Water channel AQP-1 in the primary cell culture of rat peritoneum.
To analyze the regulation of water channels in the peritoneum, we tried to establish a primary mesothelial cell culture system. Male Sprague-Dawley rats weighing about 250 g were anesthetized, and 10 mL of phosphate-buffered saline (PBS) containing 0.25% trypsin and 1 mmol/L ethylenediamine tetraacetic acid (EDTA) was infused into the peritoneal cavity for 15 minutes. Sediments from the recovered fluid were cultured in medium M199 supplemented with 10% fetal bovine serum (FBS). The culture was succeeded 4-6 times before experiments commenced. After exposure to the test medium, RNA was extracted and subjected to reverse transcriptase polymerase chain reaction (RT-PCR) for 10-19 cycles, then was measured by Southern blot analysis with a digoxin-labeled probe. Cultured cells were positively stained with mouse monoclonal anti-cytokeratin antibody, confirming their characteristics as mesothelial cells. Aquaporin-1 (AQP-1) message in the cultured cells increased with increases in glucose and mannitol concentrations when beta-actin message was used as an internal control. Tranexamic acid effected no change in AQP-1 message in the cultured mesothelial cells. This system offers potential as a simple approach to test the effects of osmolytes, cytokines, and vasoactive hormones on aquaporin expression and water transport in the peritoneum.
Akiba
T
T
Department of Blood Purification, Tokyo Medical and Dental University, Japan.
Ota
T
T
Fushimi
K
K
Shimamura
H
H
Tamura
H
H
Sasaki
S
S
Marumo
F
F
D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins / Q000502:physiology*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000502:physiology; D008297:Male; D010537:Peritoneum / Q000166:cytology / Q000502:physiology*; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley
C496022:Aqp1 protein, rat; D020346:Aquaporins; D051398:Aquaporin 1