Maintenance of connexin 32 and 26 expression in primary cultured rat hepatocytes treated with 3-acetylpyridine.
OBJECTIVE
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.


CONCLUSIONS
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.
Higaki
N
N
Department of Pathology, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo, Japan.
Mitaka
T
T
Sato
F
F
Hirata
K
K
Mochizuki
Y
Y
D000818:Animals; D015152:Blotting, Northern; D002450:Cell Communication / Q000502:physiology; D002478:Cells, Cultured; D000072259:Connexin 26; D017630:Connexins / Q000032:analysis* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017629:Gap Junctions / Q000648:ultrastructure; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D007120:Immunochemistry; D008297:Male; D008854:Microscopy, Electron; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011725:Pyridines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D000097002:Gap Junction beta-1 Protein
D000072259:Connexin 26; D017630:Connexins; D004791:Enzyme Inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011725:Pyridines; D012333:RNA, Messenger; D000097002:Gap Junction beta-1 Protein; C000607044:Gjb2 protein, rat; C010835:3-acetylpyridine