For studies on apical NHE3 exocytosis, cell monolayers were stimulated with AII for varying times with or without pretreatment with inhibitors as designated. AII was added directly into the basolateral medium. Monolayers were rapidly cooled by placing on ice, changing medium to Lenalidomide solubility phosphate buffered saline with 0. 5 mg/ml sulfo NHS biotin only on the apical side. Monolayers were incubated for 30 min with the apical biotinylation solution. Over this period, we had previously shown that biotinylation of basolateral and intracellular proteins does not occur. Biontinylation was terminated by the addition of 10l of 1 M Tris pH 8. 0 which will reacts rapidly with all free biotin. Cell monolayers were scraped off the filters, pel leted and resuspended in immunoprecipitation buffer and 1% Triton X 100.

Samples were solubilized, an aliquot removed to measure protein and total NHE3, and, to the remainder. streptavidin agarose was added. Samples were rotated for 120 min, washed 3 times with IP buffer, and samples eluted by boiling in 1�� Laemmli buffer. Biotinylated apical surface proteins as well as total NHE3 were analyzed by Western blotting. Total cellular protein or IP samples were separated on 7. 5% SDS PAGE and immediately transferred to PVDF membranes in 1�� Towbins buffer. Membranes were blocked in T TBS containing 5% wt/vol nonfat dry milk for 60 min at room temp. Blots were incubated overnight at 4 C with affinity purified specific rabbit polyclonal antisera to NHE2 and NHE3 developed and characterized by our laboratory. Blots were developed using an enhanced chemilumines cence system.

RNA isolation, reverse transcription, and real time PCR Caco 2BBE cells were treated with AII for varying times. Total RNA was isolated using TRIzol reagent. Oneg RNA was reverse transcribed by random priming and one twentieth used for real time PCR performed on an I Cycler using SybrGreen Mix and primers for human NHE3. Rela tive mRNA levels were calculated using the comparative threshold cycle method. Each PCR reaction was performed in triplicate, and all experiments were repeated three times. For each sample, mRNA levels of both NHE3 and GAPDH were measured and the cycle threshold of NHE3 subtracted from that of GAPDH. This value was set to one for untreated control conditions at zero time and other time points are calculated relative to this change.

For analysis of AII receptor type, one twenti eth of the reverse transcription reaction was used for amplification with primers for human ATGR1 and AGTR2. AV-951 PCR reactions were amplified for 30 cycles and the PCR products were first analyzed by agarose gel for confirmation of correct size and then subcloned into pCR2. 1 TOPO and sequenced. Luciferase reporter activity A 2200 bp region of the rat NHE3 promoter was a generous gift of Dr. A. Cano. This promoter was linked to firefly luciferase in the plasmid pGL3.