Transepithelial transport in cell culture.

Abstract

In cell culture a kidney epithelial cell line, MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions. The reconstructed epithelial membrane has transport and permeability qualities of in vivo transporting epithelia.The cell layer can e readily manipulated when cultured on a freely permeable membrane filter and, when placed in an Ussing chamber, electrophysiological measurements can be taken. In the absence of a chemical gradient, the cell layer generates an electrical potential of 1.42 mV, the apical surface negative. It is an effective permeability barrier and lacks significant shunting at the clamped edge, as indicated by a resistance of 84 ohms.cm2, which increased when bulk flow from basolateral to apical was induced by an osmotic gradient or electroosmosis. The MDCK cell layer is cation selective with a relative permeability ratio, PNa/PCI, of 1.7. Net water flux, apical to basolateral, was 7.3 Ml cm2 hr'1 in the absence of a chemical gradient. The morphological and functional qualities of a transporting epithelium are stable in cell culture, and the potentia use of a homogeneous cell nopulation in cell culture would enhance studies of epithelial transport at the cellular and subcellular levels. In 1969 Leighton et al. (1) and Auersperg (2) described the occurrence of domes, turgid, fluid-filled, blister-like hemicysts, in cell cultures of renal and uterine cervix epithelia, respectively. Subsequently, domes have been reported in cell cultures of normal (3) and. neoplastic (4) mouse mammary epithelia, mouse liver (5), human breast adenocarcinoma (6), pig kidney (7), and frog urinary bladder epithelium (unpublished observation). In each instance, the cells cultured were from transporting epithelia. That domes represent a transport phenomenon is suggested by the presence of morpho--logical polarity unique to transporting epithelia (8), apical microvilli extending upward into the medium and occluding junctions joining adjacent cells at the apical-basolateral membrane border. As well, time-lapse photography revealed domes to be localized regions of the cell layer that lift off the culture dish substratum, gradually expand to a maximum, and then rapidly collapse (9). The establishment and characterization of epithelial transport function in cell culture has both experimental and biological significance. The cells were cultured in Waymouth's 752/1 medium (GIBCO) supplemented with penicillin (100 units/ml), streptomycin (100,gg/ml), insulin (26 IU/ml) (11), and 10% (vol/vol) fetal calf serum. Experiments were performed on cultures 4-21 days after plating. Membrane filters, 25 mm (Millipore HAMK 02512), were boiled for 5-10 min to remove the wetting agent and sterilize. The wet filters were affixed to plastic culture dishes by droplets of Millipore Cement Formulation no. 1 applied around the edge to hold the filter flat. The cultured cells were plated directly into the culture dish over the filter. When the cell layer and underlying filter w...

Secretion of endothelin and related peptides from renal epithelial cell lines

“…Taken together, these data suggest that renal epithelial cell lines have an ET processing mechanism different from that of endothelial cells, possibly by other proteolytic enzymes responsible for further processing of big ET, other than ET-converting enzyme. The MDCK cell line retains many of the differentiated properties typical of renal tubular cells in the loop of Henle or in the distal tubule [4][5][6], whereas the LLCPK1 cell line exhibits some of the properties of proximal tubular cells [7][8][9]. We observed dome formations in all confluent cultures of the two cell lines, suggesting functional plasma membrane polarization with the apical (microvillus) cell surface in contact with the medium and the formation of occluding junctions.…”

Mechanism of milk secretion: milk composition in relation to potential difference across the mammary epithelium

“…in an animal cell, the mammary gland is, of course, itself unusual in that during lactation it continuously synthesizes and secretes lactose -a highly osmotically active, impermeant molecule. Definitive electrophysiological evidence is difficult to obtain because of the glandular nature of the mammary epithelium but a hope for the future is that a method will be developed for growing sheets of lactating mammary cells in culture and studying transport across them, as has been done with neoplastic kidney and mammary cells (Misfeldt, Hamamoto & Pitelka, 1974;D. Misfeldt & D. R. Pitelka, personal communication).…”

Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence.

“…These cells display both structural and functional polarity when grown in culture (24,32). The microvillar THE JOURNAL OF CELL BIOLOGY .…”