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Department of Medical Cell Biology, Biomedicum, Uppsala University, SE-751 23 Uppsala, Sweden

(Requests for offprints should be addressed to B Hellman; Email: Bo.Hellman{at}medcellbiol.uu.se)

	Abstract

Top Abstract Introduction Material and Methods Results Discussion References

 
External ATP is supposed to trigger short-lived increases (transients) of cytoplasmic Ca²⁺ important for entraining insulin-secreting ß-cells into a common rhythm. To get insight into this process, rises of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) induced by external ATP were compared with those obtained with acetylcholine, another neurotransmitter with stimulatory effects on the inositol trisphosphate (IP₃) production. A ratiometric fura-2 technique was used for measuring [Ca²⁺]_i in individual ß-cells and small aggregates isolated from ob/ob mouse islets and superfused with a medium containing methoxyverapamil. ATP and acetylcholine induced temporary rises of [Ca²⁺]_I from a basal level manifested as solitary transients (<20 s) and bumps (20 s) superimposed or not with transients. Addition of ATP (1–100 µM) usually triggered transients whereas acetylcholine induced bumps lacking superimposed transients. After the initial rise there was a steady-state elevation of [Ca²⁺]_i in ß-cells exposed to acetylcholine but not to ATP. Similar differences were seen comparing the responses of rat ß-cells to 100 µM ATP and acetylcholine. Inhibition of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump (with 50 µM cyclopiazonic acid) prevented both the ATP-induced rise of [Ca²⁺]_i and the spontaneous firing of transients. Similar effects were seen after activation of protein kinase C (10 nM phorbol-12-myristate-13-acetate), whereas an inhibitor of this enzyme (2 µM bisindolylmaleimide) promoted the generation of transients. The results indicate that ATP fulfils the demands for a coordinator of the secretory activity of ß-cells by generating distinct [Ca²⁺]_i transients without sustained elevation of basal [Ca²⁺]_i.

	Introduction

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The cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_i) is a key regulator of cellular events, including the release of insulin from pancreatic ß-cells. Like other secretory processes, exocytotic release of insulin is subject to periodic variations (Stagner & Samols 1985, Lefèbvre et al. 1987, Hellman et al. 1992, Bergsten & Hellman 1993, Pørksen et al. 1995). It was shown in 1988 that cyclic variations of [Ca²⁺]_i is an intrinsic property of the ß-cells, occurring also in the absence of neural pacemaker activity (Grapengiesser et al. 1988). Indeed, each ß-cell is a biological oscillator, responding to glucose stimulation with slow oscillations of [Ca²⁺]_i resulting from rhythmic depolarization with subsequent entry of Ca²⁺ through voltage-activated channels (Hellman et al. 1992). Individual ß-cells differ considerably with regard to duration and frequency of the [Ca²⁺]_i oscillations.

Within the islets both gap junctions (Meda 2003) and diffusible messengers (Grapengiesser et al. 2004) entrain the [Ca²⁺]_i oscillations into a common rhythm. Recent studies suggest that the coordinating action of diffusible messengers is linked to inositol trisphosphate (IP₃)-induced generation of short-lived increases (transients) of [Ca²⁺]_i temporarily interrupting the Ca²⁺ entry into the ß-cells by activating a repolarizing K⁺ current (Grapengiesser et al. 1999, 2001, 2003, Lundquist et al. 2003, Hellman et al. 2004). Another effect of the [Ca²⁺]_i transients is to activate exocytosis, a process coupled to intermittent release of ATP (Hellman et al. 2004). The efficiency of the mechanisms for coordinating ß-cells within an islet is illustrated by the observation that individual islets release insulin in pulses with a frequency unaffected by the islet size (Bergsten & Hellman 1993). Periodic variations of circulating insulin require that the ß-cell oscillations of [Ca²⁺]_i appear in the same phase in the numerous islets of the pancreas. The data obtained so far make it attractive to postulate that neural activity with intermittent discharge of ATP accounts for the entrainment of the differently phased islets into a common rhythm (Grapengiesser et al. 2004).

