Flavonoid galetin 3,6-dimethyl ether attenuates guinea pig ileum contraction through K(+) channel activation and decrease in cytosolic calcium concentration

Abstract

Flavonoid galetin 3,6-dimethyl ether (FGAL) has been isolated from the aerial parts of Piptadenia stipulaceae and has shown a spasmolytic effect in guinea pig ileum. Thus, we aimed to characterize its relaxant mechanism of action. FGAL exhibited a higher relaxant effect on ileum pre-contracted by histamine (EC50=1.9±0.4×10(-7) M) than by KCl (EC50=2.6±0.5×10(-6) M) or carbachol (EC50=1.8±0.4×10(-6) M). The flavonoid inhibited the cumulative contractions to histamine, as well as to CaCl2 in depolarizing medium nominally Ca(2+)-free. The flavonoid relaxed the ileum pre-contracted by S-(-)-Bay K8644 (EC50=9.5±1.9×10(-6) M) but less potently pre-contracted by KCl or histamine. CsCl attenuated the relaxant effect of FGAL (EC50=1.1±0.3×10(-6) M), but apamin or tetraethylammonium (1mM) had no effect (EC50=2.6±0.2×10(-7) and 1.6±0.3×10(-7) M, respectively), ruling out the involvement of small and big conductance Ca(2+)-activated K(+) channels (SKCa and BKCa, respectively). Either 4-aminopyridine or glibenclamide attenuated the relaxant effect of FGAL (EC50=1.8±0.2×10(-6) and 1.5±0.5×10(-6) M, respectively), indicating the involvement of voltage- and ATP-sensitive K(+) channels (KV and KATP, respectively). FGAL did not alter the viability of intestinal myocytes in the MTT assay and decreased (88%) Fluo-4 fluorescence, indicating a decrease in cytosolic Ca(2+) concentration. Therefore, the relaxant mechanism of FGAL involves pseudo-irreversible noncompetitive antagonism of histaminergic receptors, KV and KATP activation and blockade of CaV1, thus leading to a reduction in cytosolic Ca(2+) levels.

Keywords: Calcium; Flavonoid; Galetin 3,6-dimethyl ether; Galetin-3,6-dimethyl ether (PubChem CID: 5352032); Ileum; Potassium channels; S-(−)-Bay K8644 (PubChem CID: 2303); and 4-aminopyridine (PubChem 4-AP) (CID: 1727).; apamin (PubChem CID: 16218850); calcium chloride (PubChem CID: 6093260); carbachol (PubChem CID: 5831); cesium chloride (PubChem CID: 24293); glibenclamide (PubChem CID: 3488); histamine (PubChem CID: 774); potassium chloride (PubChem CID: 4873); tetraethylammonium chloride (CID: 5413); verapamil (PubChem CID: 62969).