Pancreatic islets exposed to 11 mM glucose exhibited complex variations of cytoplasmic Ca2+ concentration ([Ca2+]i) with slow (0.3‐0.9 min−1) or fast (2‐7 min−1) oscillations or with a mixed pattern. Using digital imaging and confocal microscopy we demonstrated that the mixed pattern with slow and superimposed fast oscillations was due to separate cell populations with the respective responses. In islets with mixed [Ca2+]i oscillations, exposure to the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (DTBHQ) resulted in a selective disappearance of the fast pattern and amplification of the slow pattern. In addition, the protein kinase A inhibitor RP‐cyclic adenosine 3′,5′‐monophosphorothioate sodium salt transformed the mixed [Ca2+]i oscillations into slow oscillations with larger amplitude. Islets exhibiting only slow oscillations reacted to low concentrations of glucagon with induction of the fast or the mixed pattern. In this case the fast oscillations were also counteracted by DTBHQ. The spontaneously occurring fast oscillations seemed to require the presence of cAMP‐elevating glucagon, since they were more common in large islets and suppressed during culture. Image analysis revealed [Ca2+]i spikes occurring irregularly in time and space within an islet. These spikes were preferentially observed together with fast [Ca2+]i oscillations, and they became more common after exposure to glucagon. Both the slow and fast oscillations of [Ca2+]i in pancreatic islets rely on periodic entry of Ca2+. However, the fast oscillations also depend in some way on paracrine factors promoting mobilization of Ca2+ from intracellular stores. It is proposed that such a mobilization in different cells within a tightly coupled islet syncytium generates spikes which co‐ordinate the regular bursts of action potentials underlying the fast oscillations.

The kinetics of insulin, glucagon and somatostatin release was studied in human pancreatic islets. Batches of 10-15 islets were perifused and the hormones measured with RIA in 30-sec fractions. Increase of glucose from 3 to 20 mm resulted in a brief pulse of glucagon coinciding with suppression of basal insulin and somatostatin release. There was a subsequent drop of glucagon release concomitant with the appearance of a pronounced pulse of insulin and a slightly delayed pulse of somatostatin. Continued exposure to 20 mm glucose generated pulsatile release of the three hormones with 7- to 8-min periods accounting for 60-70% of the secreted amounts. Glucose caused pronounced stimulation of average insulin and somatostatin release. However, the nadirs between the glucagon pulses were lower than the secretion at 3 mm glucose, resulting in 18% suppression of average release. The repetitive glucagon pulses were antisynchronous to coincident pulses of insulin and somatostatin. The resulting greater than 20-fold variations of the insulin to glucagon ratio might be essential for minute-to-minute regulation of the hepatic glucose production.

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Mechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1-10 mumol/l carbachol) and of purinergic P2 receptors (0.01-1 mumol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 mumol/l GTP-gamma-S into beta cells hyperpolarized to -70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1-0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2-5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca(2+)-induced secretory pulses.

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The purine nucleotide ATP is known to act as an extracellular messenger, generating its effects via a family of distinct cell surface receptors (7, 45). It was early established that external ATP stimulates the release of insulin from pieces of rabbit pancreas (9) and the efflux of radioactivity from isolated mouse islets preloaded with 45 Ca 2ϩ (20). Subsequent studies indicated that ATP mobilization of intracellular Ca 2ϩ stores is coupled to a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (27). It was found by measuring [Ca 2ϩ ] i that activation of purinoceptors elicits distinct transients in insulinsecreting RINm5F cells (10), as well as in mouse ␤-cells (4). If sufficiently pronounced, a transient increase of [Ca 2ϩ ] i is known to induce a temporary interruption of the Ca 2ϩ entry into glucose-stimulated ␤-cells by activating a repolarizing K ϩ current (12). This effect, together with the observation that the transients often appear in synchrony in the absence of cell contacts, has resulted in the proposal that transients entrain the [Ca 2ϩ ] i oscillations and, consequently, pulses of insulin release into a rhythm common for the islets in the pancreas (16,17,25,35). We have recently been able to demonstrate that an increase in the number of synchronized transients has a coordinating effect on the [Ca 2ϩ ] i oscillations (15). It was suggested from insulin release studies with the isolated perfused rat pancreas that the islets communicate via nonadrenergic, noncholinergic (NANC) neurons (44). Evidence has been provided that both nitric oxide (16, 25) and carbon monoxide (35) elicit transients of [Ca 2ϩ ] i , often occurring in synchrony in ␤-cells lacking physical contact. Another candidate for a neurotransmitter that generates transients with a coordinating effect on the [Ca 2ϩ ] i oscillations is ATP. This nucleotide has been reported to propagate mechanically induced [Ca 2ϩ ] i rises in confluent monolayers of rat insulinoma cells (10) and normal mouse ␤-cells (4). Previous studies in our laboratory have shown that the purinoceptor antagonist suramin has a suppressive action on spontaneous [Ca 2ϩ ] i transients in ␤-cells from ob/ob mice (17). However, there are also reports that dephosphorylation of external ATP with apyrase fails to affect the propagation of glucose-induced rises of [Ca 2ϩ ] i in rat ␤-cells (4). The purpose of the present study was to examine whether external ATP acts as a diffusible messenger generating the [Ca 2ϩ ] i transients supposed to coordinate the rhythmicity of the ␤-cells within and among the islets in the pancreas. In support for this idea, we now demonstrate that ␤-cells not only are recipients of an ATP signal inducing [Ca 2ϩ ] i transients but can also propagate this message to neighboring cells via intermittent release of ATP. MATERIALS AND METHODSChemicals. Reagents of analytical grade and deionized water were used. ATP (ultragrade), ADP, UTP, apyrase, carbachol, suramin, epinephrine, somatostatin, glucagon, 8-bromoadenos...

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