Quantal ATP release in rat beta-cells by exocytosis of insulin-containing LDCVs

Quantal release of adenosine triphosphate (ATP) was monitored in rat pancreatic beta-cells expressing P2X(2) receptors. Stimulation of exocytosis evoked rapidly activating and deactivating ATP-dependent transient inward currents (TICs). The unitary charge (q) of the events recorded at 0.2 microM [Ca(2+)](i) averaged 4.3 pC. The distribution of the 3 square root q of these events could be described by a single Gaussian. The rise times averaged approximately 5 ms over a wide range of TIC amplitudes. In beta-cells preloaded with 5-hydroxytryptamine (5-HT; accumulating in insulin granules), ATP was coreleased with 5-HT during >90% of the release events. Following step elevation of [Ca(2+)](i) to approximately 5 microM by photo release of caged Ca(2+), an increase in membrane capacitance was observed after 33 ms, whereas ATP release first became detectable after 43 ms. The step increase in [Ca(2+)](i) produced an initial large TIC followed by a series of smaller events that echoed the changes in membrane capacitance (DeltaC(m)). Mathematical modeling suggests that the large initial TIC reflects the superimposition of many unitary events. Exocytosis, measured as DeltaC(m) or TICs, was complete within 2 s after elevation of [Ca(2+)](i) with no sign of endocytosis masking the capacitance increase. The relationship between total charge (Q) and DeltaC(m) was linear with a slope of approximately 1.2 pC/fF. The latter value predicts a capacitance increase of 3.6 fF for the observed mean value of q, close to that expected for exocytosis of individual insulin granules. Our results indicate that measurements of ATP release and DeltaC(m) principally (> or =85-95%) report exocytosis of insulin granules.