Recent demonstration of immunoreactive (IR) atrial natriuretic factor (ANF) and β-endorphin (β-EP) in the thymus prompted a reexamination of the distribution and cellular localization of the two peptides within that tissue. Double labeling immunohistochemistry was carried out on gelatin-embedded cryostat thymic sections of adult male Sprague-Dawley rats. Cells stained positive with antiserum (S118), raised against rANF(1-28), were colocalized in > 95% of cases with immunofluorescent staining of IR-β-EP(1-31). The cells were found sparsely distributed along the corticomedullary junction and in subcapsular regions. In 1- or 5-day monolayer cultures of adherent thymic cells, 15-20% of the cells stained positive for either IR-ANF or IR-β-EP. Under these conditions, > 95% of IR-ANF or IR-β-EP positive cells were also fluorescence stained for the rat macrophage marker ED-1. Thus, taken together with previous reports, our present findings suggest that, in the rat thymus, both ANF and β-EP are produced by the same population of macrophages. To further investigate their presence in the thymus, the contents and molecular species of the two peptides were compared over the developmental period of the animal using well-characterized radioimmunoassays (RIA). Both peptides significantly increased their contents between day 2 and day 60. However, in terms of concentration, IR-ANF at day 2 was ∼50% higher than day 16 and five times greater that at day 60; in comparison the concentration of IR-β-EP remained relatively constant and the only significant difference from day 2 being a slight increase in the day 16 animals. The molecular species of the two peptides were further characterized with Sephadex G-50 gel chromatography and RIA. In neonatal animals, both ANF and β-EP immunoreactivity were predominantly eluted at positions equivalent to proANF and POMC, respectively. In the adult, a significant proportion of IR-ANF was eluted in the same fractions as the mature form of approximately 3K, Mr, ANF(1-28), although there was little change in the molecular species of β-EP. Thus, it appears that whereas the two peptides are colocalized by the same population of thymic macrophages, they differ in respect to their constitutive regulation in the thymus.
- Atrial natriuretic peptide
- Cell culture