SGC in SMC acts as Nitrite Reductase leading to NO formation

Abstract

Several heme proteins including hemoglobin and nitric oxide synthase (NOS) have been documented to serve as nitrite reductase under anoxia. Since sGC is also a heme protein and an important signaling biomolecule, we hypothesized that it may function as nitrite reductase in cultured smooth muscle cells. Millimolar levels of nitrite were required to detect cellular NO as measured by NO specific fluorescent dye (CuFL2E). However, in chemiluminescence analysis of cell lysates, addition of 400–600 μM NO2− was sufficient to detect measurable NO formation under anoxic conditions. This NO formation was inhibited with ODQ showing sGC in the cell lysate as the source of nitrite reduction. CO is a known activator of sGC and thus CO donor (50 μM CORM‐2) significantly activated nitrite reduction to NO. The cellular uptake of NO2− was studied using the AE‐1 inhibitor, DIDS and intracellular NO2− was measured by tri‐iodide assay. DIDS (300 μM) significantly inhibited NO2− uptake and showing that NO2− uses AE‐1 transport mechanism. These results demonstrate that sGC mediates NO2− reduction to form NO and that the AE‐1 channel is one of the pathways for NO2− entry into SMC. This work was supported by NIH grant SC1HL095101.