Cell line models of colonic electrolyte transport have been used extensively, despite lacking some characteristics of the native tissue. While native colonic crypts absorb or secrete NaCl, immortalized cell lines only retain the secretory phenotype. In this study we characterized functionally and molecularly fluid and electrolyte transport in the morphologically differentiated human colonic cell line LIM1863 (Whitehead et al. 1987).
Net fluid transport was evaluated by the morphometric measurement of lumens (expressed in pixels) formed in LIM organoids in response to secretagogues. To normalize the differences in organoid size, the lumen area was divided by the initial organoid radius. Functional profiling of the channels and transporters involved in fluid and electrolyte transport was determined pharmacologically, while the molecular basis was determined by RT-PCR and/or immunofluorescence confocal microscopy.
Similar to native tissue, LIM1863 cells secrete fluid and electrolytes across the apical membrane into a central lumen when exposed to cAMP-mediated secretagogues such as forskolin (10 µM, lumen size 82 ± 3 pixels (mean ± S.E.M.); n = 95). Lumen formation was markedly reduced by 100 µM azosemide (29 ± 2 pixels; n = 33), consistent with Cl– uptake across the basolateral membrane through a Na+-K+-2Cl– cotransporter. Cl– exit across the apical membrane into the lumen appears to be via the cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel, as the lumen formation was reduced by 100 µM glibenclamide, an inhibitor of CFTR (42 ± 3 pixels; n = 21) but unaffected by 100 µM DIDS (83 ± 6 pixels; n = 11), an inhibitor of Ca2+-activated Cl– channels.
Electrogenic NaCl absorption occurs throughout the human colon, and involves the aldosterone-regulated epithelial sodium channel (ENaC) (Sandle, 1989). Colonic cell lines are thought to lack ENaC due to their insufficient level of differentiation (Greger, 2000). Interestingly, we have shown that LIM1863 cells express ENaC (RT-PCR and immunocytochemistry studies) and reduce their lumen size attributable to NaCl absorption, a response that is increased significantly by aldosterone (1 µM; P < 0.001 vs. control, unpaired t test). To our knowledge, this is the first example of the functional expression of ENaC in a colonic epithelial cell line, introducing LIM1863 cells as a new model for further delineation of colonic secretion and absorption.
This work was funded by Ministerio de Ciencia y Tecnología (Spain) and The King’s Medical Research Trust, UK.