GRANULOSA CELL-CONTROL OF NA+/H+ ANTIPORT AND K+ CHANNEL

细胞外区室中K+和Na+的缺乏导致细胞外膜中K+和Na+的缺乏。 促性腺激素和生长因子影响的类固醇生成和 卵巢颗粒细胞的其他生物合成功能。 这些 观测结果已被解释为反映了这些离子的重要性 在颗粒细胞功能中。然而，人们对它知之甚少。 调节和途径，这些离子进入或离开这个内分泌 细胞的类型 本提案的目的是检验所提出的假设 根据我们的初步数据，Na+/H+逆向转运蛋白是EGF的介导剂， 在颗粒细胞中的作用。我们还将测试假设， 兴奋，K+和C1-通道和膜电位起着密切的作用。 在LH诱导的孕酮产生中的作用，以及在生长因子- (EGF、IGF-1、TGF α和TGF β）影响类固醇生成和其他 颗粒细胞的生物合成功能。具体目标是：（1） 确定Na+/H+反向转运蛋白介导的细胞质碱化是否 生长因子激活的颗粒细胞中的一个强制性事件， 使用pH敏感荧光染料进行卵泡发育。 (2)确定 未成熟颗粒细胞长期（3-4天）暴露于 生长因子对Na+/H+逆向转运蛋白活性的影响 文化模式(3)研究LH和生长因子调节（直接或间接） 信使介导）的K+和Cl-通道的颗粒细胞分离， 使用膜片钳技术观察不同成熟阶段的卵泡。 (4)检查长期（3-4天）暴露于未成熟的 颗粒细胞对LH和生长因子的发生和活性的影响 K+和Cl-通道。 研究将通过使用膜片钳技术完成， 鸡颗粒细胞荧光分光光度法研究 在卵泡发育的不同阶段。 鲜明 应用膜片钳技术研究鸡卵巢的解剖学特征 和荧光光谱技术使详细研究 卵泡成熟过程中颗粒细胞离子流的调节 排卵前卵泡我们的结果将解释离子 酶催化重要过程所必需的微环境 (such如腺苷酸环化酶和胆固醇侧链裂解酶）被 控制。这些研究还将提供有关如何 促性腺激素和生长因子调节颗粒细胞分化 在卵泡成熟期。此外，他们还将提供有关 颗粒细胞中生长因子的作用机制。

The absence of K+ and Na+ in extracellular compartment leads to the aberration of gonadotropin- and growth factor-influenced steroidogenic and other biosynthetic functions of ovarian granulosa cells. These observations have been interpreted to reflect the importance of these ions in granulosa cell function. However, very little is known about the regulation and pathways by which these ions enter or leave this endocrine type of cell. The objective of the present proposal is to test the hypothesis suggested by our preliminary data, that the Na+/H+ antiporter is a mediator of EGF action in granulosa cells. We will also test the hypothesis that electrical excitation, K+ and C1- channels and membrane potential play an intimate role in the LH-induced production of progesterone, and in growth factor- (EGF, IGF-1, TGFa and TGFb) influenced steroidogenesis and other biosynthetic functions in granulosa cells. The specific aims are: (1) Determine if the Na+/H+ antiporter-mediated cytoplasmic alkalinization is an obligatory event in growth factor-activated granulosa cells, and on follicular development using pH sensitive flourochrome. (2) Determine the effect of long term (3-4 days) exposure of immature granulosa cells to growth factor on the Na+/H+ antiporter activity using an in vitro cell culture mode. (3) Study the LH and growth factor regulation (direct or messenger-mediated) of K+ and Cl- channels in granulosa cells isolated from follicles at different stages of maturation using patch clamp techniques. (4) Examine the influence of long term (3-4 days) exposure of immature granulosa cells to LH and growth factor on the occurrence and activity of K+ and Cl- channels. The studies will be accomplished by using patch clamp techniques and spectrofluorometry on granulosa cells isolated from domestic hen (Gallus domesticus) at different stages of follicular development. The distinctive anatomical features of the hen ovary coupled with the powerful patch clamp and spectroflourometric techniques make possible a detailed study of the regulation of ion fluxes in granulosa cells during the maturation of ovarian preovulatory follicles. Our results will explain how the ionic microenvironment necessary for important processes catalysed by enzymes (such as adenylate cyclase and cholesterol side chain cleavage enzyme) are controlled. These studies will also provide information on how gonadotropins and growth factors regulate granulosa cell differentiation during follicular maturation. In addition, they will give knowledge about the mechanism of action growth factors in granulosa cells.