title: A caffeine- and ryanodine-sensitive intracellular Ca2+ store can act as a Ca2+ source and a Ca2+ sink in PC12 cells.
abstract: We have investigated the modulation of stimulus-induced changes in intracellular Ca2+ concentration ([Ca2+]i) by a caffeine-and ryanodine-sensitive Ca2+ store in PC12 cells. In populations of fura-2-loaded cells, caffeine cause a concentration-dependent increase in [Ca2+]i that was saturable, reversible and inhibited in a use-dependent fashion by ryanodine. Maximal Ca2+ release occurred with 40 mM caffeine, with an EC50 of 13 mM caffeine and a Hill coefficient (h) of 2.7, indicating that the release mechanism was co-operative. Pretreatment of intact cell populations with increasing concentrations of caffeine in nominally Ca(2+)-free medium inhibited the subsequent Ca2+ response to a maximal concentration of ATP, in a dose-dependent manner. In permeabilized cells, a maximal concentration (40 microM) of InsP3 still released Ca2+ in the presence of a supramaximal concentration (50 mM) of caffeine, whereas caffeine was unable to release Ca2+ after the InsP3-sensitive store had been completely emptied. These data suggest that PC12 cells contain a uniquely InsP3-sensitive Ca2+ store, and a store that is sensitive to both InsP3 and caffeine. Depletion of the caffeine-sensitive Ca2+ store by caffeine and ryanodine pretreatment in intact cells attenuated the Ca2+ response to ATP, but not to 55 mM K+, suggesting that the caffeine-sensitive Ca2+ store acts as a Ca2+ source after ATP stimulation, but not after depolarization with 55 mM K+. Pretreatment of intact cells with ATP and ryanodine resulted in a use-dependent block of both caffeine- and ATP-mediated Ca2+ release, confirming that ATP stimulation of PC12 cells brings about activation of ryanodine receptors. The rate of recovery, but not the magnitude or rate of onset, of the depolarization-induced [Ca2+]i transient was modulated by the state of filling of the caffeine-sensitive Ca2+ store such that recovery was prolonged if the store was either full, or empty and unable to refill. We conclude that the caffeine- and ryanodine-sensitive Ca2+ store can act as a Ca2+ source and a Ca2+ sink in PC12 cells, and that its role may in part be governed by the nature of the stimulating agent.
authors.affiliation: AFRC Laboratory of Molecular Signalling, Department of Zoology, Cambridge, U.K.
authors.forename: V A
authors.initials: VA
authors.lastname: Barry
authors.forename: T R
authors.initials: TR
authors.lastname: Cheek
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D008564:Membrane Potentials; D009124:Muscle Proteins / Q000187:drug effects / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D012433:Ryanodine / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel; D013806:Theophylline / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D013806:Theophylline; D002118:Calcium