1 Department of Medical Cell Biology, Uppsala University, Biomedicum, Box 571, SE-751 23 Uppsala, Sweden
2 Department of Biophysics, National T. Shevchenko University of Kiev, Kiev, Ukraine
*Author for correspondence (e-mail: erik.gylfe{at}medcellbiol.uu.se)
Accepted March 5, 2001
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SUMMARY |
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Key words: Pancreatic ß-cell, Store-operated, Calcium channels, Insulin secretion
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INTRODUCTION |
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The role of the store-operated current in the physiology of the glucose-stimulated ß-cell ultimately depends on how it is regulated by store filling. In some types of cells Ca2+ influx is activated in an all-or-none fashion after almost complete emptying of the intracellular Ca2+ stores (Fierro and Parekh, 2000; Fierro et al., 2000), whereas in others there is gradual activation with increasing depletion of the stores (Hofer et al., 1998; Sedova et al., 2000). The present study provides the first evidence that the store-operated entry of Ca2+ into the ß-cell exhibits a graded dependence on Ca2+ filling of the ER. Small variations in the ER Ca2+ concentration may consequently contribute to the regulation of the membrane potential and [Ca2+]i determining insulin release.
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MATERIALS AND METHODS |
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Preparation of pancreatic islets and ß-cells
Islets of Langerhans were isolated by collagenase digestion from the pancreas of adult ob/ob mice from a local colony (Hellman, 1965). These islets consist of more than 90% ß-cells, which respond normally to glucose and other regulators of insulin release (Hahn et al., 1974). Free cells were prepared by shaking the islets in a Ca2+-deficient medium (Lernmark, 1974). The cells were suspended in RPMI 1640 medium containing 11 mM glucose and supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin and 30 µg/ml gentamicin and allowed to attach to circular 25 mm coverslips during culture for 4-7 days in an atmosphere of 5% CO2 in humidified air. Further experimental handling of cells was performed with a medium containing 25 mM HEPES (pH 7.40), 1 mg/ml bovine serum albumin, 137 mM Na+, 5.9 mM K+, 1.2 mM Mg2+, and <1 nM, 1.28 or 10 mM Ca2+ with Cl- as the sole anion. The lowest Ca2+ concentration was obtained by including 2 mM EGTA in a Ca2+-deficient medium. When testing the effects of Gd3+, bovine serum albumin and EGTA were omitted.
Measurements of cytoplasmic Ca2+ and Mn2+
In most experiments, loading of the cells with the fluorescent indicator fura-2 was performed in the presence of 1.28 mM Ca2+ during a 40 minute incubation at 37°C in a medium supplemented with 1 µM fura-2 acetoxymethyl ester, 400 µM diazoxide and 20 mM glucose. However, when testing the effect of K+ depolarization, fura-2 loading was made in medium lacking diazoxide and containing 3 mM glucose. With these procedures 90±0.5% (n=4) of the fura-2 is cytoplasmic as judged from the release of indicator in response to plasma membrane permeabilization using a previously described technique (Tengholm et al., 2000) with 1250 hemolytic units/ml -toxin. Calculations of [Ca2+]i and [Mn2+]i (see below) were compensated for this compartmentalization of fura-2. The coverslips with attached cells were used as exchangeable bottoms of an open chamber containing 50 µl medium. The chamber was placed on the stage of an inverted microscope (Nikon Diaphot) within a climate box maintained at 37°C by an air stream incubator, and the cells were superfused at a rate of 0.3 ml/minute with similar indicator-free medium. When studying store-operated Ca2+ influx this medium was supplemented with 50 µM methoxyverapamil.
The microscope was equipped with an epifluorescence illuminator and a 100x UV fluorite objective. A filter changer of a time-sharing multichannel spectrophotofluorometer (Chance et al., 1975) provided excitation light flashes of 1 millisecond duration every 10 milliseconds at 340 and 380 nm, and the emission was measured at 510 nm with a photomultiplier. A computer recorded the electronically separated fluorescence signals at the two wavelengths.
[Ca2+]i values were obtained according to a previously described method (Grynkiewicz et al., 1985) using Equation 1:
![]() | (Equation 1) |
KDCa2+ is 224 nM. F0 and Rmin are the fura-2 fluorescence at 380 nm and the 340/380 nm fluorescence excitation ratio, respectively, in an intracellular K+-rich calibration solution lacking Ca2+. FS and Rmax are the corresponding data obtained with saturating concentrations of Ca2+.
