Colonic-crypt-derived epithelia express induced ion transport differentiation in monolayer cultures on permeable...

The processes of transport differentiation from stem cell to the terminally differentiated cell in intact colonic crypts are difficult to study because access to the lumen is limited. Colonocytes were isolated from the lower two-thirds of rat distal colon crypts and grown to confluence on reconstituted basement membranes and permeable support in primary culture. Crypt and surface cells were distinguished by the uptake of [3H]thymidine and [3H]leucine and by brushborder fluorescence binding. Ion concentrations in apical and basolateral compartments of filter monolayer cultures after 48 h of incubation on days 16-18 were (in mM): apical, Na+ 116 +/- 4 (n = 48) and K+ 6 +/- 1 (n = 48); basolateral, Na+ 151 +/- 3 and K+ 3.7 +/- 0.5, respectively (mean +/- SE). Aldosterone (10(-8) M), added to the basolateral compartment from days 10-18, changed apical Na+ to 72 +/- 6 mM and apical K+ to 13 +/- 4 mM (n = 23). Dexamethasone (10(-8) M) changed apical Na+ to 84 +/- 7 mM but did not influence apical K+ (n = 22). Transmonolayer electrical potential difference (VtM; control medium; days 8-10) was 5 +/- 1 mV (n = 16; apical compartment negative); electrical resistance (RtM) was 217 +/- 21 omega.cm2 and short circuit current (ISC) was 21 +/- 5 microA.cm-2. Amiloride (0.1 mM; n = 12) in the apical medium decreased VtM to 2 +/- 1 mV and ISC to 11 +/- 4 microA.cm-2. Aldosterone (10(-8) M) after 1 week in the basolateral compartment (n = 21) changed VtM to 12.3 +/- 3 mV, RtM to 92 +/- 9 omega.cm2, and ISC to 138 +/- 23 microA.cm-2. Apical amiloride (0.1 mM; n = 9) decreased the induced VtM to -3 +/- 1 mV and ISC to -13 +/- 7 microA.cm-2. Colonic-crypt-derived epithelial cells proliferate and differentiate in primary culture, when grown on reconstituted basement membrane substratum and in supplemented medium, to form monolayers that express net Na+ absorption and net K+ secretion after 1 week. Na+ and K+ vectorial transport differentiation is primarily regulated by aldosterone, which specifically induces apical conductive Na+ transfer. Mineralocorticoid and glucocorticoid hormones appear to have differing actions on ion transport in functionally surface-type colonocytes derived in culture from isolated crypt-type cells.