Establishment and long-term maintenance of primary intestinal epithelial cells cultured from the rainbow trout,Oncorhynchus mykiss

DRA (downregulated in adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl− absorption in intestinal epithelial cells. Disturbances in DRA function and expression have been implicated in diarrheal conditions such as congenital chloride diarrhea and inflammatory bowel diseases. Previous studies have shown that DRA is subject to regulation by short-term and transcriptional mechanisms. In this regard, we have recently shown that short-term treatment by lysophosphatidic acid (LPA), an important bioactive phospholipid, stimulates Cl−/HCO3−(OH−) exchange activity via an increase in DRA surface levels in human intestinal epithelial cells. However, the long-term effects of LPA on DRA at the level of gene transcription have not been examined. The present studies were aimed at investigating the effects of LPA on DRA function and expression as well as elucidating the mechanisms underlying its transcriptional regulation. Long-term LPA treatment increased the Cl−/HCO3− exchange activity in Caco-2 cells. LPA treatment (50–100 μM) of Caco-2 cells significantly stimulated DRA mRNA levels and DRA promoter activity (−1183/+114). This increase in DRA promoter activity involved the LPA2 receptor and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Progressive deletions from −1183/+114 to −790/+114 abrogated the stimulatory effects of LPA, indicating that the −1183/−790 promoter region harbors LPA response elements. Utilizing EMSA and mutational studies, our results showed that LPA induced the DRA promoter activity in a c-Fos-dependent manner. LPA also increased the protein expression of c-Fos and c-Jun in Caco-2 cells. Furthermore, overexpression of c-Fos but not c-Jun enhanced the DRA promoter activity. This increase in DRA transcription in response to LPA indicates that LPA may act as an antidiarrheal agent and could be exploited for the treatment of diarrhea associated with inflammatory or infectious diseases of the gut.

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Branchial Morphological and Endocrine Responses of Rainbow Trout (Oncorhynchus mykiss) to a Long-Term Sublethal Acid Exposure In Which Acclimation Did Not Occur

Canadian Journal of Fisheries and Aquatic Sciences ◽

10.1139/f93-022 ◽

1993 ◽

Vol 50 (1) ◽

pp. 198-209 ◽

Cited By ~ 13

Author(s):

Céline Audet ◽

Chris M. Wood

Keyword(s):

Rainbow Trout ◽

Oncorhynchus Mykiss ◽

Chloride Cell ◽

Chloride Cells ◽

Physiological Data ◽

Mucous Cells ◽

Diffusion Distance ◽

Cell Numbers ◽

Rainbow Trout Oncorhynchus Mykiss

Changes in branchial morphology and in plasma Cortisol, adrenaline, and noradrenaline were quantified throughout an 81 -d exposure of rainbow trout (Oncorhynchus mykiss) to sublethal acidity (pH 4.8) in artificial soft water and after a 5-h acid challenge (pH 4.0) of naive fish and 81-d acid-preexposed fish. Changes in branchial morphology at pH 4.8 were generally very mild and characterized by slight increases in filamental mucous cells and decreases in lamellar mucous cells. Chloride cell numbers and branchial Na+–K+- and total ATPase activities did not change. The filamental epithelium thickened, but the water–blood diffusion distance in the lamellae decreased during chronic exposure. Cortisol was significantly elevated throughout whereas catecholamines exhibited relatively little response. Response to acute pH 4.0 challenge was similar in naive and 81-d acid-exposed fish: epithelial damage, increase in visible mucous cells, loss of chloride cells by necrosis, and high cortisol levels but no changes in lamellar or filamental epithelial thickness, diffusion distance, ATPase activities, or catecholamine levels. Previously reported physiological data from these same trout demonstrated that sensitization rather than acclimation had occurred. Therefore, these observations support the view that acclimation does not occur in the absence of significant branchial damage and repair.

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