Neuroendocrine-modulated epithelial HCO3- transport

神经内分泌调节上皮 HCO3- 转运

• 批准号: 7564804
• 负责人: BRUCE D SCHULTZ
• 金额: $ 32.85万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7564804
• 关键词: Abdomen; Accounting; Acute; Address; Adenosine; Adenylate Cyclase; Agonist; Androgens; Animals; Anions; Apical; Bicarbonates; Bradykinin; Bronchi; Cell Line; Cell model; Cells; Complex; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Development; Disease; Duct (organ) structure; Duodenum; Endocrine; Environment; Epithelial; Epithelial Cells; Epithelium; Exhibits; Family suidae; Fertility; Foundations; Gene Family; Genes; Goals; Hormones; Human; Human Cell Line; Human Development; Infertility; Intervention; Intestines; Ion Transport; Ions; Liquid substance; Male Contraceptions; Male Infertility; Messenger RNA; Methods; Modeling; Molecular; Na(+)-K(+)-Exchanging ATPase; Neurosecretory Systems; Neurotransmitters; Norepinephrine; Oxytocin; Pancreas; Pancreatic duct; Pathway interactions; Pharmaceutical Preparations; Physiological; Potassium Channel; Primary Cell Cultures; Property; Prostaglandin-Endoperoxide Synthase; Proteins; Protocols documentation; RNA Splicing; Rattus; Relative (related person); Reporting; Reproductive Physiology; Research; Rest; Signal Pathway; Solutions; Specimen; Sperm Capacitation; Sperm Maturation; Stimulus; Structure; System; Techniques; Telomerase; Testing; Testosterone; Tissues; Transfection; Variant; Vas deferens structure; Vasopressins; Work; apical membrane; base; basolateral membrane; cytosolic receptor; design; direct application; ductile; human tissue; in vitro Assay; in vivo; instrument; male; receptor; reproductive; research study; response; sodium-potassium-chloride cotransporter 1 protein; sperm cell; tool

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.

描述（由申请人提供）： 该项目的长期目标是确定解释上皮HCO3的机制和相关的调节级联反应，特别关注男性生殖道。精子成熟，储存和激活需要不同的pH值。上皮阴离子转运（例如囊性纤维化）的疾病导致男性不育症。我们建立了在完整的猪VAS延迟，新鲜分离的人类和猪组织，原代细胞培养物以及永生上皮细胞中研究离子转运的方案。这些细胞系统中的每一个都表现出在完整组织中观察到的特性。这套非凡的实验系统将用于实现以下特定目标。目标1：测试HCO3传输的替代模型。最简单的模型有NA？共转运者和NA ??基底外侧膜中的共转运蛋白，以及顶膜中的CFTR。 VAS deferens细胞中存在其他HCO3-转运蛋白。我们将确定这些组件的功能贡献，并明确确定VAS deferens分泌函数是否存在分段变化。目标2：确定调节HCO3-跨VAS延迟上皮的途径。我们假设生理发射机对Cl-和HCO3分泌有差异化，以实现具有不同pH的腔体流体体积。去甲肾上腺素和腺苷刺激PKA，而催产素刺激PKC进行阴离子分泌。松曲蛋白通过不确定的机制刺激阴离子分泌，该机制需要环氧酶活性，并且通过睾丸激素预处理增强了反应。我们将首先关注心动激肽，以阐明解释这些观察值的受体和胞质途径，并确定对HCO3和/或CL-分泌的相对影响。此外，我们将确定集成的激动剂刺激的信号通路，以积极修改精子暴露的流体环境。目标3：开发永生的人体VAS延迟细胞系。我们开发的猪VAS递延上皮细胞系是一种有价值的研究工具。但是，更大的价值将与类似的人类细胞系有关，因为有许多用于与人体组织一起使用的工具。因此，我们将开发另一条细胞系进行研究，可以推断出对人类管道的推断。目标4：评估上皮HCO3传输的动态变化。尽管该参数对于精子储存和激活至关重要，但VAS递延腔液pH尚未在任何物种中进行系统评估。结果将为通过体外测定鉴定的运输机制提供生殖生理的基础。这些研究的结果将确定药物干预措施的靶标，以调节腔pH，最直接地应用于男性生育能力。我们将建立一个机械模型，以说明上皮HCO3转运的急性调节，该传输可以扩展到并与其他身体系统扩展并进行比较。 内衬雄性生殖道的细胞积极调节内部溶液的pH值，这对于精子成熟和激活很重要。我们开发并将使用来自人类和猪生殖管的许多实验系统来确定这些细胞如何调节精子暴露的液体的pH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方法的方法。