Studies on Epithelial Cells Isolated From Guinea Pig Small Intestine

Abstract

Sheets of mucosal epithelial cells were released from guinea pig small intestine after incubation with ethylenediaminetetraacetate . Cells in sheets retained their columnar shape for 24 hr at room temperature, and exclusion of nigrosine suggested they had intact plasma membranes . When sheets were disaggregated individual cells had normal morphology for at least 4 hr. During isolation 16% of the total protein and 24% of the total lactic dehydrogenase were lost from the cells, but subsequent enzyme leakage was low . Leakage increased with shaking, incubation at 37°C, or increasing the oxygen tension of the suspending medium, but was minimal when the Na+ : K+ ratio in the medium was 8 :1 and the osmolarity was high . Losses of particulate enzyme activities were negligible . Respiration was constant for up to 4 hr and was insensitive to calcium, bicarbonate, oxygen tension, and pH . It was inhibited by cyanide and iodoacetate and varied with the Na+ : K+ ratio of the extracellular fluid and the structural integrity of the cells . All preparations concentrated potassium and excluded sodium, but lost this ability if ouabain was added or cells were broken . Potassium-42 uptake was also sensitive to temperature, ouabain, and structural integrity. The preparations are being used to study cell metabolism in the intestinal epithelium.

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

“…The brush-border fractions obtained from rat small intestine consisted of relatively pure apical portions from absorptive cells, comparable in purity to preparations described in the literature from intestine of rat (21,51), mouse (52), guinea pig (37), and chicken (19, 20, 22, 31; for biochemical criteria of purity see also reference 38). The preservation of the typical brush-border structures such as microvilli, terminal web, and associated apico-lateral plasma membrane, including zonula adhaerens junctions, was as good as that described for similar preparations by other authors (19-22, 37, 51), and the ultrastructure of the desmosomes and tonofilaments was indistinguishable from that described in intact cells (e.g., 5, 9, 21, 28, 53).…”

“…Isolation of intestinal brush borders and microvilh followed the protocols used by previous authors (19)(20)(21)(22)37) with some modifications (38) : The rinsed intestinal tube pieces were filled with buffer A (96 mM NaCl, 8 mM KH 2PO,, 5.6 mM Na2HP04, 1.5 mM KCI, 10 mM EDTA, 0.1 mM dithioerythritol, 0.1 mM PMSF, and 1 mM e-aminocaproic acid, pH 6.8), the ends were tied with thread, and the intestines were immersed and incubated in 0.3 M sucrose for 15 min at 0°C. The epithelial cell layer was released from the lamina propria by gentle rubbing of the intestines, and the cells were harvested by flushing the lumen with buffer A followed by centrifugation at 300 g for 10 min.…”

Adhesion of Enteropathogenic Escherichia Coli to Pig Intestinal Brush Borders: The Existence of Two Pig Phenotypes

“…Brush borders were prepared from the small intestine of pigs. The procedure was adapted from those of Miller and Crane (1961) and Evans et al (1971). Lengths of small intestine were excised from freshly killed animals and washed free of contents with 0.154 NaCl.…”

The use of isolated cells to assess the contribution of the mucosal epithelium to the metabolism of the intestinal wall

“…withdrawal through a syringe needle of 0.7 mm bore (Evans et al, 1971) to increase their membrane permeability (Fig. 2) Sodium iodoacetate (10-4M) and sodium fluoride (10-4M) completely inhibited lactic acid production from endogenous and exogenous substrates.…”