Variations in the influx through the store-operated pathway were estimated more directly by a Mn2+ quench approach. However, instead of measuring only the reduction in fura-2 fluorescence in ß-cells exposed to this cation (Liu and Gylfe, 1997) we introduced a novel approach linearizing the data by calculating the cytoplasmic Mn2+ concentration ([Mn2+]i). Because Mn2+ quenches the fluorescence of fura-2, irrespective of excitation wavelength, a single wavelength technique is used. To make such measurements independent of changes in [Ca2+]i the isosbestic wavelength of fura-2 is utilized. However, instead of measuring the fluorescence excited at the isosbestic wavelength, a Ca2+ insensitive isosbestic fluorescence signal was calculated as Fi=F340+·F380. In this equation,
is the isocoefficient that scales the negative F380 response to compensate exactly for the positive F340 response (fluorescence excited at 340 and 380 nm, respectively) when [Ca2+]i is increased (Zhou and Neher, 1993). The effectiveness of this procedure is illustrated in Fig. 1, in which panel C shows lack of effect of carbachol on the calculated isosbestic fluorescence despite a pronounced carbachol-induced [Ca2+]i response (panel D). Owing to photobleaching and loss of indicator from the cells there is a slow gradual decrease of the Ca2+-independent fluorescence even in the absence of Mn2+ (Fig. 1C, broken line 0). After compensating for this decrease (Fig. 1B), [Mn2+]i can be calculated in analogy to the method previously described (Grynkiewicz et al., 1985) using Equation 2:
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![]() | (Equation 2) |
KDMn2+ is 2.8 nM (Kwan and Putney, 1990). Fmax is the unquenched (Fig. 1B, line 0) and Fmin the maximally quenched fura-2 fluorescence in the presence of Mn2+, which was set to 1% of Fmax (Kwan and Putney, 1990). Fig. 1A illustrates the slow rise of [Mn2+]i upon introduction of the ion (broken line 1) and acceleration of this effect after stimulation with carbachol (broken line 2). Although the apparent KDMn2+ may be expected to change slightly with the Ca2+ concentration, we found no evidence for such interference because [Ca2+]i peaks occurred without fluctuations in the Mn2+ signal.
Presentation of data and statistical analysis
Results are presented as means±s.e.m. Differences were statistically evaluated by the two-tailed Students t test. The dose-response data (Fig. 2B; Fig. 4B) were fitted to a sigmoidal equation (logistic function) using the Marquart-Levenberg algorithm (SigmaPlot, SPSS Inc. Chicago, IL). The linear curve fits (Fig. 1; Fig. 3; Fig. 5) and all illustrations were made with the Igor Pro software (Wavemetrics Inc., Lake Oswego, OR).
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RESULTS |
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After exposure to Mn2+ in the absence of agonist there was a linear rise in [Mn2+]i in the hyperpolarized and glucose-exposed ß-cells. This rise can be expected to represent entry of the ion through pathways other than the voltage-dependent and store-operated Ca2+ channels (Fig. 3A). Subsequent addition of carbachol dose-dependently accelerated the rate of [Mn2+]i increase, owing to activation of the store-operated pathway. In a series of three experiments, the acceleration obtained with 3.6 µM carbachol was 29.9±9.5% of the maximal activation obtained with 30 µM of the drug.
During maximal SERCA inhibition the ER is rapidly depleted owing to leakage of Ca2+ (Liu and Gylfe, 1997; Tengholm et al., 1999). We then used increasing concentrations of the SERCA inhibitor CPA to gradually deplete the ER in individual ß-cells. The protocol shown in Fig. 4A is similar to that used for carbachol in Fig. 2A except that extracellular Ca2+ was varied only between <1 nM and 1.28 mM. Like carbachol, CPA caused some increase of [Ca2+]i in Ca2+-deficient medium and a dose-dependent, more pronounced rise in the presence of extracellular cation. The latter effect was half-maximal and maximal at 1.94±0.23 and 10 µM CPA, respectively, whereas 30 µM gave a slightly smaller response (Fig. 3B). Using influx of Mn2+ as measure of the store-operated pathway, 2 µM CPA accelerated the influx by 23.6±2.7% (n=5) of the maximal activation obtained with 20 µM of the drug (Fig. 5A).
Gd3+ at a concentration of 1 µM has been found to inhibit the store-operated Ca2+ entry in a smooth muscle cell line without affecting vasopressin-stimulated influx of the ion (Broad et al., 1999). We now find that 1 µM Gd3+ does not interfere with mobilization of ER Ca2+ in response to 100 µM carbachol (Fig. 6A) or 50 µM CPA (Fig. 6B) in hyperpolarized ß-cells exposed to Ca2+-deficient medium. In accordance with an inhibitory effect on store-operated Ca2+ influx, subsequent restoration of a physiological Ca2+ concentration (1.28 mM) in the continued presence of carbachol or CPA resulted in elevation of [Ca2+]i only when Gd3+ was absent. When the store-operated influx in response to carbachol was monitored with Mn2+, it was completely abolished by Gd3+, which even reduced the basal Mn2+ influx (Fig. 7). In addition, Gd3+ was an effective blocker of the voltage-dependent rise of [Ca2+]i in response to K+ depolarization (Fig. 6C). Other experiments indicated that the effect of Gd3+ is not reversible and that 0.1-5 µM of this ion fails to discriminate between the store-operated and voltage-dependent entry of Ca2+ (data not shown).
