This study reveals that quercetin induced vasorelaxation and increased cGMP levels in rat aortic vessels. Moreover, in vitro experiments indicated that quercetin treatment rapidly increased Ca2+, stimulated eNOS phosphorylation at Ser1179 resulting in NO generation in BAECs. Quercetin-stimulated eNOS phosphorylation was completely blocked by increasing cellular catalase enzymatic activity with the transduction of Ad.Cat.
Investigation into the underlying mechanism(s) revealed that quercetin-induced vasorelaxation could be regulated, in part, by the formation of an oxidant. More studies are needed to verify these findings; however cell culture experiments provided some insight. The transduction of catalase activity via Ad.Cat ameliorated quercetin stimulated eNOS phosphorylation in BAECs.
New evidence supports that flavonoids, such as quercetin, not only have antioxidant capabilities but more importantly may act as signaling molecules [37
]. In fact, it has been shown that quercetin can act as an antioxidant leading to the formation of quinones and prooxidants [38
]. Furthermore, quercetin can be metabolized to a quinone/quinone methide metabolite such as o-semiquinone and o-quinone [39
]. O-semiquinone has the capability to generate O2•−
in cell culture [42
] whereas o-quinone depletes glutathione (GSH) in the presence of excess ascorbate [41
]. The depletion of GSH and the generation O2•−
through these quercetin metabolites suggest a prooxidant effect. Moreover, these quinone/quinone methide metabolites may trigger signaling pathways [37
]. In this context, quercetin and its cellular metabolites act more like signaling molecules than prooxidants that cause cell death per se. The formation of quinone/quinone methide metabolites may lead to a low level production of oxidants, which may in turn activate certain signaling cascades.
By acting as a sensor as well as a signaling molecule, H2
is an important regulator of signal transduction. An additional mechanism of action of quercetin then is through regulating H2
regulates endothelial cell function such as proliferation, inflammatory responses, apoptosis, and endothelium-dependent vasorelaxation [44
]. The generation of H2
to act as a signaling molecule is tightly regulated and depends mainly on location of production and the surrounding antioxidant enzymatic activity. The dismutation of O2•−
can occur spontaneously or catalytically by SOD generating H2
. Interestingly, endogenously produced H2
is more effective in eliciting a signaling response compared to exogenously added H2
]. Moreover, minute intracellular fluxes in H2
concentrations may rapidly alter cell signaling responses in the endothelium resulting in endothelium-derived vasorelaxation.
Vascular endothelial cells have the capacity to release H2
]. Endothelium-derived H2
stimulates EDHF. The endothelium produces and releases several vasoactive mediators involved in vessel tone, such as NO, prostacyclins, and EDHF. RWPs have been shown to induce EDHF-dependent vasorelaxation in porcine coronary arteries [22
]. Herein, quercetin stimulates vasodilation in intact vessels which is significantly attenuated by the pretreatment of ChTx. Quercetin does not stimulate relaxation significantly in denuded vessels (). These results demonstrate that quercetin stimulates endothelium-dependent vasorelaxation. The inhibition of BKCa
, and some voltage-dependent K+
-channels with ChTx blunts the response suggesting EDHF playing a role in quercetin-induced vasorelaxation.
Studies demonstrated that grape seed extracts (which are high in flavonoids and used to make red wine) and RWPs, stimulate endothelium-dependent relaxation through NO/NOS-dependent pathways by increasing cGMP levels in rat aortic vessels [19
]. The precise mechanism still remains to be elucidated. Additional studies demonstrated that RWP-induced vasorelaxation was actually impaired by the preincubation of porcine vessels with PEG-SOD or SOD mimetic (MnTMPyP). Surprisingly, PEG-catalase (but not native catalase) also impaired RWP-induced vasorelaxation suggesting that RWP potentially generate H2
or peroxide-like molecules [22
]. Additionally, RWPs stimulate vasorelaxation in small mesenteric arteries of rats is endothelium- and NOS-dependent. These effects were inhibited by SOD plus catalase or using the NADPH oxidase inhibitor DPI suggesting that RWP generate oxidants. In the presence of Ca2+
, RWPs induced superoxide production [49
]. The subsequent formation of H2
or the peroxide-like molecule may indeed mediate a signaling response resulting in RWP-induced vasorelaxation.
