The characterization and regional distribution of neuromedin N-like immunoreactivity in rat brain using a highly sensitive and specific radioimmunoassay. Comparison with the distribution of neurotensin

Abstract

Neuromedin N is a hexapeptide that shares a 4 amino acid homology with the C-terminus of neurotensin and exhibits neurotensin-like effects in the central nervous system. Both peptides were recently shown to be encoded in the same precursor molecule. In this study, a radioimmunoassay for neuromedin N was developed using monoiodo [ 125I-Tyr 4]neuromedin N as the tracer and a rabbit antiserum raised against synthetic [Cys 6]neuromedin N coupled to ovalbumin through its Cys residue. The antiserum showed strong structural requirement for the N-terminal sequence of neuromedin N and did not cross-react with neurotensin and other related peptides. The limit of detection of the radioimmunoassay was 0.5 fmol/tube and the IC 50 was 5 fmol/tube. Neuromedin N-like immunoreactivity was present in 0.1 N HCl extracts of rat brain at a concentration of 9.3 ± 1.3pmol/g of tissue and behaved like synthetic neuromedin N on HPLC. Its concentration was significantly lower than that of neurotensin assayed in the same extracts (15.1 ± 1.4pmol/g, and this was not the consequence of lower extraction yield or lower post-mortem stability of neuromedin N as compared to neurotensin. The regional rat brain distribution of neuromedin N-like immunoreactivity paralleled that of neurotensin-like immunoreactivity, being highest in the hypothalamus and lowest in the cerebellum. These data support the proposal of a neuromodulator role for neuromedin N. The highly specific and sensitive radioimmunoassay described here will make it possible to investigate in more detail the regional brain distribution of neuromedin N and to study its release from brain tissues.