Antisense targeting of delta opioid receptors in NG 108-15 cells: direct correlation between oligodeoxynucleotide uptake and receptor density.
Antisense oligodeoxynucleotides (ODN) have been used to inhibit the function of a number of structurally defined neurotransmitter receptors in vivo by transiently disrupting their expression in the CNS. However, issues concerning the cellular and molecular mechanisms of these ODN often raise questions about the specificity of such ODN-mediated "knock-down" of target proteins. This study sought to extend our in vivo "knock-down" of the delta opioid receptor (DOR) by targeting this receptor in the NG 108-15 cells with an antisense ODN for the DOR and by using a polyclonal antibody raised against this receptor to determine the efficiency and selectivity of the antisense ODN in inhibiting expression of the DOR. By fluorescence tagging the ODN and immunofluorescence labeling the DOR, we monitored the uptake efficiency of the ODN and the DOR density in individual cells that had been treated with the antisense ODN or with a mismatch control. Quantitative fluorescence image analysis showed that the uptake of ODN by NG 108-15 cells was time- and concentration-dependent and that it was not uniform within a population. Treatment with the antisense ODN elicited an inverse correlation between DOR immunoreactivity and the ODN fluorescence in individual cells. No correlation was found in cells treated with the mismatch control. These findings suggest that the antisense ODN-mediated "knock-down" of the DOR is governed by the sequence specificity of the ODN and the efficiency of its uptake by the target cells in a time- and concentration-dependent manner. These data provide further evidence in support of the selectivity of antisense ODN targeting and the utility of these molecules as an effective tool in neuropharmacological studies.
Lai
J
J
Department of Pharmacology, University of Arizona Health Sciences Center, Tucson 85724, USA.
Crook
T J
TJ
Payne
A
A
Lynch
R M
RM
Porreca
F
F
D000818:Animals; D000906:Antibodies / Q000276:immunology; D005455:Fluorescent Antibody Technique; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics* / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000235:genetics; D014407:Tumor Cells, Cultured
D000906:Antibodies; D016376:Oligonucleotides, Antisense; D017465:Receptors, Opioid, delta