The concept that coordination of the ß-cell rhythmicity is linked to mobilization of intracellular Ca²⁺ raises the question whether ATP is more suited as a trigger of a synchronizing Ca²⁺ signal than other external activators of phospholipase C (PLC). We now demonstrate that micromolar concentrations of ATP differ from acetylcholine in essentially generating transients without subsequent elevation of basal [Ca²⁺]_i.

	Material and Methods

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Chemicals

Reagents of analytical grade and deionized water were used. ATP (ultragrade), acetylcholine, glucagon and methoxyverapamil were obtained from Sigma Chemical Co. (St Louis, MO, USA). Collagenase, BSA and Hepes were provided by Roche Diagnostics GmbH (Mannheim, Germany). Calbiochem (La Jolla, CA, USA) supplied cyclopiazonic acid (CPA), bisindolylmaleimide (BIM) and phorbol-12-myristate-13-acetate (PMA). The acetoxymethyl ester of fura-2 was bought from Molecular Probes (Eugene, OR, USA).

Preparation of ß-cells

Adult ob/ob mice were taken from a non-inbred local colony (Hellman 1965) and killed by decapitation. Islets were isolated with the aid of collagenase from the splenic part of the pancreas. These islets contain >90% ß-cells, known to have a normal secretory response to glucose (Hahn et al. 1974). Single cells and small aggregates were prepared by shaking the islets in a Ca²⁺-deficient medium. In some experiments the effects of ATP and acetylcholine were tested with cells prepared from islets of adult Sprague–Dawley rats. After suspension in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin and 30 µg/ml gentamicin, the cells were allowed to attach to the central part of circular coverslips during 2–5 days of culture at 37°C in an atmosphere of 5% CO₂ in humidified air. The identification of the ß-cells was based on their large size and low nucleus/cytoplasm volume ratio compared with the islet cells secreting glucagon and somatostatin (Berts et al. 1995).

Measurements of cytoplasmic Ca²⁺

The experiments were performed with a basal medium containing 0.5 mg/ml BSA and 125 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl₂, 2.6 mM CaCl₂ and 25 mM Hepes with pH adjusted to 7.40 using NaOH. After rinsing, the cells were loaded with 0.5–1.0 µM fura-2 acetoxymethyl ester during 30–40 min incubation in the presence of 3 mM glucose. The coverslips with the attached cells were then washed and used as exchangeable bottoms of an open chamber connected to a two-channel peristaltic pump. The cells were superfused at a rate of 0.8 ml/min with a medium containing 20 mM glucose. The Ca²⁺-channel blocker methoxyverapamil (50 µM) was included in the medium to allow recognition of Ca²⁺ transients without the background disturbance of slow oscillations. If not otherwise stated glucagon (20 nM) was present to counteract the depletion of cAMP known to occur in isolated ß-cells (Schuit & Pipeleers 1985). The studies were performed with an inverted microscope (Nikon Diaphot) using a climate box maintained at 37 °C. The microscope was equipped for epifluorescence fluorometry with a 400 nm dichroic mirror and a 40 x Fluor oil immersion objective.

A xenon arc lamp was used for excitation of fura-2 at 340 and 380 nm with the aid of a monochromator (Cairn; Optoscan, Faversham, Kent, UK). Images were collected through a 30 nm half-bandwidth filter at 510 nm with an intensified CCD camera (Extended Isis-M; Photonic Science, Robertsbridge, East Sussex, UK). Pairs of 340 and 380 nm images, consisting of 10–16 accumulated video frames, were captured, followed by a delay resulting in measuring cycles of 2–5 s. The specimens were illuminated only during image capture and excitation light was kept at a minimum. Ratio frames were calculated after background subtraction, and [Ca²⁺]_i was estimated as described previously (Grynkiewicz et al. 1985, Gylfe et al. 1991).

Evaluation of data

Each experiment refers to analyses of 4–12 cells or small aggregates (<10 cells) attached to coverslips. Temporary increases of [Ca²⁺]_i extending 50 nM were recognized as transients (<20 s) or bumps (20 s). Moreover, we distinguished between bumps superimposed or not with transients. Early effects of additions to the superfusion medium were evaluated by comparing the proportion of cells/aggregates generating an increase of [Ca²⁺]_i during a 60-s period preceding and following the exposure to the compounds. When the late effects were studied, the 60-s period following the exposure was excluded. Results are presented as means ± S.E.M Statistical significances were evaluated by Student’s t-test for paired and unpaired data.

