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The regulation of plasma membrane Na(+)-K(+)-ATPases (NKA) expression by aldosterone and arginin vasopressin (AVP) in the cortical collecting duct (CCD) has been examined in a new rat CCD cell line, designated as RCCD(2). This cell line has maintained many characteristics of the CCD-in particular, the expression of the mineralocorticoid receptor. Mineralocorticoid receptor is expressed at the protein level and binds (3)H-aldosterone (approximately 15 to 20 fmol/mg protein). Short-circuit current (Isc) experiments showed approximately a twofold increase in Isc associated with a decrease in transepithelial resistance when cells were treated with aldosterone concentrations as low as 10(-9) M. This effect on Isc was significant 2 h after aldosterone addition and was still present after 24 h. It was accompanied by an increase in the amount of mRNA encoding for the alpha subunit of the epithelial sodium channel (sixfold) and the alpha1 subunit of NKA (fourfold) after 24 h of hormone treatment. In addition, mRNA expression of the serum- and glucocorticoid-induced kinase (Sgk) was increased by 10(-9) M aldosterone treatment as early as 45 min after hormone addition. As had already been documented in native CCD obtained by microdissection, incubation of RCCD(2) cells for 24 h with aldosterone resulted in the constitution of a latent pool of NKA that could be rapidly recruited by AVP (15 min). NKA biotinylation experiments and preparation of membrane fractions show that this latent pool of NKA is present in the intracellular compartment of the cells and is recruited by AVP in the basolateral membrane through a translocation process. This mechanism is accompanied by dephosphorylation of the alpha(1) catalytic subunit of NKA.

This study examines whether serine proteases can activate the amiloride-sensitive sodium channel (ENaC) in mammalian kidney epithelial cells. The transepithelial sodium transport assessed by amiloride-sensitive short-circuit current appears to be sensitive to aprotinin, a protease inhibitor in a mouse cortical collecting duct cell line (mpkCCD(c14)). This result indicated that serine proteases may be implicated in the regulation of ENaC-mediated sodium transport. Using degenerated oligonucleotides to a previously isolated serine protease from Xenopus, xCAP1 (channel activating protease), a novel full-length serine protease (mCAP1), has been isolated and characterized. RNA analysis showed a broad pattern of expression in tissues (kidney, lung, colon, and salivary glands) expressing ENaC. Reverse transcription-PCR experiments also showed that mCAP1 was abundantly expressed in proximal tubule cells and was also expressed in intact and cultured collecting duct cells. Coexpression of the Xenopus, rat, or human alpha-, beta-, and gamma-ENaC subunits in Xenopus oocytes also showed that mCAP1 induces a significant increase in ENaC-mediated current accompanied by a decrease of channel molecules at the cell surface. It is proposed that this novel mouse channel activating protease may act as a regulator of ENaC within the kidney.