Granule-specific ATP requirements for Ca2+-induced exocytosis in human neutrophils. Evidence for substantial ATP-independent release

Abstract

Ca2+-induced exocytosis in neuronal and neuroendocrine cells involves ATP-dependent steps believed to `prime' vesicles for exocytosis. Primed, docked vesicles are released in response to Ca2+ influx through voltage-gated Ca2+ channels. Neutrophils, however, do not possess voltage-gated Ca2+ channels and appear to have no docked vesicles. Furthermore, neutrophils have several types of granules with markedly different Ca2+ requirements for exocytosis. These differential Ca2+ dependencies were used as a tool to investigate the ATP dependence of different granule populations. Here we demonstrate distinct ATP requirements for release of neutrophil granule populations, with respect to rate as well as amplitude. Intracellular ATP was depleted to various levels, and exocytosis was stimulated with different Ca2+concentrations and measured with the patch-clamp capacitance technique or by detecting enzyme release. Primary granule exocytosis displayed a distinct ATP dependence with an apparent Km of ∼80 μM ATP and no ATP-independent exocytosis. Release of secondary and tertiary granules displayed a more shallow ATP dependence (Km∼330 μM), and more than 50% of secondary and tertiary granules appeared not to need ATP at all for their release. Individual granules in human neutrophils have distinct ATP requirements for exocytosis, suggesting that the ATP-sensitive elements are localised to the granules. Primary granule exocytosis has a very low affinity for ATP. Furthermore, substantial ATP-independent exocytosis of secondary and tertiary granule occurs despite the absence of docked granules. These characteristics should help neutrophils to fulfil their bactericidal functions at poorly irrigated sites of infection with low glucose supply.