Primary colonic epithelial cell culture of the rabbit producing prostaglandins.
We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E2 production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35% collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10% fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE2 and PGF2 alpha were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF1 alpha, thromboxane B2, PGF2 alpha, PGE2, and PGD2 by cultured cells. Quantitatively, 117 +/- 7 ng/mg-protein/hour PGE2 and 7.4 +/- 0.7 ng/mg-protein/hour PGF2 alpha were produced. While hydrocortisone (10(-4) - 10(-2) M) did not show a significant effect on PGE2 production, indomethacin (10(-8) - 10(-6) M), and 5-aminosalicylic acid (2X10(-4)-5X10(-3) M) inhibited PGE2 production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE2 synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.
Hata
Y
Y
2nd Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Ota
S
S
Nagata
T
T
Uehara
Y
Y
Terano
A
A
Sugimoto
T
T
D015121:6-Ketoprostaglandin F1 alpha / Q000096:biosynthesis; D000636:Aminosalicylic Acids / Q000494:pharmacology; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015237:Dinoprost / Q000096:biosynthesis; D015232:Dinoprostone / Q000096:biosynthesis; D004848:Epithelium / Q000378:metabolism; D005260:Female; D007213:Indomethacin / Q000494:pharmacology; D008297:Male; D019804:Mesalamine; D015230:Prostaglandin D2 / Q000096:biosynthesis; D011453:Prostaglandins / Q000096:biosynthesis*; D011817:Rabbits; D013929:Thromboxane B2 / Q000096:biosynthesis
D000636:Aminosalicylic Acids; D000894:Anti-Inflammatory Agents, Non-Steroidal; D011453:Prostaglandins; D019804:Mesalamine; D013929:Thromboxane B2; D015121:6-Ketoprostaglandin F1 alpha; D015237:Dinoprost; D015232:Dinoprostone; D015230:Prostaglandin D2; D007213:Indomethacin