CAFFEINE MEDIATES CATION INFLUX AND INTRACELLULAR CA2-NEURONS( RELEASE IN LEECH P)

We investigated the effect of caffeine on the intracellular free Ca2concentration ([Ca2+](i)) of leech P neurones by using the fluorescent indicator Fura-2. Caffeine induced a [Ca2+](i) increase that was stro ngly reduced, but not abolished, in Ca2+-free solution. The effect of caffeine on [Ca2+](i) was dose-dependent: while 5 mM caffeine evoked a persistent [Ca2+](i) increase that could be elicited repetitively, 10 mM caffeine or more induced a transient [Ca2+](i) increase that was s trongly reduced upon subsequent applications at the same concentration . Surprisingly, the cells remained fully responsive to a moderately in creased caffeine concentration. The caffeine-induced [Ca2+](i) increas e was not blocked by millimolar concentrations of La3+, Mg2+, Cd2+, Zn 2+, Co2+, Ni2+, or Mn2+. While La3+ and Mg2+ had no effect on the caff eine response, the other cations caused irreversible changes in the Fu ra-2 fluorescence. The inhibitors of intracellular Ca2+ pumps - thapsi gargin, cyclopiazonic acid (CPA), and 2,5-di-(t-butyl)-1,4-hydroquinon e (BHQ) - had no effect on the caffeine-induced [Ca2+](i) increase at normal extracellular Ca2+ concentration, but they reduced it in Ca2+ f ree solution. Ryanodine had no effect on the caffeine-induced [Ca2+](i ) increase at normal extracellular Ca2+ concentration, and also in Ca2 +-free solution it seemed to be largely ineffective. Caffeine evoked c omplex fluctuations of the membrane potential. The effect in Ca2+-free and in Na+-free solution suggests that the depolarizing response comp onents were mainly due to Na+ influx, while Ca2+ reduced the Na+ influ x and/or activated mechanisms which re-or hyperpolarize the cells. It is concluded that leech P neurones possess caffeine-sensitive intracel lular Ca2+ stores, as well as caffeine-sensitive ion channels, in the plasma membrane that are activated by a voltage-independent mechanism. The plasma membrane channels are permeable to various divalent cation s including Ca2+, and possibly also to Na+.