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A store-operated mechanism determines the activity of the electrically excitable glucagon-secreting pancreatic α-cell
Cell Calcium 35 (2004) 357–365
A store-operated mechanism determines the activity of the electrically excitable glucagon-secreting pancreatic ␣-cell Yi-Jia Liu1 , Elaine Vieira1 , Erik Gylfe∗ Department of Medical Cell Biology, Uppsala University Biomedical Centre, Husargatan 3, Box 571, SE-752 37 Uppsala, Sweden Received 7 August 2003; received in revised form 3 October 2003; accepted 15 October 2003
Abstract The glucagon-releasing pancreatic ␣-cells are electrically excitable cells but the signal transduction leading to depolarization and secretion is not well understood. To clarify the mechanisms we studied [Ca2+ ]i and membrane potential in individual mouse pancreatic ␣-cells using fluorescent indicators. The physiological secretagogue l-adrenaline increased [Ca2+ ]i causing a peak, which was often followed by maintained oscillations or sustained elevation. The early effect was due to mobilization of Ca2+ from the endoplasmic reticulum (ER) and the late one to activation of store-operated influx of the ion resulting in depolarization and Ca2+ influx through voltage-dependent L-type channels. Consistent with such mechanisms, the effects of adrenaline on [Ca2+ ]i and membrane potential were mimicked by inhibitors of the sarco(endo)plasmic reticulum Ca2+ ATPase. The ␣-cells express ATP-regulated K+ (KATP ) channels, whose activation by diazoxide leads to hyperpolarization. The resulting inhibition of the voltage-dependent [Ca2+ ]i response to adrenaline was reversed when the KATP channels were inhibited by tolbutamide. However, tolbutamide alone rarely affected [Ca2+ ]i , indicating that the KATP channels are normally closed in mouse ␣-cells. Glucose, which is the major physiological inhibitor of glucagon secretion, hyperpolarized the ␣-cells and inhibited the late [Ca2+ ]i response to adrenaline. At concentrations as low as 3 mM, glucose had a pronounced stimulatory effect on Ca2+ sequestration in the ER amplifying the early [Ca2+ ]i response to adrenaline. We propose that adrenaline stimulation and glucose inhibition of the ␣-cell involve modulation of a store-operated current, which controls a depolarizing cascade leading to opening of L-type Ca2+ channels. Such a control mechanism may be unique among excitable cells. © 2003 Elsevier Ltd. All rights reserved. Keywords: Glucagon secretion; Store-operated Ca2+ channel; Endoplasmic reticulum calcium sequestration; Endoplasmic reticulum calcium release; Membrane potential
1. Introduction Diabetes mellitus is characterized by hyperglycemia due to insulin deficiency, which is caused by pancreatic -cell destruction or disturbed secretion of the hormone. In both types 1 and 2 diabetes, failure of glucose to inhibit the secretion of blood glucose-elevating glucagon aggravates the hyperglycemia [1–3]. Moreover, glucagon secretion is not appropriately stimulated when blood glucose falls to low concentrations, a potentially life-threatening condition in insulin-treated diabetic subjects [4]. Whereas considerable attention has been focussed on insulin secretion from the -cell, much less is known about glucagon release from the pancreatic ␣-cell. It has been proposed that insulin [5,6], ∗ Corresponding
author. Tel.: +46-18-4714428; fax: +46-18-4714059. E-mail address: [email protected] (E. Gylfe). 1 Drs. Vieira and Liu contributed equally to the article.
0143-4160/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2003.10.002
GABA [7], or Zn2+ [8] co-secreted with insulin from the -cell, mediates glucose inhibition of glucagon secretion. Although such mechanisms may contribute, they cannot explain why glucose inhibits glucagon secretion from purified ␣-cells [9], and why secretion is inhibited at lower sugar concentrations than those stimulating the secretory activity of the -cell [10]. Metabolism of glucose is essential both for stimulation of insulin release [11] and inhibition of glucagon secretion [12,13]. In the -cell the closure of the ATP-sensitive K+ (KATP ) channels has a central role in stimulus-secretion coupling, transducing the increase in ATP/ADP ratio obtained with glucose metabolism into depolarization with resulting opening of voltage-dependent L-type Ca2+ channels [11]. There are diverging opinions about the presence of KATP channels in the ␣-cell and their possible involvement in the regulation of glucagon secretion [6,14–18]. Since glucose inhibits glucagon release, it is apparent that a fun-
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damentally different process is operating in the pancreatic ␣-cell. Nevertheless, it has been proposed that depolarization by glucose-induced closure of the KATP channels inhibits glucagon release [17]. This paradox was attributed to voltage-dependent steady-state inactivation of voltage-gated Na+ , T-type Ca2+ and A-type K+ channels participating in action potential generation thereby preventing the opening of L-type Ca2+ channels. The latter concept is difficult to reconcile with preservation of glucose-inhibited glucagon secretion in islets isolated from KATP channel knockout mice [19]. Like the secretion of most other hormones, exocytosis of glucagon from the ␣-cells is triggered by an increase of [Ca2+ ]i [20]. Studying guinea pig ␣-cells, we have previously proposed that glucose inhibits glucagon release by lowering [Ca2+ ]i after promoting intracellular sequestration and outward transport of Ca2+ [21,22]. Extending this hypothesis we show that the glucose-induced Ca2+ sequestration in the endoplasmic reticulum (ER) hyperpolarizes mouse ␣-cell by shutting off a store-operated current. This depolarizing current, which is activated by adrenaline and other agents mobilizing intracellular Ca2+ , may be essential for glucagon secretion by contributing to the opening of the L-type Ca2+ channels. The proposed mechanism can explain the paradox that lowering of ATP by inhibitors of metabolism results in stimulated glucagon secretion [23] by activation of the depolarizing store-operated current after Ca2+ store-depletion.
2. Materials and methods 2.1. Reagents and solutions Reagents of analytical grade and deionized water were used. Fura-2 and its acetoxymethyl ester as well as bis-(1,3dibutylbarbituric acid)trimethine oxonol (bis-oxonol) were from Molecular Probes Inc. (Eugene, OR). Sigma Chemical Co. (St. Louis, MO) provided atropine, biotinylated rabbit anti-guinea pig immunoglobulin, BSA (fraction V), carbachol, l-adrenaline, HEPES, nifedipine, poly-l-lysine, and thapsigargin. Cyclopiazonic acid (CPA) was from Calbiochem (La Jolla, CA), 2,5-di-tert-butylhydroquinone (DTBHQ) from Aldrich-Chemie (Steinheim, Germany) and 2-aminoethoxydiphenyl borate (2-APB) from Aldrich (Gillingham, UK). DAKO Corp. (Carpenteria, CA) supplied rabbit anti-human glucagon serum, biotinylated goat antirabbit immunoglobulin, normal goat serum and 5-bromo4-chloro-3-indoxyl phosphate plus nitro blue tetrazolium chloride (BCIP/NBT). Fetal calf serum was bought from Gibco Ltd. (Paisley, Scotland) and collagenase was from Boehringer Mannheim GmbH (Mannheim, Germany). Biolog Life Science Institute (Bremen, Germany) supplied adenosine 3 ,5 -cyclic monophosphorothioate Rp-isomer (Rp-cAMPS). Diazoxide and methoxyverapamil were kindly donated by Schering-Plough Int. (Kenilworth, NJ)
and Knoll AG (Ludwigshafen, Germany), respectively, and forskolin plus tolbutamide by Hoechst Marion Roussel AB, (Stockholm, Sweden). 2.2. Preparation of pancreatic islet cells Islets of Langerhans were collagenase-isolated from the splenic part (lacking pancreatic polypeptide-secreting cells) of the pancreas of NMRI mice. The animals were placed in sealed container into which as stream of CO2 was delivered. The experimental procedures were approved by the Uppsala Ethics Committee. When the animals became unconscious, they were killed by decapitation. The peritoneal cavity was opened and the pancreas was excised and cut into small pieces, which were digested with collagenase to obtain free islets of Langerhans. Free cells were then prepared by shaking the islets in a Ca2+ -deficient medium [24]. To increase the yield of the superficially located ␣-cells, the shaking was interrupted before the entire islets had fragmented. The cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 g/ml streptomycin and 30 g/ml gentamicin and allowed to attach to poly-l-lysine-coated circular 25 mm cover slips during 1–3 days culture at 37 ◦ C in RPMI 1640 medium in an atmosphere of 5% CO2 . 2.3. Image analysis of [Ca2+ ]i Loading of cells with the indicator fura-2 was performed during 40-min incubation at 37 ◦ C in a HEPES-buffered medium (25 mM, pH 7.4) containing 0.5 mg/ml bovine serum albumin, 138 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl2 , 1.28 mM CaCl2 , 3 mM glucose and 1 M fura-2 acetoxymethyl ester (0.1% dimethylsulphoxide). The cover slips with the attached cells were then used as exchangeable bottoms of an open chamber thermostated at 37 ◦ C. The chamber volume was 0.16 ml and the cells were superfused at a rate of 1 ml/min with a medium lacking indicator. Addition of thapsigargin, which sticks to plastic, was made directly to the superfusion chamber with a pipette. The superfusion flow was then interrupted for 2–3 min to ascertain an effect of the drug. [Ca2+ ]i measurements were conducted using a Nikon Diaphot microscope equipped with an epifluorescence illuminator and a 40× oil immersion fluorescence objective. A monochromator, which is part of a Quanticell 700 imaging system (VisiTech International, Sunderland, UK), provided excitation light flashes at 340 and 380 nm, and the emission was measured at 515 nm using a 400-nm dichroic beam splitter and an intensified CCD camera. Image pairs were taken every 1–2 s and 340/380 nm ratio (R) images were calculated after subtraction of background images. [Ca2+ ]i images were calculated using a dissociation constant (KD ) of 224 nM [25] and the equation: [Ca2+ ] = KD
