Differential knockdown of delta-opioid receptor subtypes in the rat brain by antisense oligodeoxynucleotides targeting mRNA.
Two antisense oligodeoxynucleotides (A-ODN), targeting delta-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 delta-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 delta-opioid receptor density, calculated by Bmax values of the four delta-opioid receptor ligands, was: [D-Ala2, Glu4]deltorphin approximately Dmt-Tic-OH approximately naltrindole (86-118 fmo/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 280299 (A-ODN280-299), the Bmax 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 Kd value for DPDPE saturation binding and the Ki value for Dmt-Tic-OH displacement by DPDPE were halved. In contrast, an A-ODN treatment targeting exon 3 (A-ODN741-760) 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 Ki and KD values. No A-ODN treatment modified the specific binding of the micro-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 delta-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 delta1 and delta2 binding sites in the rat brain.
Negri
L
L
Institute of Medical Pharmacology, University La Sapienza, Rome, Italy.
Lattanzi
R
R
Borsodi
A
A
Toth
G
G
Salvadori
S
S
D000818:Animals; D001921:Brain / Q000187:drug effects*; D003342:Corpus Striatum / Q000187:drug effects; D008024:Ligands; D008297:Male; D009043:Motor Activity / Q000187:drug effects*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000187:drug effects* / Q000235:genetics; D013873:Thionucleotides / Q000494:pharmacology
D008024:Ligands; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D017465:Receptors, Opioid, delta; D013873:Thionucleotides