The undecapeptide substance P (SP) is found in high concentrations in brain and spinal cord, and has been intimately associated with endogenous opioid peptides in the modulation of sensory and affective experience. Although extensive information exists on the processing of opioid peptide precursors, little is known about the maturation of SP from precursor forms. Identification of precursor forms of SP has proven to be difficult, primarily due to the lack of suitable means of detection. Antisera generated to authentic SP recognize only mature undecapeptide or its C-terminal fragments. We have therefore generated antisera to unamidated C-terminal extensions of SP that are internal sequences in larger precursor forms of the peptide, i.e., SP-Gly-Lys (SP-G-K) and SP-Gly (SP-G), and have developed biochemical and immunohistochemical procedures for detecting these determinants in nervous tissue. Our immediate goal is to provide detailed biochemical characterization of SP precursor forms containing SP-G-K and SP-G sequences. In order to examine dynamic aspects of neuropeptide processing, the effects of morphine tolerance and withdrawal and of hormonal manipulations on levels of SP, SP-G-K and SP-G determinants, endogenous opioids, and biogenic amines will be quantified in discrete brain and spinal cord areas. Collaborative immunohistochemical localization studies are planned to complement these biochemical analyses. Biosynthetic studies comparing the effects of capsaicin and of acute and chronic morphine administration on isotope incorporation into SP precursor determinants in isolated dorsal root ganglia maintained in organotypic culture will provide additional information about narcotic influences on neuropeptide processing. Characterizing of SP precursor forms in nervous tissue will contribute to understanding basic mechanisms of SP processing. Overall, these studies will strengthen the molecular foundation for research on the interaction of SP- and opioid peptide-synthesizing neural systems, and on the biochemical mechanisms by which narcotics can influence sensory and affective processes.
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