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 1 nM was significantly higher in hKOR cells and comparable to DynA (1–13) than that shown at 20 nM 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.