Neuroendocrine-modulated epithelial HCO3- transport

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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.