The use of radioimmunoassay to compare the tissue and subcellular distributions of neurotensin and neuromedin N in the cat.

A RIA for neuromedin N (NMN) was developed using 125I-labeled NMN and rabbit antisera raised against a synthetic NMN coupled to NH2-enriched putrescine-thyroglobulin. One of the antisera (TG-B), which cross-reacted less than 3% with the related substances, neurotensin (NT), Lys8,Asn9-NT8-13, and xenopsin, was used to compare the distribution of immunoreactive NMN (iNMN) in the cat to that of immunoreactive NT (iNT). Although both iNMN and iNT were found primarily in the mucosa of the ileum, brain, pituitary, and adrenal, the ratio of the two activities varied, depending upon the tissue. Feline iNMN from brain, intestine, and adrenal was indistinguishable from synthetic NMN by HPLC. In contrast, extracts of stomach and pancreas contained primarily NMN-related cross-reacting substances, the concentrations of which increased up to 500-fold within minutes of extraction in aqueous acid. Since these peptides were also present to some extent in acid/acetone extracts, they appear to be endogenous variants of NMN stored in precursor forms which could be activated by acid-proteases. During sucrose gradient centrifugation of isotonic homogenates of feline brain, intestine, and adrenal, iNMN and iNT comigrated in association with synaptosome-like and vesicle-like particles, as identified by electron microscopy. These results demonstrate the presence of a peptide similar to porcine NMN in extracts of feline brain, adrenal, and small intestine and its association with signaling vesicles. In addition, the ability of gastric and pancreatic extracts to generate variants of NMN in large quantity is described.