Analysis of inverse agonism at the delta opioid receptor after expression in Rat 1 fibroblasts.

A cDNA encoding the mouse delta opioid receptor was expressed stably in a Rat 1 fibroblast cell line. Expression of this receptor was demonstrated both in ligand binding studies and by reverse transcriptase-PCR. In membranes of clone D2 cells the opioid peptide [D-Ala(2)]-leucine enkephalin (DADLE) produced a robust, concentration-dependent, stimulation of basal high-affinity GTPase activity; the prototypic opioid antagonist naloxone and the highly selective and potent delta opioid ligands H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-Tic[Ch2-NH]Phe-Phe-OH (TIPP[psi]) had little effect but N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI174864) caused a marked dose-dependent inhibition of this activity (Tic, 1,2,3,4-tetrahydroisoquinolin-2-yl-carbonyl]; Aib, alpha-aminobutyric acid). This effect of ICI174864 was reversed by TIPP[psi] and attenuated after treatment of the cells with pertussis toxin. No stimulation by DADLE or inhibition by ICI174864 was observed in Rat 1 fibroblasts that did not express the delta opioid receptor. Basal binding of [(35)S]guanosine 5'-O-(3-thio-triphosphate) to membranes of clone D2 cells was also stimulated by DADLE and inhibited by ICI174864; both of these effects were reversed by co-incubation with TIPP[psi]. When cholera toxin-catalysed [(32)P]ADP-ribosylation was performed on membranes of clone D2 cells in the absence of guanine nucleotides, a 40 kDa G1-family polypeptide was labelled in addition to both the long and short isoforms of Gsalpha. Labelling of the 40 kDa polypeptide was enhanced by addition of DADLE and fully attenuated by addition of ICI174864. In contrast, labelling of the isoforms of Gsalpha was unaffected by either opioid ligand. Again, both the positive effect of DADLE and the inhibitory effect of ICI174864 were prevented by co-incubation with TIPP[psi] which, in isolation, had little effect on cholera toxin-catalysed [(32)P]ADP-ribosylation of either Gs or Gi. These data demonstrate that the delta opioid receptor displays a spontaneous activity when expressed in this genetic background. Attenuation of this activity is produced by ICI174864, which by acting as an 'inverse agonist' in this system, functionally uncouples the expressed receptor from the cellular G-protein population. The complete attenuation of agonist-independent cholera toxin-catalysed [(32)P]ADP-ribosylation of Gi demonstrated that ICI174864 acts as an inverse agonist with high intrinsic activity at this receptor.