title: Identification of endomorphin-1 and endomorphin-2 binding sites in human mu-opioid receptor by antisense oligonucleotide strategy.
abstract: The effects of phosphorothioate antisense oligodeoxynucleotides against exons-1, -2, -3 and -4 of the human mu-opioid receptor were studied in the CHO-mu-opioid receptor cells using aequorin luminescence-based calcium assay. All four antisense oligodeoxynucleotides significantly decreased the level of mu-opioid receptor mRNA in comparison with the non-treated cells, used as control. However, no statistically significant differences between antisense oligodeoxynucleotides were observed. antisense oligodeoxynucleotides against exon-2 attenuated endomorphin-1-induced intracellular calcium response in a concentration-dependent manner. antisense oligodeoxynucleotides against exons-1, -2, -3 and -4 inhibited endomorphin-2-induced intracellular calcium response in a concentration-dependent manner and the effect of antisense oligodeoxynucleotides against exons-3 and -4 was most pronounced. The mismatch oligodeoxynucleotides against respective exons failed to exert any effect. The selective actions of antisense probes directed against different exons of the human mu-opioid receptor gene, that resulted, at the protein level, in attenuation of calcium responses induced by endomorphin-1 and endomorphin-2, suggest that the binding sites for endomorphins are structurally and functionally different. The presence of functionally distinct binding sites might play a crucial role in the modulation of pain and may be important clinically.
authors.affiliation: Laboratory of Biomolecular Chemistry, Institute of Biomedicinal Chemistry, Medical University, Mazowiecka St. 6/8, Lodz, Poland.
authors.forename: Jakub
authors.initials: J
authors.lastname: Fichna
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Gach
authors.forename: Renata
authors.initials: R
authors.lastname: Perlikowska
authors.forename: Jeroen
authors.initials: J
authors.lastname: Poels
authors.forename: Jozef
authors.initials: J
authors.lastname: Vanden Broeck
authors.forename: Janusz
authors.initials: J
authors.lastname: Szemraj
authors.forename: Anna
authors.initials: A
authors.lastname: Janecka
mesh_terms: D000331:Aequorin / Q000032:analysis / Q000737:chemistry; D000701:Analgesics, Opioid / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D005091:Exons / Q000235:genetics; D008163:Luminescent Measurements; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D009842:Oligopeptides / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
chemical_list: D000701:Analgesics, Opioid; D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D009842:Oligopeptides; D012333:RNA, Messenger; D017450:Receptors, Opioid, mu; C105293:endomorphin 1; C105294:endomorphin 2; D000331:Aequorin; D002118:Calcium