECFP 24–48 h before imaging. A and B, Effects of KCl on the fluorescence of EYFP (A) and ECFP (B) during excitation at 440 nm (emission 480 nm) and excitation at 505 (emission 535), respectively. Data are the means ± SEM of measurements on 21–28 separate cells in each case. *, P < 0.05; **, P < 0.01 for the effects of KCl. Con, Prestimulation condition in each case (100%).

 
Changes of [Ca2+]ER in response to KCl
The steady-state Ca2+ concentration within the ER lumen is likely to reflect intralumenal Ca2+ binding and the balance of ER Ca2+ uptake and release. Thus, a net fall in ER [Ca2+] may not necessarily occur in response to the activation of an ER Ca2+ release pathway if the stimulation of Ca2+ uptake is greater. To test the possibility that ER stores may contribute to the increase in [Ca2+]c after depolarization with KCl, we applied caffeine to activate ER ryanodine receptors (8, 9) and deplete ER Ca2+. These experiments were performed in the presence of high (24 mM) HCO3– concentrations (buffer BBM) to abrogate any effects of KCl on pHi. When cells were preincubated in the absence of 10 mM caffeine (Fig. 5BGo), the increase in FRET prompted by KCl was approximately 22% (Fig. 5Go, B and E). By contrast, addition of 10 mM caffeine caused a 10–12% decrease in apparent [Ca2+]ER, and under these conditions the response to 25 mM KCl was reduced by more than 2-fold, compared with the control case (~10%; Fig. 5Go, C and E). As expected, emptying of the ER Ca2+ store with 10 mM caffeine was accompanied by the appearance of a cytosolic [Ca2+] transient (Fig. 5AGo). We next used 1 mM caffeine, which does not itself empty the ER Ca2+ stores (Fig. 5DGo) or affect cytosolic Ca2+ concentrations (Fig. 5AGo), to sensitize the Ca2+ release mechanism. Cells were preincubated with 1 mM caffeine, and then 25 mM KCl was applied. In 76% of cells (130 of 175), a severely attenuated increase in FRET was observed [Fig. 5Go, D (a) and E], which was approximately 7-fold smaller than that apparent in the control (caffeine-free) condition (3 vs. 22%, Fig. 5EGo). Moreover, 24% of cells (45 of 175) now showed a clear decrease in FRET in response to KCl (Fig. 5Go, D (b) and E). By contrast, we never observed a significant decrease in apparent free ER Ca2+ concentration when cells were pretreated with ryanodine and then stimulated with caffeine and KCl (n = 26 cells in three independent experiments). Moreover, preincubation of cells with 100 µM ryanodine significantly reduced the peak cytosolic Ca2+ concentration after cell depolarization with KCl (Fig. 6AGo) and had a tendency to slow down the recovery of ER Ca2+ concentration (Fig. 5BGo, n = 28 cells in five experiments). By contrast, addition of ryanodine had no effect on the ability of 100 µM acetylcholine (ACh) to mobilize Ca2+ from inositol 1,4,5-triphosphate (IP3)-sensitive stores, as expected (Fig. 6BGo). Together, the above observations thus suggest that, when sensitized, ryanodine receptors are able to mediate Ca2+ release from the ER in response to Ca2+ influx.



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  		FIG. 5. [Ca2+]ER changes induced by KCl in BBM. Cytosolic and ER Ca2+ concentrations were monitored in fura 2-loaded cells (A) or Ycam-4ER-transfected cells (B–D), respectively. Cells were maintained at 3 mM glucose- containing growth medium for 12 h before Ca2+ imaging. A, Cytosolic Ca2+ concentration changes in response to 1 or 10 mM caffeine. B, Ycam-4ER ratio changes after the addition of 25 mM KCl in control cells (triangles) and cells preincubated with 100 µM ryanodine for 45 min (rectangles). Data are the means of five experiments involving 34 control cells and 28 ryanodine-treated cells, and error bars show SEM at selected time points. C, Ryanodine receptors were activated and ER Ca2+ mobilized with 10 mM caffeine before the addition of 25 mM KCl in the continued presence of caffeine (triangles); the trace with squares shows the stable FRET level apparent after the addition of caffeine but with no addition of KCl. Data are the means ± SEM of at least three experiments involving 13 cells treated with caffeine alone or 18 cells sensitized with caffeine and then stimulated with KCl. **, P < 0.01 for the effect of KCl. D, Ryanodine receptors were sensitized by addition of 1 mM caffeine before stimulation with 25 mM KCl. a and b, Demonstration of the two chief response types observed in different individual cells. E, Ratio changes of Ycam-4ER; data are means ± SEM from three to six independent experiments involving at least 18 cells in total. Images were captured at 5- (A) or 10-sec (B–D) intervals. **, P < 0.01 for the effect of caffeine and KCl (E).

