A study on thapsigargin-induced calcium ion and cation influx pathways in vascular endothelial cells.
To investigate Ca2+/cation entry pathway in vascular endothelial cells, we examined the effects of thapsigargin on [Ca2+]i and Mn2+ entry in cultured porcine aortic endothelial cells. Thapsigargin inhibits the activity of endoplasmic reticulum (ER, intracellular Ca2+ pool) Ca(2+)-ATPase, and stimulates Ca2+ entry from extracellular space by depleting intracellular Ca2+ pool. Cultured endothelial cells were loaded with fura-2/AM, and [Ca2+]i was measured by the ratios of fluorescence at 340/380 nm excitation, and Mn2+ entry was observed by the quenching of fluorescence at 360 nm excitation. Thapsigargin elevated [Ca2+]i in a time- and dose-dependent manner. The increase in [Ca2+]i caused by thapsigargin was lowered in Ca(2+)-free solution containing 3 mM EGTA. Verapamil (10(-5) M) and equimolar replacement of extracellular NaCl by LiCl had no effects on the maximum elevation of [Ca2+]i by thapsigargin. The increase in [Ca2+]i by thapsigargin was significantly inhibited by either NiCl2 (10(-3) M) or membrane depolarization using 50 mM KCl. Thapsigargin stimulated Mn2+ entry concomitantly with the increase in [Ca2+]i. Mn2+ entry was augmented in Ca(2+)-free solution. These results suggested that (1) the increase in [Ca2+]i by thapsigargin consisted of both Ca2+ release from ER and Ca2+ entry from extracellular space, and (2) thapsigargin also stimulated Mn2+ entry, which was interfered with in the presence of extracellular Ca2+.
Yamamoto
N
N
Department of Internal Medicine III, Hamamatsu University School of Medicine, Japan.
Watanabe
H
H
Kakizawa
H
H
Hirano
M
M
Kobayashi
A
A
Ohno
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D009532:Nickel / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013552:Swine; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014700:Verapamil / Q000494:pharmacology
D015220:Calcium Channels; D013729:Terpenes; D008345:Manganese; D012965:Sodium Chloride; D019284:Thapsigargin; D009532:Nickel; D014700:Verapamil; D002118:Calcium