mediates vasorelaxation in aortas and is blunted by catalase but not SOD [50
treatment increases eNOS activity and NO bioavailability in primary cultured endothelial cells through the phosphorylation of eNOS at Ser1179
and the concomitant dephosphorylation at Thr495
. The phosphorylation of eNOS at Ser1179
, and not Thr495
, is mediated by Ca2+
and the PI3K/Akt pathway [36
]. Additionally, H2
dose-dependently increases Ca2+
in endothelial cells [51
]. The removal of Ca2+
from buffers and/or aorta rings pretreated with BAPTA/AM significantly reduced H2
-mediated vasorelaxation [35
Other studies have shown that cellular responses to RWPs, such as resveratrol, are mediated through redox-sensitive pathways such as p38 MAPK, ERK1/2, PI3K [48
]. Resveratrol can increase eNOS mRNA, protein, and promoter activity resulting in an increase eNOS activity and NO [54
]. Regardless of these mechanism(s), the physiological relevance cannot be overlooked. Herein, quercetin stimulated vasorelaxation in rat aortic vessels through the phosphorylation of eNOS at ser1179
. Quercetin treatment did not alter Thr497
and Ser 116
levels (not shown) suggesting that the quercetin-mediated NO production may be due to the stimulation of eNOS phosphorylation at Ser1179
. Several studies have shown that PI3K inhibitors block agonist-stimulated eNOS phosphorylation. At higher concentrations, quercetin has been shown to partially block the PI3K pathway [56
]. Moreover, pretreatment of the PI3K pathway inhibitor wortmannin (100 nM) or LY294002 (10 μM) for 30 min failed to attenuate quercetin-stimulated eNOS phosphorylation at Ser1179
suggesting that this process is not mediated by the PI3K pathway. Moreover, quercetin did not significantly increase eNOS mRNA or protein (not shown).
Flavonoid and/or RWP studies report vasorelaxation through a NO synthase (NOS)- and PI3K-dependent pathway [19
], although the precise mechanism(s) still remains unclear. Black tea polyphenols (BTP) induced eNOS phosphorylation at Ser1177
and dephosphorylation of Thr495
, increased cGMP levels, and enhanced conversion of L-[3
H] arginine to L-[3
H] citrulline in porcine aortic endothelial cells (PAECs) within 5 min [57
]. The BTP-induced cGMP and conversion of arginine to citrulline was blocked by L-nitro-arginine methyl ester (L-NAME), the PI3K inhibitor LY294002, and a dominant negative p38 MAP kinase (MAPK) α suggesting that this effect is NOS, PI3K, and p38 MAPK dependent. Additionally, RWP-induced vasorelaxation was ablated by PI3K inhibitors (wortmannin and LY294002) and not affected by ERK1/2 (PD98059) nor p38 MAPK (SB203580) inhibitors [48
]. These studies are highly suggestive that polyphenols mainly activate the redox-sensitive PI3K pathway inducing endothelium-dependent NO-mediate relaxation through polyphenol-stimulated eNOS phosphorylation.
Quercetin stimulates eNOS phosphorylation at ser1179
and perhaps sensitizes eNOS to lower concentrations of Ca2+
. Moreover, quercetin increases the intracellular concentration of Ca2+
, whereas Ca2+
chelation with BAPTA/AM inhibits quercetin-stimulated phosphorylation of Ser1179
in BAEC (). In cultured endothelial cells, quercetin-mediated increases in Ca2+
may be responsible for the stimulation of eNOS phosphorylation at ser1179
eventually resulting in NO production. Not to diminish the fact that polyphenols are also widely accepted antioxidants and can scavenge ROS and reactive nitrogen species (RNS) such as O2•−
and peroxynitrite (ONOO−
]. BTPs and RWPs can inhibit lipid peroxidation and increase the antioxidant capacity in human plasma in vitro
]. Additionally, lipid radicals serve as a sink for NO [61
] suggesting that polyphenolic antioxidant properties lead to an increase in NO bioavailability by reducing ROS and RNS, which are known sinks for NO consumption. This may help explain the inverse relationship between flavonoid intake and cardiovascular risk.
In summary, endothelial dysfunction is often associated with the lower NO bioavailability. We have shown that quercetin improves endothelial function by stimulating endothelium-dependent vasorelaxation ex vivo, eNOS phosphorylation in vitro thereby resulting in an increase in NO bioavailability. This suggests that NO bioavailability may play an important role in the quercetin-dependent attenuation of vascular dysfunction through endothelial dependent NO producing enzymes and EDHF. These findings serve to further elucidate potential mechanisms regarding the epidemiological evidence from several cohort studies demonstrating an inverse correlation with dietary intake of flavonoids and CVD.