Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells.
Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+ channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni(2+)-, La(3+)-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 mumol l-1 and 2.5 mumol l-1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mumol l-1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mumol l-1 and 95.29 mumol l-1, respectively. The depolarization was significantly inhibited at 50 mumol l-1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.
Tiemann
U
U
Unit of Reproductive Biology, Research Institute for the Biology of Farm Animals, Dummerstorf, Germany.
Neels
P
P
Pöhland
R
R
Walzel
H
H
Löhrke
B
B
D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects*; D002417:Cattle / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005187:Fallopian Tubes / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005439:Flufenamic Acid / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006706:Homeostasis; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D010972:Platelet Activating Factor / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010972:Platelet Activating Factor; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005439:Flufenamic Acid; D005707:Gallic Acid; D002118:Calcium; D016257:Fura-2