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

Abstract

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 P4-10. Designated A47, this reagent bound a variety of related peptide species containing the substance P4-10 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 anti-substance 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.

Olfactory marker protein (OMP) gene deletion causes altered physiological activity of olfactory sensory neurons.

“…In the assay '4C-dopa generated from 14C-tyrosine substrate is separated on alumina, quantified by scintillation spectrometry, and reported as nmol dopa/bulb/15 min. For quantification of levels of cholecystokinin (CCK)-like immunoreactivity (CCK-LI) by RIA, the second member of each pair of frozen olfactory bulbs, stored at -80°C, was extracted using procedures previously described in detail (16). Briefly, bulbs were each homogenized in 2 M acetic acid followed by the addition of 20 volumes of cold 50% acetonitrile/0.1% trifluoroacetic acid.…”

“…The RIA procedure for quantification of CCK-LI uses a highly selective antiserum generated against synthetic sulfated CCK-8 coupled to keyhole limpet hemocyanin using previously described conjugation procedures (16). The antibody recognizes sulfated CCK-8 and sulfated gastrin 17 on an equimolar basis, with nonsulfated CCK-8 and gastrin 17 displaying <10% cross-reactivity.…”