 


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  		FIG. 6. [Ca2+]ER changes induced by glucose and ACh in BBM. Cells were loaded with fura 2 (see Materials and Methods), and cytosolic Ca2+ concentration changes in response to 25 mM KCl (A) or 100 µM ACh (B) were monitored in control cells and cells preincubated with 100 µM ryanodine for 45 min. Data are the means of five experiments involving 78 control cells and 98 ryanodine-treated cells, and error bars show SEM at selected time points. **, P < 0.01 for the effect of ryanodine (A). C, Effect of preincubation with ryanodine (100 µM) on glucose-induced increases in [Ca2+]ER. Traces are derived from the mean of four separate experiments involving 13 (control) or 12 (plus ryanodine) cells; error bars show SEM. D, Typical trace of changes in Ycam-4ER fluorescence ratio in response to stimulation 100 µM Ach. Images were captured at 5- (A and B) or 10-sec (C and D) intervals.

 
Effect of glucose on [Ca2+]ER
We previously reported, using targeted cameleons, that elevated glucose concentrations increase ER lumenal-free Ca2+ concentration (12). Because the previous data were obtained in a medium containing low bicarbonate concentrations (KRH), in which small changes in pHi (16) could conceivably affect Ycam-4ER fluorescence, we reassessed the response of [Ca2+]ER to elevated glucose concentrations in the presence of a high HCO3– concentration (buffer BBM) (16). Under these conditions, as in KRH (12), an increase in glucose concentration from 3 to 30 mM increased the Ycam-4ER FRET signal (Figs. 5EGo and 6CGo), indicative of an increase in [Ca2+]ER. In agreement with these data, a recent study also showed that administration of 20 mM glucose increased the average FRET ratio of Ycam-3ER by 79% in primary mouse ß-cells (13). Furthermore, pretreatment of cells with 1 mM caffeine (Fig. E) or ryanodine (Fig. 6CGo) had no significant effect on Ycam-4ER fluorescence after glucose stimulation. As expected, IP3-generating agents ACh (Fig. 6DGo) or carbachol (12) caused a marked lowering of ER Ca2+ concentration.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The principal aims of the present study were to reassess the utility of ER-targeted cameleons to measure changes in free Ca2+ concentration in this organelle in single clonal ß-cells and to use these probes to determine whether CICR may play a role in ß-cell Ca2+ homeostasis under a range of different conditions.

