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We have isolated and characterized a rat delta-opioid receptor. The deduced amino acid sequence (372 aa) closely resembles the murine delta-opioid receptor, DOR-1. In fact, 97% of the amino acid residues are conserved between the two species, while 93% of the nucleic acid residues are identical. A 6 kb mRNA was detected in rat cortex using rat DOR-1 as a probe. When expressed in COS cells, the clone shows high-affinity opioid binding with selectivity for delta-opioids. The rat delta-opioid receptor cDNA clone will be a useful tool for studying the function of delta-opioid receptor in rats.

To study whether weaning stimulates the development of a subtype of the delta-opioid receptor we have measured antinociceptive activity of putative delta 1 (DELT I) and delta 2 (DSLET) agonists in weaned and non-weaned 25-day-old rats. In weaned rats, DSLET produced antinociception reversed by the delta 2-antagonist naltriben, but in non-weaned rats DSLET had no effect. Responses to DELT I were evident in both weaned and non-weaned rats and were not antagonised by naltriben. This is the first evidence that delta-receptor subtypes develop independently and suggests that weaning is the stimulus for the expression of the delta 2-subtype.

The recent cloning of the mouse delta opioid receptor (Evans et al., 1992; Kieffer et al., 1992) has demonstrated it to be a member of the seven transmembrane G-protein coupled family of neurotransmitter receptors. The present study describes the cellular localization in the central nervous system (CNS) of an mRNA encoding this receptor and compares it with the distribution of delta receptor binding and proenkephalin mRNA using a combination of in situ hybridization and receptor autoradiographic techniques. Delta receptor mRNA was visualized with a cRNA probe (472-903 bp) corresponding to transmembrane domains III-VI of the receptor, while proenkephalin mRNA was labeled with a cRNA probe to exon 3 (139-832 bp). A high level of correspondence was observed between the distribution of delta receptor mRNA and delta receptor binding as defined by the selective ligand [3H]D-Pen2-Pen5-enkephalin. Delta receptor mRNA and binding were expressed in the neocortex, caudate-putamen, nucleus accumbens, olfactory tubercle, diagonal band of Broca, amygdala and the nucleus of the solitary tract. Discrepancies in the distribution of delta receptor mRNA and binding in the olfactory bulb, hippocampus, globus pallidus and substantia nigra pars reticulata, may in part be due to differential receptor synthesis and transport. These results are discussed in relation to the distribution of proenkephalin mRNA and how this may affect our understanding of opioid circuitry in the CNS.

Cloned mouse delta-subtype opioid receptor (DOR1) was expressed in Xenopus oocytes to study the signal transduction. Opioid delta-agonists evoked a calcium-dependent chloride current in oocytes injected with mRNA derived from DOR1, together with that from the alpha subunit of Gi1. The delta-agonist-induced current was blocked by naltrindol, a delta-specific antagonist. The delta-agonist evoked no or very weak currents in oocytes with the alpha subunit of Gq or G(o). These findings indicate the functional coupling between the opioid delta-receptor and phospholipase C through an activation of Gi.

Functional delta opioid receptors were expressed in Xenopus oocytes following injection of poly(A+)RNA isolated from the mouse neuroblastoma X rat glioma hybrid cell line, NG108-15. Oocytes coinjected with in vitro transcribed beta 2-adrenergic receptor mRNA and NG108-15 cell mRNA had 3-fold elevated cAMP levels following isoproterenol treatment. Application of delta opioids to these oocytes caused dose-dependent inhibition of isoproterenol-induced increase of cAMP, which was antagonized by naltrexone. This is the first demonstration of the expression of opioid receptors in Xenopus oocytes. The ability to assay expression of delta opioid receptors functionally coupled to the inhibition of adenylate cyclase in Xenopus oocytes following injection of exogenous mRNA will provide a system to investigate the relationship of molecular structure to function of opioid receptors.