B radykinin Receptor-Meadiated Intracellular Calcium Mobilization and ATP Changes in Cultured Bovine Corneal Endothelial Cell

To clarify the effect of bradykinin(Bk)on cultured bovine corneal endothelial cells(BCEC), cytosolic free calcium([Ca2+])mobilization and cell proliferation were investigated. The [Ca2+] was determined using a Ca2+ sensitive indicator, Fura-2/AM, and cell proliferation was evaluated by counting the cell number. Bk induced the transient increase of [Ca2+] in a concentration-dependent manner(10(-11)M~10(-7)M)and its 50% effective concentration was about 5x10(-11)M. The basal [Ca2+] with 1mM CaCl2 in the bathing solution was 87+/-9nM. Transient Bk(10(-8)M)-induced [Ca2+] increase was inhibited slightly but significantly by the pretreatment with EGTA. The pretreatment with U-73122(5x10(-6)M), an inhibitor of phospholipase C, also attenuated Bkinduced [Ca2+] mobilization. To identify and characterize the Bk receptor subtype in BCEC, Bk1 and Bk2 antagonists were investigated. Transient Bk(10(-8)M)-induced [Ca2+] increase was almost absolutely attenuated by the pretreatment with Bk2 antagonist for 10 minutes. To investigate the physiological effect of Bk, Bk-induced mitogenic effect was studied. 10(-8)M of Bk produced significant increase of intracellular ATP levels from the day 2 to the day six of culture period. This Bk-induced mitogenic effect was inhibited by the treatment with Bk2 antagonist. Bk-induced ion transport was determined by measuring intracellular ATP contents. Intracellular ATP content([ATP]i)was decreased by the treatment with 10(-8)M Bk for 10 minutes. Bk-induced [ATP]i decrement was significantly restored by the pretreatment with ouabain for 30 minutes. In summary, stimulation of intracellular signal transduction by Bk in BCEC is coupled with Bk2 type receptor. And also, Bk produces mitogenic effect and enhancesion and fluid transport in BCEC.

Endothelin-mediated Intracellular Calcium Mobilization in Cultured Bovine Corneal Endothelium

To investigate the effect of endothelin on intracellular free calcium ([Ca(2+)]i) mobilization and the existence of the endothelin receptor in cultured bovine corneal endothelial cells(BCEC), endothelin-1(ET-1) -induced [Ca(2+)]i increase was measuredby using calcium sensitive indicator, fura-2/AM, and the studies on its desensitizaton and receptor antagonists were also performed. ET-1 increased [Ca(2+)]i in a concentration-dependent manner(10(-9)M-10(-5)M) and ET-1 -unduced [Ca(2+)]i transient increase was significantly ingibited (~50%) by the pretreatment with EGTA for 1 min. Similarly, neomycin also attenuated ET-1 -unduced [Ca(2+)]i transient increase in a concentration-dependent mannet. Desensitizatin study with successive treatment of ET-1 and ET-3, and the experiments of antagonists(BQ-610 for the ET(A) receptor and BQ-788 for the ET(B) receptor) showed the presence of ET(B) receptor in BCEC. In addition, ET-1(10(-6)M) accumulated inositol trisphosphate about two folds (310+/-6.8 cpm) comparing to control(154+/-11.6 cpm) and this accumulation was significantly inhibited by the treatment with neomycin (187+/-28 cpm). These results suggest that endothelin-induced calcium mobilization is receptor-mediated response and TE(B) receptor exists in BCEC.