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

Abstract

External ATP is supposed to trigger short-lived increases (transients) of cytoplasmic Ca2+ important for entraining insulin-secreting β-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 β-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 β-cells exposed to acetylcholine but not to ATP. Similar differences were seen comparing the responses of rat β-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 β-cells by generating distinct [Ca2+]i transients without sustained elevation of basal [Ca2+]i.