In contrast to a recent report (14), we show here that, unless ER Ca2+ release mechanisms are sensitized, low concentrations of external KCl (15 mM or less) increase [Ca2+]ER, as reported with Ycam-4ER (Fig. 1BGo) when this is measured at either low or high external HCO3– concentrations. These results are thus in line with our previous findings (12). However, both the present studies and the work of Graves and Hinkle (14) demonstrate that higher KCl concentrations (20 mM or above) cause an apparent decrease in the FRET ratio of Ycam-4ER (Figs. 1Go, D and E, and 3Go, B, E, and J), when this is measured in medium containing low (≤5 mM) bicarbonate concentrations. Whereas the latter authors (14) interpreted these changes as indicating a Ca2+-induced decrease in [Ca2+]ER, the measurements of cytosolic and ER lumen pH reported here suggest an alternative explanation. Thus, in medium containing 2.5 mM HCO3–, increases in [KCl] above 20 mM progressively decreased cytosolic pH, with a drop of approximately 0.3 pH units at 30 mM KCl (Fig. 2AGo). Similarly, under conditions identical with those used in the study of Graves and Hinkle (14), addition of 25 mM KCl to a buffer containing 5 mM HCO3– caused an apparent decrease in the pH of both the cytosol (assessed with BCECF; Fig. 3FGo) and ER lumen (assessed with ER-targeted EYFP; Fig. 4AGo), which were similar in magnitude (0.1–0.2 pH units for cytosolic pH) to those observed in medium containing 2.5 mM HCO3– (12) (Figs. 3Go and 4Go). By contrast, the KCl-induced decreases in cytosolic and ER lumen pH were almost completely abrogated when intracellular pH was strongly buffered by a high extracellular concentration of HCO3– ions (Figs. 3Go, I and K, and 4AGo).

What mechanisms may underlie the depolarization- stimulated intracellular acidification at low HCO3– concentrations? Several may be involved, including: 1) activation of the plasma membrane Na+/K+-ATPase, an increase in intracellular Na+ concentration, and stimulation of an amiloride-sensitive Na+/H+ exchange mechanism (22); 2) displacement of H+ from intracellular binding sites by Ca2+ ions; and 3) Ca2+-dependent changes in mitochondrial metabolism (29). Consistent with the absence of a substantial pH gradient between the cytosol and ER (30), we also show, using ER lumen-targeted EYFP, that elevated KCl concentrations decrease the pH within this compartment, and are thus likely to affect the fluorescence of Ycam-4ER (11). Importantly, examined in either compartment, KCl-induced decreases in pH caused a more marked drop in EYFP fluorescence than in ECFP fluorescence (as observed with the separately expressed cytosolic GFP mutants; Fig. 2Go, B vs. C and Fig. 4Go, A vs. B) (11). These changes can easily be misinterpreted as a decrease in intramolecular FRET in measurements involving Ycam-4ER. Indeed, the profound sensitivity to pH of the cameleon reporters used by ourselves (12) and others (14), and relatively small changes in FRET induced by Ca2+ (12, 14), mean that a small alteration in pHi can dramatically affect [Ca2+] measurements. Indeed, after correcting for the pH-dependent decreases in the fluorescence of EYFP (Fig. 2BGo), we calculate (data not shown) that both Ycam-2 and Ycam-4ER report increases in [Ca2+]c and [Ca2+]ER, in response a KCl concentration of 25 mM or more, even at low extracellular bicarbonate concentrations. Moreover, a direct demonstration of 25 mM KCl-induced increases in [Ca2+]ER is now provided under circumstances where intracellular pH is tightly buffered by the presence in the medium of 24 mM HCO3– (Fig. 3Go, H and J).

The principal finding of the present report is that, under conditions in which pHi is stable (11), the ER-targeted probe Ycam-4ER (12, 14) reports glucose and depolarization- induced increases, rather than decreases, in [Ca2+]ER in clonal ß-cells. These findings are consistent with previous measurements of [Ca2+]ER in ß-cells by other techniques (2, 3). Thus, it appears likely that KCl-induced decreases in Ycam-4ER FRET, reported recently (14), may be due, at least in large part, to the effects of intracellular acidification (Fig. 3Go, D–F and J). Although the latter authors reported that the effect of KCl to decrease Ycam-4ER FRET was reversed by ryanodine, implicating a CICR mechanism, no direct, parallel measurements of cytosol-free [Ca2+] were presented under identical conditions (25 mM KCl alone) (14).

