External ATP triggers Ca2+ signals suited for synchronization of pancreatic beta-cells.

External ATP is supposed to trigger short-lived increases (transients) of cytoplasmic Ca2+ important for entraining insulin-secreting beta-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 beta-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 (> or =20 s) superimposed or not with transients. Addition of ATP (1-100 microM) usually triggered transients whereas acetylcholine induced bumps lacking superimposed transients. After the initial rise there was a steady-state elevation of [Ca2+]i in beta-cells exposed to acetylcholine but not to ATP. Similar differences were seen comparing the responses of rat beta-cells to 100 microM ATP and acetylcholine. Inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump (with 50 microM 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 microM bisindolylmaleimide) promoted the generation of transients. The results indicate that ATP fulfils the demands for a coordinator of the secretory activity of beta-cells by generating distinct [Ca2+]i transients without sustained elevation of basal [Ca2+]i.

Synchronization of pancreatic beta-cell rhythmicity after glucagon induction of Ca2+ transients.

Pancreatic beta-cells are biological oscillators requiring a coupling force for the synchronization of the cytoplasmic Ca(2+) oscillations responsible for pulsatile insulin release. Testing the idea that transients, superimposed on the oscillations, are important for this synchronization, the concentration of cytoplasmic Ca(2+) ([Ca(2+)](i)) was measured with ratiometric fura-2 technique in single beta-cells and small aggregates prepared from islets isolated from ob/ob-mice. Image analyses revealed asynchronous [Ca(2+)](i) oscillations in adjacent beta-cells lacking physical contact. The addition of glucagon stimulated the firing of [Ca(2+)](i) transients, which appeared in synchrony in adjacent beta-cells. Moreover, the presence of glucagon promoted synchronization of the [Ca(2+)](i) oscillations in beta-cells separated by a distance <100 microm but not in those >200 microm apart. The results support the proposal that the repolarizing effect of [Ca(2+)](i) transients provides a coupling force for co-ordinating the pulses of insulin release generated by pancreatic beta-cells.

Nitric oxide induces synchronous Ca2+ transients in pancreatic beta cells lacking contact.

AIMS: To evaluate the role of nitric oxide (NO) in the coordination of the Ca2+ signals generating pulsatile insulin release in pancreatic beta cells isolated from ob/ob mice. METHODOLOGY: Using ratiometric fura-2 technique for recording glucose-induced cytoplasmic Ca2+ transients, it was possible to demonstrate a synchronization of beta cells lacking contact. RESULTS: The frequency of the transients increased 10-fold in the presence of 20 n M glucagon. Additional increase in frequency with maintenance of synchronization was observed when the beta cells were exposed to 100 microM of the NO donors sodium nitroprusside and hydroxylamine. Bolus additions of 0.1-10 microM gaseous NO resulted in prompt appearance of cytoplasmic Ca2+ transients. An activator of soluble guanylate cyclase (mesoporphyrin) increased the frequency of the transients, and inhibition of this enzyme with 1H-(1,2,4) oxadiazolo [4,3-a] quinoxalin-1-one had the opposite effect. CONCLUSION: The results support the idea that nitrergic nerves generate beta-cell transients of Ca2+ synchronizing the activity of the numerous islets in the pancreas.