ABSTRACT
Endothelial dysfunction, characterized by decreased production or availability of nitric oxide (NO), is widely believed to be the hallmark of early-stage atherosclerosis. In addition, hypercholesterolemia is considered a major risk factor for development of atherosclerosis and is associated with impaired flow-induced dilation. However, the mechanism by which elevated cholesterol levels leads to decreased production of NO is unclear. NO is released in response to shear stress and agonist-evoked changes in intracellular calcium. Although calcium signaling is complex, we have previously shown that NO production by endothelial nitric oxide synthase (eNOS) is preferentially activated by calcium influx via store-operated channels. We hypothesized that cholesterol enrichment altered this signaling pathway (known as capacitive calcium entry; CCE) ultimately leading to decreased NO. Our results show that cholesterol enrichment abolished ATP-induced eNOS phosphorylation and attenuated the calcium response by the preferential inhibition of CCE. Furthermore, cholesterol enrichment also inhibited shear stress-induced NO production and eNOS phosporylation, consistent with our previous results showing a significant role for ATP autocrine stimulation and subsequent activation of CCE in the endothelial flow response.
ABSTRACT
Nitric oxide (NO) generated from nitrite through nitrite reductase activity in red blood cells has been proposed to play a major role in hypoxic vasodilation. However, we have previously predicted from mathematical modeling that much more NO can be derived from tissue nitrite reductase activity than from red blood cell nitrite reductase activity. Evidence in the literature suggests that tissue nitrite reductase activity is associated with xanthine oxidoreductase (XOR) and/or aldehyde oxidoreductase (AOR). We investigated the role of XOR and AOR in nitrite-mediated vasodilation from computer simulations and from in vivo exteriorized rat mesentery experiments. Vasodilation responses to nitrite in the superfusion medium bathing the mesentery equilibrated with 5% O2 (normoxia) or zero O2 (hypoxia) at either normal or acidic pH were quantified. Experiments were also conducted following intraperitoneal (IP) injection of nitrite before and after inhibiting XOR with allopurinol or inhibiting AOR with raloxifene. Computer simulations for NO and O2 transport using reaction parameters reported in the literature were also conducted to predict nitrite-dependent NO production from XOR and AOR activity as a function of nitrite concentration, PO2 and pH. Experimentally, the largest arteriolar responses were found with nitrite >10 mM in the superfusate, but no statistically significant differences were found with hypoxic and acidic conditions in the superfusate. Nitrite-mediated vasodilation with IP nitrite injections was reduced or abolished after inhibiting XOR with allopurinol (p < 0.001). Responses to IP nitrite before and after inhibiting AOR with raloxifene were not as consistent. Our mathematical model predicts that under certain conditions, XOR and AOR nitrite reductase activity in tissue can significantly elevate smooth muscle cell NO and can serve as a compensatory pathway when endothelial NO production is limited by hypoxic conditions. Our theoretical and experimental results provide further evidence for a role of tissue nitrite reductases to contribute additional NO to compensate for reduced NO production by endothelial nitric oxide synthase during hypoxia. Our mathematical model demonstrates that under extreme hypoxic conditions with acidic pH, endogenous nitrite levels alone can be sufficient for a functionally significant increase in NO bioavailability. However, these conditions are difficult to achieve experimentally.
