title: Intracellular cAMP assay and Eu-GTP-γS binding studies of chimeric opioid peptide YFa.
abstract: In our previous studies chimeric peptide of Met-enkephalin and FMRFa, YGGFMKKKFMRFamide (YFa), demonstrated concentration dependent κ- and μ-opioid receptor mediated antinociception without tolerance development. To gain further insight of the observed behavior of YFa, the present study was undertaken. The effect of chimeric peptide on forskolin-stimulated cAMP formation under acute and chronic treatment and stimulation of Eu-GTP-γS binding in CHO cells stably expressing κ- and μ-opioid receptors was assessed. YFa showed concentration dependent inhibition of forskolin-stimulated cAMP in both hKOR and hMOR-CHO cells; however, the inhibition at 1nM was significantly higher in hKOR cells and comparable to DynA (1-13) than that shown at 20nM in hMOR cells. Chronic treatment of YFa, similar to DynA (1-13), did not show significant change in forskolin-stimulated cAMP level in both hKOR and hMOR cells. However, chronic treatment of morphine and DAMGO showed an increase in forskolin-stimulated cAMP level in hMOR-CHO cells indicating superactivation of adenylyl cyclase. Eu-GTP-γS binding studies of YFa showed a concentration dependent adherent binding with κ- and μ-opioid receptors; however, the latter demonstrated significant binding at higher concentration. Thus the study indicates the chimeric opioid peptide YFa as a potent κ- receptor specific antinociceptive moiety, showing no tolerance and hence may serve as a lead in understanding the mechanism of tolerance development, antinociception and its modulation.
authors.affiliation: Institute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi, India.
authors.forename: Krishan
authors.initials: K
authors.lastname: Kumar
authors.forename: Sambuddha
authors.initials: S
authors.lastname: Kumar
authors.forename: Raj Kumar
authors.initials: RK
authors.lastname: Kurupati
authors.forename: Mahesh Kumar
authors.initials: MK
authors.lastname: Seth
authors.forename: Anita
authors.initials: A
authors.lastname: Mohan
authors.forename: M Ejaz
authors.initials: ME
authors.lastname: Hussain
authors.forename: Santosh
authors.initials: S
authors.lastname: Pasha
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005063:Europium / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects* / Q000378:metabolism*; D018847:Opioid Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D017473:Receptors, Opioid, kappa / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015290:Second Messenger Systems / Q000187:drug effects
chemical_list: D018847:Opioid Peptides; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005063:Europium; D000242:Cyclic AMP