In line with a limited role for ER Ca2+ stores in modulating depolarization-induced increases in [Ca2+]c, the addition of 30 (31) or 45 mM KCl (32) to mouse ß-cells was unaffected by store depletion with thapsigargin. Whereas Ca2+ uptake by sarco(endo)plasmic reticulum Ca2+-ATPase (33) and other ER pumps (6) is likely to be accelerated in response to increases in [Ca2+]c and ATP (2), increases in the rate of ER Ca2+ efflux, albeit of smaller magnitude than those of ER Ca2+ influx, may still occur under these conditions (5, 6, 8, 9, 12, 33). Indeed, we observed here that increases in [Ca2+]ER in response to 25 mM KCl (measured in BBM; Fig. 3HGo) were more transient than those in the cytosol (Fig. 3GGo), suggesting a time-dependent activation of ER Ca2+ release. Furthermore, we also show that KCl-induced increases in [Ca2+]ER are decreased by almost 90% when Ca2+ efflux from the ER is enhanced in the presence of caffeine (34). Whereas this decrease may be due in part to an inhibition of Ca2+ influx via L-type Ca2+ channels (expected to be less than 10% at 10 mM caffeine) (34), it seems likely that direct activation of ER-localized ryanodine (9, 10, 35) or an indirect effect on inositol 1,4,5-Trisphosphate (7) receptors (the latter as a result of potential small elevations in intracellular cAMP concentration due to the inhibition by caffeine of phosphodiesterases; this may lead to phosphorylation of IP3 receptors by protein kinase A and their sensitization to Ca2+) (36) may sensitize the corresponding ER Ca2+ release pathways, thus attenuating the increases in [Ca2+]ER.

By contrast, the present and five previous studies (2, 3, 5, 12, 14) failed to reveal any decrease (rather an increase) (2, 3, 5, 12) in [Ca2+]ER in response to elevated glucose concentrations, arguing against net Ca2+ release from this organelle in response to the sugar. A similar response was also found here in the presence of a low concentration of caffeine (Fig. 5EGo), a condition in which CICR pathways were shown to be active after cell depolarization or after treatment with ryanodine. These data argue that CICR from the ER plays a limited if any role in generating cytosolic [Ca2+] increases in response to glucose in clonal MIN6 cells under normal circumstances, possibly reflecting relatively small and slow increases in cytosolic [Ca2+] observed in response to the sugar and enhanced ER Ca2+ uptake driven by ATP-mediated increases in sarco(endo)plasmic reticulum Ca2+-ATPase pump activity (37, 38, 39). However, previous measurements with cytosolically targeted aequorin (5, 40) indicate that cytosolic-free [Ca2+] increases in MIN6 cells in response to 30 mM glucose either monotonically to approximately 200 nM above a resting value of approximately 100 nM (i.e. ~300 nM final) (5, 40) or with oscillations (41) peaking at approximately 600 nM, ranges comparable with those reported in primary ß-cells using fluorescent indicators (31). Moreover, because the MIN6 cells used in the present work (see Materials and Methods) and in many previous studies (5, 6, 40, 41, 42, 44) responded to elevated glucose concentrations (11–30 mM) with a 3- to 10-fold increase in insulin secretion, the present data indicate that this stimulation of hormone release can occur without substantial calcium-induced calcium release from the ER. This finding does not, however, exclude highly localized changes in free cytosolic [Ca2+] as a result of Ca2+-induced Ca2+ mobilization from the ER or other organelles such as secretory granules (5, 6, 18). Moreover, whether CICR from the ER can be induced by glucose in primary ß-cells in the context of the intact islet, in which the stimulation of insulin secretion by glucose is usually greater than in MIN6 cells (5- to 30-fold), remains a possibility. However, a very recent report (44) failed to detect any effect of 100 µM ryanodine on glucose-induced insulin secretion from primary mouse or human ß-cells, whereas slightly reducing KCl-induced cytosolic [Ca2+] increases (as also reported here in MIN6 cells; Fig. 6AGo), arguing against this view. Studies of islets derived from mice expressing a relatively pH-insensitive cameleon, YC3.3-er (14), selectively in ß-cells may offer further insights into this question.


   Acknowledgments
 
We thank Professor Andrew P. Halestrap, Dr. Mark Jepson, and Alan Leard (Bristol MRC Imaging Facility, Bristol, UK) for advice and assistance, and Dr. Nicolas Demaurex (University of Geneva, Geneva, Switzerland) for providing constructs.


