REGULATION OF EXCITATION-SECRETION COUPLING MECHANISMS

递质和激素的囊泡释放是所有神经元和分泌细胞的基本功能，但其潜在的机制仍然知之甚少。在可兴奋的细胞中，细胞内钙的升高是分泌的触发因素。去极化诱发的钙通过电压门控通道进入是钙升高的最佳表征途径。在许多细胞中，也可以通过刺激G蛋白偶联受体来刺激分泌，G蛋白偶联受体从对IP3敏感的细胞内储存物中释放钙。商店中的钙含量非常有限，因此很快就会耗尽。最近，人们已经认识到，储存耗尽会产生一种信号，激活钙跨质膜内流。在血源性细胞中，钙库操作的钙内流途径已被发现，并被称为CRAC电流(即“钙释放激活的钙电流”)。在其他电池中也发现了相关但不相同的存储操作电流。利用膜片钳技术结合细胞内钙的同步测量，我们在分泌儿茶酚胺的嗜铬细胞中发现了一种可能由thapsigargin激活的储藏操作电流。在没有去极化的情况下，通过这一途径进入的钙离子可以触发胞吐作用。此外，电流似乎对传统的去极化引起的胞吐有很强的促进作用。我们的假设是，G蛋白偶联受体的刺激通过一种新的途径有效地调节可兴奋细胞的分泌：激活储存操作电流。这些特定的目标将检验假设：1)thapsigargin诱发的电流是商店操作的电流；2)生理激动剂可以激活商店操作的电流；3)生理激动剂可以通过激活商店操作的电流触发或调节胞吐。确定G蛋白偶联受体在胞吐作用中的作用将为了解包括神经元突触传递在内的分泌过程提供重要的基础。这些洞察力可以导致在正常和病理条件下调节分泌的新策略的开发。

The vesicular release of transmitters and hormones is a fundamental function of all neurons and secretory cells, but the underlying mechanisms are still poorly understood. In excitable cells, elevation of intracellular calcium (Ca) is the trigger for secretion. Depolarization-evoked Ca entry through voltage-gated channels is the best characterized pathway for Ca elevation. In many cells, secretion can also be evoked by stimulating G-protein coupled receptors that release Ca from IP3-sensitive intracellular stores. The amount of Ca contained within stores is very limited and therefore rapidly depleted. Recently, it has been recognized that store depletion generates a signal that activates Ca influx across the plasma membrane. In blood-derived cells, the pathway for store-operated Ca influx has been identified and called CRAC current (for "Ca Release Activated Ca current). Related, but non-identical store-operated currents have been found in other cells. Using patch clamp techniques with simultaneous measurements of intracellular Ca, we have identified a putative store-operated current activated by thapsigargin in catecholamine-secreting chromaffin cells. Ca entry through this pathway can trigger exocytosis in the absence of depolarization. Additionally, the current appears to have a strong facilitatory effect on conventional depolarization-evoked exocytosis. Our hypothesis is that stimulation of G -protein coupled receptors potently regulates secretion in excitable cells via a novel pathway: activation of a store-operated current. The specific aims will test the hypotheses: 1) that the thapsigargin-evoked current is a store-operated current; 2) that physiological agonists can activate a store-operated current; 3) that physiological agonists can trigger or modulate exocytosis via activation of a store-operated current. Determining the role of G-protein coupled receptors in exocytosis will provide important fundamental understanding of secretory processes, including neuronal synaptic transmission. These insights can lead to development of novel strategies for modulation of secretion in normal and pathological conditions.