Transmitter release: ruthenium red used to demonstrate a possible role of sialic acid containing substrates.

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Abstract
The possible function of sialic acid-containing substrates (SACS) in synaptic terminals of Aplysia was studied by intracellular injection of ruthenium red and of neuraminidase. Ruthenium red, a dye having sialic acid as a molecular target, blocked transmission irreversibly in cholinergic (buccal ganglion) and non-cholinergic (cerebral ganglion) synapses. An intracellular site of action is likely because much less ruthenium red is necessary to block transmission when it is injected intracellularly than when it is presented by bath perfusion. Ca2+ spikes recorded in the presence of tetrodotoxin or in Na+-free solution were not modified by ruthenium red or neuraminidase injections or perfusions. These substances probably did not block transmission by blocking voltage-dependent Ca2+ influx. Strong electrotonic depolarization of a pre-synaptic interneuron in the presence of 10-4 M tetrodotoxin caused a sustained post-synaptic response, which was abolished by ruthenium red. This result eliminated axonal conduction block as the principal mechanism of ruthenium red action. Post-synaptic responses to ionophoretically applied acetylcholine (ACh) were not modified by bath perfusion of 2 .times. 10-2 M ruthenium red. Biochemical analysis of pools of [3H]ACh was performed after injection of a precursor, [3H]acetate, into an identified interneuron. Ruthenium red apparently increased significantly the free (cytoplasmic) ACh pool without any change of bound (vesicular) [3H]ACh-pool. A model is proposed in which SACS act as intracellular Ca2+ receptors involved in transmitter release.