Effects of thiorphan, bestatin and a novel metallopeptidase inhibitor JMV 390-1 on the recovery of neurotensin and neuromedin N released from mouse hypothalamus.

The effects of the endopeptidase 24.11 ('enkephalinase') inhibitor thiorphan, the aminopeptidase inhibitor bestatin and a novel metallopeptidase inhibitor JMV 390-1 on the K(+)-evoked release of immunoreactive neurotensin and neuromedin N (iNT and iNN) from mouse hypothalamic slices were examined. (JMV 390-1 inhibits several metallopeptidases including endopeptidases 24.11, 24.15 and 24.16, and aminopeptidase N equipotently with Ki values around 50 nM.) Thiorphan increased the recovery of released iNT nearly 2-fold and had no effect on iNN. Bestatin produced a 4-fold increase in iNN recovery and was inactive on iNT. Finally, iNT and iNN recoveries were increased up to 4- and 5-fold, respectively, by JMV 390-1. These results show that in the mouse hypothalamus endopeptidase 24.11 participates with other metalloendopeptidases to the degradation of endogenously released NT while endogenously released NN is principally degraded by aminopeptidase(s).

Calcium-dependent release of neuromedin N and neurotensin from mouse hypothalamus.

Neuromedin N (NN), a hexapeptide, was isolated from porcine spinal cord. Its C-terminal tetrapeptide sequence is identical to that of neurotensin (NT) and it exhibits NT-like effects when injected in the central nervous system. Both peptides were recently shown to be encoded in the same precursor molecule. We have just developed a sensitive and specific radioimmunoassay (RIA) for NN and showed that the peptide central nervous system distribution paralleled that of NT, the highest concentrations being found in the hypothalamus. Using this assay and a specific RIA for NT, we show here that NN and NT were simultaneously released from slices of mouse hypothalamus by K(+)-induced depolarization in a Ca(++)-dependent manner. The ratio of released NN over NT was 0.3 and was identical to the ratio of endogenous NN over NT. For both NN and NT, the releasable peptide pool represented 2% of the endogenous peptide pool. HPLC characterization of the releasable and endogenous immunoreactive material reacting with the NN and NT antisera showed that it coeluted with synthetic NN and NT, respectively. The present data further support the hypothesis that NN acts as a neuromodulator in the central nervous system.