F0 (R − Rmin ) Fs (Rmax − R)
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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 medium lacking Ca2+ . Fs and Rmax are the corresponding data obtained with a saturating concentration of Ca2+ . By selecting image fields dominated by small cells, it was possible to obtain recordings from more non--cells than expected from their percentage in the preparation [26]. Usually, two to five small cells were present in each image field. Simultaneous recordings from 2 ␣-cells were not uncommon and rarely up to four ␣-cells were studied in parallel. Some of the stimuli applied were helpful to preliminarily classify the cells before the final identification by immunostaining. 2.4. Changes in membrane potential Changes in membrane potential were recorded using the fluorescent probe bis-oxonol added to a final concentration of 150 nM from a 1000-fold concentrated stock solution in ethanol. After 10 min equilibration with the bis-oxonol containing medium at 37 ◦ C, the fluorescence was recorded from individual ␣-cells. The cells were studied in a similar microscopic system as used for imaging of Ca2+ . However, in this case a photometric approach was employed using a monochromator-based photon-counting Deltascan system with Felix software (Photon Technology International, Lawrenceville, NJ). The excitation and emission wavelengths were 485 and 520 nm and a 505 nm dichroic beam splitter was used. The anionic dye enters the depolarized cells where it binds to intracellular proteins or membranes and exhibit enhanced fluorescence and red spectral shifts [27]. Depolarization and hyperpolarization are consequently reflected as increases and decreases in fluorescence, respectively.
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heterogeneity, and even those on the same cover glass often reacted differently to the same stimulus. Due to the qualitative differences in cellular responses the results have been presented as proportions of cells reacting in different ways. Statistical analyses of the proportion of cells with a certain response were made with two-tailed Fisher’s exact test or χ2 test with Yates’ correction. Wilcoxon’s signed rank test was used when qualitatively different responses were found ranking no response to a stimulus as 0, a single [Ca2+ ]i peak as 1, oscillations as 2 and sustained elevation as 3. Paired Student’s t-test was used to assess quantitative differences. Statistical significance was set at a P value of <0.05. Due to the scarcity of ␣-cells, one to two cells were studied in most experiments (see above), and cells from one cell isolation were used for four to six experiments.
3. Results 3.1. The KATP channels are functionally inactive in most α-cells In >100 experiments on cells from >20 islet isolations, individual mouse pancreatic ␣- and -cells exposed to 3 mM glucose typically exhibit stable basal [Ca2+ ]i levels in the 60–100 nM range. Only 7% of the ␣-cells (16 of 244) displayed slow oscillations of [Ca2+ ]i under these conditions (see Fig. 2D). Whereas 134 of 136 -cells reacted to 0.5–1 mM of the depolarizing KATP channel inhibitor tolbutamide with elevation of [Ca2+ ]i (Fig. 1B), 94% of the ␣-cells (261 of 277) remained silent (Figs. 1A, 5 and 6A). The tolbutamide-induced elevations of [Ca2+ ]i in the 16 responsive ␣-cells were modest. Fig. 2A and B shows the most prominent effect of tolbutamide. Introduction of
2.5. Identification of α-cells Each experiment was terminated by immunostaining the cells in the experimental chamber. The cells were superfused with albumin-free medium and fixed with 95% ethanol. After rinsing with distilled water and Tris buffer (0.5 M, pH 7.6), normal goat serum (diluted 1:10) was added to reduce background staining. After 10 min, rabbit anti-human glucagon (1:200) was added for 20–30 min followed by rinsing with Tris buffer. Biotinylated goat anti-rabbit immunoglobulin (1:500) was then introduced for 10 min, followed by rinsing and addition of alkaline phosphatase-conjugated streptavidine (1:200) for a further 10 min. The BCIP/NBT colour reagent was then added for 2–5 min. 2.6. Statistical analysis Like many cell types, the ␣-cells responded with a single peak, oscillations and sustained elevation of [Ca2+ ]i with increasing concentrations of agonists. However, the sensitivity of individual ␣-cells was characterized by considerable
Fig. 1. Effects of tolbutamide, adrenaline, diazoxide and methoxyverapamil on [Ca2+ ]i of an ␣-cell and a simultaneously studied -cell. The fura-2-loaded cells were initially exposed to a medium containing 3 mM glucose. Tolbutamide (Tol; 500 M), Adrenaline (5 M), diazoxide (Diaz; 400 M) and methoxyverapamil (MV; 50 M) were then added as indicated. The ␣-cell is shown in panel (A) and the -cell in panel (B) as indicated.
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Fig. 2. Effects of adrenaline, tolbutamide and diazoxide on [Ca2+ ]i of four separately studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 3 mM glucose. Adrenaline (A, Adr; 5 M), Tolbutamide (Tolb; 1 mM, panels A and B; 500 M, panels C and D) and diazoxide (400 M) were then added as indicated.
5 M adrenaline did not affect previously silent -cells (Fig. 1B) but caused elevation of [Ca2+ ]i in 86% of the ␣-cells (219 of 255; P < 0.001, χ2 or Wilcoxon test). Among the responsive ␣-cells 40% (64 cells) reacted with a single [Ca2+ ]i peak (not shown), there were slow [Ca2+ ]i oscillations in 46% (75 cells; Figs. 1A and 6) and 14% (23 cells) displayed sustained elevation of [Ca2+ ]i (not shown). In support for the presence of KATP channels, the hyperpolarizing channel activator diazoxide almost completely inhibited the late adrenaline effect (P < 0.001) leaving only a small suprabasal elevation in 91% (20 cells; Figs. 1A, 2C and D) and was partially inhibitory in the remaining 2 ␣-cells. Tolbutamide counteracted this effect by promptly reversing diazoxide inhibition of the [Ca2+ ]i response to adrenaline (all six ␣-cells; P < 0.01; Fig. 2C and D). 3.2. Glucose stimulates Ca2+ removal from the cytoplasm and enhances the initial effect of adrenaline on [Ca2+ ]i Using guinea pig ␣-cells, we have previously found that pre-exposure to glucose enhances the subsequent [Ca2+ ]i peak response to adrenaline by promoting intracellular Ca2+ sequestration [22]. Since the inhibitory effect of diazoxide indicates that adrenaline depolarizes the ␣-cells, we now studied the effect of glucose on the initial [Ca2+ ]i peak response to adrenaline under conditions preventing voltagedependent Ca2+ entry. Fig. 3A shows an initial adrenaline response of an ␣-cell equilibrated in 3 mM glucose. The elevation of [Ca2+ ]i was terminated immediately upon removal of adrenaline. After subsequent omission of glucose and addition of 400 M hyperpolarizing diazoxide short restimulation with adrenaline gave a smaller peak in all 16 ␣-cells (P < 0.001). When 3 mM glucose was reintroduced in the continued presence of diazoxide, a third adrenaline stimulation resulted in a 4.3 ± 0.6-fold (P < 0.05, n = 6) greater integrated [Ca2+ ]i peak response than in the absence
Fig. 3. Effect of glucose concentration on the early depolarization-independent [Ca2+ ]i response to adrenaline in two separately studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 3 mM (A) or 20 mM (B) glucose. Adrenaline (Adr; 5 M), 100 or 400 M diazoxide, 10 M nifedipine and 0, 3 or 20 mM glucose were then present as indicated.
