Superoxide negatively modulates angiotensin II‐elicited calcium response via increasing nitric oxide uptake in vascular smooth muscle cells

Abstract

Angiotensin II (Ang II), a multifunctional hormone, increases cytosolic calcium concentration via inositol 1,4,5-trisphosphate (IP3) signal pathway in vascular smooth muscle cells (SMCs), resulting in cell contraction. In addition, Ang II can also raise the level of intracellular superoxide (O2−) via activation of NAD(P)H oxidase. Nitric oxide (NO), on the other hand, has been shown to inhibit mobilization of IP3-gated calcium channel through increased intracellular cGMP. We have previously demonstrated that O2− determines NO uptake rate by vascular SMCs. Thus, it is likely that Ang II may use O2− to increase the SMC-NO uptake rate, which may in turn serve as a negative feedback circuitry of calcium response. To demonstrate the role of O2− in the Ang II-elicited calcium response, first we showed that the calcium response is in an Ang II dependent manner using fluorescent dye, Fura-2, with a fluorescence spectrophotometer. After verifying the assay for calcium responses, we had SMCs compete with oxyhemoglobin for NO during the Ang II challenge. Increasing Ang II-enhanced intracellular O2− content by pretreatment of SMCs with NADPH or SOD inhibitors decreases the Ang II-elicited calcium response. In contrary, pretreatment of tiron, a O2− scavenger, increases the Ang II-stimulated calcium response. Taken together, our results demonstrate that O2− in vascular SMCs, at least in a certain range of concentrations, modulates negatively the Ang II-elicited calcium response via increasing the SMC-NO uptake rate, suggesting a beneficial role of O2− in SMCs in the blood vessel response.