Resolution of two [S-35]GTP-gamma-S binding sites and their response to chronic morphine treatment: A binding surface analysis

The mechanisms by which prolonged exposure to morphine leads to tolerance a re not fully understood. We investigated the effects of etorphine (ET) on [ S-35]guanosine 5'-(thio)-triphosphate ([S-35]GTP-gamma-S) binding in brains of rats made tolerant to morphine via the implantation of morphine (or pla cebo) pellets. Binding surface analysis was used to characterize the intera ctions of ET, Gpp(Np)H and GTP-gamma-S with sites labeled by [S-35]GTP-gamm a-S. Data sets were fitted to one- and two-site binding models using the no nlinear least squares curve fitting program MLAB-PC (Civilized Software, Be thesda, MD, USA). Two binding sites were readily resolved. Chronic morphine significantly increased the B-max and K-d of the high affinity binding sit e, ET stimulated [S-35]GTP-gamma-S binding in placebo membranes via an incr ease in the B-max of the high affinity binding site. In contrast, ET stimul ated [S-35]GTP-gamma-S in chronic morphine membranes via a large decrease i n the K-d of the high affinity site. These results suggest that chronic mor phine treatment alters the mechanism by which ET stimulates [S-35]GTP-gamma -S binding to G-proteins. Since proper G-protein/receptor coupling increase s [S-35]GTP-gamma-S binding via an increase in B-max values, these results suggest that opioid receptors in chronic morphine membranes are not normall y coupled to G-proteins. These findings corroborate earlier studies that re ported changes in G-protein function in morphine tolerant animals. publishe d by Elsevier Science Inc.