Quantitative HPLC analysis of rat neurophysin processing

Neuropeptides exist in multiple bioactive forms and it is important to understand quantitative aspects of variations in the metabolism of the different forms. Towards this end a sensitive HPLC procedure, utilizing on-line reaction with fluorescamine, was developed for the quantitative analysis of the major neural lobe peptides of male Wistar rats. Peptide peaks were defined by amino acid analysis, carboxypeptidase digestion, gel electrophoresis and, in the case of the glycopeptide, gel filtration and Edman degradation. Control experiments were done to show that in vivo amounts of all of the peptides were being measured. Quantitation of the extent of conversion of the oxytocin neurophysin to the form missing the carboxyl-terminal glutamate residue provided an indirect measure of secretory granule age in (1) single neural lobes from animals of different ages; (2) single neural lobes from animals at various stages of dehydration and rehydration; (3) single neural lobes from animals subjected to chronic osmotic stress by providing 1.5% NaCl as drinking water; (4) high potassium perfusates from pooled isolated neural lobes; (5) isolated secretory granules of varying densities. Relative to homogenates of control neural lobes, the extent of conversion of the oxytocin neurophysin is increased in neural lobes from animals of larger size, in secretory granules of greater than the mean density, and in the neural lobes taken from animals during the early stage of dehydration. The extent of conversion is decreased in neural lobes from animals of smaller sizes, in the later stages of dehydration, after a chronic osmotic stress and in secretory granules of greater than mean density. No large change relative to control homogenates was seen in the high potassium perfusates. We argue from the data that the peptide content of axon terminals (putative release sites) and swellings (putative storage sites) is relatively homogeneous and that rates of secretory granule transfer between axonal compartments are, therefore, fast relative to rates of secretory granule turnover. Granule movements between release and storage sites will be of fundamental importance in determining the types of changes in secreted peptide content which will occur with changes in secretory demand.