Ryanodine-, IP₃- and NAADP-dependent calcium stores control acetylcholine release

Abstract

Injections of inositol trisphosphate (IP₃) or nicotinamide adenine dinucleotide phosphate (NAADP) into the presynaptic neurone of an identified cholinergic synapse in the buccal ganglion of Aplysia californica increased the amplitude of the inhibitory postsynaptic current evoked by a presynaptic action potential. This suggests that Ca²⁺ release from various Ca²⁺ stores can modulate acetylcholine (ACh) release. Specific blockade of the calcium-induced calcium release (CICR) mechanism with ryanodine, or of IP₃-induced calcium release with heparin, abolished the effects of IP₃, but not the effects of NAADP, suggesting the presence of an intracellular Ca²⁺ pool independent of those containing ryanodine receptors (RyR) or IP₃ receptors. To reinforce electrophysiological observations, intracellular [Ca²⁺]_i changes were measured using the fluorescent dye rhod-2. Injections of cyclic ADP-ribose (an activator of RyR), IP3 or NAADP into the presynaptic neurone induced transient increases in the free intracellular Ca²⁺ concentration. RyR- and IP₃-induced increases were prevented by application of respective selective antagonists but not NAADP-induced increases. Our results show that RyR-dependent, IP₃-dependent, and NAADP-dependent Ca²⁺ stores are present in the same presynaptic terminal but are differently involved in the regulation of the presynaptic Ca²⁺ concentration that triggers transmitter release.