Characterization of [3H]morphine binding to interleukin-1-activated thymocytes.

We have previously reported that interleukin-1-induced proliferation of thymocytes is accompanied by the appearance of [3H]morphine binding sites on these cells. In the present study, we have characterized these binding sites. They differ from classical opioid receptors in the brain in several ways, including: 1) lack of stereoselectivity; 2) relatively low affinity (Kd = 50 nM) and high capacity (Bmax = 3 pmol/mg of protein); 3) binding is strongly inhibited by Ca++, Mg++, Mn++ and Cl- ions and 4) binding is inhibited by proteinase K or E and by phospholipase A2 but not trypsin treatment of thymocyte membranes. The binding sites, which were found largely on the CD4+ subset of T-cells, also showed a preference for opioid alkaloids over peptides. These [3H]morphine binding sites may mediate a negative feedback effect on interleukin-1-induced proliferation of thymocytes in vivo.

[3H]morphine binding is enhanced by IL-1-stimulated thymocyte proliferation.

Mouse thymocytes incubated in vitro with increasing concentrations of interleukin-1 (IL-1) in the presence of phytohemagglutinin (PHA) exhibited a dose-dependent increase in cell proliferation, as measured by [3H]thymidine incorporation. Under these conditions, there was a parallel dose-dependent increase in specific [3H]morphine binding, with a maximum increase of approximately 5-fold over basal levels. The binding sites differ from classical opioid receptors in that they are not stereo-selective. Interleukin-2 was ineffective in promoting either cell proliferation or enhanced opioid binding, but the effects of IL-1 could be mimicked by phorbol myristate acetate (PMA), suggesting the involvement of tyrosine phosphorylation. These results indicate that morphine-binding sites on immune cells can be regulated by cytokine activation.

[P-8502--a new mu selective opioid receptor ligand].

The analgesic potencies of 3-[beta-(p-amino)-phenethyl)-9 beta-methoxy-9 alpha-(m-methoxyphenyl)-3- azabicyclo [3,3,1] nonane (P-8502) and 3-[beta-(p-monoester fumarylamido)-phenethyl]-9 beta-methoxy-9 alpha-(m-methoxyphenyl)-3-azabicyclo [3,3,1] nonane (P-8511) were examined. The analgesic ED50 of P-8502 and P-8511 were 55 and 200 micrograms/kg (mice, ip, hot plate), and 30 and 95 micrograms/kg (rat, sc, tail flick), respectively. The duration of the analgesic action of P-8511 (about 4 h) was longer than that of P-8502 (about 1.5 h, rat, sc, tail flick). Binding assay showed that P-8502 had a high ratio of delta/mu, kappa/mu: IC50 (DPDPE)/IC50 (DAGO) = 399; IC50(DAD-LE)/IC50 (DAGO) = 1498; IC50 (kappa)/IC50 (DAGO) = 159. In conclusion, P-8502 appears to be a new mu selective opioid receptor ligand, whereas P-8511 has no such selectivity.

Purification to apparent homogeneity of a mu-type opioid receptor from rat brain.

A mu-opioid-specific receptor was purified to apparent homogeneity from rat brain membranes by 6-succinylmorphine affinity chromatography, gel filtration, wheat germ agglutinin affinity chromatography, and isoelectric focusing. The purified receptor had a molecular weight of 58,000 as determined by NaDodSO4/polyacrylamide gel electrophoresis and was judged to be homogeneous by the following criteria: (i) a single band was detected by autoradiography after NaDodSO4/polyacrylamide gel electrophoresis of 125I-labeled receptor and (ii) the purified preparation had a specific opioid-binding activity of 17,720 pmol/mg of protein, close to the theoretical value. In addition, the Mr 58,000 value agrees closely with that determined by covalently labeling purified receptor with bromoacetyl-[3H]dihydromorphine or with 125I-labeled beta-endorphin and dimethyl suberimidate.

Isolation and purification of morphine receptor by affinity chromatography.

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.

Isolation of opiate binding components by affinity chromatography and reconstitution of binding activities.

Rat brain membranes exhibiting stereospecific opiate binding activity were solubilized by sonication and detergent treatment. The active material could be bound to an affinity column containing 6-succinylmorphine but could not be eluted with free agonist. Although two protein peaks could be eluted with NaCl, neither possessed binding activity; however, one of the peaks (A), in combination with an acidic lipid fraction, eluted subsequently from the column, showed stereospecific binding. Opiate ligands of the mu type bound to this protein/lipid mixture with an order of affinities closely correlating with those of the original membrane but one to two orders of magnitude lower; binding of delta, kappa, and sigma prototype opioids was considerably less. The protein/lipid mixture also competed with the membranes for mu ligands. These results suggest that the isolated protein-lipid complex may be a component of the opiate receptor and, specifically, the mu receptor or binding site. However, because of the lower affinities of mu opiates for this complex, it is conceivable that some essential membrane component is still missing. Preliminary analysis of peak A indicates that it contains a broad spectrum of protein bands, but it remains to be seen which of these are essential for activity.

Studies on synthesis and relationship between analgesic activity and receptor affinity for 3-methyl fentanyl derivatives.

In the present paper, the synthesis and analgesic activity (mice, i.p. hot plate test) of the derivatives of 3-methyl fentanyl are briefly described. Compound 7302, cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N-phenylpropionamide (cis: 3-methyl/4-N-phenylpropionamide) is found to be the most potent analgesic agent in this series synthesized by our laboratory (ED50 = 0.0022 mg/kg). The analgesic activity of 7302 is 28 times more potent than that of fentanyl and 6300 times more than that of morphine. The partition coefficients of 10 compounds in the series are determined by high performance liquid chromatography (HPLC) and their log p values are about 3. There are no regular relationships between the analgesic activity and partition coefficients. Study on the specific binding of 8 out of the above 10 compounds to crude synaptic plasma membrane (P2-fraction) of mouse brain demonstrates that there is an excellent statistical linear correlation (r = 0.998) between the analgesic potency and the specific binding affinity. The result shows that the analgesic potency of the derivatives of this series is mainly dependent on binding affinity for opiate receptor.