Isolation of neuropeptide-containing vesicles from the guinea pig ileum

Three distinct vesicle fractions enriched 40-60 times in the neuropeptides substance P, somatostatin, and vasoactive intestinal peptide (VIP) were prepared from the myenteric plexus of guinea pig ileum by density gradient centrifugation in a small zonal rotor. Mean densities (in g X ml-1) and diameters (in nm) of the three classes of vesicles were: substance P, 1.123, 65; somatostatin, 1.138, 37; VIP, 1.148, 110; standard deviations were about 5%. These peaks were distinct from the peak of acetylcholine-containing vesicles of density 1.066 g X ml-1 and diameter 61 nm. When a relatively mild method of homogenization was used a second peak of acetylcholine appeared in the same region of the gradient as VIP and the VIP was larger. This may represent a class of vesicles containing both acetylcholine and VIP, though cosedimentation of two classes of vesicles of almost the same density and similar fragility, one containing VIP and the other acetylcholine, cannot be entirely excluded on present evidence.