of the sugar. A similar or slightly greater [Ca2+ ]i peak response to adrenaline was observed after elevation of glucose to 20 mM (all 14 ␣-cells; Fig. 3A). The stimulatory effect of glucose on the initial [Ca2+ ]i response to adrenaline was apparent also when switching between 0 and 20 mM glucose in six experiments performed in the presence of 100 M diazoxide and 10 M of the voltage-dependent Ca2+ channel blocker nifedipine (Fig. 3B; P < 0.01). Supporting the idea that glucose stimulates Ca2+ removal from the cytoplasm the sugar concentration also affected the basal [Ca2+ ]i level. Omission of glucose consequently resulted in a small gradual elevation of basal [Ca2+ ]i in all 11 ␣-cells and reintroduction of 3 (Fig. 3A) or 20 (not shown) mM glucose restored the initial [Ca2+ ]i level in all 11 cells (P < 0.001). However, most of the effect of glucose was obtained already at 3 mM, since basal [Ca2+ ]i was little affected by further elevation to 20 mM of the sugar (all five ␣-cells; Fig. 3A). Although most of the late [Ca2+ ]i response to adrenaline disappeared when preventing Ca2+ influx through the voltage-dependent channels, there was a small sustained elevation as long as hormone was present (Figs. 3 and 4A). The store-operated Ca2+ channel blocker 2-APB reduced the remaining elevation by 63 ± 5% (P < 0.01; n = 10; Fig. 4), but there was little effect on the initial [Ca2+ ]i response to adrenaline (all five ␣-cells; Fig. 4A). [Ca2+ ]i of the ␣-cells was resistant to changes in the extracellular Ca2+ concentration when no stimulus was present, but became markedly sensitive to extracellular Ca2+ when adrenaline stimulation was combined with inhibition of voltage-dependent Ca2+ entry (all six ␣-cells; P < 0.01; Fig. 4B). This sensitivity probably represents activation of store-operated Ca2+ influx, since it was also found in all six ␣-cells after releasing Ca2+ from the ER with 50 M of the sarco(endo)plasmatic reticulum Ca2+ ATPase (SERCA)
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Fig. 4. Effects of Ca2+ concentration and 2-APB on the depolarization-independent [Ca2+ ]i responses to adrenaline and CPA in two separately studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 20 mM (A) or 3 mM (B) glucose. Adrenaline (5 M), 100 M diazoxide (Diaz), 10 M nifedipine (Nifed), 100 M 2-APB (APB), 50 M CPA and 0 mM Ca2+ + 2 mM EGTA, 1.28 or 10 mM Ca2+ were then present as indicated.
inhibitor CPA (P < 0.01; Fig. 4B). Moreover, 2-APB inhibited the CPA-induced elevation of [Ca2+ ]i depending on Ca2+ influx. 3.3. The effects of adrenaline are mimicked by other agents mobilizing intracellular Ca2+ Fig. 5 shows two ␣-cells, one with little change of [Ca2+ ]i during exposure to adrenaline but a clear reactivity to carbachol (Fig. 5A), and the other with the opposite response pattern (Fig. 5B). Among 54 ␣-cells challenged with 20 M carbachol, 56% (30 cells) reacted with elevation of [Ca2+ ]i (P < 0.001), which was manifested as a single peak (6 cells), oscillations (20 cells) or sustained elevation (4 cells).
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The carbachol effect was completely prevented by 10 M atropine in two cells with oscillations and two with sustained elevation (P < 0.03; not shown). Moreover, diazoxide (400 M) reduced the late response to carbachol into a small sustained suprabasal elevation of [Ca2+ ]i in all two cells (not shown). After SERCA inhibition with 50 M CPA, 64% of the ␣-cells (7 of 11) exhibited [Ca2+ ]i oscillations (Fig. 5), and 4 ␣-cells exhibited a small sustained suprabasal elevation (not shown). Another SERCA inhibitor DTBHQ (10 M) induced [Ca2+ ]i oscillations in 55% of the ␣-cells (6 of 11) and a small sustained suprabasal elevation of [Ca2+ ]i in the remaining 5 cells (not shown). A third SERCA inhibitor thapsigargin (0.5 M) induced [Ca2+ ]i oscillations in all five cells, and 1 M caused a small sustained suprabasal elevation of [Ca2+ ]i in another two cells (not shown). In accordance with the idea that the late response to SERCA inhibition involves depolarization with voltage-dependent influx of Ca2+ , diazoxide blocked the [Ca2+ ]i oscillations in all five CPA-stimulated (P < 0.01; Fig. 5) and all three DTBHQ-stimulated (not shown) ␣-cells. The CPA-induced [Ca2+ ]i oscillations were also blocked by methoxyverapamil in one tested cell. However, diazoxide failed to affect the small sustained suprabasal elevation of [Ca2+ ]i in all two DTBHQ-stimulated and two thapsigargin-stimulated (not shown) ␣-cells. Moreover, methoxyverapamil had no effect on the small sustained suprabasal elevation of [Ca2+ ]i in two cells stimulated with CPA and DTBHQ, respectively. 3.4. Glucose inhibits the late [Ca2+ ]i responses to adrenaline, forskolin and carbachol Under non-hyperpolarizing conditions, the late [Ca2+ ]i response to adrenaline was partially inhibited after raising the glucose concentration from 3 to 20 mM in 90% of the ␣-cells (9 of 10; P < 0.001; Fig. 6A). Like previously demonstrated in guinea pig ␣-cells [22], forskolin mimics the [Ca2+ ]i elevating action of adrenaline indicating involvement of a -adrenergic mechanism with elevation of cAMP (Fig. 6B). Also this response as well as that to carbachol (Fig. 6C) was completely or partially inhibited by 20 mM glucose in two of three and three of five ␣-cells, respectively. 3.5. Adrenaline and SERCA inhibition depolarizes and glucose hyperpolarizes the α-cells
Fig. 5. Effects of adrenaline, tolbutamide, carbachol, CPA and diazoxide on [Ca2+ ]i in two simultaneously studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 3 mM glucose. Adrenaline (Adr; 5 M), tolbutamide (Tol; 500 M), carbachol (Car; 20 M), CPA (50 M) and diazoxide (Diaz; 400 M) were then added as indicated.
Using a fluorescent indicator we tested how adrenalin, glucose and the SERCA inhibitor CPA affect the membrane potential of the ␣-cells. In the presence of 3 mM glucose, the introduction of 5 M adrenaline resulted in marked depolarization in all four ␣-cells (P < 0.03; Fig. 7A). However, this effect was smaller than that obtained with subsequent exposure to 30.9 mM K+ (Fig. 7B). Raising the glucose concentration to 20 mM suppressed the adrenalineinduced depolarization in all four ␣-cells (P < 0.03). When the depolarizing influence of Ca2+ influx through the L-
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depolarizing effect than glucose omission, and this depolarization was fully reversible (all five ␣-cells; P < 0.01; Fig. 7D). Due to interference with the bis-oxonol indicator, it was not possible to test the effect of the store-operated Ca2+ channel inhibitors 2-APB and Gd3+ .
4. Discussion
Fig. 6. Effects of glucose on the late [Ca2+ ]i response to adrenaline, forskolin and carbachol in three separately studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 3 mM glucose. Adrenaline (Adr; 5 M), tolbutamide (Tol; 500 M), forskolin (5 M), carbachol (20 M) and 20 mM glucose were then present as indicated.
type channels was eliminated by 50 M methoxyverapamil, omission of 20 mM glucose resulted in slight depolarization, which was completely reversed after reintroduction of the sugar in all five ␣-cells (P < 0.01; Fig. 7C). Under similar conditions (20 mM glucose and 50 M methoxyverapamil), the SERCA inhibitor CPA (50 M) had a more pronounced
Fig. 7. Effects of adrenaline, glucose, K+ and CPA on membrane potential in four separately studied ␣-cells. Before the start of the traces, the cells were equilibrated with 150 nM bis-oxonol medium containing 3 mM glucose (A and B) or 20 mM glucose plus 50 M methoxyverapamil (C and D). The glucose concentration was then changed to 20 or 0 mM, the K+ concentration was increased to 30.9 mM and 5 M adrenaline or 50 M CPA were added as indicated. The relative fluorescence is shown as counts per second (CPS) with depolarization corresponding to an increase (arrow). Note the different scale in panel (B).
