Next, we measured the downstream target of Akt/eNOS phosphorylation that is NO production (Fig

Next, we measured the downstream target of Akt/eNOS phosphorylation that is NO production (Fig. action of NM may contribute to the regulation of cardiovascular function. and NO production, arginase activity in vitro; mediated through the Akt/eNOS phosphorylation dependent signaling pathway. METHODS Cell culture Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego, CA, USA) and cultured in endothelial growth A 83-01 medium (EGM-2). Sub-confluent, proliferating HUVECs at passages 2~8 were used. Western blot analysis Anti-phospho-eNOS antibody was purchased from Cell Signaling (Beverly, MA, USA). Anti-NOS3, anti–actin, anti-phospho-Akt and total Akt antibodies were purchased from Santa Cruz Biotechnology (Santa A 83-01 Cruz, CA, USA). Western blot analysis was performed by boiling 30 g of whole cell lysate or 30 g of tissue homogenate (obtained from rat A 83-01 aorta) in sodium dodecyl sulfate-polyacrylamide gel Rabbit polyclonal to AML1.Core binding factor (CBF) is a heterodimeric transcription factor that binds to the core element of many enhancers and promoters. electrophoresis (SDSCPAGE) loading buffer, before separation by electrophoresis and transfer to A 83-01 a nitrocellulose membrane. After incubation in appropriate primary and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), chemiluminescent signaling was developed using Super Signal West Pico or Femto Substrate from Thermo Fisher Scientific (Pierce, Rockford, IL, USA). Blots were imaged and band densities quantified with a Gel Doc 2000 Chemi Doc system using Quantity One software from Bio-Rad (Hercules, CA, USA). Values were normalized to a -actin loading control. Animals The present study utilized 6 week aged male SpragueCDawley (SD) rats (Samtako, Osan, Korea) with body weights of between 250 and 280 g. All experimental procedures adhered to the guidelines of Chungnam National University regarding the use and care of animals. All animals were housed in a standard environment with a 12:12 h light/dark cycle, a constant room temperature maintained at 20~25, and 40~60% humidity. Food and water were supplied ad libitum. Nitrite and nitrate measurements Two NO metabolites, nitrite (NO2C) and nitrate (NO3C), the stable breakdown products of NO, were quantified using a commercially available Nitrate/Nitrite Fluorometric Assay Kit from Cayman Chemicals (Lexington, KY, USA), as per the manufacturer’s instructions. Plasma obtained from the rat blood was deproteinized using a 10 kDa cutoff filter (Microcon YM-10, Millipore, USA) and used for the quantification of NO. DAF-FM DA staining DAF-FM DA is usually a cell-permeable fluorescent probe for the detection of NO. This was chosen in preference to the widely used DAF-2 as DAF-FM and DAF-FM DA are more sensitive to NO, more photo-stable, and less pH sensitive. DAF-FM DA permeates living cells and is rapidly transformed into water-soluble DAF-FM by cytosolic esterases. Aortic sections stained with DAF-FM DA were imaged using a fluorescence microscope. All images from control and NM treated rings were captured using identical laser intensity, brightness, and contrast settings. Arginase activity assay Arginase activity was measured using Quanti Chrom Arginase Assay Kit (DARG-200) from Bioassay systems (Hayward, CA, USA) following the manufacturer’s instructions. Vascular reactivity Rats were sacrificed via sodium pentobarbital overdose. A mid-sternal split was quickly performed, A 83-01 and the descending thoracic aorta was carefully excised and placed in ice-cold Krebs buffer (118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, 1.2 mM MgSO4, 11 mM glucose, and 0.0026 mM EDTA-CaNa 2). The aorta was cleaned of excess fat, cut transversely into 5~10 rings (2.0~3.0 mm), and maintained at 37 and pH 7.4. Endothelium-dependent vasodilation was determined by generating doseCresponse curves in aortic rings pre-constricted with phenylephrine. Statistical analysis All experiments were performed at least three times. All data are expressed as meansstandard deviations. Statistical analysis was performed using Sigma Stat (Systat Software, La Jolla, CA, USA)..