Theander Sten, Lew Daniel P, Nüsse Oliver
Division of Clinical Biochemistry, University of Geneva Medical School, Switzerland.
J Cell Sci. 2002 Jul 15;115(Pt 14):2975-83. doi: 10.1242/jcs.115.14.2975.
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 K(m) of approximately 80 microM ATP and no ATP-independent exocytosis. Release of secondary and tertiary granules displayed a more shallow ATP dependence (K(m) approximately 330 microM), 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.
神经元和神经内分泌细胞中钙离子诱导的胞吐作用涉及依赖ATP的步骤，这些步骤被认为是使囊泡为胞吐作用做好“准备”。准备好的、停靠的囊泡会响应通过电压门控钙离子通道的钙离子内流而释放。然而，中性粒细胞不具备电压门控钙离子通道，而且似乎没有停靠的囊泡。此外，中性粒细胞有几种类型的颗粒，它们胞吐作用所需的钙离子明显不同。这些不同的钙离子依赖性被用作一种工具来研究不同颗粒群体对ATP的依赖性。在这里，我们证明了中性粒细胞颗粒群体释放时在速率和幅度方面对ATP有不同的需求。细胞内ATP被消耗到不同水平，用不同的钙离子浓度刺激胞吐作用，并通过膜片钳电容技术或检测酶释放来进行测量。初级颗粒胞吐作用表现出明显的ATP依赖性，其表观米氏常数约为80微摩尔ATP，且不存在不依赖ATP的胞吐作用。次级和三级颗粒的释放表现出对ATP的依赖性较弱（米氏常数约为330微摩尔），超过50%的次级和三级颗粒似乎在释放时根本不需要ATP。人类中性粒细胞中的单个颗粒胞吐作用对ATP有不同的需求，这表明对ATP敏感的元件定位于颗粒上。初级颗粒胞吐作用对ATP的亲和力非常低。此外，尽管没有停靠的颗粒，但次级和三级颗粒仍会发生大量不依赖ATP的胞吐作用。这些特性应有助于中性粒细胞在葡萄糖供应低、灌溉不良的感染部位发挥其杀菌功能。