	Results

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Initial response with temporary rise of Ca²⁺

In the absence of glucagon, spontaneous transients of [Ca²⁺]_i were only seen occasionally. Addition of ATP or acetylcholine to medium lacking glucagon resulted in temporary rises of [Ca²⁺]_i (Fig. 1). The proportion of cells responding with solitary transients and/or bumps, superimposed or not with transients, is shown in Table 1. ATP was more effective than acetylcholine as a promoter of transients superimposed on bumps or starting from the basal level. Comparing the effects of different concentrations of ATP, it was found that 100 µM generated more solitary transients than 1 µM.

View larger version (20K): [in this window] [in a new window]   Figure 1 Effects of micromolar concentrations of ATP and acetylcholine (ACh) on [Ca2+]i in mouse ß-cells superfused with a glucagon-free medium containing 20 mM glucose and 50 µM methoxyverapamil. Typical responses to ATP are a bump with a superimposed transient (A) or a solitary transient (B). Responses to acetylcholine were often manifested as a bump lacking transients followed by a sustained elevation of [Ca2+]i (C, D).

View larger version (22K): [in this window] [in a new window]   Figure 2 Effects of ATP (A) and acetylcholine (ACh; B) on [Ca2+]i in mouse ß-cells superfused with a medium containing 20 mM glucose, 20 nM glucagon and 50 µM methoxyverapamil. Both agonists induced transients and/or bumps followed by suppression of the spontaneous transients. Solitary transients and bumps sometimes coexist as shown in (A). Prolonged exposure to acetylcholine resulted in steady-state elevation of [Ca2+]i. Representative of five experiments.

View larger version (21K): [in this window] [in a new window]   Figure 3 Effects of 50 µM CPA on [Ca2+]i in mouse ß-cells exposed or not to ATP or acetylcholine (ACh) during superfusion with a medium containing 20 mM glucose, 20 nM glucagon and 50 µM methoxyverapamil. Addition of CPA generated a bump with a superimposed transient, followed by disappearance of the spontaneous transients and steady-state elevation of [Ca2+]i (A). Both 1 µM ATP (B) and 1 µM acetylcholine (C) lacked effects in the presence of CPA. Addition of 50 µM CPA in the presence of 1 µM ATP generated a bump lacking superimposed transients but followed by steady-state elevation of [Ca2+]i (D). Addition of 50 µM CPA in the presence of 1 µM acetylcholine generated a small bump without superimposed transients or subsequent elevation of [Ca2+]i (E). All observations are representative of five experiments.

Prolonged exposure to acetylcholine, but not to ATP, induced a steady-state elevation of [Ca²⁺]_i both in the absence (Fig. 1; Table 3) and presence (Fig. 2; Table 3) of glucagon. A similar elevation was seen when blocking the SERCA pump with CPA (Fig. 3A; Table 3). Addition of CPA resulted in steady-state elevation of [Ca²⁺]_i also in the presence of 1 µM ATP (Fig. 3D; Table 2). However, there was no additional increase of steady-state [Ca²⁺]_i when CPA (Fig. 3E; Table 2) or ATP (Fig. 4A) was added to cells exposed to 1 µM acetylcholine.

View larger version (23K): [in this window] [in a new window]   Figure 4 Effects of ATP, acetylcholine (ACh) and PMA on [Ca2+]i in mouse ß-cells superfused with a medium containing 20 mM glucose, 20 nM glucagon and 50 µM methoxyverapamil. After removal of spontaneous transients by 15 min exposure to 1 µM acetylcholine, addition of 100 µM ATP induced a temporary rise of [Ca2+]i (106 ± 16 nM) in 23 ± 6% of the cells (n=5; A). After removal of spontaneous transients by 15 min exposure to 1 µM ATP, addition of 100 µM acetylcholine induced a temporary rise of [Ca2+]i (175 ± 22 nM) in 96 ± 2% of the cells (n=5; B). Spontaneous transients disappeared in all of six experiments in the presence of 10 nM PMA (C). Pulse additions (60 s) of 100 µM acetylcholine but not of ATP induced a temporary rise of [Ca2+]i in all of six experiments when spontaneous transients were removed by PMA (D).

