Somatostatin₅Receptor-Mediated [³⁵S]Guanosine-5′-O-(3-thio)triphosphate Binding: Agonist Potencies and the Influence of Sodium Chloride on Intrinsic Activity

Abstract

We studied the activation of the human somatostatin5 receptor recombinantly expressed in CHO-K1 cells by using some newly available agonists and antagonists. Somatostatin-28 bound to this receptor with a higher affinity than somatostatin-14 and was more potent in increasing [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding. Somatostatin-14-induced [35S]GTPgammaS binding to membranes from this cell line was decreased in a concentration-related manner by increasing concentrations of GDP and sodium chloride. At 50 mM (low) sodium, agonist EC50 values for stimulating [35S]GTPgammaS binding were lower than those at 150 mM (high) sodium and were closer to their respective affinity estimates (dissociation equilibrium constants) for binding to the receptor in the absence of sodium. Both agonist binding to the high affinity state of the receptor and agonist-induced [35S]GTPgammaS binding were abolished by pertussis toxin pretreatment. The putative somatostatin5 receptor-selective ligand L-362,855, unlike somatostatin-14 and somatostatin-28, showed differential intrinsic activity for stimulation of [35S]GTPgammaS binding, behaving as a partial agonist in high sodium and a full agonist in low sodium. In contrast, BIM-23056 did not behave as an agonist under any conditions studied but was able to antagonize somatostatin-14-induced [35S]GTPgammaS binding. We conclude that measurement of [35S]GTPgammaS binding mediated by somatostatin receptor activation in the presence of different concentrations of sodium chloride provides a useful functional assay for assessing the relative agonist efficacies of novel ligands identified from radioligand binding studies.

Effects of sodium on agonist efficacy for G‐protein activation in μ‐opioid receptor‐transfected CHO cells and rat thalamus

“…The eect of sodium on relative ecacy was due to a decrease in maximal stimulation by partial agonists compared to the full agonist DAMGO, rather than to a decrease in the potency of partial agonists. This ®nding agrees with previous studies of d opioid and somatostatin sst 5 receptor-stimulated [ 35 S]-GTPgS binding (Szekeres & Traynor, 1997;Williams et al, 1997), and agrees with the well-established ®nding that sodium has a greater eect on binding of full versus partial agonists to opioid receptors in radioligand binding studies (Pert & Snyder, 1974). The present study expands upon previous ®ndings by providing a quantitative examination of the eects of a range of sodium concentrations on the relative ecacy of several full and partial opioid agonists for G-protein activation.…”

Control of signalling efficacy by palmitoylation of the rat Y1 receptor

“…35 S] binding in Y1 and HAY1 membranes, mediated through the transfected Y 1 receptors under conditions known to favour agonist-mediated GPCR -G i protein interactions (100 mM NaCl and the presence of GDP; Williams et al, 1997). NPY and PYY potencies were considerably lower than their almost identical affinities in [ 125 I]PYY competition studies in the same membranes (as for SST 5 ; Williams et al, 1997), probably reflecting the low-affinity state of the Y 1 receptor.…”

“…NPY and PYY potencies were considerably lower than their almost identical affinities in [ 125 I]PYY competition studies in the same membranes (as for SST 5 ; Williams et al, 1997), probably reflecting the low-affinity state of the Y 1 receptor. This conformation is difficult to study using available Y 1 -agonist radioligands, but would be favoured by the high Na + and GDP concentrations (Kenakin, 1996).…”

“…To assess the relative efficacies of these nonpalmitoylated receptors in stably transfected Chinese hamster ovary (CHO) K1 cells, we have measured agonist-stimulated guanosine-5 0 -O-(3-thio)triphosphate (GTPg[ 35 S]) binding, a useful technique to directly determine GPCR activation of G proteins (particularly G i ; Williams et al, 1997). In addition, we have adopted a novel functional approach to investigate the Y 1 and Y 1 (C337S) receptors expressed in a human epithelial cell line, by measuring their effects on anion secretion using shortcircuit current (I SC ) techniques (Holliday & Cox, 1996).…”

Chemokine Receptor CCR3 Function Is Highly Dependent on Local pH and Ionic Strength

“…A similar link to biology is seen with major histocompatibility complex interactions with specific peptide ligands (21). While the effects of salt variations tend to be qualitatively consistent, there are even examples of increases of sodium chloride concentrations increasing ligand function, specific agonists of the somatostatin receptor being one example (22). While it is likely that pH and salt-dependent variations occur with other chemokine/receptor pairs the magnitude of these has not yet been fully characterized.…”