   Footnotes
 
This work was supported by Welcome Trust Programe Grant (067081/Z/02/Z) and the Biotechnology and Biological Sciences Research Council. G.A.R. is a Wellcome Trust Research Leave Fellow.

Present address for A.V.: Centre for Research in Biomedicine, Bristol Genomics Institute Faculty of Applied Science, University of the West of England, Bristol BS16 1QY, United Kingdom.

Abbreviations: ACh, Acetylcholine; BBM, bicarbonate-buffered medium; BCECF, 2',7'-bis(carboxyethyl)-5(6 )-carboxyfluorescein; [Ca2+]c, concentration of free Ca2+ in the cytosol; [Ca2+]ER, concentration of free Ca2+ in the ER; CICR, Ca2+-induced Ca2+ release; ECFP, enhanced cyan fluorescent protein; ER, endoplasmic reticulum; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; IP3, inositol 1,4,5-triphosphate; KBRS, Krebs-buffered Ringer solution; KRH, modified Krebs-Ringer bicarbonate buffer; pHi, intracellular pH; Ycam-2, untargeted cameleon; Ycam-4ER, targeted cameleon.

Received February 24, 2004.

Accepted for publication June 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rutter GA 2001 Nutrient-secretion coupling in the pancreatic islet ß-cell: recent advances. Mol Aspects Med 22:247–284[CrossRef][Medline]
  2. Tengholm A, Hellman B, Gylfe E 1999 Glucose regulation of free Ca2+ in the endoplasmic reticulum of mouse pancreatic ß cells. J Biol Chem 274:36883–36890[Abstract/Free Full Text]
  3. Maechler P, Kennedy ED, Sebo E, Valeva A, Pozzan T, Wollheim CB 1999 Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells—studies in aequorin-expressing intact and permeabilized INS-1 cells. J Biol Chem 274:12583–12592[Abstract/Free Full Text]
  4. Tsuboi T, da Silva X, Holz GG, Jouaville LS, Thomas AP, Rutter GA 2003 Glucagon-like peptide-1 mobilizes intracellular Ca2+ and stimulates mitochondrial ATP synthesis in pancreatic MIN6 ß-cells. Biochem J 369:287–299[CrossRef][Medline]
  5. Mitchell K, Pinton P, Varadi A, Ainscow EK, Pozzan T, Rizzuto R, Rutter GA 2001 Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a VAMP2.aequorin chimaera. J Cell Biol 155:41–51[Abstract/Free Full Text]
  6. Mitchell KJ, Lai FA, Rutter GA 2003 Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate (NAADP) receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic ß-cells (MIN6). J Biol Chem 278:11057–11064[Abstract/Free Full Text]
  7. Wollheim CB, Biden TJ 1986 Signal transduction in insulin secretion: comparison between fuel stimuli and receptor agonists. Ann NY Acad Sci 488:317–333[Abstract]
  8. Holz GG, Leech CA, Heller RS, Castonguay M, Habener JF 1999 cAMP-dependent mobilization of intracellular Ca2+ stores by activation of ryanodine receptors in pancreatic ß-cells. A Ca2+ signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7–37). J Biol Chem 274:14147–14156[Abstract/Free Full Text]
  9. Islam MS, Leibiger I, Leibiger B, Rossi D, Sorrentino V, Ekstrom TJ, Westerblad H, Andrade FH, Berggren PO 1998 In situ activation of the type 2 ryanodine receptor in pancreatic ß cells requires cAMP-dependent phosphorylation. Proc Natl Acad Sci USA 95:6145–6150[Abstract/Free Full Text]
  10. Islam MS 2002 The ryanodine receptor calcium channel of ß-cells: molecular regulation and physiological significance. Diabetes 51:1299–1309[Abstract/Free Full Text]
  11. Miyawaki A, Griesbeck O, Heim R, Tsien RY 1999 Dynamic and quantitative Ca2+ measurements using improved chameleons. Proc Natl Acad Sci USA 96:2135–2140[Abstract/Free Full Text]
