Functional ryanodine receptors in the membranes of neurohypophysial secretory granules

Highly localized Ca²⁺ release events have been characterized in several neuronal preparations. In mouse neurohypophysial terminals (NHTs), such events, called Ca²⁺ syntillas, appear to emanate from a ryanodine-sensitive intracellular Ca²⁺ pool. Traditional sources of intracellular Ca²⁺ appear to be lacking in NHTs. Thus, we have tested the hypothesis that large dense core vesicles (LDCVs), which contain a substantial amount of calcium, represent the source of these syntillas. Here, using fluorescence immunolabeling and immunogold-labeled electron micrographs of NHTs, we show that type 2 ryanodine receptors (RyRs) are localized specifically to LDCVs. Furthermore, a large conductance nonspecific cation channel, which was identified previously in the vesicle membrane and has biophysical properties similar to that of an RyR, is pharmacologically affected in a manner characteristic of an RyR: it is activated in the presence of the RyR agonist ryanodine (at low concentrations) and blocked by the RyR antagonist ruthenium red. Additionally, neuropeptide release experiments show that these same RyR agonists and antagonists modulate Ca²⁺-elicited neuropeptide release from permeabilized NHTs. Furthermore, amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Collectively, our findings suggest that RyR-dependent syntillas could represent mobilization of Ca²⁺ from vesicular stores. Such localized vesicular Ca²⁺ release events at the precise location of exocytosis could provide a Ca²⁺ amplification mechanism capable of modulating neuropeptide release physiologically.