Both ATP and acetylcholine interfered with the generation of spontaneous [Ca²⁺] transients (Fig. 2). The suppression obtained after addition of the agonists to a glucagon-containing medium is shown in Table 4. Whereas prolonged exposure to 0.1 µM acetylcholine lacked significant effect, the same concentration of ATP removed 81% of the transients. Studying the combined effects of the agonists it was found that 100 µM ATP induced a temporary rise of [Ca²⁺]_i in 23 ± 6% (n=5) of the cells lacking spontaneous transients due to the presence of 1 µM acetylcholine (Fig. 4A). However, when the transients were removed with 1 µM ATP almost all cells (96 ± 2%; n=5) responded to 100 µM acetylcholine with an initial rise of [Ca²⁺]_i followed by sustained elevation (Fig. 4B).

View larger version (23K): [in this window] [in a new window]   Figure 5 Effects of BIM, ATP and acetylcholine (ACh) on [Ca2+]i in mouse ß-cells superfused with a medium containing 20 mM glucose, 20 nM glucagon and 50 µM methoxyverapamil. The number of spontaneous [Ca2+]i transients increased in the presence of 2 µM BIM (A). The presence of 1 µM ATP (B) or 1 µM acetylcholine (C) resulted in disappearance of spontaneous [Ca2+]i transients in a medium containing 2 µM BIM. Representative of four–six experiments.

Differences in the effects of ATP and acetylcholine on rat ß-cells were found both for the initial responses and the presence of a sustained elevation of [Ca²⁺]_i (Fig. 6). The proportion of rat ß-cells generating transients and/or bumps in the presence of 100 µM of the agonists is shown in Table 5. Whereas most cells (82%) rapidly responded to ATP with transients superimposed or not on bumps, the addition of acetylcholine usually resulted in bumps lacking superimposed transients (83%). Accordingly, the ATP-induced rise of [Ca²⁺]_i above the basal level often exceeded that found with acetylcholine. The proportion of cells responding with a temporary rise of [Ca²⁺]_i above 1000 nM was 27 ± 7% (n=6) for ATP and 4 ± 3% (n=8) for acetylcholine.

View larger version (11K): [in this window] [in a new window]   Figure 6 Effects of ATP and acetylcholine (ACh) on [Ca2+]i in rat ß-cells superfused with a glucagon-free medium containing 20 mM glucose and 50 µM methoxyverapamil. Addition of 100 µM ATP usually resulted in transients superimposed on bumps (A). Most ß-cells responded to 100 µM acetylcholine with a bump lacking transients but followed by sustained elevation of [Ca2+]i (B). Representative of six–eight experiments.

	Discussion

Top Abstract Introduction Material and Methods Results Discussion References

It is likely that IP₃-induced mobilization of Ca²⁺ from endoplasmic reticulum (ER) is important for coordinating the [Ca²⁺]_i rhythmicity in pancreatic ß-cells. Evidence has been provided that spontaneous transients of [Ca²⁺]_i appear in synchrony in ß-cells lacking contact (Grapengiesser et al. 1999), and that these transients entrain the oscillatory activity into a common rhythm (Grapengiesser et al. 2003). In the present study initial rises of [Ca²⁺]_i >50 nM were classified as transients and bumps. The observation that the bumps were often superimposed with one or several transients justifies their recognition as a distinct type of [Ca²⁺]_i increase. As indicated from the CPA experiments, inhibition of the SERCA pump was sufficient to induce bumps with superimposed transients. Similar bumps can be evoked by gaseous NO, a messenger supposed to be complementary to ATP as a coordinator of the ß-cell rhythmicity (Grapengiesser et al. 2001).