Although it was early reported that the KATP channel is expressed in clonal mouse ␣-cells [28], other studies gave no support for the presence of KATP channels in normal ␣-cells from guinea pig [14,22] and mouse [6,15]. More recent studies have identified KATP channels in hamster glucagonoma [29] as well as in rat [16] and mouse [17,18] ␣-cells. The present results explain why the KATP channels easily escape detection, demonstrating that channel closure by tolbutamide increased [Ca2+ ]i in only 6% of the mouse ␣-cells. In the latter cells, tolbutamide had a modest effect, probably indicating that only few channels are active and that their closure barely causes sufficient depolarization to open the voltage-dependent Ca2+ channels. However, it was possible to activate the KATP channels with diazoxide, and such activation inhibited the [Ca2+ ]i response to adrenaline. The fact that this inhibition was incomplete in occasional ␣-cells is consistent with reports that the number of KATP channels is only 10–15% of that in -cells [15,17]. The concept that tolbutamide promotes and diazoxide inhibits voltagedependent [Ca2+ ]i signalling was further supported by the observation that tolbutamide readily restored an adrenaline response in the presence of diazoxide. It has been suggested that moderate depolarization by glucose-induced closure of the KATP channels inhibits glucagon secretion by inactivation of the voltage-gated Na+ , T-type Ca2+ and A-type K+ channels participating in the generation of action potentials with accompanying influx of Ca2+ through the L-type channels [17]. Showing that the KATP channels are closed in most ␣-cells and that glucose is hyperpolarizing rather than depolarizing, the present data offer little support for such a paradoxical regulation by glucose. Even if we cannot exclude that the KATP channels are open to a greater extent in ␣-cells located within the pancreatic islets, the maintenance of a glucose-inhibited glucagon secretion from pancreatic islets isolated from KATP channel knockout mice [19] argues against a role of this channel in the nutrient sensing of the ␣-cell. Among blood glucose-elevating hormones adrenaline is important both by mobilizing hepatic glycogen directly and by stimulating glucagon secretion. It is clear from the present data that adrenaline induces intracellular mobilization of Ca2+ in ␣-cells. The initial [Ca2+ ]i response was consequently maintained even when hyperpolarization with diazoxide was combined with the voltage-dependent Ca2+ channel blocker nifedipine. However, most of the late [Ca2+ ]i response to adrenaline depended on depolarization, since it was usually prevented by diazoxide and always
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inhibited by the voltage-dependent Ca2+ channel blocker methoxyverapamil. The present and previous [22] data indicate that the effect of adrenaline involves a -adrenergic mechanism with elevation of cAMP [22]. Although cAMP has been found to enhance the Ca2+ current through L-type channels in depolarized rat ␣-cells [30], such a mechanism cannot account for the depolarization, which opens the channels. We now suggest that emptying of the Ca2+ stores in the ER is the mechanism causing the depolarization by activating a store-operated depolarizing current, which is at least in part carried by Ca2+ . In mouse -cells, the storeoperated current is not sufficiently pronounced to depolarize the cells from the resting level of −70 mV to the −40 to −50 mV required for opening the voltage-dependent L-type Ca2+ channels [31,32]. A small store-operated current can be expected to be more important in ␣-cells due to their higher input resistance [20] and the fact that the action potentials start at voltages as negative as −60 mV [14,16,17]. Carbachol has previously been found to mobilize intracellular Ca2+ in mouse ␣-cells by an atropine-sensitive muscarinic mechanism [33]. In support for the involvement of a store-operated mechanism leading to depolarization, the late effect of carbachol on [Ca2+ ]i was now found to be inhibited by diazoxide. Moreover, depletion of the ER by SERCA inhibition depolarized the ␣-cells and caused [Ca2+ ]i oscillations, which were inhibited by diazoxide and methoxyverapamil. Sometimes the SERCA inhibition caused only a small sustained suprabasal elevation of [Ca2+ ]i . Being insensitive to diazoxide and methoxyverapamil this elevation was probably due to store-operated influx alone. It remains to elucidate whether such a failure to activate Ca2+ influx through the L-type channels is related to depolarizationdependent inactivation of the voltage-gated Na+ , T-type Ca2+ and A-type K+ channels as previously suggested [17]. In mouse pancreatic -cells, Ca2+ -mobilizing agonists activate a non-voltage-dependent store-operated influx of Ca2+ , which is blocked by the inhibitor 2-APB [34]. This agent has been found to be a reliable blocker of store-operated Ca2+ and only inconsistently inhibit InsP3 -mediated Ca2+ release [35]. In accordance with such actions we found that 2-APB had little effect on the initial ␣-cell response to adrenaline but inhibited the small sustained elevation, which remains when preventing Ca2+ influx through the voltage-dependent channels. It is well established that glucose stimulates Ca2+ sequestration in the ER of pancreatic [32,36,39] as well as clonal cells [37,38], and that such filling turns off the store-operated entry of Ca2+ [40]. The present observation that Ca2+ incorporated in response to glucose is mobilized with adrenaline extends previous observations in guinea pig ␣-cells [22], indicating that the Ca2+ sequestration results in hyperpolarization by shutting off a store-operated current. Ca2+ at least in part, mediates this current, since omission of the sugar resulted in depolarization and increase of basal [Ca2+ ]i under conditions preventing influx of the ion through the L-type channels. Future studies will have to clarify the detailed re-
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lationship between the glucose concentration and the membrane potential as well as [Ca2+ ]i . The observation that both the lowering effect on basal [Ca2+ ]i and that on store filling were close to maximal at 3 mM glucose distinguishes the ␣- from the -cell, in which 20 mM of the sugar is required [36,39,41]. This difference is consistent with the observation that glucagon release is partially inhibited already at 3 mM glucose, which is below the 4–5 mM threshold for the stimulation of insulin secretion [42]. Due to the presence of a depolarizing cascade with activation of low threshold Ttype Ca2+ , voltage-dependent Na+ as well as L-type Ca2+ channels the ␣-cells respond to injection of small currents with electrical activity [17]. The present data indicate that mobilization of intracellular Ca2+ by adrenaline, forskolin or carbachol induces a store-operated current sufficient for ␣-cell activation, which may be a unique mechanism among excitable cells. Glucose, which fills the Ca2+ stores, inhibits secretion by shutting off this current. This model for glucose inhibition at the level of the pancreatic ␣-cell is more intuitive than that proposed by Göpel et al. [17] and does not involve the KATP channels. Moreover, it explains the paradox that reduction of ATP by inhibitors of metabolism results in stimulated glucagon secretion [23] by activation of the depolarizing cascade after Ca2+ store-depletion. The direct effects of adrenaline and glucose on the regulation of [Ca2+ ]i in the pancreatic ␣-cell do not exclude that glucose sensing in the hypothalamus [19] and paracrine events within the islets of Langerhans [5–8] are also important for the regulation of glucagon secretion as previously suggested. Different models have suggested a role of a store-operated current for the generation of the characteristic electrophysiology of the -cell [31,43–46]. These studies are all based on measurements of [Ca2+ ]i in intact pancreatic islets. Since the Ca2+ indicators load preferentially the cells located in the islet periphery, leaving the core of -cells essentially unlabelled [47], it is pertinent to note that ␣-cells are located in the islet periphery [48]. With the demonstration of a store-operated pathway in ␣-cells, caution is warranted when drawing conclusions about this mechanism in -cells from [Ca2+ ]i measurements in islets.
Acknowledgements This work was supported by grants from the Swedish Medical Research Council (12X-6240), the Swedish Foundation for Strategic Research, the Swedish Foundation for International Cooperation in Research and Higher Education, the Wenner-Gren Center Foundation, the Swedish Diabetes Association, Novo-Nordisk Foundation, Family Ernfors foundation, the Scandinavian Physiological Society and the Swedish Society for Medical Research. Dr. Vieira received a scholarship from the Amerique Latine Formation Academique (ALFA) Program Islet Research European and Latin American Network (IRELAN) of the European Union.
