Internalization and recycling of human mu opioid receptors expressed in Sf9 insect cells.

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists.

3-Pyrroline containing arylacetamides: a novel series of remarkably selective kappa-agonists.

A series of 2-(substituted phenyl)-N-methyl-N-[(1S)-1-(substituted alkyl)-2-(1-(3-pyrrolinyl))ethyl]acetamides were synthesized and evaluated as highly selective kappa-agonists with K(i) values in low nanomolar range. 3-Pyrroline incorporated into the basic amino functionality in combination with 2-(methylthio)ethyl substituent on the carbon adjacent to the amide nitrogen remarkably enhanced the kappa-selectivity. 3,4-Dichlorophenyl derivative 1e was found the most potent and selective analgesic in this series with ED(50) value of 0.023 mg/kg.

Human &mgr; Opioid Receptor Expressed in Sf9 Insect Cells was Functionally Coupled to Endogenous G Protein.

Human &mgr; opioid receptor(H&mgr;OR) with a tag of six consecutive histidines at its carboxyl terminus overexpressed in recombinant baculovirus infected Sf9 insect cells was demonstrated to be functionally coupled to endogenous G proteins. Na(+) and GTP could reduce the affinity binding of etorphine and Ohmefentanyl(Ohm) to H&mgr;OR overexpressed in Sf9. The binding of (35)S GTPgammaS to Sf9 cell membranes containing H&mgr;OR was stimulated by DAGO and Ohm. This stimulation could be blocked by naloxone or the pretreatment of PTX. Also, DAGO and Ohm could inhibit the increasing of intracellular cAMP stimulated by forskolin. The results showed H&mgr;OR expressed in Sf9 insect cells were functionally coupled to endogenous PTX sensitive G(i/o) proteins.