First and second messenger role of calcium. Survival versus apoptosis in serum-free cultured granulosa explants.
In order to elucidate the causal relationship between increased intracellular free calcium ([Ca2+]i) and induction of apoptosis, serum-free cultured granulosa cell (GC) explants were subjected to various experimental protocols interfering with normal Ca2+ homeostasis. Modulation of apoptotic indices was calculated on DAPI-stained GC explants. In some experiments [Ca2+]i was measured with the Ca2+ probe fura-2 in combination with epifluorescence microscopy. Buffering of [Ca2+]i with BAPTA-AM resulted in inhibition of apoptosis, while increasing extracellular Ca2+ (otherwise called [Ca2+]e load) resulted in a biphasic response characterized by an initial inhibitory effect on apoptosis followed by a delayed phase of increased apoptosis that became apparent at 4 h after withdrawal of the [Ca2+]e load. The initial inhibitory effect of the [Ca2+]e load on apoptosis was dependent on the concentration of the load (range 2-50 mM), was augmented when the [Ca2+]e load was applied in the presence of the Ca2+ channel blocker methoxyverapamil, and was mimicked by applying Mg2+ and Gd3+, two Ca(2+)-receptor agonists. These observations point towards the involvement of an extracellular Ca(2+)-sensing receptor (CaR). Measurements of [Ca2+]i showed that the ion was increased just after [Ca2+]e load, followed by recovery that was complete at 2 h after the load. Collectively these data suggest that a [Ca2+]e load initiates apoptosis, becoming manifest 4 h later, by the provoked [Ca2+]i increase, and this effect is preceded by an apoptosis-inhibiting phase presumably involving CaR activation. We conclude that Ca2+ may act as a first (extracellular) messenger promoting cell survival and as a second (intracellular) messenger activating the cell death pathway.
Mussche
S
S
Department of Anatomy, Embryology, Histology and Medical Physics Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium. s.mussche@rug.ac.be
Leybaert
L
L
D'Herde
K
K
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000378:metabolism; D003370:Coturnix; D016895:Culture Media, Serum-Free; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D005711:Gallopamil / Q000494:pharmacology; D006062:Gonadotropins / Q000378:metabolism; D006107:Granulosa Cells / Q000187:drug effects / Q000502:physiology*; D066298:In Vitro Techniques; D007211:Indoles / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006062:Gonadotropins; D007211:Indoles; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D005711:Gallopamil; C007293:DAPI; D004533:Egtazic Acid; D019284:Thapsigargin; D002118:Calcium