| Background:Hyperuricemia has become an important risk factor for health of residents in China during the past 30 years. Researchers have recognized that the harmful of uric acid (UA) is not limited in its causing gout, urinary stones, and so called "uric acid nephropathy" only, on the contrary, it was found that UA per se is a risk factor in cardiovascular and renal damage, in which endothelial dysfunction plays a critical role. In the process of endothelial dysfunction, mitochondrial calcium homeostasis imbalance can increased intracellular reactive oxygen species (ROS) production, inhibit endothelial nitric oxide synthase (eNOS) production and nitric oxide (NO) release, and lead to increased inflammatory cytokine production. As a result, the main purpose of this study is to clarify whether UA induced endothelial injury and inflammation were also caused by this classical process, or have another special mechanism.Objective:To investigate the mechanisms of intracellular calcium in the process of UA induced endothelial dysfunction and inflammation.Methods:Human umbilical vein endothelial cells (HUVEC-C) cultured in vitro were stimulated with UA in different concentrations and different time points. The cytoplasmic calcium ([Ca2+]cyt) specific probe, Fluo-3 AM, and the mitochondrial calcium ([Ca2+]mito) specific probe, Rhod-2 AM, were employed to study the intracellular calcium changing after the UA stimulation, using confocal laser microscopy. The intracellular superoxide anion (·O2-) specific probe CM-H2DCFDA were employed to detect the changes in total·O2-, and the mitochondria·O2- specific probe, cpYFP, to detect the mitochondrial·O2-changes. Furthermore, we used reverse transcription-polymerase chain reaction (RT-PCR), Western Blot and enzyme-linked immunosorbent assay (ELISA) to detect eNOS and a series of inflammatory factors such as C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6) in mRNA and protein levels expression, respectively. In the second part of this study, different specific blockers for intracellular calcium channels were employed to inhibit the transduction through these channels. These blockers are, Nicardipine for L-type calcium channel, KB-R7943 for plasma membrane Na+/Ca2+exchanger (NCXpm), Ru360 for mitochondrial calcium uniporter (MCU), CGP-37157 for mitochondrial Na+/Ca2+exchanger (NCXmito), Thapsigangin for sarcoplasmic reticulum/endoplasmic reticulum calcium ATPase (SERCA),2-APB for 1,4,5-triphosphate receptor (IP3R), and high dose of Ryanodine for Ryanodine receptor (RyR).Results:High concentration of UA induced endothelial dysfunction in a time and concentration dependent manner, marked as reduced eNOS production and NO release. In this process, [Ca2+]mito was significantly increased, while [Ca2+]cyt did not changed; ROS production was significantly increased, so as the release of inflammatory factors. When the blockers for IP3R and NCXmito were used, UA induced [Ca2+]mito, total·O2-levels and mitochondrial·O2-elevations were significantly decreased, while the reduction of NO release increased, and the elevated inflammatory factors generation were significantly decreased. However, when SERCA and MCU were blocked, we only observed the reduction of elevation of the [Ca2+]mito and the total·O2-levels, but not the protection effect for the endothelial dysfunction and inflammation.Conclusion:High concentration of UA can induce endothelial dysfunction and inflammation, in which the the [Ca2+]mito were increased significantly (mitochondrial calcium overload). ROS, in particular,·O2-production, plays a mediating role in this process. Calcium transport through IP3R and NCXmito may play an important role. The results of this study clarified the molecular mechanisms of UA induced endothelial dysfunction and inflammation, and may provide a new target for clinics.
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