The long term goal of this project is to establish the feasibility of using non-vertebrate organisms as model systems for the study of hormonal mechanisms. These systems offer the advantage of relative simplicity and ease of manipulation, and can thus be used as tools to answer both medical and general questions in endocrinology and developmental biology. Opioids have been found to inhibit phagocytosis in Tetrahymena thermophila, and this inhibition shall be studied in detail. Phagocytosis is of great importance in higher organisms in a wide variety of medical problems such as parasitic infections and defense mechanisms, and is similar in many respects to the process of digestive vacuole formation in Tetrahymena. This ciliate offers the advantage that it can be grown in pure culture, and in large homogeneous populations. The putative opioid receptor will be characterized by testing the effect of a wide variety of opioid agonists on the process. This will establish if it is similar to the mammalian opioid receptor. Biogenic amines have been found to stimulate phagocytosis in Tetrahymena and to counteract the inhibitory effect of opioids. The interaction of these regulatory molecules will be studied in detail to determine if they exert a dual control on phagocytosis. The effect of opioids and biogenic amines on phagocytosis by mouse macrophages will be tested for comparative purposes; an inhibition of this process by opioids would explain at least in part the lowering of the immune defenses in drug addicts. The mechanism of action of opioids on phagocytosis will be studied by measuring their effect on uptake of calcium 45 and the levels of cyclic nucleotides during the process. Cyclic nucleotides will be purified by column chromatography and measured by radio immunoassays. The receptors will be localized in the Tetrahymena cells by means of autoradiography; and endogenous opioids by means of indirect immunofluorescence. The levels of endogenous opioids will be measured by radioimmunoassay. Some aspects of hormonal mechanisms will also be studied in embryos of the sea urchin Litechinus variegatus, mostly during the Spring and Summer months. The serotonin receptor will be characterized in embryo membranes by means of standard filtration and centrifugation assays. The activity of adenylate cyclase in these organisms will also be studied, particularly its regulation by biogenic amines and guanine nucleotides. These data should enable us to compare the mechanism of hormone action in sea urchin embryos with that postulated for mammalian systems.
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