The tetrapeptide phe-met-arg-phe-NH2 (FMRF-NH2) was originally isolated from macrocallista nimboas clam and subsequently existence of FMRF-NH2-like peptides in mammalian CNS was demonstrated by the antiserum raised against FMRF- NH2. Recently, two of these FMRF-NH2-like peptides weer isolated from bovine brain and chemically characterized. In this study, monoclonal antibody against one of these FMRF-NH2-like peptides of mammalian origin, phe-leu-phe-gln-pro-gln-arg-phe- NH2 (F-8-F-NH2), was generated and will be used to study the distribution of F-8-NH2 in detail by immunohistochemical technique. By using F-8-F-NH2 conjugated to hemocyanin as antigen, three murine monoclonal antibodies of IgG isotype were produced through cell fusion and cloning. The specificity study indicated that those antibodies cross-reacted with peptides having arg-phe-NH2 at their c-termini but showed no cross-reaction with NPY, PYY or met5-enk-arg6-phe7. The result indicates that these monoclonal antibodies will enable us to carry out the immunohistochemical localization of F-8-F-NH2 or FMRF-NH2 immunoreactivity and compare this distribution with the distribution of NPY. Some immunohistochemical studies using polyclonal antiserum indicate that it is difficult to distinguish between mammalian FMRF-NH2-like immunoreactivity from NPY due to cross-reactions of antisera. Extracts from spinal cord was analyzed by high pressure liquid chromatography coupled with radioimmunoassay using the monoclonal antibody. The major immunoreactivity was identified as F-8-F-NH2 and NPY was not recognized by the antibody, thus the specificity of the monoclonal antibody was further confirmed. In the periaqueductal gray area, the preliminary study using the monoclonal antibody showed numerous immunoreactive nerve fibers around blood vessels and capillaries. Network of F-8-F-NH2 positive nerve fibers was found surrounding the cerebral aqueduct and running in the epithelium. We plan to extend this immunohistochemical study to other regions of CNS and hope to provide a frame work for further understanding of possible function of F-8-F-NH2 or mammalian FMRF-NH2-like peptides.
