Characterization of prostaglandin E2-induced Ca2+ mobilization in single bovine adrenal chromaffin cells by digital image microscopy.
We recently reported that prostaglandin E2 (PGE2) stimulates phosphoinositide metabolism accompanied by an increase in intracellular free Ca2+ concentration ([Ca2+]i) in cultured bovine adrenal chromaffin cells. In the present study, temporal and spatial changes in [Ca2+]i induced by PGE2 in fura-2-loaded individual cells were investigated by digital image microscopy and were compared with those induced by nicotine and histamine. Image analysis of single cells revealed that responses to PGE2 showed asynchrony with the onset of [Ca2+]i changes. After a lag time of 10-30 s, PGE2-induced [Ca2+]i changes took a similar prolonged time course in almost all cells: a rapid rise followed by a slower decline to the basal level over 5 min. Few cells exhibited oscillations in [Ca2+]i. In contrast, nicotine and histamine induced rapid and transient [Ca2+]i changes, and these [Ca2+]i changes were characteristic of each stimulant. Whereas pretreatment of the cells with pertussis toxin (100 ng/ml, 6 h) did not block the response to any of these stimulants, treatment with 12-O-tetradecanoylphorbol 13-acetate (100 nM, 10 min) completely abolished [Ca2+]i changes elicited by PGE2 and histamine. In a Ca2(+)-free medium containing 3 mM EGTA, or in medium to which La3+ was added, the [Ca2+]i response to nicotine disappeared, but that to histamine was not affected significantly. Under the same conditions, the percentage of the cells that responded to PGE2 was reduced to 37% and the prolonged [Ca2+]i changes induced by PGE2 became transient in responding cells, suggesting that the maintained [Ca2+]i increase seen in normal medium is the result of a PGE2-stimulated entry of extracellular Ca2+. Whereas the organic Ca2(+)-channel blocker nicardipine inhibited [Ca2+]i changes by all stimulants at 10 microM, these [Ca2+]i changes were not affected by any of the organic Ca2(+)-channel blockers, i.e., verapamil, diltiazem, nifedipine, and nicardipine, at 1 microM, a concentration high enough to inhibit voltage-sensitive Ca2+ channels. These results demonstrate that PGE2 may promote Ca2+ entry with concomitant release of Ca2+ from intracellular stores and that the mechanism(s) triggered by PGE2 is apparently different from that by histamine or nicotine.
Ito
S
S
Department of Cell Biology, Osaka Bioscience Institute, Japan.
Mochizuki-Oda
N
N
Hori
K
K
Ozaki
K
K
Miyakawa
A
A
Negishi
M
M
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002417:Cattle; D002838:Chromaffin System / Q000187:drug effects / Q000378:metabolism*; D015232:Dinoprostone / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016257:Fura-2; D018738:Hexamethonium; D006584:Hexamethonium Compounds / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009529:Nicardipine / Q000494:pharmacology; D009538:Nicotine / Q000494:pharmacology; D018733:Nicotinic Antagonists; D037342:Pertussis Toxin; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D006584:Hexamethonium Compounds; D018733:Nicotinic Antagonists; D010566:Virulence Factors, Bordetella; D018738:Hexamethonium; D007811:Lanthanum; D009538:Nicotine; D006632:Histamine; D009529:Nicardipine; D037342:Pertussis Toxin; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2