Type III CD38 is present in the membrane of neurosecretory vesicles and has a cytosol-facing catalytic domain in primate oxytocin neurons

CD38, an ADP-ribosyl cyclase that generates cyclic ADP-ribose (cADPR), is essential for Ca2+-dependent oxytocin release. However, its subcellular localisation and membrane topology within oxytocin neurones have remained unclear. We investigated the distribution and orientation of CD38 in oxytocin-producing neurones of Japanese macaques (Macaca fuscata) using immunoelectron microscopy combined with biochemical isolation of neurosecretory vesicles (NSVs). CD38 immunoreactivity was selectively detected on oxytocin-containing NSVs in axon terminals in the posterior pituitary and dendrites of the supraoptic nucleus, whereas vasopressin vesicles and the plasma membrane lacked detectable labelling. Cryo-electron microscopy confirmed the structural integrity of purified NSV fractions, and Western blotting verified the presence of CD38 protein within these fractions. Permeabilisation-dependent immunogold labelling further demonstrated that the NSV membrane localisation of CD38 and that the N-terminal region of CD38 is oriented toward the vesicle lumen, consistent with a type III membrane topology in which the catalytic domain faces the cytosol. This arrangement positions the active site near cytosolic NAD+, enabling localised cADPR production adjacent to Ca2+-mobilising channels that support regulated exocytosis. These findings identify, in primate oxytocin neurones, a previously unrecognised, vesicle-associated pool of CD38 with a cytosol-facing catalytic domain and provide a structural framework for understanding how intracellular type III CD38 contributes to neuropeptide release.