Regulation of serine proteases and their role in sodium transport in cortical collecting duct.

摘要

The amiloride-sensitive epithelial sodium channel (ENaC), located at the apical membrane of collecting duct, plays a major role in the regulation of sodium and fluid reabsorption, thus, in the control of blood pressure. An apical serine protease, channel activating protease 1 (CAP1), has been shown to activate ENaC in kidney cells. Prostasin, a novel serene protease originally purified from seminal fluid, has been proposed to be the mammalian ortholog of CAP1.; Functional evidence for a similar process was found in the M-1 cortical collecting duct (CCD) cell line: sodium transport was inhibited with the serine protease inhibitor aprotinin, and was subsequently stimulated with apical trypsin (a seine protease). Afterwards prostasin cDNA was cloned from M-1 cells, and the proteolytic activity of serine protease was detected in M-1 cell media using an amidolytic assay. Prostasin mRNA was also detected in most nephron segments in rats by in situ hybridization.; Since prostasin has been suggested to be upregulated by aldosterone, the issue of whether prostasin is regulated by aldosterone (Aldo) and dexamethasone (Dex) in M-1 cells and rat kidneys was investigated. Using Northern analysis, studies were demonstrated that neither Aldo, Dex, nor the combination, aldosterone+dexamethasone (A+D), significantly increased prostasin mRNA in M-1 cells. Also, the functional activity of prostasin, indicated by aprotinin sensitive equivalent current, was not altered by Aldo, Dex, or A+D. Moreover, the amidolytic assay with the fluorogenic substrate D-Pro-Phe-Arg-AMC showed that the proteolytic activity of seine protease in M-1 cell apical media was also not regulated by steroids In situ hybridization demonstrated that the level of prostasin mRNA and its segmental distribution were not regulated by steroids in proximal tubules (PTs) or CCDs in rats.; In conclusion, prostasin is present in M-1 cells, secreted by these cells, and augments sodium transport by ENaC in these cells. Prostasin is expressed in most nephron segments in rats. Mineralocorticoid and glucocorticoid, at the dose used in our studies, do not regulate prostasin mRNA and functional activity in M-1 cells, and do not regulate prostasin mRNA in PTs or CCDs in rats.