KCa3.1 upregulation preserves endothelium‐dependent vasorelaxation during aging and oxidative stress

Summary Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium‐dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial KCa3.1, which contributes to EDR, is upregulated by H2O2. We investigated whether KCa3.1 upregulation compensates for diminished EDR to NO during aging‐related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1‐phosphate were increased in aged wild‐type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild‐type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age‐matched wild‐type mice. Increased H2O2 levels induced Fyn and extracellular signal‐regulated kinases (ERKs) phosphorylation and KCa3.1 upregulation. Catalase/GPX1 double knockout (catalase−/−/GPX1−/−) upregulated KCa3.1 in MAECs. NO production was decreased in aged wild‐type, CerS2 null, and catalase−/−/GPX1−/− MAECs. However, KCa3.1 activation‐induced, NG‐nitro‐l‐arginine‐, and indomethacin‐resistant EDR was increased without a change in acetylcholine‐induced EDR in aortic rings from aged wild‐type, CerS2 null, and catalase−/−/GPX1−/− mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1‐phosphate induced similar changes in levels of the antioxidant enzymes and upregulated KCa3.1. Our findings suggest that, during aging‐related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H2O2 and thereby upregulate KCa3.1 expression and function via a H2O2/Fyn‐mediated pathway. Altogether, enhanced KCa3.1 activity may compensate for decreased NO signaling during vascular aging.

with a 10× eyepiece. Confocal laser-scanning microscopic images of DHE were acquired in an excitation wavelength of 543 nm, a rhodamine emission filter, and a Zeiss Plan-Apochromat 63× oil immersion objective with a 10× eyepiece. Fluorescent intensities acquired from confocal laser-scanning microscopy were quantified by image processing software Image J (http://imagej.nih.gov/ij).

Measurement of intracellular nitric oxide (NO)
To monitor quantitative changes in NO production from primary MAECs, the NO fluorescence detection probe kit (Enzo Life Sciences, Farmingdale, NY) was used according to the manufacturer's instruction (Wardman 2007). Briefly, MAECs plated on 96-well microplates were incubated under normal cell-culture conditions with non-fluorescent cell-permeable NO detection dye (10 M) for 20 min that react with NO in the presence of oxygen with high specificity and accuracy, yielding a red fluorescent product. Levels of fluorescence were measured by using a filter set of 643 nm (ex)/670 nm (em) in fluorescence reader (model SpectraMax, Molecular Devices, Sunnyvale, CA).

Polymerase chain reaction (PCR)
RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen Inc, Valencia, CA), and then reverse transcribed using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). PCR was performed on PCR Thermal Cycler (BioRad, Hercules, CA) or ABI 7000 sequence detection system (Applied Biosystems) using a SYBR Green PCR Master Mix (Applied Biosystems). The primers used were listed in Table 1. Table 1 Primers used in current study.
Gene Sequence
Sphingosine, S1P and sphinganine were extracted with the mixture of 50 µL cell lysate (50 μg) or 10 mg tissue, 100 pM internal standard C17-sphingosine and C17-S1P, 1.2 mL chloroform/methanol (2:1, v/v) and 0.3 mL 0.1N HCl. The lower phase was collected after repeated extraction and dried under a vacuum. The resulting residue was re-dissolved in 100 μL methanol, and 10 μL was then injected into the LC-ESI-MS/MS machine. For optimization, a mixture of ceramide standards or S1P and sphingosine was infused directly into the mass spectrometer and all source parameters and ionization conditions were adjusted to improve the sensitivity of the assay. Extracted samples (10 μL     (100-week-old) and young (15-week-old) wild-type mice. Aortic tissue from a mouse was used in each experiment (A-C). Bar graphs were made with pooled data from three experiments performed with three different cultures or aortas. Results were normalized to GAPDH levels. *P < 0.05, **P < 0.01 versus age-matched wild-type.