A CAFFEINE-SENSITIVE AND RYANODINE-SENSITIVE INTRACELLULAR CA2+ STORECAN ACT AS A CA2+ SOURCE AND A CA2+ SINK IN PC12 CELLS

We have investigated the modulation of stimulus-induced changes in int racellular Ca2+ concentration ([Ca2+](i)) by a caffeine- and ryanodine -sensitive Ca2+ store in PC12 cells. In populations of fura-2-loaded c ells, caffeine caused a concentration-dependent increase in [Ca2+](i) that was saturable, reversible and inhibited in a use-dependent fashio n by ryanodine. Maximal Ca2+ release occurred with 40 mM caffeine, wit h an EC(50) of 13 mM caffeine and a Hill coefficient (h) of 2.7, indic ating that the release mechanism was co-operative. Pretreatment of int act cell populations with increasing concentrations of caffeine in nom inally Ca2+-free medium inhibited the subsequent Ca2+ response to a ma ximal concentration of ATP, in a dose-dependent manner. In permeabiliz ed cells, a maximal concentration (40 mu M) of InsP(3) still released Ca2+ in the presence of a supramaximal concentration (50 mM) of caffei ne, whereas caffeine was unable to release Ca2+ after the InsP(3)-sens itive store had been completely emptied. These data suggest that PC12 cells contain a uniquely InsP(3)-sensitive Ca2+ store, and a store tha t is sensitive to both InsP(3) 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+, sugge sting that the caffeine-sensitive Ca2+ store acts as a Ca2+ source aft er ATP stimulation, but not after depolarization with 55 mM K+. Pretre atment of intact cells with ATP and ryanodine resulted in a use-depend ent block of both caffeine- and ATP-mediated Ca2+ release, confirming that ATP stimulation of PC12 cells brings about activation of ryanodin e receptors. The rate of recovery, but not the magnitude or rate of on set, of the depolarization-induced [Ca2+](i) transient was modulated b y the state of filling of the caffeine-sensitive Ca2+ store such that recovery was prolonged if the store was either full, or empty and unab le to refill. We conclude that the caffeine- and ryanodine-sensitive C a2+ 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.