Differential Knockdown of δ-Opioid Receptor Subtypes in the Rat Brain by Antisense Oligodeoxynucleotides Targeting mRNA

Abstract

Two antisense oligodeoxynucleotides (A-ODN), targeting δ-opioid receptor mRNA (DOR) and two mismatch ODN sequences (mODN) were continuously infused for 24 days into the lateral brain ventricles of Wistar rats. The density of δ-opioid receptors in rat brain homogenates was measured by saturation binding experiments using four selective ligands, two agonists ([D-Ala², Glu⁴]-deltorphin and DPDPE) and two antagonists (Dmt-Tic-OH and naltrindole), and by immunoblotting SDS solubilized receptor protein. In brain membranes of mODN or saline-infused rats, the rank order of δ-opioid receptor density, calculated by B_max values of the four δ-opioid receptor ligands, was: [D-Ala², Glu⁴]deltorphin ≅ Dmt-Tic-OH ≅ naltrindole (86–118 fmol/mg protein)> DPDPE (73.6 ± 6.3 fmol/mg protein). At the end of the 24 day infusion of A-ODN targeting DOR nucleotide sequence 280–299 (A-ODN_280–299), the B_max of DPDPE (62.4 ± 3.2 fmol/mg protein) was significantly higher than that of Dmt-Tic-OH (31.5 ± 3.9 fmol/mg protein). Moreover, both the K_d value for DPDPE saturation binding and the K_i value for Dmt-Tic-OH displacement by DPDPE were halved. In contrast, an A-ODN treatment targeting exon 3 (A-ODN_741–760) decreased the specific binding of [D-Ala², Glu⁴]deltorphin and Dmt-Tic-OH significantly less (67%–81%) than the binding of DPDPE (53%), without changes in DPDPE K_i and K_D values. No A-ODN treatment modified the specific binding of the μ-opioid agonist DAMGO and of the k-selective opioid receptor ligand U69593. On the Western blot of solubilized striatum proteins, A-ODN_280–299 and A-ODN_741–760 downregulated the levels of the DOR protein, whereas the corresponding mODN were inactive. The 24-day infusion of A-ODN_280–299 inhibited the rat locomotor response to [D-Ala², Glu⁴]deltorphin but not to DPDPE. Intracerebroventricular (i.c.v.) infusion of A-ODN_741–760 reduced the locomotor responses to both δ-opioid receptor agonists, whereas mODN infusion never affected agonist potencies. In conclusion, these results demonstrate that 24-day continuous i.c.v. infusion of A-ODN targeting the nucleotide sequence 280–299 of DOR can differentially knockdown δ₁ and δ₂ binding sites in the rat brain.