DEVELOPMENT OF AN ANTISERUM TO THE MIDPORTION OF SUBSTANCE-P - APPLICATIONS FOR BIOCHEMICAL AND ANATOMICAL STUDIES OF SUBSTANCE P-RELATED PEPTIDE SPECIES IN CNS TISSUES

This report describes the generation and biochemical characterization of a high-affinity antiserum that recognizes an epitope contained in the midportion sequence of substance P, i.e., substance P-4-10. Designated A47, this reagent bound a variety of related peptide species containing the substance P-4-10 sequence with apparent equipotency. A double radioimmunoassay procedure was developed 'that utilized A47, in combination with a traditional high-affinity COOH-terminally directed anti-substance P serum. to provide quantification of mature and immature forms of substance P in CNS tissues. Across most rat CNS areas, levels of substance P-like immunoreactivity were consistently 15% higher when monitored by analyses using A47 versus antisubstance P serum. In the dorsal root ganglia, an apparent enhancement in levels of substance P-like immunoreactivity of approximately 40%, when quantified by analyses using A47 versus anti-substance P serum, was observed: this most likely reflected the presence of an active biosynthetic pool of intermediate processing forms of substance P in this tissue. Coordinated HPLC/radioimmunoassay analyses of extracted dorsal root ganglia tissues demonstrated multiple peaks of immunoreactivity corresponding to mature substance P and to several of its precursor forms found in the normal biosynthetic pathway. Of the total recovered HPLC-fractionated immunoreactivities. that corresponding to the putative immediate precursor to substance P, i.e., substance P-glycine. was the predominant peak. In an additional series of HPLC/radioimmunoassay analyses, selective decreases in immunoreactive peaks corresponding to precursor forms of substance P were observed in dorsal root ganglia tissues from rats treated with the neurotoxic agent capsaicin. These results indicated decreased turnover of substance P as a consequence of drug treatment. Finally. initial immunohistochemical analyses employing affinity-purified A47 produced an unusual pattern of labeling characterized by well defined punctate terminal elements within the superficial aspects of the dorsal horn of the spinal cord.