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We investigated the effect of L-type Ca2+ channel antagonists on the Ca2+ influx through voltage-gated Ca2+ channels in leech Retzius, Leydig, AP, AE, P, and N neurons. The efficacy of the antagonists was quantified by monitoring their effect on the increase in the intracellular free Ca2+ concentration ([Ca2+]i; measured by Fura-2) that was induced by depolarizing the cell membrane by raising the extracellular K+ concentration. This K+-induced [Ca2+]i increase was blocked by the phenylalkylamines verapamil, gallopamil, and devapamil, the benzothiazepine diltiazem, as well as by the 1,4-dihydropyridine nifedipine. The blocking effect of the three phenylalkylamines was similar, being most pronounced in P and N neurons and smaller in Leydig, Retzius, AP, and AE neurons. Contrastingly, diltiazem and nifedipine were similarly effective in the neurons investigated, whereby their efficacy was like that of the phenylalkylamines in Retzius, Leydig, AP, and AE neurons. Depending on cell type and blocking agent, the concentrations necessary to suppress the K+-induced [Ca2+]i increase by 50% were estimated to vary between 5 and 190 microM. At high concentrations, the phenylalkylamines and diltiazem by themselves caused a marked [Ca2+]i increase in Leydig, P, and N neurons, which is probably due to activation of the caffeine-sensitive ion channels present in the plasma membrane of these cells. Together with previous observations, the results indicate a distant relationship of the voltage-gated Ca2+ channels present in many if not all leech neurons to vertebrate L-type Ca2+ channels.

Extracellular 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA) and quinine depolarized the neuropile glial cell membrane and decreased its input resistance. As 4-AP induced the most pronounced effects, we focused on the action of 4-AP and clarified the ionic mechanisms involved. 4-AP did not only block glial K + channels, but also induced Na + and Ca 2+ influx via other than voltage-gated channels. The reversal potential of the 4-AP-induced current was −5 mV. Application of 5 mM Ni 2+ or 0.1 mM d-tubocurarine reduced the 4-AP-induced depolarization and the associated decrease in input resistance. We therefore suggest that 4-AP mediates neuronal acetylcholine release, apparently by a presynaptic mechanism. Activation of glial nicotinic acetylcholine receptors contributes to the depolarization, the decrease in input resistance, and the 4-AP-induced inward current. Furthermore, the 4-AP-induced depolarization activates additional voltage-sensitive K + and Cl − channels and 4-AP-induced Ca 2+ influx could activate Ca 2+ -sensitive K + and Cl − channels. Together these effects compensate and even exceed the 4-AP-mediated reduction in K + conductance. Therefore, the 4-AP-induced depolarization was paralleled by a decreasing input resistance.

We determined the relationships between the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca2+]i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K+ concentration ([K+]o) up to 89 mM caused a persistent increase in [Ca2+]i, which was abolished in Ca(2+)-free solution indicating that it was due to Ca2+ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca2+]i increase ranged between -40 and -25 mV. The different threshold potentials as well as differences in the relationships between [Ca2+]i and EM were partly due to the cell-specific generation of action potentials. In Na(+)-free solution, the action potentials were suppressed and the [Ca2+]i/Em relationships were similar. The K(+)-induced [Ca2+]i increase was inhibited by the polyvalent cations Co2+, Ni2+, Mn2+, Cd2+, and La3+, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K(+)-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca2+ channels with similar properties.