Effects of Pb 2+ and Cd 2+ on acetylcholine release and Ca 2+ movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

Abstract

In this work we examined the effects of Pb 2+ and Cd 2+ on (a) [ 3H]ACh release and voltage-sensitive Ca 2+ channels in rat brain synaptosomes, and (b) 45Ca 2+ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb 2+ ( K i ≈ 1.1 μM) and Cd 2+ ( K i ≈ 2.2) competitively block the K +-evoked influx of 45Ca 2+ through the ‘fast’ calcium channels in synaptosomes. The K is obtained with synaptosomes are in good agreement with the K i values obtained from electrophysiological experiments at the frog neuromuscular junction ( K Pb:0.99 μM, K Cd: 1.7 μM) 7. The K i for the inhibition of ACh release from synaptosomes by Cd 2+ is 4.5 μM. Pb 2+ is a less effective inhibitor of transmitter release ( K i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd 2+ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb 2+ on spontaneous release is (a) not abolished by omission of Ca 2+ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb 2+ exposure, (c) is reversed upon the removal of Pb 2+. In the presence of physiological concentrations of ATP (1 mM), Mg 2+ (1 mM) and P i (2 mM), 1–10 μM Pb 2+ inhibits calcium uptake but Pb 2+ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb 2+-induced enhancement of P i uptake. Cd 2+ inhibits Ca 2+ binding at all concentrations tested (1–50 μM) and reduces the Pb 2+-induced Ca 2+-binding to organelles. Neither Pb 2+ nor Cd 2+ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with 45Ca. In summary, these data are consistent with the notion that Pb 2+ and Cd 2+ are potent blockers of presynaptic voltage-sensitive Ca 2+ channels and the evoked release of transmitter which is contingent on Ca 2+ influx through these channels. Our results are not consistent with the hypothesis that Pb 2+ augments spontaneous release by interfering with intraterminal Ca 2+-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.