Maintenance of connexin 32 and 26 expression in primary cultured rat hepatocytes treated with 3-acetylpyridine.
BACKGROUND AND AIM: We recently reported that primary rat hepatocytes treated with 3-acetylpyridine (3-AP), an analog of nicotinic acid, could maintain hepatic differentiated functions such as albumin, tryptophan 2,3-dioxygenase, and connexin 32 (Cx32) mRNA expressions for more than a week. In the present experiment, we investigated the expression of not only Cx32, but also Cx26 in cells treated with 10 mmol/L 3-AP in detail. METHODS: We examined the expression of Cx32 and Cx26 in primary rat hepatocytes by using the methods of immunocytochemistry, immunoelectron microscopy, northern blotting, and dye-transfer. RESULTS: The hepatocytes treated with 3-AP were polygonal with a large cytoplasm from day 3, and were maintained for approximately 2 weeks, whereas the cells without 3-AP began to die from day 4. Immunocytochemically in the cells with 3-AP, many Cx32- and Cx26-positive spots were observed between most adjacent cells, and the intensity of positive spots increased with time in culture, whereas in the cells without 3-AP, Cx32- and Cx26-positive spots disappeared at day 4. Furthermore, most Cx26-positive spots were colocalized with Cx32-positive ones. The amounts of Cx32 and Cx26 mRNA transcripts in the cells with 3-AP at day 14 were more than 80% and approximately 30% of those of Cx32 and Cx26 mRNA transcripts in the cells at day 1, respectively. Gap junctional intercellular communication was maintained in the cells treated with 3-AP at day 8, although it was lost in the cells without 3-AP. CONCLUSION: Thus, the addition of 10 mmol/L 3-AP to the medium enhanced the maintenance of Cx32 and Cx26 expression, which is one of the hepatic differentiated functions, in primary rat hepatocytes for a long time.