Membrane retrieval in the guinea-pig neurohypophysis. Isolation and characterization of secretory vesicles and coated microvesicles after radiolabel incorporation in vivo.
We have developed small-scale methods for the isolation and biochemical characterization of subcellular fractions from single guinea-pig posterior-pituitary glands. Secretory vesicles and coated microvesicles produced in this way were of similar purity to those isolated from large amounts of tissue by conventional ultracentrifugation. [35S]Cysteine injected into the hypothalamus was found in the soluble contents of secretory vesicles isolated from the neural lobes 24 h later. High-pressure liquid-chromatographic analysis revealed that the radiolabel was incorporated into the expected neurosecretory products (oxytocin, vasopressin and neurophysin) and also into a biosynthetic intermediate in the vasopressin system. The membranes of secretory vesicles were labelled with [3H]choline 24 h after its hypothalamic injection. Little or no [3H]choline could be demonstrated in coated microvesicles at this time, although these structures were labelled 5 days after injection. Stimulating hormone secretion by chronic dehydration produced a significant fall in [3H]choline content of the secretory-vesicle membranes without any transfer of label into coated microvesicles, suggesting that coated microvesicles are not involved in membrane retrieval in the neurohypophysis.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D002794:Choline / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000648:ultrastructure; D003681:Dehydration / Q000378:metabolism; D006168:Guinea Pigs; D008565:Membrane Proteins / Q000378:metabolism; D010904:Pituitary Gland, Posterior / Q000378:metabolism / Q000648:ultrastructure*; D013347:Subcellular Fractions / Q000378:metabolism
D008565:Membrane Proteins; D003545:Cysteine; D002794:Choline