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 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.
Shimonaka
H
H
Anesthesia Research, Tufts University School of Medicine, Boston, Massachusetts 02111.
Marchand
J E
JE
Connelly
C S
CS
Kream
R M
RM
D000818:Animals; D002211:Capsaicin / Q000494:pharmacology; D002490:Central Nervous System / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005724:Ganglia / Q000187:drug effects / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology*; D007150:Immunohistochemistry; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D011863:Radioimmunoassay; D012677:Sensation / Q000502:physiology; D013373:Substance P / Q000737:chemistry / Q000276:immunology*
D007106:Immune Sera; D009479:Neuropeptides; D013373:Substance P; D002211:Capsaicin