CONICET | Buscador de Institutos y Recursos Humanos

Flavonoids and calcium transport through biologicalmembranes Ontiveros, M1;Rinaldi, D1; Marder, M1; Espelt M1; Vigil, M;Mangialavori, I1; Rossi, RC1; Rossi, JP1;Ferreira-Gomes, M11Institutode Química y Fisicoquímica Biológicas. ?Prof. Paladini?. Departamento deQuímica Biológica, Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires. Flavonoids arepolyphenolic compounds synthesized by plants that are widely distributed infruits and vegetables. These compounds have been reported to reach a micromolarconcentration in human blood plasma. Flavonoids have a broad bioactivity,mainly antioxidant, modulate the catalytic activity of enzymes like P-typeATPase and trigger the apoptosis of malignant cells through the Ca2+-dependentmitochondrial pathway. Some flavonoids, like quercetin, inhibit Ca2+removal systems, as the sarcoplasmic/endoplasmic reticulum (SERCA) and theplasma membrane (PMCA) calcium ATPases. However, the molecular mechanism ofthis effect is not yet defined. PMCA is aP-type ATPase regulated by calmodulin that maintains the homeostasis ofintracellular Ca2+ in eukaryotic cells, coupling the transport of Ca2+with ATP hydrolysis. The presentstudy seeks to investigate the effect of several natural flavonoids on hPMCA4(human Plasma Membrane Calcium Pump isoform 4) both in the purified system andin living cells, and to establish the relationship between the flavonoidstructure and its inhibitory activity. Resultsobtained with purified PMCA show that some flavonoids inhibit the PMCA activityand that the increase in the number of -OH in the B ring enhances the potencyof the inhibition. The best inhibitors were quercetin, a flavonol aglycone, andgossypin, a flavonol glycoside, with Ki of 0.3 and 4.1 mM,respectively. Magnesium competition assays indicate that the mechanism ofinhibition of these flavonoids is dependent on the concentration of Mg2+,suggesting that the inhibitor is a flavonoid-Mg2+ complex. On theother hand, when phosphorylated intermediates (EP) were measured,quercetin led to the increase whereas gossypin induced a decrease of EP.The dephosphorylation of EP formed in the presence of quercetin wassensitive to ADP indicating that this flavonoid could stabilize an E1Pintermediate. These results suggest that gossypin could affect the binding ofATP while quercetin could prevent the conformational change E1P → E2P.Altogether, these flavonoids inhibit PMCA through different mechanisms.In order toassess whether the effects of quercetin and gossypin on the purified hPMCA4could occur in living cells, we evaluated the effect of these flavonoidsmonitoring changes in the cytoplasmic calcium in HeK293T cells that overexpresshPMCA4. Results show that quercetin and gossypin inhibited the PMCA activity in HeK293Tcells. Fluorescence microscopy images indicate that both flavonoids distributedwidely in the cytoplasm of HEK293T cells, suggesting that it is possible adirect interaction between these flavonoids and the cytoplasmic domains ofPMCA. Acknowledgement, with grants of CONICET, ANPCYT andUBACYT

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