Mechanisms of bradykinin-mediated Ca(2+) signalling in canine cultured corneal epithelial cells.
Experiments were designed to differentiate the mechanisms of bradykinin receptors mediating the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in canine cultured corneal epithelial cells (CECs). Bradykinin and Lys-bradykinin caused an initial transient peak of [Ca(2+)](i) in a concentration-dependent manner, with half-maximal stimulation (pEC(50)) obtained at 6.9 and 7.1, respectively. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin (CTX) for 24 h did not affect the bradykinin-induced [Ca(2+)](i) changes. Application of Ca(2+) channel blockers, diltiazem and Ni(2+), inhibited the bradykinin-induced Ca(2+) mobilization, indicating that Ca(2+) influx was required for the bradykinin-induced responses. Addition of thapsigargin (TG), which is known to deplete intracellular Ca(2+) stores, transiently increased [Ca(2+)](i) in Ca(2+)-free buffer, and subsequently induced Ca(2+) influx when Ca(2+) was readded to this buffer. Pretreatment of CECs with TG completely abolished bradykinin-induced initial transient [Ca(2+)](i), but had slight effect on bradykinin-induced Ca(2+) influx. Pretreatment of CECs with 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF96365) and 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) inhibited the bradykinin-induced Ca(2+) release and Ca(2+) influx, consistent with the inhibition of receptor-gated Ca(2+) channels and phospholipase C (PLC) in CECs, respectively. These results demonstrate that bradykinin directly stimulates B(2) receptors and subsequently Ca(2+) mobilization via a PTX-insensitive G protein in canine CECs. These results suggest that bradykinin-induced Ca(2+) influx into the cells is not due to depletion of these Ca(2+) stores, as prior depletion of these pools by TG has no effect on the bradykinin-induced Ca(2+) influx that is dependent on extracellular Ca(2+) in CECs.
Huang
S C
SC
Department of Ophthalmology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan, ROC.
Chien
C
C
Hsiao
L
L
Wang
C
C
Chiu
C
C
Liang
K
K
Yang
C
C
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D037342:Pertussis Toxin; D011760:Pyrrolidinones / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D004963:Estrenes; D007093:Imidazoles; D011760:Pyrrolidinones; D010566:Virulence Factors, Bordetella; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D019284:Thapsigargin; D002772:Cholera Toxin; D037342:Pertussis Toxin; D010738:Type C Phospholipases; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D001920:Bradykinin; D002118:Calcium