Effects of low doses of caffeine on [Ca2+]i in voltage-clamped snail (Helix aspersa) neurones.

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1. We have measured cytosolic free Ca2+ concentrations ([Ca2+]i) in voltage-clamped snail neurones using fura-2. Transient increases in [Ca2+]i were induced by depolarizing voltage steps of 20-60 mV for 0.1-10 s from a holding potential of -50 or -60 mV. 2. Low doses of caffeine, 0.2-1 mM, increased the size of the [Ca2+]i transients by both increasing the peak and producing an undershoot. 3. Ryanodine, an inhibitor of Ca2+ release from the intracellular Ca2+ stores, and cyclopiazonic acid (CPA), an inhibitor of the Ca(2+)-ATPase of the intracellular Ca2+ stores, both reduced the size of the [Ca2+]i transients and blocked the effects of caffeine on the transients. 4. The effects of caffeine and CPA were greater on transients produced by long, small, rather than short, large depolarizations. This suggests that calcium-induced calcium release (CICR) played a greater role in the [Ca2+]i increase resulting from longer, smaller depolarizations. 5. Increasing the extracellular pH from 7.5 to over 9, which inhibits the plasmalemmal Ca(2+)-H(+)-ATPase, increased the resting [Ca2+]i level. Depolarization-induced [Ca2+]i transients became much larger while the two effects of caffeine remained. CPA was ineffective at high pH. 6. In some experiments the increase in basal [Ca2+]i caused by alkaline pH was reduced by 0.2 or 0.5 mM caffeine. The increase in basal [Ca2+]i caused by maintained depolarization was reduced, after a transient increase, by 0.5 mM caffeine. Both reduction and increase were blocked by CPA. 7. We conclude that low doses of caffeine can increase uptake by intracellular Ca2+ stores. Caffeine could also release Ca2+ from ryanodine-insensitive Ca(2+)-ATPase-dependent stores as well as facilitating normal ryanodine-sensitive CICR.

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