OPC-12759 increases proliferation of cultured rat conjunctival goblet cells.
OBJECTIVE
To determine if the gastroprotective drug OPC-12759 increased proliferation of rat conjunctival goblet cells in culture.


METHODS
Cultured goblet cells were incubated with 10(-12) to 10(-8) M OPC-12759 for 1 to 7 days. Fetal bovine serum (FBS) was used as a positive control. Cell proliferation was determined by a MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and by immunohistochemical staining with anti-Ki-67, a marker of cell division. Goblet cells were identified by double-labeling with anti-Ki-67, a marker of cell division, and Ulex europaeus agglutinin I lectin, anti-MUC5AC and anticytokeratin 7. Stratified squamous cells were identified by using Griffonia (Bandeiraea) simplicifolia lectin and anticytokeratin 4 antibody.


RESULTS
As determined by MTT conversion to formazan, OPC-12579 at 10(-11) M induced an almost 2-fold increase in goblet cell proliferation on Days 1 and 3 of incubation but not on Days 5 and 7. The FBS at 10% increased cell proliferation by 2- to 3-fold at each time point. Daily replenishment of OPC-12579 for 3 consecutive days induced cell proliferation at all concentrations. Proliferation as determined by the number of Ki-67 positive cells increased by 4- and 3-fold at Days 1 and 3, respectively with addition of 10(-11) M OPC-12579. The FBS at 10% induced a 10-fold increase in goblet cell proliferation on Days 1, 3, and 5. Colocalization of Ulex europaeus agglutinin I, MUC5AC and anticytokeratin 7 with Ki-67 indicated that proliferating cells were goblet cells. Proliferating cells were negative for the nongoblet cell markers Bandeiraea lectin and anticytokeratin 4.


CONCLUSIONS
The OPC-12759 stimulates proliferation of conjunctival goblet cells in primary culture.
Ríos
José D
JD
Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114-2500, USA. rios@vision.eri.harvard.edu
Shatos
Marie
M
Urashima
Hiroki
H
Tran
Hao
H
Dartt
Darlene A
DA
D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000897:Anti-Ulcer Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004396:Coloring Agents; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D020397:Goblet Cells / Q000166:cytology* / Q000378:metabolism; D006651:Histocytochemistry; D053552:Keratin-7; D007633:Keratins / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D008297:Male; D055271:Mucin 5AC; D009077:Mucins / Q000378:metabolism; D015363:Quinolones / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013778:Tetrazolium Salts; D013844:Thiazoles
D000897:Anti-Ulcer Agents; D000975:Antioxidants; D004396:Coloring Agents; D053552:Keratin-7; D019394:Ki-67 Antigen; C524680:Muc5ac protein, rat; D055271:Mucin 5AC; D009077:Mucins; D015363:Quinolones; D013778:Tetrazolium Salts; D013844:Thiazoles; D007633:Keratins; C022616:thiazolyl blue; C052785:rebamipide; D000409:Alanine