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Acid extracts of the brain of a urodele amphibian, Ambystoma tigrinum, were screened with radioimmunoassays specific for enkephalin-related products and dynorphin-related products. Following Sephadex G-50 column chromatography a peak of enkephalin-sized immunoreactive material was detected near the total volume of the column. The enkephalin-sized immunoreactivity was further analyzed by reversed phase HPLC. This analysis detected peaks of authentic Met-enkephalin and Leu-enkephalin. However, the molar ratio of Met-enkephalin to Leu-enkephalin in the brain of this amphibian was approximately 80:1. These observations would suggest that the Leu-enkephalin detected in the brain of Ambystoma may be derived from a source other than the Proenkephalin precursor. Neither Met-enkephalin-RGL or Met-enkephalin-RF were detected by radioimmunoassay in brain extracts from this urodele. However, following digestion with trypsin and carboxypeptidase B, a novel peak of C-terminally extended Met-enkephalin was detected. Two peaks of Prodynorphin-related products were also detected following gel filtration chromatography. These immunoreactive forms were detected using antisera specific for alpha-neo-endorphin and dynorphin B(1-13). No immunoreactive forms with antigenic determinants similar to mammalian dynorphin A(1-17) or dynorphin A(1-8) were detected in this species. Reversed phase HPLC analysis indicated that the major form of urodele alpha-neo-endorphin eluted with the same retention time as synthetic mammalian alpha-neo-endorphin. Urodele dynorphin B(1-13)-related immunoreactivity eluted as a single peak, however, this form did not elute with the same retention time as synthetic mammalian dynorphin B(1-13).(ABSTRACT TRUNCATED AT 250 WORDS)

Acid extracts of the brain and pars intermedia of the chestnut lamprey, Ichthyomyzon castaneus, were fractionated by a combination of gel filtration chromatography and reversed-phase HPLC, and screened with RIAs specific for Met-enkephalin and Leu-enkephalin, respectively. In the brain extract, both Met-enkephalin and Leu-enkephalin were detected in a molar ratio of approximately 4:1. These results would suggest that these two enkephalins were derived from a lamprey Proenkephalin precursor. However, reversed-phase HPLC analysis of the pars intermedia of this species revealed the presence of Met-enkephalin, but not Leu-enkephalin. Analysis of the pars intermedia of the marine lamprey, Petromyzon marinus, also indicated the presence of Met-enkephalin but not Leu-enkephalin. These results would suggest that the Met-enkephalin present in the pars intermedia of lampreys may not be derived from Proenkephalin, but may originate from another opioid precursor. This possibility and alternative hypotheses to explain these observations are discussed.

Acid extracts of the brain of the holostean fish Lepisosteus platyrhincus and the forebrain of the dipnoan fish Neoceratodus forsteri were separately fractionated by Sephadex G-50 column chromatography. For both species, Met-enkephalin-related immunoreactivity was detected coeluting with the total volume internal standard. Higher-molecular-weight Met-enkephalin-containing immunoreactive peaks were not detected in these chromatographs. Furthermore, immunoreactive forms with antigenic determinants identical to mammalian dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, or dynorphin B(1-13) were not detected in either species. Reverse-phase HPLC analysis of enkephalin-sized immunoreactive material indicated the presence of authentic Met-enkephalin and Leu-enkephalin in the extracts of both species. In the brain of L. platyrhincus the molar ratio of Met-enkephalin to Leu-enkephalin was approximately 3:1, whereas, the molar ratio of these enkephalins in the forebrain of N. forsteri was approximately 5:1 [corrected]. C-terminally extended forms of Met-enkephalin were also detected in the extracts of both species. These results suggest that the ancestral proenkephalin gene of both actinopterygian and sarcopterygian fish contained both the Met-enkephalin and Leu-enkephalin sequences.

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.

Three distinct opioid precursors have been identified in the central nervous system of mammals: proopiomelanocortin (POMC), proenkephalin, and prodynorphin. These precursors are derived from separate genes, synthesized in distinct neurons, and yield unique sets of opioid end products. This review will discuss the general mechanisms involved in the biosynthesis of neuropeptide precursors and consider the roles of posttranscriptional and posttranslational processing mechanisms in the generation of multiple sets of end products from a single gene. In addition, techniques that can be used for isolating and characterizing neuropeptide genes, mRNAs, and end products will be reviewed. These introductory comments will serve as the framework for a discussion of the phylogeny of the opioid precursors in the major groups of non-mammalian vertebrates.

Nine endogenous volatile compounds were found in rat brain tissue, and were identified by mass spectrometry as chloroform, a 5-C-aldehyde, dimethyl disulphide, 2,5-dimethyl tetrahydrofuran, a 8-C-alkane, xylene, 2-heptanone, heptaldehyde and 2-n-pentylfuran. Using gas chromatographic and gas chromatographic mass spectrometric techniques, it was established that lithium carbonate did not induce the production of detectable amounts of any new volatile compounds in brain tissue. However, after administration of chloral hydrate, trichloroethanol, a compound not normally present in rat brain tissue, was found to be present.

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.