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Immunohistochemical analysis of the pituitary of sexually mature axolotls revealed both ACTH(1-39)-related and alpha-MSH-related immunoreactivity present in corticotropic cells located in the rostral anterior pituitary. Gel filtration analysis indicated that the ACTH(1-39)-sized immunoreactivity and the alpha-MSH-sized immunoreactivity detected in acid extracts of the axolotl anterior pituitary were present in a ratio in a range between 1:1 and 1:0.6. Reversed-phase HPLC analyses indicated that the alpha-MSH-sized immunoreactivity had the same retention time as synthetic ACTH(1-13)-NH2. The corticotropic activity of the ACTH(1-39)-sized immunoreactivity and the purified ACTH(1-13)-NH2 was tested in a heterologous, larval bullfrog adrenal bioassay system. As expected, the ACTH(1-39)-sized immunoreactivity stimulated corticosterone release; however, the purified ACTH(1-13)-NH2 lacked glucocorticoid activity. The proteolytic cleavage of ACTH in corticotropes of sexually mature axolotls was identical to the cleavage events observed in neotenic Ambystoma tigrinum that had not reached sexual maturity. These studies indicate that the transient expression of ACTH cleavage activity is not affected by the reproductive state of the animal. Since axolotls do not undergo metamorphosis, it is possible that events associated with metamorphosis may induce the decline in ACTH cleavage activity observed in amphibian corticotropes.

Acid extracts of the brains of the American eel, Anguilla rostrata, and the coho salmon, Oncorhynchus kisutch, were screened for enkephalin-related products and dynorphin-related products. Following Sephadex G-50 column chromatography, a peak of Met-enkephalin-related immunoreactivity was detected near the total volume of the column for both species. No higher molecular weight forms of Met-enkephalin-related material were detected, nor were any immunoreactive forms with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) or alpha-neo-endorphin detected for either species. The enkephalin-sized immunoreactivity was further analyzed by reverse phase HPLC. For both species, a peak of authentic Met-enkephalin was detected. However, Leu-enkephalin, Met-enkephalin-RGL and Met-enkephalin-RF were not detected by RIA in either species. In addition, no novel C-terminally extended forms of Met-enkephalin were detected in either species. Finally, opiate receptor binding activity was only found associated with the peak of immunoreactive Met-enkephalin.

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 (n = 4). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8) and dynorphin B(1-13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.