The presence of morphine-like and codeinelike substances was demonstrated in the pedal ganglia, hemolymph, and mantle tissues of the mollusc Mytilus edulis. The pharmacological activities of the endogenous morphine-like material resemble those of authentic morphine. Both substances were found to counteract, in a dose-dependent manner, the stimulatory effect of tumor necrosis factor ax or interleukin la on human monocytes and Mytilus immunocytes, when added simultaneously to the incubation medium. The immunosuppressive effect of this opiate material expresses itself in a lowering of chemotactic activity, cellular velocity, and adherence. Codeine mimics the activity of authentic morphine, but only at much higher concentrations. Specific high-affinity receptor sites (jx3) for morphine have been identified on human monocytes and Mytilus immunocytes. In Mytilus recovering from experimentally induced stress, the return of "alerted" immunocytes to a more inactive state appears to be due to a significant rise in the content of morphine-like material in the pedal ganglia and hemolymph at this time. Thus, morphine may have a role in calming or terminating the state of immune alertness.The occurrence throughout the animal kingdom of messenger molecules identical with, or closely related to, those known in mammals has been amply documented by biochemical identification, functional analysis, and the demonstration of specific receptor sites (1-6). This applies in particular to classical neurotransmitters, neuropeptides, and cytokines. The demonstration by Spector and coworkers (9-11) and others (7, 8) added to the sample in a 6:10 (vol/vol) ratio (HCl/sample) to a final strength of 10%]. The samples were then heated at 100°C for 30 min, cooled, and centrifuged at 10,000 x g for 20 min. The resulting precipitate was discarded and the supernatant was extracted with 5 vol of 10% (vol/vol) 1-butanol in chloroform. The supernatant was adjusted to pH 8.8-9.0. The organic phase was back-extracted into 2 vol of 0.01 M HCl. Morphine recovery was 50-70%. The aqueous phase was evaporated to dryness and dissolved in 10 ml of phosphate-buffered saline (PBS, pH 7.4) and then the pH was adjusted to 8.5-9.0. Samples were passed through a Sep-Pak C18 cartridge. Morphine and codeine were eluted from the cartridge with 7 ml of 0.1 M pyridine in acetic acid (pH 6.2) containing 2.5% (vol/vol) 1-propanol. The eluate was evaporated to dryness and dissolved in 250 ,ul of 1 mM HCl. An aliquot of 200 ,ul was injected onto a C18 reverse-phase HPLC column (LiChrosorb, 0.4 x 25 cm, Merck). The samples were eluted with 0.1 M pyridine in acetic acid (pH 6.2) followed by a linear gradient of 0-25% 1-propanol/0.1 M pyridine in acetic acid at a flow rate of 1.5 ml/min. Four 1-min fractions were collected during morphine and codeine elution as determined by known standards. These fractions were then evaporated, dissolved, and assayed for morphine and codeine by a sensitive radioimmunoassay (RIA) described by . Polyclonal antibodies generated against 3...
The specific binding of benzodiazepines (BZDs) has been observed in many tissues and cell types and can be pharmacologically separated into two classes. The first and most studied are the central-type receptors in brain that are thought to mediate the known clinical effects of the BZDs (1-3). The other class is the peripheral-type binding site found in a variety of tissues and cells, including kidney (4, 6), heart (5, 6), platelets (7), mast cells (8), lymphocytes (9), many cell lines (10), and brain (11,12). BZD binding to this site has no known physiological consequences. Several pharmacological effects of the BZDs have been reported (13-15); however, none has been supported by a positive correlation between the peripheral-type binding and the biological response. We now report the presence of specific, (New England Nuclear, 101.0 Ci/mmol) and unlabeled 0.5 ,uM thymidine. After 2 hr, the cells were harvested and washed over glass fiber filters by a Microharvester (Bellco Glass). The filters were dried and placed in 4 ml of Aquasol (New England Nuclear) in minivials for scintillation spectroscopy.Binding Studies. The thymnoma cells were centrifuged at 100 x g and washed once in balanced glucose salt buffer (5.0 mM KCI/120.0 mM NaCI/5.0 mM Na2HPO4/5.0 mM Tris/0.6 mM CaCl2/1.0 mM MgSO4/5.5 mM glucose, final pH = 7.4). Binding of [3H]Ro5-4864 (New England Nuclear, 73.8 Ci/mmol) to the cells was carried out in this buffer at 0°C for 40 min with the indicated concentrations of labeled ligand in a total volume of 250 ,ul.[3H]Diazepam (New England Nuclear, 87.6 Ci/mmol) binding was also carried out at 0°C but the incubation time was 15 min. Binding reaction was terminated by the addition of 3 ml of ice-cold Dulbecco's phosphate-buffered saline and rapid filtration over Whatman GF/B glass fiber filters, followed by two washes of 3 ml of ice-cold phosphate-buffered saline. The filters were air dried and placed in 4 ml of Aquasol for liquid scintillation spectroscopy. Specific binding was defined as the difference between total binding (in the absence of unlabeled ligand) and nonspecific binding (in the presence of unlabeled 10 ,M diazepam).The benzodiazepine compounds were obtained from Hoffmann-La Roche, Inc. All reagents were obtained from commercial sources. RESULTSThe specific binding of [3H]Ro5-4864, a BZD selective for peripheral-type sites (4), was saturable, whereas nonspecific binding increased linearly with increasing concentrations of the labeled ligand (Fig. 1). Scatchard analysis of the specific binding showed a single class of sites with a Kd (mean ±
The development of a radioimmunoassay for morphine is described. The hapten morphine is made antigenic by coupling it to a protein at the phenolic group of the molecule. Extremely low concentrations of morphine (0.5 nanogram) can be measured by this assay procedure.
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