  12. Varadi A, Rutter GA 2002 Dynamic imaging of endoplasmic reticulum [Ca2+] in MIN6 ß-cells using recombinant chameleons: roles of SERCA2 and ryanodine receptors. Diabetes 51(Suppl 1):S190–S201
  13. Hara H, Bindokas V, Lopez JP, Kaihara K, Landa LR, Harbeck M, Roe MW 2004 Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice. Am J Physiol Cell Physiol 10.1152/ajpcell.00151
  14. Graves TK, Hinkle PM 2003 Ca2+-induced Ca2+ release in the pancreatic ß-cell: direct evidence of endoplasmic reticulum Ca2+ release. Endocrinology 144:3565–3574[Abstract/Free Full Text]
  15. Varadi A, Rutter GA 2002 GFP chameleons for Ca2+ imaging. In: Hicks BW, ed. Green fluorescent protein: methods and protocols. Totowa, NJ: Humana Press Inc.; 255–264
  16. Shepherd RM, Henquin JC 1995 The role of metabolism, cytoplasmic Ca2+, and pH-regulating exchangers in glucose-induced rise of cytoplasmic pH in normal mouse pancreatic islets. J Biol Chem 270:7915–7921[Abstract/Free Full Text]
  17. Kennedy HJ, Viollet B, Rafiq I, Kahn A, Rutter GA 1997 Upstream stimulatory factor-2 (USF2) activity is required for glucose stimulation of L-pyruvate kinase promoter activity in single living islet ß cells. J Biol Chem 272:20636–20640[Abstract/Free Full Text]
  18. Emmanouilidou E, Teschemacher A, Pouli AE, Nicholls LI, Seward EP, Rutter GA 1999 Imaging [Ca2+] changes at the secretory vesicle surface with a recombinant targeted cameleon. Curr Biol 9:915–918[CrossRef][Medline]
  19. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY 1997 Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887[CrossRef][Medline]
  20. Tsuboi T, Lippiat J, Ashcroft F, Rutter GA2004 ATP-dependent interaction of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by fluorescence resonance energy transfer. Proc Natl Acad Sci USA 101:76–81
  21. Wang X, Levi AJ, Halestrap AP 1996 Substrate and inhibitor specificities of the monocarboxylate transporters of single rat heart cells. Am J Physiol 270(2 Pt 2):H476–H484
  22. Lynch AM, Meats JE, Best L, Tomlinson S 1989 Effects of nutrient and non-nutrient stimuli on cytosolic pH in cultured insulinoma (HIT-T15) cells. Biochim Biophys Acta 1012:166–170[Medline]
  23. Kennedy HJ, Pouli AE, Jouaville LS, Rizzuto R, Rutter GA 1999 Glucose-induced ATP microdomains in single islet ß-cells. J Biol Chem 274:13281–13291[Abstract/Free Full Text]
  24. Ainscow EK, Rutter GA 2002 Glucose-stimulated oscillations in free cytosolic ATP concentration imaged in single islet ß cells: evidence for a Ca2+-dependent mechanism. Diabetes 51:S162–S170
  25. Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY 1998 Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci USA 95:6803–6808[Abstract/Free Full Text]
  26. Eisner DA, Kenning NA, O’Neill SC, Pocock G, Richards CD, Valdeolmillos M 1989 A novel method for absolute calibration of intracellular pH indicators. Pflugers Arch 413:553–558[CrossRef][Medline]
  27. Juntti-Berggren L, Civelek VN, Berggren PO, Schultz V, Corkey BE, Tornheim K 1994 Glucose-stimulated increase in cytoplasmic pH precedes increase in free Ca2+ in pancreatic ß-cells. A possible role for pyruvate. J Biol Chem 1269:14391–14395
  28. Grapengiesser E, Gylfe E, Hellman B 1989 Regulation of pH in individual pancreatic ß-cells as evaluated by fluorescence ratio microscopy. Biochim Biophys Acta 1014:219–224[Medline]
