Neuroendocrine-modulated epithelial HCO3- transport

描述（由申请人提供）： 该项目的长期目标是确定机制和相关的监管级联，占上皮HCO 3-运输，特别关注男性生殖道。精子在输精管中的成熟、储存和激活需要不同的pH值。上皮阴离子转运疾病如囊性纤维化导致男性不育。我们建立了完整的猪输精管，在新鲜分离的人类和猪组织，在原代细胞培养，并在永生化上皮细胞的离子转运研究方案。这些细胞系统中的每一种都表现出在完整组织中观察到的特性。这套出色的实验系统将用于实现以下具体目标。目标1：测试HCO 3运输的替代模型。最简单的模型有一个Na？协同转运蛋白和一个纳？？基底外侧膜中的协同转运蛋白，和顶膜中的CFTR。输精管细胞中存在额外的HCO 3转运蛋白。我们将确定这些成分的功能贡献，特别是确定是否有输精管分泌功能的节段性变化。目的2：确定调节输精管上皮HCO 3-分泌的途径。我们假设，生理递质差异调节Cl-和HCO 3-分泌，以实现不同的pH值的管腔流体体积。去甲肾上腺素和腺苷刺激PKA，而催产素刺激PKC，以实现阴离子分泌。缓激肽刺激阴离子分泌的机制尚未确定，需要环氧合酶的活性和反应增强睾酮预处理。我们将首先集中在缓激肽阐明受体（S）和胞质途径（S），占这些观察结果，并确定对HCO 3-和/或Cl-分泌的相对影响。此外，我们将确定激动剂刺激的信号通路，这些信号通路被整合以积极地改变精子所暴露的流体环境。目的3：建立永生化人输精管细胞系。我们建立的猪输精管上皮细胞系是一个很有价值的研究工具。然而，更大的价值将与类似的人类细胞系相关，因为有许多靶向用于人类组织的工具可用。因此，我们将开发另一种细胞系用于研究，可以推断人类导管。目的4：评估，在体内，上皮HCO 3-运输的动态变化。输精管内腔pH值尚未在任何物种中进行系统评估，尽管该参数对精子储存和激活至关重要。结果将提供一个基础，在生殖生理学的运输机制，在体外试验中确定。这些研究的结果将确定药物干预的靶点，以调节管腔pH值，最直接应用于男性生育力。我们将建立一个机制模型（S）来解释上皮HCO 3运输的急性调节，可以扩展到其他身体系统，并与之进行比较。 男性生殖道内的细胞主动调节内部溶液的pH值，这对精子的成熟和激活很重要。我们开发并将使用许多来自人类和猪生殖道的实验系统，以确定这些细胞如何调节精子暴露的液体的pH值和体积。这些研究的结果将确定治疗男性不育症或实施男性避孕的方法。

DESCRIPTION (provided by applicant): The long-term goal of this project is to determine the mechanisms and associated regulatory cascades that account for epithelial HCO3- transport with particular focus on the male reproductive tract. Distinct pHs are required for sperm maturation, storage and activation in the deferent duct. Diseases of epithelial anion transport such as cystic fibrosis result in male infertility. We established protocols to study ion transport in the intact porcine vas deferens, in freshly isolated human and porcine tissues, in primary cell cultures, and in immortalized epithelial cells. Each of these cell systems exhibits properties observed in the intact tissue. This remarkable set of experimental systems will be used to achieve the following specific aims. Aim 1: To test alternative models of HCO3- transport. The simplest model has a Na? cotransporter and a Na?? cotransporter in the basolateral membrane, and CFTR in the apical membrane. Additional HCO3- transporters are present in vas deferens cells. We will determine the functional contribution of these components and specifically determine whether there is segmental variation in vas deferens secretory function. Aim 2: To determine pathways that modulate HCO3- secretion across vas deferens epithelium. We hypothesize that physiological transmitters differentially regulate Cl- and HCO3- secretion to achieve luminal fluid volumes with distinct pH. Norepinephrine and adenosine stimulate PKA whereas oxytocin stimulates PKC to achieve anion secretion. Bradykinin stimulates anion secretion by an undetermined mechanism that requires cyclooxygenase activity and the response is enhanced by testosterone pretreatment. We will focus first on bradykinin to elucidate the receptor(s) and cytosolic pathway(s) that accounts for these observations and determine the relative effects on HCO3- and/or Cl- secretion. Further, we will determine the agonist-stimulated signaling pathways that are integrated to actively modify the fluid environment to which sperm are exposed. Aim 3: To develop an immortalized human vas deferens cell line. The porcine vas deferens epithelial cell line that we developed is a valuable research tool. However, greater value will be associated with an analogous human cell line because numerous tools that are targeted for use with human tissues are available. Thus, we will develop another cell line for studies that can be extrapolated for inferences to the human duct. Aim 4: To assess, in vivo, dynamic changes in epithelial HCO3- transport. Vas deferens lumen pH has not been systematically assessed in any species although this parameter is critical for sperm storage and activation. Results will provide a foundation in reproductive physiology for the transport mechanisms that are identified by in vitro assays. 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 HCO3- transport that can be extended to, and compared with, other bodily systems. Cells lining the male reproductive tract actively regulate the pH of the internal solution, which is important for sperm maturation and activation. We developed and will use a number of experimental systems from human and pig reproductive duct to determine how these cells regulate the pH and volume of the fluid to which sperm are exposed. Results from these studies will identify methods to treat male infertility or to implement male contraception.