The long term goal of this project is to characterize the opioid mechanism in the protozoan ciliate Tetrahymena. A beta-endorphin-like peptide has been reported in this organism and mammalian beta-endorphin inhibits phagocytosis in these cells with a high potency and efficacy. These are important findings because it is believed that the mechanisms of signal transduction in mammalian organisms originated from autocrine and paracrine mechanisms in unicellular eukaryotes. Therefore these simple systems could serve as models to study problems that are difficult to elucidate in more complex organisms. We now propose to characterize two important elements of the opioid mechanism in Tetrahymena: the endogenous opioid and its receptor. We have confirmed by means of a radioimmunoassay (RIA) the presence of a beta-endorphin-like peptide in Tetrahymena. We now wish to establish the optimal conditions for the maximal expression of this peptide by studying how different factors, both environmental and biochemical, modulate its synthesis and secretion. The peptide will then be purified from cell extracts by various kinds of chromatography in combination with the RIA. The effect of the purified peptide on phagocytosis in Tetrahymena and in metazoan models will be determined to see if it is similar to that of mammalian beta-endorphin. The primary sequence of the purified peptide will be studied to determine its degree of homology with mammalian beta- endorphin or other peptides that have been reported to have opioid-like activities. We shall also initiate studies on the putative beta-endorphin gene in Tetrahymena by standard recombinant DNA techniques; this will determine if the putative beta-endorphin gene in Tetrahymena is coded independently or is part of a part of a POMC gene, as in vertebrates. We shall also perform a pharmacological study of the opioid receptor in Tetrahymena in order to obtain information about the degree of micro- heterogeneity. In addition we shall determine how the properties of the receptor, including desensitization, are affected by changes in the ionic environment and by G-proteins. This research will contribute to establishing if the Tetrahymena opioid mechanism may be used as a model system to study general aspects of opioid physiology. Furthermore, our findings will be relevant to our understanding of other systems in advanced eukaryotes, such as macrophages, in which phagocytosis is also modulated by opioids.
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