Daf 2

The reagents used were: NA, ACh, L-NAME and DAF-2 were obtained from Sigma-Aldrich, and human recombinant EGF was from PeproTech EC. Reagents were prepared in distilled water except NA that was dissolved in a solution of 0.9% (w/v) NaCl and 0.01% (w/v) ascorbic acid, and EGF that was dissolved in 25 mM HEPES-NaOH (pH 7.4). Reagent stock solutions were kept at −20°C and appropriate dilutions were made in KHS or HEPES-buffer on the day of the experiment.

NO production in the thoracic aorta was measured by using the NO-sensitive fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2, 8 μM, Sigma-Aldrich). Images were obtained with a fluorescence microscope (Nikon, Eclipse E600). Data were analyzed using the NIH Image J software.

Nitric oxide detection was performed as in Horchani et al. (2011) using the 4,5‐diaminofluorescein probe (DAF‐2; Sigma‐Aldrich) with the following changes. Either nodules (20–30 mg FW) or root segments (50–100 mg FW) were incubated in 1 ml of detection buffer (10 mM Tris‐HCl pH 7.4, 10 mM KCl) in the presence of 10 μM DAF‐2. As a control, NO production was measured in the same experimental system through the use of the Cu(II) fluorescein (CuFL) fluorescent probe (Strem Chemicals, Bischheim, France) instead of DAF‐2 in the detection buffer as described in Horchani et al. (2011). Similar results were obtained with both probes. The production of NO was measured with a spectrofluorimeter‐luminometer (Xenius, Safas, Monaco).

Mammalian Ringer solution (He et al., 1996) was used for dissecting mesenteries, superfusing tissues, and preparing perfusion solutions. The composition of the mammalian Ringer solution was (in mmol/L) 132 NaCl, 4.6 KCl, 2 CaCl₂, 1.2 MgSO₄, 5.5 glucose, 5.0 NaHCO₃, 20 N‐2‐hydroxyethylpiperazine‐N’‐2‐ethanesulfonic acid (HEPES), and Na‐HEPES. All perfusates used for control and test perfusion contained BSA (10 mg/ml). Fura‐2 AM was purchased from Invitrogen. H₂O₂ (30%), DAF‐2, DAF‐2 DA, LaCl₃, L‐NMMA, DPI, and DHE were purchased from Sigma. All the fluorescent dyes except for Alexa488‐conjugated secondary antibody (in BSA‐Ringer solution) were prepared in DMSO for stock solution, and at least 1:1000 dilution was made for the final working solutions. ONOO⁻ and degraded ONOO⁻ were purchased from EMD Millipore and were diluted in the BSA‐Ringer solution into final concentrations of 10 or 50 µmol/L. All perfusates containing the test reagents were freshly prepared before each cannulation.

HAEC were seeded into a 96 well plate and transfected as described above. At time of the assay, cells were incubated with Krebs buffer containing 0.25 µM 4,5-diaminofluorescein diacetate (DAF-2, Sigma) in presence or absence of 10 µM of the unspecific nitric oxide synthase (NOS) inhibitor l-N⁵-(1-Iminoethyl)ornithine hydrochloride (l-NIO, Sigma), or 0.25 µM of the DAF-2 negative control 4-aminofluorescein diacetate (4-AF-DA, Merck Millipore), respectively, for 20 min at 37 °C. Then l-arginine with or without calcium-ionophore (positive control) was added to the wells and fluorescence read at 490/525 nm (excitation/emission) to set the baseline. After 30 min the fluorescence was measured again and the percentage of nitric oxide increase calculated.

The drugs used were as follows: DAF-2, HE, L-NA hydrochloride, ACh chloride, potassium chloride, SNP, and pinacidil (Sigma-Aldrich). The stock solutions (10 mM) of drugs were prepared in distilled water, except for NA which was dissolved in NaCl (0.9%)-ascorbic acid (0.01% w/v) solution. These solutions were kept at -20°C, and appropriate dilutions were made in KHS on the day of the experiment.

A NO-sensitive fluorescent probe, diaminofluorescein 2 (DAF-2) (Sigma-Aldrich), was employed for the quantification of NO released by RAECs as previously described (Toral et al., 2015 (link)). Succinctly, RAECs were incubated in 96-well plates, as already mentioned. Then, cells were washed with phosphate-buffered saline (PBS) + 0.9 mM Ca²⁺ and then were preincubated with L-arginine (100 μM in PBS, 5 min, 37°C) (Sigma-Aldrich). Afterward, DAF-2 (0.1 μM) was added for 2 min, and then a calcium ionophore, calimycin (A23187, 1 μM) (Sigma-Aldrich), was incubated for 30 min. Subsequently, fluorescence intensity [expressed as arbitrary units (AU)] was determined at excitation and emission wavelengths of 490 and 510 nm, respectively, using a spectrofluorometer (Fluorostart; BMG Labtechnologies, Offenburg, Germany). Autofluorescence was subtracted from all values. To determine NO-independent fluorescence signal induced by A23187, N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) was added to some wells 15 min before the addition of L-arginine. The difference between the fluorescence signal with and without L-NAME was considered as NO production.