Role of the NHE₃ isoform of the Na⁺/H⁺ exchanger in sodium absorption by the rabbit gallbladder

Abstract

The absorption of water and electrolytes by the gallbladder seems to be largely dependent upon a Na⁺/H⁺ exchange at the apical membrane of the gallbladder epithelium. To find out if the exchanger involved is the NHE₃ isoform, as in other absorbing epithelia, two studies were performed using the rabbit gallbladder. First, we studied ²²Na absorption in Ussing chambers with Krebs buffer as a control solution, and in the presence of amiloride (100, 200 or 1000 μM), ethyl-isopropyl-amiloride (EIPA, 1 or 5 μM), or the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 μM). A net mucosal-to-serosal Na⁺ flux was observed with control buffer. No inhibition of this net flux was observed with 5 μM EIPA, and the IC₅₀ for amiloride was found to be 200 μM. PMA induced a reduction of absorption by 30% that was prevented by incubation with calphostin C. Resistance to amiloride and EIPA, and inhibition by PMA are consistent with the involvement of the NHE₃ isoform. The second study involved reverse-transcriptase polymerase chain reaction (RT-PCR) of total gallbladder RNA, with two primers designed to amplify a 645-base-pair fragment from NHE₃ mRNA. A cDNA fragment of the expected size was actually obtained from gallbladder RNA, while RT-PCR of RNA from the liver, which does not contain NHE₃, gave negative 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 NHE₃. It is concluded that, in rabbit gallbladder, neutral NaCl absorption is, at least in part, dependent on the NHE₃ isoform of the Na⁺/H⁺ exchanger.