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A store-operated mechanism determines the activity of the electrically excitable glucagon-secreting pancreatic ␣-cell Yi-Jia Liu1 , Elaine Vieira1 , Erik Gylfe∗ Department of Medical Cell Biology, Uppsala University Biomedical Centre, Husargatan 3, Box 571, SE-752 37 Uppsala, Sweden Received 7 August 2003; received in revised form 3 October 2003; accepted 15 October 2003
Abstract The glucagon-releasing pancreatic ␣-cells are electrically excitable cells but the signal transduction leading to depolarization and secretion is not well understood. To clarify the mechanisms we studied [Ca2+ ]i and membrane potential in individual mouse pancreatic ␣-cells using fluorescent indicators. The physiological secretagogue l-adrenaline increased [Ca2+ ]i causing a peak, which was often followed by maintained oscillations or sustained elevation. The early effect was due to mobilization of Ca2+ from the endoplasmic reticulum (ER) and the late one to activation of store-operated influx of the ion resulting in depolarization and Ca2+ influx through voltage-dependent L-type channels. Consistent with such mechanisms, the effects of adrenaline on [Ca2+ ]i and membrane potential were mimicked by inhibitors of the sarco(endo)plasmic reticulum Ca2+ ATPase. The ␣-cells express ATP-regulated K+ (KATP ) channels, whose activation by diazoxide leads to hyperpolarization. The resulting inhibition of the voltage-dependent [Ca2+ ]i response to adrenaline was reversed when the KATP channels were inhibited by tolbutamide. However, tolbutamide alone rarely affected [Ca2+ ]i , indicating that the KATP channels are normally closed in mouse ␣-cells. Glucose, which is the major physiological inhibitor of glucagon secretion, hyperpolarized the ␣-cells and inhibited the late [Ca2+ ]i response to adrenaline. At concentrations as low as 3 mM, glucose had a pronounced stimulatory effect on Ca2+ sequestration in the ER amplifying the early [Ca2+ ]i response to adrenaline. We propose that adrenaline stimulation and glucose inhibition of the ␣-cell involve modulation of a store-operated current, which controls a depolarizing cascade leading to opening of L-type Ca2+ channels. Such a control mechanism may be unique among excitable cells. © 2003 Elsevier Ltd. All rights reserved. Keywords: Glucagon secretion; Store-operated Ca2+ channel; Endoplasmic reticulum calcium sequestration; Endoplasmic reticulum calcium release; Membrane potential
1. Introduction Diabetes mellitus is characterized by hyperglycemia due to insulin deficiency, which is caused by pancreatic -cell destruction or disturbed secretion of the hormone. In both types 1 and 2 diabetes, failure of glucose to inhibit the secretion of blood glucose-elevating glucagon aggravates the hyperglycemia [1–3]. Moreover, glucagon secretion is not appropriately stimulated when blood glucose falls to low concentrations, a potentially life-threatening condition in insulin-treated diabetic subjects [4]. Whereas considerable attention has been focussed on insulin secretion from the -cell, much less is known about glucagon release from the pancreatic ␣-cell. It has been proposed that insulin [5,6], ∗ Corresponding
author. Tel.: +46-18-4714428; fax: +46-18-4714059. E-mail address: [email protected] (E. Gylfe). 1 Drs. Vieira and Liu contributed equally to the article.
0143-4160/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2003.10.002
GABA [7], or Zn2+ [8] co-secreted with insulin from the -cell, mediates glucose inhibition of glucagon secretion. Although such mechanisms may contribute, they cannot explain why glucose inhibits glucagon secretion from purified ␣-cells [9], and why secretion is inhibited at lower sugar concentrations than those stimulating the secretory activity of the -cell [10]. Metabolism of glucose is essential both for stimulation of insulin release [11] and inhibition of glucagon secretion [12,13]. In the -cell the closure of the ATP-sensitive K+ (KATP ) channels has a central role in stimulus-secretion coupling, transducing the increase in ATP/ADP ratio obtained with glucose metabolism into depolarization with resulting opening of voltage-dependent L-type Ca2+ channels [11]. There are diverging opinions about the presence of KATP channels in the ␣-cell and their possible involvement in the regulation of glucagon secretion [6,14–18]. Since glucose inhibits glucagon release, it is apparent that a fun-
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damentally different process is operating in the pancreatic ␣-cell. Nevertheless, it has been proposed that depolarization by glucose-induced closure of the KATP channels inhibits glucagon release [17]. This paradox was attributed to voltage-dependent steady-state inactivation of voltage-gated Na+ , T-type Ca2+ and A-type K+ channels participating in action potential generation thereby preventing the opening of L-type Ca2+ channels. The latter concept is difficult to reconcile with preservation of glucose-inhibited glucagon secretion in islets isolated from KATP channel knockout mice [19]. Like the secretion of most other hormones, exocytosis of glucagon from the ␣-cells is triggered by an increase of [Ca2+ ]i [20]. Studying guinea pig ␣-cells, we have previously proposed that glucose inhibits glucagon release by lowering [Ca2+ ]i after promoting intracellular sequestration and outward transport of Ca2+ [21,22]. Extending this hypothesis we show that the glucose-induced Ca2+ sequestration in the endoplasmic reticulum (ER) hyperpolarizes mouse ␣-cell by shutting off a store-operated current. This depolarizing current, which is activated by adrenaline and other agents mobilizing intracellular Ca2+ , may be essential for glucagon secretion by contributing to the opening of the L-type Ca2+ channels. The proposed mechanism can explain the paradox that lowering of ATP by inhibitors of metabolism results in stimulated glucagon secretion [23] by activation of the depolarizing store-operated current after Ca2+ store-depletion.
2. Materials and methods 2.1. Reagents and solutions Reagents of analytical grade and deionized water were used. Fura-2 and its acetoxymethyl ester as well as bis-(1,3dibutylbarbituric acid)trimethine oxonol (bis-oxonol) were from Molecular Probes Inc. (Eugene, OR). Sigma Chemical Co. (St. Louis, MO) provided atropine, biotinylated rabbit anti-guinea pig immunoglobulin, BSA (fraction V), carbachol, l-adrenaline, HEPES, nifedipine, poly-l-lysine, and thapsigargin. Cyclopiazonic acid (CPA) was from Calbiochem (La Jolla, CA), 2,5-di-tert-butylhydroquinone (DTBHQ) from Aldrich-Chemie (Steinheim, Germany) and 2-aminoethoxydiphenyl borate (2-APB) from Aldrich (Gillingham, UK). DAKO Corp. (Carpenteria, CA) supplied rabbit anti-human glucagon serum, biotinylated goat antirabbit immunoglobulin, normal goat serum and 5-bromo4-chloro-3-indoxyl phosphate plus nitro blue tetrazolium chloride (BCIP/NBT). Fetal calf serum was bought from Gibco Ltd. (Paisley, Scotland) and collagenase was from Boehringer Mannheim GmbH (Mannheim, Germany). Biolog Life Science Institute (Bremen, Germany) supplied adenosine 3 ,5 -cyclic monophosphorothioate Rp-isomer (Rp-cAMPS). Diazoxide and methoxyverapamil were kindly donated by Schering-Plough Int. (Kenilworth, NJ)
and Knoll AG (Ludwigshafen, Germany), respectively, and forskolin plus tolbutamide by Hoechst Marion Roussel AB, (Stockholm, Sweden). 2.2. Preparation of pancreatic islet cells Islets of Langerhans were collagenase-isolated from the splenic part (lacking pancreatic polypeptide-secreting cells) of the pancreas of NMRI mice. The animals were placed in sealed container into which as stream of CO2 was delivered. The experimental procedures were approved by the Uppsala Ethics Committee. When the animals became unconscious, they were killed by decapitation. The peritoneal cavity was opened and the pancreas was excised and cut into small pieces, which were digested with collagenase to obtain free islets of Langerhans. Free cells were then prepared by shaking the islets in a Ca2+ -deficient medium [24]. To increase the yield of the superficially located ␣-cells, the shaking was interrupted before the entire islets had fragmented. The cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 g/ml streptomycin and 30 g/ml gentamicin and allowed to attach to poly-l-lysine-coated circular 25 mm cover slips during 1–3 days culture at 37 ◦ C in RPMI 1640 medium in an atmosphere of 5% CO2 . 2.3. Image analysis of [Ca2+ ]i Loading of cells with the indicator fura-2 was performed during 40-min incubation at 37 ◦ C in a HEPES-buffered medium (25 mM, pH 7.4) containing 0.5 mg/ml bovine serum albumin, 138 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl2 , 1.28 mM CaCl2 , 3 mM glucose and 1 M fura-2 acetoxymethyl ester (0.1% dimethylsulphoxide). The cover slips with the attached cells were then used as exchangeable bottoms of an open chamber thermostated at 37 ◦ C. The chamber volume was 0.16 ml and the cells were superfused at a rate of 1 ml/min with a medium lacking indicator. Addition of thapsigargin, which sticks to plastic, was made directly to the superfusion chamber with a pipette. The superfusion flow was then interrupted for 2–3 min to ascertain an effect of the drug. [Ca2+ ]i measurements were conducted using a Nikon Diaphot microscope equipped with an epifluorescence illuminator and a 40× oil immersion fluorescence objective. A monochromator, which is part of a Quanticell 700 imaging system (VisiTech International, Sunderland, UK), provided excitation light flashes at 340 and 380 nm, and the emission was measured at 515 nm using a 400-nm dichroic beam splitter and an intensified CCD camera. Image pairs were taken every 1–2 s and 340/380 nm ratio (R) images were calculated after subtraction of background images. [Ca2+ ]i images were calculated using a dissociation constant (KD ) of 224 nM [25] and the equation: [Ca2+ ] = KD
