Stability of enterocytes and certain enzymatic activities in suspensions of cells from the villous tip to the crypt of Lieberkühn of the mouse small intestine

Abstract

A series of studies in this laboratory have focused on how an indigenous microbiota influences the activities of alkaline phosphatase, phosphodiesterase I, and thymidine kinase in the enterocytes of the upper small intestine of mice. To draw conclusions about the role of the microflora in determining levels of enzymatic activity, we found it necessary to develop a procedure by which cell suspensions could be obtained containing enterocytes isolated sequentially from the villous tip to the crypt of Lieberkuhn. The procedure was modified from the one developed for rats by Weiser (J. Biol. Chem. 248:2536-2541, 1973), involved a minimum number of interfering factors (e.g., proteolytic enzymes and mechanical agitation), and worked reproducibly for mice. During development of the procedure, some variables affecting the assays of the enzymes known to be present in enterocytes were also explored. Rods to which were tied everted segments of gut were incubated in a series of tubes containing a solution of EDTA the concentration of which was changed from 1.5 to 5.0 mM, thus giving a greater yield of enterocytes at every step. The cells incubating in the chelating solution were most stable when 0.23 M sucrose was included in the EDTA solutions. Success in assaying enzymatic activities in the cell suspensions depended on (i) how the cells were isolated, (ii) the assay procedure for thymidine kinase, and (iii) whether cellular suspensions or extracts were assayed.

Growth phase, cellular hydrophobicity, and adhesion in vitro of lactobacilli colonizing the keratinizing gastric epithelium in the mouse

“…Cells of strains of Lactobacillus spp. isolated from animals of various taxonomic groups were grown to stationary phase in MRS broth, harvested into homogenization buffer (16), and assayed for the capacity to partition into hexadecane. Most of the strains more or less partitioned into the organic solvent ( Table 2).…”

Influence of indigenous microbiota on activities of alkaline phosphatase, phosphodiesterase I, and thymidine kinase in mouse enterocytes

“…In that study, the cells were isolated under harsher conditions (i.e., incubation in 5 mM EDTA). To assure ourselves that the results we had obtained reflected the conditions in the intact animal, we did a series of experiments with ex-germfree mice associated with a complex flora to determine the conditions most favorable for stabilizing the cells and for optimizing the assay systems for the enzymes of interest (26). The refined techniques, which are effective for both germfree and associated mice, were briefly described in an earlier publication (25).…”

“…Intestinal cell preparation. Intestinal cell suspensions were prepared as previously described (18,25,26). A 3-cm (-200 mg) segment of small intestine extending distally from the pylorus was removed from each mouse, everted on a glass rod, rinsed with cold 0.9% NaCl, blotted, and placed in 4 ml of solution A (1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, 8 mM KH2PO4, 5.6 mM Na2HPO4, pH 7.3 [22]) for 30 min at 37°C.…”

“…Whole cells were used in all assays. The activities of alkaline phosphatase, phosphodiesterase, and thymidine kinase and the protein content were assayed as previously described (25,26).…”

Effect of clotrimazole on microsomal metabolism and pharmacokinetics of albendazole

“…The bioconversion of albendazole was calculated by measuring albendazole sulfoxide and albendazole sulfone formation through the amount of sulfoxide and sulfone produced by microsomes. The method used to obtain enterocytes was that described by Weiser (1973) and optimized by Whitt & Savage (1988) and Villaverde et al (1995). Cells from the upper and midvillus regions were used as a source of intestinal material.…”