Two- and three-dimensional distributions of opioid receptors on NG108-15 cells visualized with the aid of fluorescence confocal microscopy and anti-idiotypic antibodies.
Polyclonal anti-idiotypic antibodies previously shown to be specific for mu- and delta-opioid receptor subclasses on rat brain membrane preparations using radioreceptor assays have been labeled with fluorescent conjugates of avidin. The resulting complexes have been used to study the distribution and properties of opioid receptors on living and fixed NG108-15 cells using normal fluorescence, and confocal fluorescence, microscopy. From the confocal data three-dimensional views of immunofluorescent distributions have been obtained using a computer transformation of the data. From experiments involving co-incubation with media containing naloxone, selective opioid peptides and stereoisomeric synthetic opioid analogs as blocking agents, the specificity and stereospecificity of immunofluorescent binding is reported. The effects on immunofluorescent binding by co-incubation with media containing a sulfhydryl reagent, 100 mM-NaCl and sodium azide are also reported. The results have implications concerning the use of immunoprecipitation in the biochemical isolation of the receptors. Finally, the confocal two-dimensional sections and two-dimensional reconstructions obtained from fixed cells show internal immunofluorescence in the form of cytoplasmic clusters. The reagents and methods should be applicable to visualizing native opioid receptor distributions on neurons.
Ornatowska
M
M
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
Glasel
J A
JA
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D001386:Azides / Q000494:pharmacology; D002460:Cell Line; D004723:Endorphins / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D004745:Enkephalins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009270:Naloxone / Q000494:pharmacology; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011869:Radioligand Assay; D011957:Receptors, Opioid / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D019810:Sodium Azide; D012965:Sodium Chloride / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000888:Antibodies, Anti-Idiotypic; D001386:Azides; D004723:Endorphins; D004745:Enkephalins; D011957:Receptors, Opioid; D013438:Sulfhydryl Compounds; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D012965:Sodium Chloride; D016308:Enkephalin, Leucine-2-Alanine; D019810:Sodium Azide