enkephalin--ala(2)-mephe(4)-gly(5)- and Pituitary-Neoplasms

The differential ability of various mu-opioid receptor (MOP) agonists to induce rapid receptor desensitization and endocytosis of MOP could arise simply from differences in their efficacy to activate G proteins or, alternatively, be due to differential capacity for activation of other signaling processes. We used AtT20 cells stably expressing a low density of FLAG-tagged MOP to compare the efficacies of a range of agonists to 1) activate G proteins using inhibition of calcium channel currents (ICa) as a reporter before and after inactivation of a fraction of receptors by beta-chlornaltrexamine, 2) produce rapid, homologous desensitization of ICa inhibition, and 3) internalize receptors. Relative efficacies determined for G protein coupling were [Tyr-D-Ala-Gly-MePhe-Glyol]enkephalin (DAMGO) (1) > or = methadone (0.98) > morphine (0.58) > pentazocine (0.15). The same rank order of efficacies for rapid desensitization of MOP was observed, but greater concentrations of agonist were required than for G protein activation. By contrast, relative efficacies for promoting endocytosis of MOP were DAMGO (1) > methadone (0.59) >> morphine (0.07) > or = pentazocine (0.03). These results indicate that the efficacy of opioids to produce activation of G proteins and rapid desensitization is distinct from their capacity to internalize mu-opioid receptors but that, contrary to some previous reports, morphine can produce rapid, homologous desensitization of MOP.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Electric Conductivity; Endocytosis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Methadone; Mice; Morphine; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Pentazocine; Pituitary Neoplasms; Receptors, Opioid, mu; Transfection; Tumor Cells, Cultured

AtT-20 cells, which make and release beta-endorphin, or AtT-20/hENK cells, an AtT-20 cell line transfected with the human proenkephalin gene and secreting enkephalin as well as presumably beta-endorphin, were implanted in mouse spinal subarachnoid space. Cell implants did not affect the basal response to thermal nociceptive stimuli. Administration of isoproterenol, believed to stimulate secretion from these cells, produced antinociception in groups receiving AtT-20 or AtT-20/hENK cell implants but not in control groups receiving no cells. The antinociceptive effect of isoproterenol was dose related and could be blocked by the opioid antagonist naloxone. Implantation of these cells offers a novel approach for the study of tolerance. Mice receiving AtT-20 cell implants developed tolerance to beta-endorphin and the mu-opioid agonist DAMGO, whereas mice receiving genetically modified AtT-20/hENK cell implants developed tolerance to the delta-opioid agonist DPDPE. Genetically modified AtT-20/hENK cell implants, but not AtT-20 cell implants, reduced the development of acute morphine tolerance in the host mice. This finding is consistent with the suggestion that enkephalin alters development of opioid tolerance. These results suggest that opioid-releasing cells implanted around mouse spinal cord can produce antinociception and may provide an alternative therapy for chronic intractable pain.

Topics: Animals; beta-Endorphin; Cell Line; Cell Transplantation; Dose-Response Relationship, Drug; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Isoproterenol; Male; Mice; Mice, Inbred ICR; Narcotics; Nociceptors; Pituitary Neoplasms; Protein Precursors; Spinal Cord; Transfection; Tumor Cells, Cultured

Pituitary 7315c tumor cells maintained in culture were treated with varying concentrations of morphine from 10 nM to 300 microM, for periods of five or forty-eight hours. The ability of the mu-opioid receptor agonist, DAMGO, to inhibit forskolin-stimulated adenylyl cyclase in washed membrane preparations from the treated cells was compared with its activity in membranes from cells incubated in the absence of added morphine. In the same membrane preparations, the number and affinity of mu-opioid receptors was estimated by measurements of [3H]diprenorphine binding. After 5 hr of treatment with morphine concentrations of 100 nM or higher, a significant reduction in inhibition of adenylyl cyclase by DAMGO was observed. Little further loss of agonist activity was observed when the incubations were extended to 48 hr. After 5 hr of morphine treatment, there was no change in either the number of receptors, or their affinity for [3H]diprenorphine. However, after 48 hr of morphine treatment, greater than 25% reductions in receptor number were apparent with morphine pretreatment concentrations of 10 microM or higher. These results suggest that opioid tolerance in this system is primarily associated with a reduced ability of agonist-occupied receptor to activate the effector system. Receptor down-regulation was not necessary for loss of agonist response, although a reduction in receptor number occurred after exposure to high concentrations of morphine for periods longer than 5 hr.

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Colforsin; Diprenorphine; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Morphine; Pituitary Neoplasms; Rats; Receptors, Opioid; Receptors, Opioid, mu; Tumor Cells, Cultured

The effects of varying the sodium concentration (at constant ionic strength) on opioid binding at mu- and delta-opioid receptors in 7315c and NG108-15 cells has been examined. The binding of [3H]etorphine to mu-receptors on 7315c cells was increased by replacing the sodium in the incubation medium with potassium or N-methyl-D-glucamine. This effect was shown to be attributable to an increase in affinity, with no change in the maximum number of binding sites, both in cell membrane suspensions and in intact 7315c cells. Replacement of sodium with potassium or N-methyl-D-glucamine in NG108-15 membrane or intact cell suspensions also resulted in an increase in [3H]etorphine binding, but in these cells the effect was associated with an increase in the number of binding sites measurable under these experimental conditions. The effects of sodium on opioid inhibition of adenylate cyclase in membrane preparations from 7315c and NG108-15 cells also differed. Sodium reduced apparent agonist affinity in 7315c membranes. In NG108-15 cell membranes, sodium was essential for the demonstration of opioid inhibition of cyclase activity. Increasing the sodium concentration above 0.5 mM resulted in an increase in the fraction of total enzyme activity inhibited by opioid, but the opioid IC50 did not change. In the companion paper, it is shown that the effects of sodium removal on mu- and delta-receptor binding in guinea pig brain neural membranes were similar to those observed in the cell preparations. An increase in intracellular sodium concentration without change in extracellular concentration was effected by incubation of 7315c and NG108-15 cells with the sodium-selective ionophore, monensin. When sodium was present in the extracellular medium, monensin reduced [3H]etorphine binding by 50% or more, both at mu-receptors in 7315c cells and at delta-receptors in NG108-15 cells. In the absence of sodium, however, monensin treatment produced only a small inhibition of binding. These results suggest that sodium acts at an intracellular site to regulate opioid agonist binding at both mu- and delta-receptors, but that the mode of regulation is not identical at each site. Since a reduction in intracellular sodium concentration by removal of extracellular sodium increases agonist binding, and an increase in intracellular sodium following monensin treatment reduces agonist binding, it is probable that the intracellular sodium concentration is a critical regulator of opioid agonist b

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Cell Membrane; Cyclazocine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Etorphine; Glioma; Guinea Pigs; Monensin; Naloxone; Neuroblastoma; Pituitary Neoplasms; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sodium