F0 (R − Rmin ) Fs (Rmax − R)
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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 medium lacking Ca2+ . Fs and Rmax are the corresponding data obtained with a saturating concentration of Ca2+ . By selecting image fields dominated by small cells, it was possible to obtain recordings from more non--cells than expected from their percentage in the preparation [26]. Usually, two to five small cells were present in each image field. Simultaneous recordings from 2 ␣-cells were not uncommon and rarely up to four ␣-cells were studied in parallel. Some of the stimuli applied were helpful to preliminarily classify the cells before the final identification by immunostaining. 2.4. Changes in membrane potential Changes in membrane potential were recorded using the fluorescent probe bis-oxonol added to a final concentration of 150 nM from a 1000-fold concentrated stock solution in ethanol. After 10 min equilibration with the bis-oxonol containing medium at 37 ◦ C, the fluorescence was recorded from individual ␣-cells. The cells were studied in a similar microscopic system as used for imaging of Ca2+ . However, in this case a photometric approach was employed using a monochromator-based photon-counting Deltascan system with Felix software (Photon Technology International, Lawrenceville, NJ). The excitation and emission wavelengths were 485 and 520 nm and a 505 nm dichroic beam splitter was used. The anionic dye enters the depolarized cells where it binds to intracellular proteins or membranes and exhibit enhanced fluorescence and red spectral shifts [27]. Depolarization and hyperpolarization are consequently reflected as increases and decreases in fluorescence, respectively.
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heterogeneity, and even those on the same cover glass often reacted differently to the same stimulus. Due to the qualitative differences in cellular responses the results have been presented as proportions of cells reacting in different ways. Statistical analyses of the proportion of cells with a certain response were made with two-tailed Fisher’s exact test or χ2 test with Yates’ correction. Wilcoxon’s signed rank test was used when qualitatively different responses were found ranking no response to a stimulus as 0, a single [Ca2+ ]i peak as 1, oscillations as 2 and sustained elevation as 3. Paired Student’s t-test was used to assess quantitative differences. Statistical significance was set at a P value of <0.05. Due to the scarcity of ␣-cells, one to two cells were studied in most experiments (see above), and cells from one cell isolation were used for four to six experiments.
3. Results 3.1. The KATP channels are functionally inactive in most α-cells In >100 experiments on cells from >20 islet isolations, individual mouse pancreatic ␣- and -cells exposed to 3 mM glucose typically exhibit stable basal [Ca2+ ]i levels in the 60–100 nM range. Only 7% of the ␣-cells (16 of 244) displayed slow oscillations of [Ca2+ ]i under these conditions (see Fig. 2D). Whereas 134 of 136 -cells reacted to 0.5–1 mM of the depolarizing KATP channel inhibitor tolbutamide with elevation of [Ca2+ ]i (Fig. 1B), 94% of the ␣-cells (261 of 277) remained silent (Figs. 1A, 5 and 6A). The tolbutamide-induced elevations of [Ca2+ ]i in the 16 responsive ␣-cells were modest. Fig. 2A and B shows the most prominent effect of tolbutamide. Introduction of
2.5. Identification of α-cells Each experiment was terminated by immunostaining the cells in the experimental chamber. The cells were superfused with albumin-free medium and fixed with 95% ethanol. After rinsing with distilled water and Tris buffer (0.5 M, pH 7.6), normal goat serum (diluted 1:10) was added to reduce background staining. After 10 min, rabbit anti-human glucagon (1:200) was added for 20–30 min followed by rinsing with Tris buffer. Biotinylated goat anti-rabbit immunoglobulin (1:500) was then introduced for 10 min, followed by rinsing and addition of alkaline phosphatase-conjugated streptavidine (1:200) for a further 10 min. The BCIP/NBT colour reagent was then added for 2–5 min. 2.6. Statistical analysis Like many cell types, the ␣-cells responded with a single peak, oscillations and sustained elevation of [Ca2+ ]i with increasing concentrations of agonists. However, the sensitivity of individual ␣-cells was characterized by considerable
Fig. 1. Effects of tolbutamide, adrenaline, diazoxide and methoxyverapamil on [Ca2+ ]i of an ␣-cell and a simultaneously studied -cell. The fura-2-loaded cells were initially exposed to a medium containing 3 mM glucose. Tolbutamide (Tol; 500 M), Adrenaline (5 M), diazoxide (Diaz; 400 M) and methoxyverapamil (MV; 50 M) were then added as indicated. The ␣-cell is shown in panel (A) and the -cell in panel (B) as indicated.
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Fig. 2. Effects of adrenaline, tolbutamide and diazoxide on [Ca2+ ]i of four separately studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 3 mM glucose. Adrenaline (A, Adr; 5 M), Tolbutamide (Tolb; 1 mM, panels A and B; 500 M, panels C and D) and diazoxide (400 M) were then added as indicated.
5 M adrenaline did not affect previously silent -cells (Fig. 1B) but caused elevation of [Ca2+ ]i in 86% of the ␣-cells (219 of 255; P < 0.001, χ2 or Wilcoxon test). Among the responsive ␣-cells 40% (64 cells) reacted with a single [Ca2+ ]i peak (not shown), there were slow [Ca2+ ]i oscillations in 46% (75 cells; Figs. 1A and 6) and 14% (23 cells) displayed sustained elevation of [Ca2+ ]i (not shown). In support for the presence of KATP channels, the hyperpolarizing channel activator diazoxide almost completely inhibited the late adrenaline effect (P < 0.001) leaving only a small suprabasal elevation in 91% (20 cells; Figs. 1A, 2C and D) and was partially inhibitory in the remaining 2 ␣-cells. Tolbutamide counteracted this effect by promptly reversing diazoxide inhibition of the [Ca2+ ]i response to adrenaline (all six ␣-cells; P < 0.01; Fig. 2C and D). 3.2. Glucose stimulates Ca2+ removal from the cytoplasm and enhances the initial effect of adrenaline on [Ca2+ ]i Using guinea pig ␣-cells, we have previously found that pre-exposure to glucose enhances the subsequent [Ca2+ ]i peak response to adrenaline by promoting intracellular Ca2+ sequestration [22]. Since the inhibitory effect of diazoxide indicates that adrenaline depolarizes the ␣-cells, we now studied the effect of glucose on the initial [Ca2+ ]i peak response to adrenaline under conditions preventing voltagedependent Ca2+ entry. Fig. 3A shows an initial adrenaline response of an ␣-cell equilibrated in 3 mM glucose. The elevation of [Ca2+ ]i was terminated immediately upon removal of adrenaline. After subsequent omission of glucose and addition of 400 M hyperpolarizing diazoxide short restimulation with adrenaline gave a smaller peak in all 16 ␣-cells (P < 0.001). When 3 mM glucose was reintroduced in the continued presence of diazoxide, a third adrenaline stimulation resulted in a 4.3 ± 0.6-fold (P < 0.05, n = 6) greater integrated [Ca2+ ]i peak response than in the absence
Fig. 3. Effect of glucose concentration on the early depolarization-independent [Ca2+ ]i response to adrenaline in two separately studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 3 mM (A) or 20 mM (B) glucose. Adrenaline (Adr; 5 M), 100 or 400 M diazoxide, 10 M nifedipine and 0, 3 or 20 mM glucose were then present as indicated.
