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上皮 HCO3- 转运的机制和相关的调控级联，特别关注男性生殖道。精子在输精管中成熟、储存和激活需要不同的 pH 值。囊性纤维化等上皮阴离子转运疾病会导致男性不育。我们建立了研究完整猪输精管、新鲜分离的人和猪组织、原代细胞培养物和永生化上皮细胞中离子转运的方案。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具体目标。目标 1：测试 HCO3- 运输的替代模型。最简单的模型有一个Na？协同转运蛋白和Na??协同转运蛋白位于基底外侧膜，CFTR 位于顶膜。其他 HCO3- 转运蛋白存在于输精管细胞中。我们将确定这些成分的功能贡献，并具体确定输精管分泌功能是否存在分段变异。目标 2：确定调节输精管上皮 HCO3- 分泌的途径。我们假设生理递质差异性地调节 Cl- 和 HCO3- 的分泌，以实现具有不同 pH 值的管腔液体体积。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。缓激肽通过一种未确定的机制刺激阴离子分泌，该机制需要环氧合酶活性，并且睾酮预处理可增强该反应。我们将首先关注缓激肽，以阐明解释这些观察结果的受体和胞质途径，并确定对 HCO3- 和/或 Cl- 分泌的相对影响。此外，我们将确定激动剂刺激的信号通路，这些信号通路被整合以主动改变精子所暴露的液体环境。目标 3：开发永生化的人类输精管细胞系。我们开发的猪输精管上皮细胞系是一种有价值的研究工具。然而，更大的价值将与类似的人类细胞系相关，因为有许多针对人体组织的工具可供使用。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。目标 4：评估体内上皮 HCO3- 转运的动态变化。尽管输精管管腔 pH 值对于精子储存和激活至关重要，但尚未对任何物种进行系统评估。结果将为体外测定确定的转运机制提供生殖生理学基础。这些研究的结果将确定药物干预的目标，以调节管腔 pH 值，最直接地应用于男性生育能力。我们将建立一个机制模型来解释上皮 HCO3- 转运的急性调节，该模型可以扩展到其他身体系统并与其他身体系统进行比较。 男性生殖道内壁细胞主动调节内部溶液的 pH 值，这对于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管的实验系统来确定这些细胞如何调节精子所接触液体的 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。