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T84 epithelial cells respond to 5-hydroxytryptamine when grown in serum-free media

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Abstract

The aim of this study was to establish the cause of insensitivity of T84 human colonic epithelial cells to 5-hydroxytryptamine (5-HT). Monolayers of T84 cells were placed in modified Ussing chambers for measurement of short-circuit current, an index of secretion. When grown in serum-supplemented media, T84 cells gave secretory responses to acetylcholine and forskolin but not to 5-HT. When grown in AIM V serum-free media, T84 cells responded to 5-HT. Chromatographic analysis with fluorimetric detection showed high levels of 5-HT (1.8 μM) in the serum. This contamination is probably responsible for subsequent desensitization of T84 cells to 5-HT.

Introduction

The T84 cell line was originally derived from lung metastases in a patient with a colonic adenocarcinoma. The cells grow as confluent monolayers, retain cellular polarity, form tight junctions, and exhibit directional ion transport when grown in serum containing media. The cells can be grown in serum-free media, but instead of monolayers, gland-like structures were formed which closely resembled the original tumour morphology (Murakami and Masui, 1980). The T84 cell shows a close structural resemblance to the human colonic crypt cell for which it serves as a model for ion transport studies Dharmsathaphorn and Madara, 1990, Dharmsathaphorn et al., 1984. T84 cells have subsequently been shown to respond to a wide variety of neurotransmitters and hormones with a chloride secretory response, and as such, have gained wide acceptance as a general model of intestinal transepithelial chloride transport (Barrett, 1993). However, T84 cells do not respond to 5-hydroxytryptamine (5-HT) (Dharmsathaphorn et al., 1984) despite the fact that 5-HT is an effective intestinal secretagogue (Cooke and Reddix, 1994) which causes non-neural secretory responses in human isolated colonic mucosa (Borman and Burleigh, 1996). A possible explanation may have been that the cells were tested for their sensitivity to 5-HT only 40–56 h after seeding onto the semi-permeable membranes used for the ion transport studies. Alternatively, it was suggested that foetal bovine serum might contain sufficient 5-HT to desensitize T84 cells to the indolealkylamine (Hamilton, personal communication). The purpose of this investigation was to determine whether T84 cells can be made to respond to 5-HT by manipulating the conditions under which they are grown.

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Materials and methods

T84 cells were obtained from the European Collection of Cell Cultures (ECACC) and used between passages 70 and 85. They were cultured in Dulbecco's modified Eagle's medium/Ham's F12 (1:1) supplemented with: N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid (HEPES) (15 mM), NaHCO3 (1.2 g l−1), l-glutamine (2 mM), penicillin (100 units ml−1), streptomycin (0.1 mg.ml−1), and 10% foetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in 95% room air. Cells were initially grown in 75 cm2

Results

When grown in serum-supplemented media, basal short-circuit current was 1.0±0.3 and conductance was 2.3±0.1 mS cm−2 (equivalent to a resistance of 441±17 Ω) after an equilibration period of 30 min (n=54). Basal short-circuit current was not affected by duration of culture whereas a maximum resistance of 538±35 Ω was attained after 7 days (n=12). When cells were grown in the presence of serum, 5-HT (1–100 μM, n=6) had no significant effect on basal short-circuit current (P>0.05, Fig. 1).

Discussion

Although T84 cells serve as a model for ion transport by human colonic mucosa, there are differences in their electrical properties. Firstly, T84 cells have the functional characteristics of intestinal crypt cells, which are mainly secretory in function. The lack of differentiation of T84 cells into absorptive cells is reflected in the virtual absence of a basal short-circuit current. Secondly, in contrast to short-circuit current, the transepithelial resistance developed by monolayers of T84

Acknowledgements

We are grateful to Dr. T. Hamilton for useful discussions.

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