Caffeine mediates cation influx and intracellular Ca 2+ release in leech P neurones

Abstract

We investigated the effect of caffeine on the intracellular free Ca 2+ concentration ([Ca 2+] i) of leech P neurones by using the fluorescent indicator Fura-2. Caffeine induced a [Ca 2+] i increase that was strongly reduced, but not abolished, in Ca 2+-free solution. The effect of caffeine on [Ca 2+] i was dose-dependent: while 5 mM caffeine evoked a persistent [Ca 2+] i increase that could be elicited repetitively, 10 mM caffeine or more induced a transient [Ca 2+] i increase that was strongly reduced upon subsequent applications at the same concentration. Surprisingly, the cells remained fully responsive to a moderately increased caffeine concentration. The caffeine-induced [Ca 2+] i increase was not blocked by millimolar concentrations of La 3+, Mg 2+, Cd 2+, Zn 2+, Co 2+, Ni 2+, or Mn 2+. While La 3+ and Mg 2+ had no effect on the caffeine response, the other cations caused irreversible changes in the Fura-2 fluorescence. The inhibitors of intracellular Ca 2+ pumps-thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-( t-butyl)-1,4-hydroquinone (BHQ)-had no effect on the caffeine-induced [Ca 2+] i increase at normal extracellular Ca 2+ concentration, but they reduced it in Ca 2+-free solution. Ryanodine had no effect on the caffeine-induced [Ca 2+] i increase at normal extracellular Ca 2+ concentration, and also in Ca 2+-free solution it seemed to be largely ineffective. Caffeine evoked complex fluctuations of the membrane potential. The effect in Ca 2+-free and in Na +-free solution suggests that the depolarizing response components were mainly due to Na + influx, while Ca 2+ reduced the Na + influx and/or activated mechanisms which re- or hyperpolarize the cells. It is concluded that leech P neurones possess caffeine-sensitive intracellular Ca 2+ 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 cations including Ca 2+, and possibly also to Na +.