These studies therefore provide compelling evidence of direct regula tion of apical NHE3 in intestinal epithelial cells by AII. Results Angiotensin II increases NHE3, but not selleck bio NHE2, activity and membrane insertion acutely and in long term Caco2BBE cell monolayers were treated on the basolateral side with 1 nM angiotensin II for times ranging from 1 48 hours. Apical NHE activities were measured as 22Na uptake sensitive to amiloride analogs HOE694 or DMA, as previously described. NHE2 and NHE3 activities were defined as the HOE694 sensitive and insensitive components of DMA inhibited 22Na uptake, respectively. After two hours, 1 nM angiotensin II significantly increased apical NHE3, but not NHE2 activ ity. The increased NHE3 activity was paralleled by increased apical surface abundance of NHE3, as assessed by apical surface biotinylation.

In previous studies we had demon strated that the conditions for apical surface biotinylation do not result in biotinylation of either basolateral surface proteins or intracellular proteins. Equivalent protein amounts were used for apical surface biotinyla tion and total NHE analyses. Apical addition of 1 nM AII did not stimulate apical 22Na transport at any time up to 48 hours. Further increases in apical NHE3 activity were observed between 4 48 hours after AII stimulation, occurring in two phases. From 1 4 hours, a smaller increase in apical NHE3 activ ity was observed with a progressive increase from 4 to 24 hours that was maintained for at least 24 hours. These changes were associated with increased apical surface NHE3 abundance.

Within one hour, however, little increase in total NHE3 protein expression was observed and from 2 48 hours, NHE3 protein expression increased. No changes were observed for apical surface or total NHE2 over this time. AII increased NHE3 expression and activity at 24 hours in a concentra tion dependent fashion with effects Brefeldin_A beginning at low pM concentrations and maximal effects near 1 nM, concentra tions that are in the physiologic range. To determine if AII stimulates Na transport in native intestine, segments of mouse jejunum were mounted in Ussing chambers and transmural 22Na fluxes measured. Addition of AII significantly increased the mucosal to sero sal absorptive flux without change in the seriosal to mucosal flux, demonstrating that the AII induced apical NHE3 activity observed in cultured Caco2BBE cells is also observed in native intestine. The increase in m to s flux is small, however, it should be noted that the incubations time with AII was limited due to the limited viability of mouse jejunum in Ussing chambers.