Agonist stimulation at human μ opioid receptors in a [(35)S]GTPγS incorporation assay: observation of "bell-shaped" concentration-response relationships under conditions of strong receptor G protein coupling.

CONTEXT

The appearance of "bell"- (or "inverted U"-) shaped agonist concentration-response curves (CRCs) in in vitro pharmacological experiments is a frequently observed but poorly communicated phenomenon. In the context of G protein coupled receptor research, it is commonly attributed to the recruitment of secondary targets or to desensitization or feedback processes, but the concrete background of these observations often remains intriguing.

OBJECTIVE

Here, we addressed the subject of bell-shaped agonist CRCs at the µ opioid receptor (µOR) by testing the impact of experimental conditions favoring G protein coupling.

METHODS

G protein activation by recombinant human µORs heterologously expressed in CHO cells was assessed in [(35)S]GTPγS binding assays using the opioid ligands DAMGO, morphine, fentanyl and naloxone. Experimental conditions were varied by changing the NaCl (10-300 mM) and the GDP concentration (0.3-30 µM).

RESULTS

Both the sodium and the GDP concentration were inversely related to G protein coupling, as evident by an increase in basal [(35)S]GTPγS incorporation at low sodium and low GDP levels and by the concomitant appearance of the partial agonist activity of the µOR antagonist, naloxone. Bell-shaped CRCs were observed for the efficacious agonists DAMGO, fentanyl and morphine, and this phenomenon was promoted by low sodium as well as by low GDP concentrations.

CONCLUSION

µOR agonist CRCs show a non-monotonic behavior with a decline of maximal stimulation under conditions of strong receptor-G protein coupling, and this behavior is visible at the level of G protein activation itself.