NHE-3 isoform of the Na+/H+ exchanger in human gallbladder. Localization of specific mRNA by in situ hybridization
V Silviani1, M Gastaldi, R Planells
1INSERM U.260, Faculté de Médecine, Marseilles, France.
Background/Aims:
Electroneutral absorption of NaCl by the gallbladder mucosa is likely to depend at least in part on a Na+/H+ exchanger. In intestine and colon, absorption due to Na+/H+ exchanger is explained by the presence of specific isoforms of the exchanger, the NHE-3 isoform and possibly the NHE-2 isoform. The aim of the present work was to determine whether the mRNAs coding for NHE-2 and NHE-3 are expressed in epithelial cells of human gallbladder.
Methods:
Total RNAs from human gallbladder were subjected to reverse transcription-polymerase chain reaction using specific primers. No message was observed with NHE-2 specific primers, showing that NHE-2 isoform plays no role in gallbladder absorption. With NHE-3 specific primers, a 239 bp cDNA fragment was obtained and showed a high homology with the NHE-3 isoform, confirming the presence of NHE-3 in the gallbladder wall. This fragment was cloned in a pLitmus vector in order to produce cRNA probes by in vitro transcription. Cellular localization of the NHE-3 mRNA was studied on cryostat sections using the cRNA probes labeled with Digoxigenin-11-UTP, controls included assays with sense probe, antibodies without probe and RNaseA treated tissue. A specific staining of the NHE-3 mRNAs was found to be strictly localized to the gallbladder epithelial cells.
Results/Conclusions:
Expression of NHE-3 in the gallbladder was found only in the absorptive epithelial cells. The NHE-3 isoform of the Na+/H+ exchanger is likely to be involved in water and electrolyte absorption from bile.
