interpreted results of experiments; H

interpreted results of experiments; H.-F.B., T.L.T., A.F.E., and D.C.E. the ENaC subunits was increased. The increase RHPS4 in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation andPoby PKC in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood RHPS4 pressure in knockout mice fed a high-salt diet. Keywords:protein kinase C, ENaC, renal tubules, single channels, knockout mice, hypertension epithelial na channels(ENaC) are sodium-permeable ion channels located in the apical membrane of polarized epithelial cells primarily in the distal nephron, lung, and distal colon. In the distal nephron, ENaC activity is RHPS4 the rate-limiting step for Na+reabsorption (16,34); therefore, ENaC activity is critical for the physiological maintenance of systemic Na+homeostasis and long-term control of blood pressure. Because of its central role in responding to changes in Na+uptake, ENaC activity is tightly regulated; dysregulation of this channel has been linked to abnormal blood pressure in several genetic disorders including Liddle’s syndrome (18,37) and pseudohypoaldosteronism type 1 (9,33,41). ENaC can be regulated either by altering the amount of time the channel spends open (open probability orPo) or by altering the density of functional channels (N) in the apical membrane of distal nephron epithelial cells. One signaling molecule that appears to alter ENaC activity is protein kinase C (PKC). Activation of PKC with phorbol esters reduces ENaC activity in the apical membrane of A6 cells, an amphibian renal cell line, and in rat principal cells (15). In contrast to the inhibitory effect on ENaC due to activating PKC, pharmacologically inhibiting PKC increases ENaCPo(23,49). A6 cells, on which many of the experiments described above were performed, contain several different PKC isoforms; so that it is difficult to determine which isoform is responsible for the changes in ENaC activity after stimulation or inhibition of all the PKC isoforms. There are conflicting reports in the literature about which Rabbit Polyclonal to TPH2 isoforms of PKC are present in principal cells of the mouse cortical collecting duct. One report suggested that, in mice, there was no PKC present in principal cells (29); subsequent work suggested that PKC was present, but no other typical or novel isoforms (22). Therefore, we made use of PKC knockout mice to examine PKC regulation of ENaC in isolated split-open collecting duct principal cells. == METHODS == == == == Animals. == PKC/mice were initially graciously obtained from the laboratory of Jeffery Molkentin at the University of Cincinnati (17) and maintained in house. Control mice were developed from backcrossing PKC/mice to SV129 controls 10 times to obtain littermate wild-type animals. Once established, RHPS4 the wild-type control line was maintained and used as controls for the PKC knockouts. Mice were kept on a 12:12-h light-dark cycle and fed standard laboratory chow and tap water ad libitum. Mice fed a high-salt diet were fed standard laboratory chow containing 8% NaCl in place of standard chow ad libitum for 1421 days before euthanasia. All of our animal protocols and procedures in this paper were approved by the Emory Institutional Animal Care and Use Committee. == Blood pressure measurements. == Systolic blood pressure and heart rate were measured by tail cuff as previously described (43). Blood pressures were measured for 5 consecutive days before and duringweeks 1and2of a high-salt diet. Data from the first 2 days of each cycle were discarded as this was considered a transition period in which the mice become accustomed to the.