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DISCUSSION |
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To selectively study the store-operated pathway in individual ß-cells without interference from voltage-dependent Ca2+ entry we employed a previously developed technique (Gylfe, 1991; Liu and Gylfe, 1997). In this approach the ß-cells are hyperpolarized with diazoxide, which activates the KATP channels (Trube et al., 1986). As an extra precaution, the medium was supplemented with methoxyverapamil, a voltage-dependent Ca2+ channel blocker lacking effects on the store-operated entry (Gylfe, 1991; Liu and Gylfe, 1997). Maximal filling of the Ins(1,4,5)P3-sensitive store of ER Ca2+ was ascertained by pre-exposure to 20 mM glucose (Gylfe, 1988; Gylfe, 1991; Tengholm et al., 1999), which was present throughout the experiments. In every cell we found that increasing concentrations of the Ca2+-mobilizing carbachol cause gradual elevation of [Ca2+]i depending on store-operated influx. In most experiments, one observation point was on the steepest part of the dose-response curve, contrary to what can be expected if the entry is regulated in an all-or-none fashion. Similar results were obtained with increasing concentrations of the SERCA inhibitor CPA, which empties the ER via a leakage pathway after inhibition of Ca2+ uptake. Unlike a previous study (Liu and Gylfe, 1997), we did not attempt to correlate the effects of carbachol and CPA on mobilization of intracellular Ca2+ with the magnitude of the store-operated influx. Such an approach requires separate experiments at each concentration to ascertain that the ER is completely filled when introducing the test substance.
Mn2+ quenching of the fura-2 fluorescence is a potent technique for more direct studies of fluxes through the voltage-dependent (Dryselius et al., 1999) and store-operated (Liu and Gylfe, 1997) pathways in the ß-cell. Because quenching exhibits a non-linear dependence on Mn2+ concentration, we introduced a novel approach calculating actual [Mn2+]i levels from the quenching curve. In all situations studied, [Mn2+]i increased linearly throughout the observation periods, although the rate varied depending on stimulation. The rate of increase can therefore be taken as a measure of influx with little interference from outward transport. Using this approach we found that 3.6 µM carbachol and 2 µM CPA, concentrations slightly higher than those giving half-maximal elevation of [Ca2+]i, induced only 30 and 24% activation of the store-operated influx, respectively. Consequently, there is no linear relationship between influx rate and elevation of [Ca2+]i. An explanation may be that, at high agonist concentrations, fura-2 in the submembrane space becomes saturated with Ca2+ resulting in underestimation of [Ca2+]i and a left shift of the dose-response relationships.
Individual pancreatic ß-cells respond to glucose with slow [Ca2+]i oscillations with a frequency of 0.2-0.5/minute (Grapengiesser et al., 1988). Similar oscillations are observed in pancreatic islets but, within the islets, the ß-cell response is dominated by about tenfold faster oscillations (Valdeolmillos et al., 1989; Bergsten et al., 1994; Gilon et al., 1994). It was previously shown that the fast oscillations depend on cAMP and that they can be transformed into slow oscillations by SERCA inhibition (Liu et al., 1998). Modeling the generation of the fast oscillatory pattern it has been suggested that release of Ca2+ from the ER causes a hyperpolarizing current, which shuts off the voltage-dependent entry of Ca2+ (Ämmälä et al., 1991; Liu et al., 1998; Dryselius et al., 1999). However, the associated emptying of the ER has been suggested to generate the fast oscillations by activating a depolarizing store-operated current (Worley et al., 1994; Bertram et al., 1995; Gilon et al., 1999). To discriminate between these seemingly inconsistent alternatives it would be valuable to have an inhibitor of the store-operated pathway, which does not affect mobilization of ER Ca2+ or voltage-dependent entry of the ion. Testing suggested inhibitors we found that Gd3+ and 2-APB lack the required Ca2+ channel specificity. The usefulness of 2-APB is limited because this Ins(1,4,5)P3 receptor inhibitor will interfere with Ca2+ mobilization from the ER.
Taken together, this study provides the first evidence that the store-operated entry of Ca2+ into the ß-cell exhibits a graded dependence on Ca2+ filling of the ER. Small variations in the ER Ca2+ concentration may consequently contribute to the regulation of the membrane potential and [Ca2+]i determining insulin release.
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ACKNOWLEDGMENTS |
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