1 Long chain fatty acids have recently been identified as agonists for the G protein-coupled receptors GPR40 and GPR120. Here, we present the first description of GW9508, a small-molecule agonist of the fatty acid receptors GPR40 and GPR120. In addition, we also describe the pharmacology of GW1100, a selective GPR40 antagonist. These molecules were used to further investigate the role of GPR40 in glucose-stimulated insulin secretion in the MIN6 mouse pancreatic b-cell line. 2 GW9508 and linoleic acid both stimulated intracellular Ca 2 þ mobilization in human embryonic kidney (HEK)293 cells expressing GPR40 (pEC 50 values of 7.3270.03 and 5.6570.06, respectively) or GPR120 (pEC 50 values of 5.4670.09 and 5.8970.04, respectively), but not in the parent HEK-293 cell line. 3 GW1100 dose dependently inhibited GPR40-mediated Ca 2 þ elevations stimulated by GW9508 and linoleic acid (pIC 50 values of 5.9970.03 and 5.9970.06, respectively). GW1100 had no effect on the GPR120-mediated stimulation of intracellular Ca 2 þ release produced by either GW9508 or linoleic acid. 4 GW9508 dose dependently potentiated glucose-stimulated insulin secretion in MIN6 cells, but not in primary rat or mouse islets. Furthermore, GW9508 was able to potentiate the KCl-mediated increase in insulin secretion in MIN6 cells. The effects of GW9508 on insulin secretion were reversed by GW1100, while linoleic acid-stimulated insulin secretion was partially attenuated by GW1100. 5 These results add further evidence to a link between GPR40 and the ability of fatty acids to acutely potentiate insulin secretion and demonstrate that small-molecule GPR40 agonists are glucose-sensitive insulin secretagogues.
Bradykinin is a small peptide that acts mainly as a hormone by activating specific receptors that confer protection against the development of hypertension. The efficacy of bradykinin is influenced by the activities of various kininases present in plasma and blood. In this study, both human and rat plasma were incubated with a labelled form of bradykinin (at 4 and 12.5 microM), that will be referred to as bromobradykinin. The metabolic fate of bromobradykinin was monitored by liquid chromatography coupled to an orthogonal acceleration time-of-flight mass spectrometer (oaTOF). Quantification measurements of the bromine-containing metabolites were performed on-line, via flow splitting, by inductively coupled plasma mass spectrometry (ICPMS). The data obtained highlighted that the mechanism(s) of bradykinin metabolism in human and rat plasma are different, with the metabolism of bradykinin in rat plasma being much more aggressive than that observed in human plasma. In addition to the known bradykinin metabolites, e.g. [1,5], [1,7] from ACE, [1,8] from carboxypeptidase and [2,9] from aminopeptidase activity, we have identified the presence of new bradykinin metabolites in both human and rat plasma. These have been identified as fragment [5], the amino acid phenylalanine, which was present in both the human and rat plasma and the fragments [2,8] and [4,8] in rat plasma. To our knowledge it is the first time that these fragments have been recorded in human and rat plasma. The occurrence of these new fragments provides evidence for the presence of potentially new enzymes and mechanisms of bradykinin metabolism. The method described here provides a powerful technique for monitoring the activity of the many kininases involved in bradykinin metabolism such as ACE (angiotensin I converting enzyme), carboxypeptidase N and aminopeptidase P. In addition, this procedure could be used as a screening assay for selecting and monitoring the actions of inhibitors of the enzymes implicated in bradykinin metabolism directly in plasma or serum.
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