This project focuses on the regulation of amiloride-sensitive Na+ channels and water channels in the rat and rabbit cortical collecting duct (CCD). Techniques to be used include: measurement of osmotic water permeability (P/f), lumen-to-bath 22/Na+ flux (J/1 to b), and transepithelial voltage (V/T) in isolated perfused rat CCDs; immunodissection of rat and rabbit CCD cells, followed by primary culture of rabbit CCD cells; reverse transcription (RT) and polymerase chain reaction (PCR) to identify expression of mRNAs, and immunocytochemistry and immuno-blotting of cell lysates to localize and quantify expression at the protein level.
Our specific aims are: a) To examine the hypothesis that the effects of increased [Ca2+]i or protein kinase C (PKC) activation on AVP-dependent P/f and J/1 to b in the rat versus the rabbit CCD depend on the specific PKC isoforms expressed. Measurements of [Ca2+]i in isolated perfused rat and rabbit CCDs will be conducted over a wide range of AVP doses, and after stimulation with A1F/4 minus. Specific PKC isoenzyme expression at the mRNA level will be determined using RT/PCR with degenerate primers and a MAPPing technique. PKC isoform expression at the protein level will be determined by immunocytochemistry and immuno-blotting of lysates of rat and rabbit CCD cells. b) To Test the hypothesis that nitric oxide (NO) and/or ANP inhibit Na+ and water transport in the rat CCD via a cGMP- dependent mechanism. In order to examine this possibility, we will look for inhibitory effects of dibutyryl-cGMP (db-cGMP) and nitroprusside (a NO donor) on basal and AVP-dependent P/f and J/1 to b, and we will measure the production of cGMP in isolated nonperfused CCD segments stimulated by ANP or nitroprusside. c) We will also test the hypothesis that tyrosine kinase acts as a Na+ channel activator in the CCD, and whether or not this enzyme or its products are involved in the stimulatory response to increased Na+ delivery, growth and development. We will first look for effects of tyrosine kinase activators and inhibitors on basal Na+ transport in the rat CCD, expecting that inhibitors will decrease and activators increase transport. If so, does AVP or db-cAMP stimulate J/1 to b of 22/Na+ and P/f in the presence of tyrosine kinase inhibitors? Because a high NaC1 (4%) diet hypertrophies rat CCD principal cells, we will test whether or not basal J/1 to b is elevated in these CCDs. If so, can this flux be inhibited by tyrosine kinase inhibitors as might be expected if trophic factors are responsible? In the absence of these inhibitors, is J/1 to b further augmented by AVP or db-cAMP? We also intend to determine if tyrosine kinase-initiated events are responsible for the change in the rabbit CCD hormone response observed in primary culture. We hypothesize activation of tyrosine kinase results in phosphorylation-mediated changes in the expression of growth factors, e.g., the ras/rab family, in both primary cultures and CCDs in vivo, and we will look for changes in expression of these factors using RT/PCR and immunocytochemical methods. Results from these studies will provide important new information on how conditions of growth and response to diet can alter the expression of regulatory mechanism that control Na+ and water reabsorption in the CCD.
Showing the most recent 10 out of 34 publications
