REGULATION OF EXCITATION-SECRETION COUPLING MECHANISMS

囊状释放递质和激素是所有神经元和分泌细胞的基本功能，但其潜在机制仍知之甚少。在可兴奋细胞中，细胞内钙（Ca）的升高是分泌的触发因素。通过电压门控通道的去极化诱发钙离子进入是钙离子升高的最佳表征途径。在许多细胞中，也可以通过刺激g蛋白偶联受体从ip3敏感的细胞内储存中释放Ca来引起分泌。贮藏物中钙的含量非常有限，因此很快就会耗尽。最近，人们已经认识到，钙储存量耗尽会产生一个激活钙跨质膜内流的信号。在血源性细胞中，储存操作的钙内流途径已被确定，并称为CRAC电流（即“钙释放激活的钙电流”）。在其他细胞中也发现了相关但不相同的存储操作电流。使用膜片钳技术同时测量细胞内钙，我们已经确定了一个假定的储存操作电流由分泌儿茶酚胺的染色质细胞中的thapsigargin激活。在没有去极化的情况下，钙通过这一途径进入可触发胞吐。此外，电流似乎对传统的去极化诱发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。具体目标将测试假设：1)该信号素诱发电流是存储操作电流；2)生理激动剂可以激活存储操作电流；3)生理激动剂可以通过激活储存操作电流来触发或调节胞吐。确定g蛋白偶联受体在胞外分泌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知识。这些见解可以导致在正常和病理条件下调节分泌的新策略的发展。

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.