Biochemical characterization and anatomical distribution of a major form of unamidated precursor of substance P in rat brain

Abstract

Previous work from this laboratory has provided biochemical characterization of several posttranslational processing intermediates of the neuropeptide substance P (SP) in central nervous system (CNS) tissues, including the COOH-terminal glycine-extended dodecapeptide Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly (SP-G). SP-G is a major species of unprocessed SP found in rodent CNS tissues, and is the likely immediate precursor form of SP in the biosynthetic scheme. Here we present extensive characterization of the normal regional distribution of SP-G, as compared to SP, throughout the rat CNS via coordinated biochemical and morphological analyses. By radioimmunoassay (RIA), an approximate 10-fold variation in regional levels of SP-G-like immunoreactivity (SP-G-LI) was observed, ranging from 0.30 pmol/g in the amygdala, to 6.49 pmol/g in the medulla. On a normalized basis, the regional variation of unamidated precursor relative to mature peptide (SP-G-LI/SP-LI molar ratio) ranged from 0.30% in the amygdala to 5.15% in the dorsal root ganglia (DRG). Overall, the highest SP-G-LI/SP-LI ratios were found in DRG, medulla, and spinal cord, i.e. CNS areas associated with primary sensory afferent innervation via capsaicin-sensitive unmyelinated small diameter fibers. In addition, chromatographic and RIA analyses of extracted brain tissues indicated that the quantified immunoreactivities corresponding to SP, SP-G, as well as an additional COOH-terminal Gly-Lys-extended precursor, i.e., SP-G-K, displayed very similar chromatographic behavior as demonstrated for chemically authentic standards. These biochemical data were complemented by immunohistochemical analyses demonstrating a pattern of immunohistochemical staining for the presence of SP-G-LI as a defined subset of SP-LI-containing neural elements. Here, reaction product was localized to dendritic, axonal, and terminal neuronal elements in representative CNS regions of the rat, with relatively high levels of SP-G-LI found within anatomical areas containing a high density of sensory terminal structures. In an attempt to provide correlative functional anatomy, a group of rats was treated with colchicine, in order to differentially localize SP-LI- and SP-G-LI-containing somata after inhibition of axoplasmic transport. Most prominently, colchicine administration engendered immunohistochemical visualization of both SP-LI- and SP-G-LI-positive cells in mesencephalic and brainstem regions associated with stress, pain responses, and central control of autonomic function. Within this context, the coordinate expression of both SP-LI- and of SP-G-LI-positive somata in discrete brain areas is probably indicative of high ongoing rates of tachykinin synthesis coupled to utilization.