of the sugar. A similar or slightly greater [Ca2+ ]i peak response to adrenaline was observed after elevation of glucose to 20 mM (all 14 ␣-cells; Fig. 3A). The stimulatory effect of glucose on the initial [Ca2+ ]i response to adrenaline was apparent also when switching between 0 and 20 mM glucose in six experiments performed in the presence of 100 M diazoxide and 10 M of the voltage-dependent Ca2+ channel blocker nifedipine (Fig. 3B; P < 0.01). Supporting the idea that glucose stimulates Ca2+ removal from the cytoplasm the sugar concentration also affected the basal [Ca2+ ]i level. Omission of glucose consequently resulted in a small gradual elevation of basal [Ca2+ ]i in all 11 ␣-cells and reintroduction of 3 (Fig. 3A) or 20 (not shown) mM glucose restored the initial [Ca2+ ]i level in all 11 cells (P < 0.001). However, most of the effect of glucose was obtained already at 3 mM, since basal [Ca2+ ]i was little affected by further elevation to 20 mM of the sugar (all five ␣-cells; Fig. 3A). Although most of the late [Ca2+ ]i response to adrenaline disappeared when preventing Ca2+ influx through the voltage-dependent channels, there was a small sustained elevation as long as hormone was present (Figs. 3 and 4A). The store-operated Ca2+ channel blocker 2-APB reduced the remaining elevation by 63 ± 5% (P < 0.01; n = 10; Fig. 4), but there was little effect on the initial [Ca2+ ]i response to adrenaline (all five ␣-cells; Fig. 4A). [Ca2+ ]i of the ␣-cells was resistant to changes in the extracellular Ca2+ concentration when no stimulus was present, but became markedly sensitive to extracellular Ca2+ when adrenaline stimulation was combined with inhibition of voltage-dependent Ca2+ entry (all six ␣-cells; P < 0.01; Fig. 4B). This sensitivity probably represents activation of store-operated Ca2+ influx, since it was also found in all six ␣-cells after releasing Ca2+ from the ER with 50 M of the sarco(endo)plasmatic reticulum Ca2+ ATPase (SERCA)
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Fig. 4. Effects of Ca2+ concentration and 2-APB on the depolarization-independent [Ca2+ ]i responses to adrenaline and CPA in two separately studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 20 mM (A) or 3 mM (B) glucose. Adrenaline (5 M), 100 M diazoxide (Diaz), 10 M nifedipine (Nifed), 100 M 2-APB (APB), 50 M CPA and 0 mM Ca2+ + 2 mM EGTA, 1.28 or 10 mM Ca2+ were then present as indicated.
inhibitor CPA (P < 0.01; Fig. 4B). Moreover, 2-APB inhibited the CPA-induced elevation of [Ca2+ ]i depending on Ca2+ influx. 3.3. The effects of adrenaline are mimicked by other agents mobilizing intracellular Ca2+ Fig. 5 shows two ␣-cells, one with little change of [Ca2+ ]i during exposure to adrenaline but a clear reactivity to carbachol (Fig. 5A), and the other with the opposite response pattern (Fig. 5B). Among 54 ␣-cells challenged with 20 M carbachol, 56% (30 cells) reacted with elevation of [Ca2+ ]i (P < 0.001), which was manifested as a single peak (6 cells), oscillations (20 cells) or sustained elevation (4 cells).
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The carbachol effect was completely prevented by 10 M atropine in two cells with oscillations and two with sustained elevation (P < 0.03; not shown). Moreover, diazoxide (400 M) reduced the late response to carbachol into a small sustained suprabasal elevation of [Ca2+ ]i in all two cells (not shown). After SERCA inhibition with 50 M CPA, 64% of the ␣-cells (7 of 11) exhibited [Ca2+ ]i oscillations (Fig. 5), and 4 ␣-cells exhibited a small sustained suprabasal elevation (not shown). Another SERCA inhibitor DTBHQ (10 M) induced [Ca2+ ]i oscillations in 55% of the ␣-cells (6 of 11) and a small sustained suprabasal elevation of [Ca2+ ]i in the remaining 5 cells (not shown). A third SERCA inhibitor thapsigargin (0.5 M) induced [Ca2+ ]i oscillations in all five cells, and 1 M caused a small sustained suprabasal elevation of [Ca2+ ]i in another two cells (not shown). In accordance with the idea that the late response to SERCA inhibition involves depolarization with voltage-dependent influx of Ca2+ , diazoxide blocked the [Ca2+ ]i oscillations in all five CPA-stimulated (P < 0.01; Fig. 5) and all three DTBHQ-stimulated (not shown) ␣-cells. The CPA-induced [Ca2+ ]i oscillations were also blocked by methoxyverapamil in one tested cell. However, diazoxide failed to affect the small sustained suprabasal elevation of [Ca2+ ]i in all two DTBHQ-stimulated and two thapsigargin-stimulated (not shown) ␣-cells. Moreover, methoxyverapamil had no effect on the small sustained suprabasal elevation of [Ca2+ ]i in two cells stimulated with CPA and DTBHQ, respectively. 3.4. Glucose inhibits the late [Ca2+ ]i responses to adrenaline, forskolin and carbachol Under non-hyperpolarizing conditions, the late [Ca2+ ]i response to adrenaline was partially inhibited after raising the glucose concentration from 3 to 20 mM in 90% of the ␣-cells (9 of 10; P < 0.001; Fig. 6A). Like previously demonstrated in guinea pig ␣-cells [22], forskolin mimics the [Ca2+ ]i elevating action of adrenaline indicating involvement of a -adrenergic mechanism with elevation of cAMP (Fig. 6B). Also this response as well as that to carbachol (Fig. 6C) was completely or partially inhibited by 20 mM glucose in two of three and three of five ␣-cells, respectively. 3.5. Adrenaline and SERCA inhibition depolarizes and glucose hyperpolarizes the α-cells
Fig. 5. Effects of adrenaline, tolbutamide, carbachol, CPA and diazoxide on [Ca2+ ]i in two simultaneously studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 3 mM glucose. Adrenaline (Adr; 5 M), tolbutamide (Tol; 500 M), carbachol (Car; 20 M), CPA (50 M) and diazoxide (Diaz; 400 M) were then added as indicated.
Using a fluorescent indicator we tested how adrenalin, glucose and the SERCA inhibitor CPA affect the membrane potential of the ␣-cells. In the presence of 3 mM glucose, the introduction of 5 M adrenaline resulted in marked depolarization in all four ␣-cells (P < 0.03; Fig. 7A). However, this effect was smaller than that obtained with subsequent exposure to 30.9 mM K+ (Fig. 7B). Raising the glucose concentration to 20 mM suppressed the adrenalineinduced depolarization in all four ␣-cells (P < 0.03). When the depolarizing influence of Ca2+ influx through the L-
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depolarizing effect than glucose omission, and this depolarization was fully reversible (all five ␣-cells; P < 0.01; Fig. 7D). Due to interference with the bis-oxonol indicator, it was not possible to test the effect of the store-operated Ca2+ channel inhibitors 2-APB and Gd3+ .
4. Discussion
Fig. 6. Effects of glucose on the late [Ca2+ ]i response to adrenaline, forskolin and carbachol in three separately studied ␣-cells. The fura-2-loaded cells were initially exposed to a medium containing 3 mM glucose. Adrenaline (Adr; 5 M), tolbutamide (Tol; 500 M), forskolin (5 M), carbachol (20 M) and 20 mM glucose were then present as indicated.
type channels was eliminated by 50 M methoxyverapamil, omission of 20 mM glucose resulted in slight depolarization, which was completely reversed after reintroduction of the sugar in all five ␣-cells (P < 0.01; Fig. 7C). Under similar conditions (20 mM glucose and 50 M methoxyverapamil), the SERCA inhibitor CPA (50 M) had a more pronounced
Fig. 7. Effects of adrenaline, glucose, K+ and CPA on membrane potential in four separately studied ␣-cells. Before the start of the traces, the cells were equilibrated with 150 nM bis-oxonol medium containing 3 mM glucose (A and B) or 20 mM glucose plus 50 M methoxyverapamil (C and D). The glucose concentration was then changed to 20 or 0 mM, the K+ concentration was increased to 30.9 mM and 5 M adrenaline or 50 M CPA were added as indicated. The relative fluorescence is shown as counts per second (CPS) with depolarization corresponding to an increase (arrow). Note the different scale in panel (B).
