Anales RANF

P.88 POLYPHENOLS INCREASE ATP RELEASE AND NO PRODUCTION BY SEPARATE MECHANISMS IN MESENTERIC ENDOTHELIAL CELL CULTURES. M. Verónica Donoso 1 , Camila Silva 1 , Francisca Donoso 1 , Camila Calfío 1 , and J. Pablo García-Huidobro 1,2 . 1 Universidad de Santiago de Chile, Santiago, CHILE. 2 Centro de NanoCiencias y Nanotecnología, CEDENNA, Santiago, Estación Central, Chile. Endothelial cells participate in vascular wall regulation through the release of multiple vasoactive chemicals such as ATP and nitric oxide (NO) the most potent endogenous dilator. In view that diet polyphenols have vasodilator effects, we assessed whether 30 µM quercetin (Q, flavonol) or 100 nM conjugated delphinidin (D3G, anthocyanin), modified ATP released and NO production in endothelial cells derived from rat mesenteric bed with and without (basal) mechanical stimulation. Cells were cultured in control media or with 25 mM glucose (mimicking diabetes) or 10 mM KCl supplement. Extracellular ATP was monitored following chloroacetaldehyde derivatization; extracellular NO was quantified by chemiluminescence while intracellular NO was determined by DAF fluorescence. Q increased basal, ATP overflow from 50±16 to 102±24 pmol/mg protein (n=28 well, p<0.05), 5 min following mechanical stimulation, extracellular ATP rose from 162±32 to 591±169 pmol/mg protein (n=16 well, p<0.05), without modifying basal or NO production 5 min post stimuli. In contrast, D3G doubled extracellular NO from 14.3±2.2 to 26.8±1.9 nmol/mg protein (n=6, p<0.01), without modifying extracellular ATP. Not all NO produced is due to ATP release, since apyrase (4 U/mL) only partially annulled both extracellular and intracellular NO production, consistent with two targets of polyphenols in these cells. In KCl enriched medium, basal ATP overflow increased 163±50 pmol/mg protein (n=12 well, p<0.01); while that evoked 1 min post mechanical stimuli caused a rise of 815±255 pmol/mg protein (n=6 well, p<0.05). Addition of Q to KCl supplemented cultures increased extracellular ATP 743±158 pmol/mg protein (n=6 well, p<0.05) 5 min post stimuli. Moreover, in glucose enriched medium, Q did not change extracellular ATP outflow. In sum, polyphenols activate separate mechanisms to increase ATP and NO overflow, consonant with vasodilator mechanism and beneficial effect of high KCl diets. Funded by FONDECYT 117-0842 and grant 21141226 to CC and CEDENNA FB0807