A specific antisense oligodeoxynucleotide to mRNAs encoding receptors with seven transmembrane spanning regions decreases muscarinic m2 and gamma-aminobutyric acidB receptors in rat cerebellar granule cells.
Many receptors that inhibit adenylyl cyclase belong to a common superfamily of receptors that couple to guanine nucleotide binding proteins. These receptors are thought to span the membrane seven times and to have a highly homologous amino acid sequence (LACADL) in the second membrane spanning region. The antisense oligodeoxynucleotide seven-transmembrane spanning receptor (7TMR), a 15-mer that binds to mRNA encoding this amino acid sequence, was added to cerebellar granule neuron cultures to decrease receptors of this superfamily. Intact antisense 7TMR was found to enter neurons. This 4- to 6-day treatment with antisense oligonucleotide decreased the total number of muscarinic receptor binding sites by about 40% and completely eliminated muscarinic m2 receptors that inhibit cyclic AMP formation. Antisense 7TMR treatment at 25 microM prevented the gamma-aminobutyric acid (GABA)B-mediated inhibition of cyclic AMP formation by about 40%. The treatment was effective in decreasing GABAA receptors only when the antisense oligonucleotide was given 1 day after plating the cells, and the receptor response assay was performed 6 days later. The half-maximal concentration of antisense 7TMR was approximately 5 microM in blocking GABAB receptors. Antisense 7TMR appeared to be specific because another antisense oligodeoxynucleotide sequence (15-mer) having four mismatches with 7TMR had no effect on either muscarinic m2 or GABAB receptor-mediated responses and did not affect the total number of muscarinic binding sites. These results are consistent with the view that antisense oligonucleotides decrease proteins in which the nucleotide sequence is known such as the muscaranic m2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Holopainen
I
I
Fidia Georgetown Institute for the Neurosciences, Georgetown University, School of Medicine, Washington, D.C.
Wojcik
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D000242:Cyclic AMP / Q000096:biosynthesis; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D058787:GABA-A Receptor Antagonists; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011963:Receptors, GABA-A / Q000096:biosynthesis* / Q000235:genetics; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000235:genetics; D012680:Sensitivity and Specificity
D058787:GABA-A Receptor Antagonists; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D011976:Receptors, Muscarinic; D000242:Cyclic AMP