Characterization, by Size, Density, Osmotic Fragility, and Immunoaffinity, of Acetylcholine‐ and Vasoactive Intestinal Polypeptide‐Containing Storage Particles from Myenteric Neurones of the Guinea‐Pig

Abstract: When cytoplasmic extracts of guinea‐pig myenteric neurones are submitted to centrifugal density gradient fractionation in a zonal rotor acetylcholine is bimodally distributed in the gradient, in a peak (I) rich in synaptic vesicles of the classic type and in a denser peak (II/VI) rich in densecored vesicles and vasoactive intestinal polypeptide (VIP). The putative stable synaptic vesicle markers synaptophysin (p38), vesicular proteoglycan, and Mg²⁺‐activated ATPase were also bimodally distributed, with a peak coincident with peak I and another, broader peak embracing peak II/VI, and neighbouring peaks of other neuropeptides resolved from peak II/VI by the density gradient separation procedure used. To establish whether the stable markers, acetylcholine and VIP in peak II/VI were present in one particle or several, attempts were made to separate them by particle‐exclusion chromatography and differential osmotic fragility. These were unsuccessful, leading us to conclude that the storage particles in peak II/VI contain both neurotransmitters and all three putative stable synaptic vesicle markers. It is suggested that such particles are the counterparts, in cholinergic neurones of the myenteric plexus, of the dense‐cored, enkephalin‐ and noradrenaline‐containing vesicles of certain adrenergic neurones and, like the latter, may exist in a precursor–product relationship with the classic synaptic vesicles containing the small‐molecular‐mass transmitters and found in the same nerve terminals.