  29. Ainscow EK, Zhao C, Rutter GA 2000 Acute overexpression of lactate dehydrogenase-A perturbs ß-cell mitochondrial metabolism and insulin secretion. Diabetes 49:1149–1155[Abstract]
  30. Wu MM, Grabe M, Adams S, Tsien RY, Moore HP, Machen TE 2001 Mechanisms of pH regulation in the regulated secretory pathway. J Biol Chem 276:33027–33035[Abstract/Free Full Text]
  31. Gilon P, Arredouani A, Gailly P, Gromada J, Henquin JC 1999 Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell. J Biol Chem 274:20197–20205[Abstract/Free Full Text]
  32. Lemmens R, Larsson O, Berggren PO, Islam MS 2001 Ca2+-induced Ca2+ release from endoplasmic reticulum amplifies Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic ß cells. J Biol Chem 276:9971–9977[Abstract/Free Full Text]
  33. Varadi A, Molnar E, Ostenson CG, Ashcroft SJH 1996 Isoforms of endoplasmic reticulum Ca2+-ATPase are differentially expressed in normal and diabetic islets of Langerhans. Biochem J 319:521–527
  34. Islam MS, Larsson O, Nilsson T and Berggren, P-O 1995 Effects of caffeine on cytoplasmic free Ca2+ concentration in pancreatic ß-cells are mediated by interaction with ATP-sensitive K+ channels and L-type voltage-gated Ca2+ channels but not the ryanodine receptor. Biochem J 306:679–686
  35. Bruton JD, Lemmens R, Shi CL, Persson-Sjogren S, Westerblad H, Ahmed M, Pyne NJ, Frame M, Furman BL, Islam MS 2003 Ryanodine receptors of pancreatic ß-cells mediate a distinct context-dependent signal for insulin secretion. FASEB J 17:301–303[Abstract/Free Full Text]
  36. Volpe P, Alderson-Lang BH 1990 Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. II. Effect of cAMP-dependent protein kinase. Am J Physiol 258(6 Pt 1):C1086–C1091
  37. Roe MW, Mertz RJ, Lancaster ME, Worley 3rd JF, Dukes ID 1994 Thapsigargin inhibits the glucose-induced decrease of intracellular Ca2+ in mouse islets of Langerhans. Am J Physiol 266:E852–E862
  38. Chow RH, Lund PE, Loser S, Panten U, Gylfe E 1995 Coincidence of early glucose-induced depolarization with lowering of cytoplasmic Ca2+ in mouse pancreatic ß-cells. J Physiol 485:607–617[Medline]
  39. Detimary P, Gilon P, Henquin JC 1998 Interplay between cytoplasmic Ca2+ and the ATP/ADP ratio: a feedback control mechanism in mouse pancreatic islets. Biochem J 333:269–274
  40. Mitchell KJ, Tsuboi T, Rutter GA 2004 Role for plasma membrane-related Ca2+-ATPase-1 (ATP2C1) in pancreatic ß-cell Ca2+ homeostasis revealed by RNA silencing. Diabetes 53:393–400[Abstract/Free Full Text]
  41. Miyazaki J, Araki K, Yamato E, Ikegami H, Asano T, Shibasaki Y, Oka Y, Yamamura K 1990 Establishment of a pancreatic ß cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127:126–132[Abstract]
  42. Zhao C, Rutter GA 1998 Overexpression of lactate dehydrogenase A attenuates glucose-induced insulin secretion in stable MIN-6 ß-cell lines. FEBS Lett 430:213–216[CrossRef][Medline]
  43. Nakazaki M, Ishihara H, Kakei M, Inukai K, Asano T, Miyazaki JI, Tanaka H, Kikuchi M, Yada T, Oka Y 1998 Repetitive mitochondrial Ca2+ signals synchronize with cytosolic Ca2+ oscillations in the pancreatic ß-cell line, MIN6. Diabetologia 41:279–286[CrossRef][Medline]
  44. Johnson JD, Kuang S, Misler S, Polonsky KS 2004 Ryanodine receptors in human pancreatic ß cells: localization and effects on insulin secretion. FASEB J 18:878–880[Abstract/Free Full Text]




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