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

Abstract

Quantal release of adenosine triphosphate (ATP) was monitored in rat pancreatic β-cells expressing P2X2 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 μM [Ca2+]i averaged 4.3 pC. The distribution of the 3ρq of these events could be described by a single Gaussian. The rise times averaged ∼5 ms over a wide range of TIC amplitudes. In β-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 [Ca2+]i to ∼5 μM by photo release of caged Ca2+, an increase in membrane capacitance was observed after 33 ms, whereas ATP release first became detectable after 43 ms. The step increase in [Ca2+]i produced an initial large TIC followed by a series of smaller events that echoed the changes in membrane capacitance (ΔC m). Mathematical modeling suggests that the large initial TIC reflects the superimposition of many unitary events. Exocytosis, measured as ΔC m or TICs, was complete within 2 s after elevation of [Ca2+]i with no sign of endocytosis masking the capacitance increase. The relationship between total charge (Q) and ΔC m was linear with a slope of ∼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 ΔC m principally (≥85-95%) report exocytosis of insulin granules. © Springer-Verlag 2008.