Discussing the physiological role of the observed [Ca²⁺]_i rises, it should be kept in mind that the presence of methoxyverapamil prevents voltage-activated Ca²⁺ entry mediated by closure of K_ATP channels. In media lacking methoxyverapamil both ATP and acetylcholine induce premature oscillations of [Ca²⁺]_i, an effect also obtained with other activators of cytoplasmic phospholipase A₂ (Grapengiesser et al. 2004). The bumps now observed sometimes mimic premature oscillations. Nevertheless, pre-treatment with CPA, which is known to leave most of the premature oscillations unaffected (Grapengiesser et al. 2004), resulted in a complete removal of the bumps. Transients are mediated by steeper increases to higher levels of [Ca²⁺]_i than seen with bumps. When superimposed on slow [Ca²⁺]_i oscillations the transients can generate a repolarizing K⁺ current, temporarily interrupting the electrical activity (Ämmälä et al. 1993, Dryselius et al. 1999). Accumulating data support the idea that this effect provides a coupling force for synchronization of pancreatic ß-cells (Grapengiesser et al. 2003), in analogy to what has been shown for other pulse-coupled oscillators (Gilbey 2001, Strogatz 2001).

Consistent with a previous report (Hellman et al. 2004) continuous exposure to ATP (0.1 µM or above) evoked initial rise of [Ca²⁺]_i followed by disappearance of spontaneously occurring transients. The presence of a suppressive component in the ATP action emphasizes the importance of an intermittent release of the nucleotide from the ß-cells. We now report that prolonged exposure to acetylcholine of 1 µM or more results in a similar loss of transients. In the case of ATP the disappearance of transients cannot be explained by depletion of Ca²⁺ stored in the ER, since the SERCA-pump inhibitor CPA evoked a pronounced increase of [Ca²⁺]_i also in the presence of the nucleotide. The observation that ATP has a suppressive action on the [Ca²⁺]_i transients may not only reflect desensitization of the purinoceptors. It has been proposed that the action of ATP on ß-cells involves autocrine feedback inhibition of exocytosis via G-protein-dependent activation of the serine/threonine phosphatase calcineurin (Poulsen et al. 1999).

Both ATP and acetylcholine activate PLC with resulting generation of diacylglycerol, the natural stimulator of PKC. It has been reported that PMA stimulation of PKC effectively removes the spontaneous [Ca²⁺]_i transients (Liu et al. 1996). We now observe that PMA is a suppressor also of the temporary [Ca²⁺]_i rises evoked by ATP and acetylcholine. The suppression was particularly pronounced in the case of ATP, as indicated by the absence of a [Ca²⁺]_i rise after addition of 100 µM of the nucleotide. BIM, an established inhibitor of PKC (Toullec et al. 1991), promoted the generation of [Ca²⁺]_i transients but did not prevent their disappearance during prolonged exposure to ATP and acetylcholine. The decisive role of cAMP for the generation of [Ca²⁺]_i transients (see above) raises the question of whether activation of PKC results in inhibition of adenylate cyclase or protein kinase A. Pancreatic islets express several subtypes of these enzymes, which are known to react differently to agents affecting PKC (Tian & Laychock 2001, Gao et al. 2002).

Like ATP, acetylcholine induced a temporary rise of [Ca²⁺]_i followed by removal of the spontaneous transients. Comparing the initial responses to the agonists in ß-cells from both ob/ob mice and rats it was found that micro-molar concentrations of ATP essentially triggered transients, whereas the main effect of acetylcholine was to generate bumps without superimposed transients. Moreover, prolonged exposure to acetylcholine, but not to ATP, resulted in a steady-state elevation of Ca²⁺ similar to what is seen when capacitative entry of Ca²⁺ is activated by depleting the ER stores of Ca²⁺ (Dyachok & Gylfe 2001). This observation should not be taken to indicate that capacitative entry of Ca²⁺ is the only mechanism by which acetylcholine induces sustained elevation of [Ca²⁺]_i. It is likely that entry of Na⁺ via non-specific cation channels is essential for the steady-state elevation of [Ca²⁺]_i observed after activation of the muscarinic receptors (Saha & Grapengiesser 1995, Gilon & Henquin 2001).

In summary, previous studies have shown improved coordination of the ß-cell rhythmicity after increasing the number of synchronous [Ca²⁺]_i transients (Grapengiesser et al. 2003). We now demonstrate that external ATP is better suited than acetylcholine for coordinating the activity of the ß-cells by generating distinct [Ca²⁺]_i transients without sustained elevation of [Ca²⁺]_i.

	Funding

 
This study was supported by the Swedish Research Council (72X-562), the Swedish Diabetes Association and the Family Enfors Fund. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received in final form 14 January 2005
Accepted 25 January 2005

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