Cross - Reactivity of Antiserum to ACTH With ACTH

Adrenocorticotropin (ACTH) and/3-1ipotropin (/3-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-~m sections were immunostained first for either ACTH or/3-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and fl-LPH demonstrated, immunocytochemicaUy as well as by radioimmunoassay, that the ACTH and/3-LPH antisera were directed to the COOHand NH2-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained/3LPH, but fl-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. /3-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and/3-LPH are contained in the same nerve cells. They strongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell. 50 J. CELL BIOLOGY 9 The Rockefeller University Press 9 0021-9525/79/04/0050/09 $1.00 Volume 81 April 1979 50-58 on A uust 8, 2017 jcb.rress.org D ow nladed fom