Functional Characterization of Na + /H + Exchangers in Primary Cultures of Prairie Dog Gallbladder

Abstract

Gallbladder Na(+) absorption is linked to gallstone formation in prairie dogs. We previously reported Na(+)/H(+) exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I(sc), (11.6 +/- 0.5 microA. cm(-2)), potential differences, V(t) (2.1 +/- 0.2 mV), and resistance, R(t) (169 +/- 12 omega. cm(2)). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable (22)Na(+) uptake under a H(+) gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of (22)Na(+) uptake was 21.4 +/- 1.3 nmol x mg prot(-1) x min(-1), of which 9.5 +/- 0.7 (approximately 45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for approximately 6%, approximately 66% and approximately 28% of GBECs' total NHE activity, respectively. GBECs exhibited saturable NHE kinetics ( V(max) 9.2 +/- 0.3 nmol x mg prot(-1) x min(-1); K(m) 11.4 +/- 1.4 m M Na(+)). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na(+) transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.

The Role of Plasma Membrane Sodium/Hydrogen Exchangers in Gastrointestinal Functions: Proliferation and Differentiation, Fluid/Electrolyte Transport and Barrier Integrity

“…The NHE1 isoform is believed to be ubiquitously expressed (although this was of course not documented in all cell types), and localized to the basolateral membrane of epithelial cells. An expression of the NHE1 isoform was documented in the esophagus ( Shallat et al, 1995 ), in the different gastric epithelial cells ( Rossmann et al, 2001 ; Stuart-Tilley et al, 1994 ; Xu et al, 2008 ), the salivary glands ( He et al, 1997 ; Robertson et al, 1997 ), the small and large intestine ( Bookstein et al, 1994a ; Dudeja et al, 1996 ), the gallbladder ( Narins et al, 2004 ), the biliary ducts ( Spirlì et al, 1998 ) and the pancreas ( Roussa et al, 2001 ) of different species ( Figures 1 – 3 ). Anti-NHE1 isoform specific antibodies were used for localization, but yielded conflicting results, because specificity of an antibody can only be tested with a high confidence level in knockout mice for the respective antigen.…”

“…NHE2 is predominantly expressed in epithelial cells of the gastrointestinal tract ( Ghishan et al, 1995 ; Hoogerwerf et al, 1996 ; Park et al, 1999 ; Lee et al, 2000 ; Rossmann et al, 2001 ; Narins et al, 2004 ). However, it is also found outside the GI tract, namely in the kidney ( Chambrey et al, 1998 ; Peti-Peterdi et al, 2000 ), endometrium and placenta ( Johansson et al, 2002 ; Wang et al, 2003 ) chondrocytes ( Trujillo et al, 1999 ), inner ear ( Bond et al, 1998 ; Goto et al, 1999 ), heart, testes, and pituitary gland ( Miller et al, 2011 ).…”

SLC9 Gene Family: Function, Expression, and Regulation

“…Na + /H + exchange, and NHE3 in particular, has also been detected in the gallbladder epithelium in human (277), calf (17), and prairie dog (1). Primary epithelial cells from the prairie dog gallbladder showed H + gradient-dependent 22 Na uptake mediated by NHE1 (6%), NHE2 (66%), and NHE3 (~28% of total uptake), as determined by studies with DMA and HOE-694 inhibitors (211). It has been hypothesized that apically expressed Na + /H + exchangers may participate in the pathogenesis of gallstone formation.…”