ROLE OF THE NHE(3) ISOFORM OF THE NA+ H+ EXCHANGER IN SODIUM-ABSORPTION BY THE RABBIT GALLBLADDER/

The absorption of water and electrolytes by the gallbladder seems to b e largely dependent upon a Na+/H+ exchange at the apical membrane of t he gallbladder epithelium. To find out if the exchanger involved is th e NHE(3) isoform, as in other absorbing epithelia, two studies were pe rformed using the rabbit gallbladder. First, we studied Na-22 absorpti on in Ussing chambers with Krebs buffer as a control solution, and in the presence of amiloride (100, 200 or 1000 mu M), ethyl-isopropyl-ami loride (EIPA, 1 or 5 mu M), or the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 mu M) A net mucosal-to-serosal Na+ flux was observ ed with control buffer. No inhibition of this net flux was observed wi th 5 mu M EIPA, and the IC50 for amiloride was found to be 200 mu M. P MA induced a reduction of absorption by 30% that was prevented by incu bation with calphostin C. Resistance to amiloride and EIPA, and inhibi tion by PMA are consistent with the involvement of the NHE(3) isoform. The second study involved reverse-transcriptase polymerase chain reac tion (RT-PCR) of total gallbladder RNA, with two primers designed to a mplify a 645-base-pair fragment from NHE(3) mRNA. A cDNA fragment of t he expected size was actually obtained from gallbladder RNA, while RT- PCR of RNA from the liver, which does not contain NHE(3), gave negativ e results. A sequence of 492 nucleotides of the amplified product was determined, which was almost superimposable onto the known sequence of the corresponding fragment of rabbit NHE(3). It is concluded that, in rabbit gallbladder, neutral NaCl absorption is, at least in part, dep endent on the NHE(3) isoform of the Na+/H+ exchanger.