Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium

Abstract

The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by RT-PCR, Western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes, and tight junctions expressing zonula occludens-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone, and insulin, monolayer transepithelial voltage was −6.8 mV (apical side), transepithelial resistance was 1,050 Ω·cm2, and short-circuit current ( Isc) was 8.1 μA/cm2. Transcellular and paracellular resistances were estimated as 14.8 and 1.1 kΩ·cm2, respectively. Serosal ouabain reduced voltage and current toward zero, as did apical amiloride. The presence of mRNA of α-epithelial Na+channel (ENaC) was confirmed. Na-d-glucose cotransport was identified with an antibody to Na+-glucose cotransporter (SGLT) 1. The unidirectional mucosa-to-serosa Na+flux (19 nmol·min−1·cm−2) was two times as large as the reverse flux, and net transepithelial Na+flux was nearly double the amiloride-sensitive Isc. In plain Ringer solution, the amiloride-sensitive Iscwent toward zero. Under these conditions plus mucosal amiloride, serosal dibutyryl-cAMP elicited a Cl−-dependent Iscconsistent with the stimulation of transepithelial Cl−secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na+absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl−secretion activated by cAMP.