CORTICOSTEROID-MODULATED EPITHELIAL NA+ & HC03- TRANSPORT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目的目标是确定皮质类固醇调节的跨输精管上皮细胞离子转运的机制。精子的成熟、储存和激活需要不同的管腔环境。我们建立了研究新鲜分离的人和猪的导管、原代延性上皮细胞培养和永生的猪输精管上皮细胞中离子转运的方案。这些系统中的每一个都表现出在完整组织中观察到的所有或许多调节特性。我将使用这套实验系统来实现以下具体目标。 目的1：明确调节输精管上皮钠转运的皮质类固醇信号通路。输精管上皮细胞只有在接触糖皮质激素时才表现出对阿米洛利敏感的离子转运。在肾脏、肺和半规管导管等组织中，皮质类固醇通过盐皮质激素或糖皮质激素受体和各种第二信使与上皮钠转运的变化有关。这些受体和信使是否影响男性生殖管上皮钠通道的活动尚不清楚。药理调节剂和分子技术将被用来测试这些胞浆调节因子的离散变化，并确定皮质类固醇调节信使的顺序或平行顺序。 目的2：明确糖皮质激素调节上皮HCO3转运的机制。糖皮质激素受体激动剂和拮抗剂调节cAMP刺激的阴离子分泌。皮质类固醇受体可以通过第二信使途径直接与HCO3-转运相连，也可以通过阳离子通道对膜电压的影响间接与HCO3-转运相连，膜电压是支持基底外侧阴离子进入和顶端阴离子退出的驱动力。我们将使用药物调节剂和分子技术来测试对HCO3-转运蛋白表达或活性的离散影响。皮质类固醇与HCO3分泌的关系尚不清楚，但在输精管的情况下，可能在男性生育中起着关键作用。 这些研究的结果将确定药物干预的靶点，以调节管腔pH值，最直接地应用于男性生育。我们将建立一个机制模型(S)来解释上皮Na和HCO3-运输的急性调节，该模型可以扩展到体内其他复杂的细胞系统。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The project goal is to define mechanisms of corticosteroid-regulated ion transport across vas deferens epithelia. Different luminal environments are required for sperm maturation, storage and activation in the male duct. We established protocols to study ion transport in freshly isolated human and porcine ducts, in primary ductile epithelial cell cultures, and in immortal porcine vas deferens epithelial cells. Each of these systems exhibits all or many of the regulatory properties observed in the intact tissue. I will use this set of experimental systems to achieve the following specific aims. Aim 1: To define corticosteroid signaling pathways that regulate Na+ transport across vas deferens epithelium. Vas deferens epithelial cells exhibit amiloride-sensitive ion transport only when exposed to glucocorticoids. In tissues such as kidney, lung, and semicircular canal duct, corticosteroids are linked to changes in epithelial Na+ transport by either mineralocorticoid or glucocorticoid receptors and by various 2nd messengers. Whether these receptors and messengers affect activity of the epithelial Na+ channel in the male reproductive duct is unknown. Pharmacological modulators and molecular techniques will be used to test for discrete changes in these cytosolic regulators and to determine the sequential or parallel order in which messengers are modulated by corticosteroids. Aim 2: To define mechanisms by which glucocorticoids regulate epithelial HCO3- transport. Glucocorticoid receptor agonists and antagonists modulate cAMP-stimulated anion secretion. Corticosteroid receptors could be linked directly to HCO3- transport by a second messenger pathway or indirectly by effects of cation channels on membrane voltage, which is a driving force that supports both basolateral anion entry and apical anion exit. We will use pharmacological modulators and molecular techniques to test for discrete effects on the expression or activity of HCO3- transporters. The relationship of corticosteroids to HCO3- secretion is unknown, but in the context of the vas deferens, likely plays a pivotal role in male fertility. Results from these studies will identify targets for pharmacological interventions to modulate luminal pH with the most direct application to male fertility. We will establish a mechanistic model(s) to account for acute modulation of epithelial Na+ and HCO3- transport that can be extended to other complex cellular systems in the body.