Although it was early reported that the KATP channel is expressed in clonal mouse ␣-cells [28], other studies gave no support for the presence of KATP channels in normal ␣-cells from guinea pig [14,22] and mouse [6,15]. More recent studies have identified KATP channels in hamster glucagonoma [29] as well as in rat [16] and mouse [17,18] ␣-cells. The present results explain why the KATP channels easily escape detection, demonstrating that channel closure by tolbutamide increased [Ca2+ ]i in only 6% of the mouse ␣-cells. In the latter cells, tolbutamide had a modest effect, probably indicating that only few channels are active and that their closure barely causes sufficient depolarization to open the voltage-dependent Ca2+ channels. However, it was possible to activate the KATP channels with diazoxide, and such activation inhibited the [Ca2+ ]i response to adrenaline. The fact that this inhibition was incomplete in occasional ␣-cells is consistent with reports that the number of KATP channels is only 10–15% of that in -cells [15,17]. The concept that tolbutamide promotes and diazoxide inhibits voltagedependent [Ca2+ ]i signalling was further supported by the observation that tolbutamide readily restored an adrenaline response in the presence of diazoxide. It has been suggested that moderate depolarization by glucose-induced closure of the KATP channels inhibits glucagon secretion by inactivation of the voltage-gated Na+ , T-type Ca2+ and A-type K+ channels participating in the generation of action potentials with accompanying influx of Ca2+ through the L-type channels [17]. Showing that the KATP channels are closed in most ␣-cells and that glucose is hyperpolarizing rather than depolarizing, the present data offer little support for such a paradoxical regulation by glucose. Even if we cannot exclude that the KATP channels are open to a greater extent in ␣-cells located within the pancreatic islets, the maintenance of a glucose-inhibited glucagon secretion from pancreatic islets isolated from KATP channel knockout mice [19] argues against a role of this channel in the nutrient sensing of the ␣-cell. Among blood glucose-elevating hormones adrenaline is important both by mobilizing hepatic glycogen directly and by stimulating glucagon secretion. It is clear from the present data that adrenaline induces intracellular mobilization of Ca2+ in ␣-cells. The initial [Ca2+ ]i response was consequently maintained even when hyperpolarization with diazoxide was combined with the voltage-dependent Ca2+ channel blocker nifedipine. However, most of the late [Ca2+ ]i response to adrenaline depended on depolarization, since it was usually prevented by diazoxide and always
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inhibited by the voltage-dependent Ca2+ channel blocker methoxyverapamil. The present and previous [22] data indicate that the effect of adrenaline involves a -adrenergic mechanism with elevation of cAMP [22]. Although cAMP has been found to enhance the Ca2+ current through L-type channels in depolarized rat ␣-cells [30], such a mechanism cannot account for the depolarization, which opens the channels. We now suggest that emptying of the Ca2+ stores in the ER is the mechanism causing the depolarization by activating a store-operated depolarizing current, which is at least in part carried by Ca2+ . In mouse -cells, the storeoperated current is not sufficiently pronounced to depolarize the cells from the resting level of −70 mV to the −40 to −50 mV required for opening the voltage-dependent L-type Ca2+ channels [31,32]. A small store-operated current can be expected to be more important in ␣-cells due to their higher input resistance [20] and the fact that the action potentials start at voltages as negative as −60 mV [14,16,17]. Carbachol has previously been found to mobilize intracellular Ca2+ in mouse ␣-cells by an atropine-sensitive muscarinic mechanism [33]. In support for the involvement of a store-operated mechanism leading to depolarization, the late effect of carbachol on [Ca2+ ]i was now found to be inhibited by diazoxide. Moreover, depletion of the ER by SERCA inhibition depolarized the ␣-cells and caused [Ca2+ ]i oscillations, which were inhibited by diazoxide and methoxyverapamil. Sometimes the SERCA inhibition caused only a small sustained suprabasal elevation of [Ca2+ ]i . Being insensitive to diazoxide and methoxyverapamil this elevation was probably due to store-operated influx alone. It remains to elucidate whether such a failure to activate Ca2+ influx through the L-type channels is related to depolarizationdependent inactivation of the voltage-gated Na+ , T-type Ca2+ and A-type K+ channels as previously suggested [17]. In mouse pancreatic -cells, Ca2+ -mobilizing agonists activate a non-voltage-dependent store-operated influx of Ca2+ , which is blocked by the inhibitor 2-APB [34]. This agent has been found to be a reliable blocker of store-operated Ca2+ and only inconsistently inhibit InsP3 -mediated Ca2+ release [35]. In accordance with such actions we found that 2-APB had little effect on the initial ␣-cell response to adrenaline but inhibited the small sustained elevation, which remains when preventing Ca2+ influx through the voltage-dependent channels. It is well established that glucose stimulates Ca2+ sequestration in the ER of pancreatic [32,36,39] as well as clonal cells [37,38], and that such filling turns off the store-operated entry of Ca2+ [40]. The present observation that Ca2+ incorporated in response to glucose is mobilized with adrenaline extends previous observations in guinea pig ␣-cells [22], indicating that the Ca2+ sequestration results in hyperpolarization by shutting off a store-operated current. Ca2+ at least in part, mediates this current, since omission of the sugar resulted in depolarization and increase of basal [Ca2+ ]i under conditions preventing influx of the ion through the L-type channels. Future studies will have to clarify the detailed re-
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lationship between the glucose concentration and the membrane potential as well as [Ca2+ ]i . The observation that both the lowering effect on basal [Ca2+ ]i and that on store filling were close to maximal at 3 mM glucose distinguishes the ␣- from the -cell, in which 20 mM of the sugar is required [36,39,41]. This difference is consistent with the observation that glucagon release is partially inhibited already at 3 mM glucose, which is below the 4–5 mM threshold for the stimulation of insulin secretion [42]. Due to the presence of a depolarizing cascade with activation of low threshold Ttype Ca2+ , voltage-dependent Na+ as well as L-type Ca2+ channels the ␣-cells respond to injection of small currents with electrical activity [17]. The present data indicate that mobilization of intracellular Ca2+ by adrenaline, forskolin or carbachol induces a store-operated current sufficient for ␣-cell activation, which may be a unique mechanism among excitable cells. Glucose, which fills the Ca2+ stores, inhibits secretion by shutting off this current. This model for glucose inhibition at the level of the pancreatic ␣-cell is more intuitive than that proposed by Göpel et al. [17] and does not involve the KATP channels. Moreover, it explains the paradox that reduction of ATP by inhibitors of metabolism results in stimulated glucagon secretion [23] by activation of the depolarizing cascade after Ca2+ store-depletion. The direct effects of adrenaline and glucose on the regulation of [Ca2+ ]i in the pancreatic ␣-cell do not exclude that glucose sensing in the hypothalamus [19] and paracrine events within the islets of Langerhans [5–8] are also important for the regulation of glucagon secretion as previously suggested. Different models have suggested a role of a store-operated current for the generation of the characteristic electrophysiology of the -cell [31,43–46]. These studies are all based on measurements of [Ca2+ ]i in intact pancreatic islets. Since the Ca2+ indicators load preferentially the cells located in the islet periphery, leaving the core of -cells essentially unlabelled [47], it is pertinent to note that ␣-cells are located in the islet periphery [48]. With the demonstration of a store-operated pathway in ␣-cells, caution is warranted when drawing conclusions about this mechanism in -cells from [Ca2+ ]i measurements in islets.
Acknowledgements This work was supported by grants from the Swedish Medical Research Council (12X-6240), the Swedish Foundation for Strategic Research, the Swedish Foundation for International Cooperation in Research and Higher Education, the Wenner-Gren Center Foundation, the Swedish Diabetes Association, Novo-Nordisk Foundation, Family Ernfors foundation, the Scandinavian Physiological Society and the Swedish Society for Medical Research. Dr. Vieira received a scholarship from the Amerique Latine Formation Academique (ALFA) Program Islet Research European and Latin American Network (IRELAN) of the European Union.
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