The epithelial sodium channel (ENaC) marks the tightly regulated rate-limiting step of sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). In the ASDN, stimulation of ENaC activity by the mineralocorticoid aldosterone and the antidiuretic hormone AVP is mediated via complex mechanisms which are still incompletely understood [1]. These include enhanced insertion of either recycled or newly synthesised channels into the plasma membrane and/or inhibition of channel retrieval via Nedd4-2 mediated ENaC ubiquitination and proteasomal degradation [2]. The aim of this study was to investigate the role of the proteasomal pathway in basal and hormonally-regulated ENaC activity. Cultured mouse cortical collecting duct cells (mCCDcl1) which respond to physiological concentrations of aldosterone and AVP [3] were used and ENaC activity was quantified by recording the equivalent short circuit current (ISC) in Ussing chambers. Biotinylation studies were performed to monitor membrane abundance of β-ENaC subunits. Values are means ± SEM, compared by Student’s t-test. Addition of 1µM MG132, a specific proteasomal inhibitor, maximally stimulated ISC from 9±1 to 23±2 µA cm-2 (n=7, p<0.01) after 3.5h. Application of 3nM aldosterone increased ISC from 10±2 to 25±4 µA cm-2 (n=7, p<0.05) reaching a plateau after 2h. Subsequent application of MG132 further increased ISC to 47±6 µA cm-2 (n=7, p<0.01). Increases in ISC correlated with an increased abundance of β-ENaC at the plasma membrane. In contrast to the stimulatory effects of aldosterone and MG132, a slight run-down of ISC ~20% was observed in matched control cells over the course of 7h. Treatment with 25pM AVP rapidly increased ISC by 13±1 µA cm-2 (n=3) and 13±0 µA cm-2 (n=3) in vehicle- and aldosterone-treated cells, respectively. This effect was significantly reduced in both groups in the presence of MG132, ~65% (vehicle-treated) and ~70% (aldosterone-treated). Application of 10µM amiloride abolished ISC in all experiments indicating that changes in ISC can be attributed to altered ENaC activity. Our results demonstrate that inhibition of proteasomal degradation in mCCDcl1 cells stimulates ENaC activity. This effect is preserved in cells pre-stimulated with aldosterone which suggests that the stimulatory effect of MG132 is additive to that of aldosterone. In contrast, the response to AVP is reduced following MG132 treatment. Interestingly, ENaC stimulation by AVP has been shown to involve channel recycling from a subapical pool to the plasma membrane. Thus, the inhibitory effect of MG132 on the AVP response may reflect altered channel recycling due to proteasomal inhibition.