Dea nonoate

L-NIL (hydrochloride) and DEANONOate were purchased from Cayman Chemical (Ann. Harbor, MI). PTIO, SNAP, and Dithiothreitol were from Sigma-Aldrich (St. Louis, MO). Vemurafenib (PLX4032/RG7204) from Selleckchem (Houston, TX). All chemicals were prepared freshly from powdered stock on the day of each experiment. All cell culture reagents were from Invitrogen.

Purified H-NOX was incubated with 20 mM potassium ferricyanide at room temperature for 20 min to generate oxidized Fe(III)-heme. The protein was desalted using a PD-10 column equilibrated in 50 mM HEPES (pH 7.5) and 200 mM NaCl to remove excess potassium ferricyanide. The Fe(III)-H-NOX was exposed to an anaerobic environment using a COY Anaerobic Chamber (Coy Laboratory Products) to deoxygenate the protein. Oxygen-free Fe(III)-H-NOX was treated with 60 mM sodium dithionite (Na₂S₂O₄) at room temperature for 20 min to generate Fe(II) bound heme protein. The protein was again desalted as previously described, but under anaerobic conditions. NO-bound Fe(II)-H-NOX was formed by incubating the protein with the NO donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, Cayman Chemicals) dissolved in 10 mM sodium hydroxide and followed by desalting. Carbon monoxide(CO)-bound Fe(II)-H-NOX was generated by bubbling CO gas into the protein for 15 min in an airtight vial. Electronic absorption spectra of all complexes were measured on a Cary 100 UV-Vis spectrophotometer equipped with a constant temperature bath set to 25°C.

The following compounds were used for myograph studies and purchased from Sigma-Aldrich (St. Louis, MO, USA): (R)-(−)-phenylephrine hydrochloride (PE), acetylcholine chloride (ACh), adenosine 5′ diphosphate sodium salt (ADP), potassium phosphate monobasic, potassium chloride, D-(+)-glucose, magnesium sulfate heptahydrate, sodium chloride, calcium chloride dehydrate, α,ß-methylene adenosine 5′-triphosphate (α,ß-MetATP), guanethidine, N_ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). DEA NONOate and NF449 were purchased from Cayman Chemicals (Ann Arbor, MI, USA), and MRS2500, 2-methylthioadenosine diphosphate trisodium salt (2-MeSADP), and tetrodotoxin citrate from Tocris (Bristol, United Kingdom). All compounds were dissolved in distilled water with the exception of atropine and DEA NONOate which were dissolved in 100% ethanol and further diluted using distilled water.

To test the effects of NO on RBC deformability, whole blood from 4 healthy volunteers was incubated with NO donors and agents that could affect nitric oxide synthase, and their deformability was measured via osmotic gradient ektacytometry. Whole blood was drawn from healthy volunteers into vacutubes with sodium heparin (BD, 367871). One milliliter samples were incubated for one hour at room temperature with 10 μM SNP (MP Biomedicals, 152061), 10 μM DEA-NONOate (Cayman, 82100), 1 mM L-NAME (Cayman, 80210) or 3 mM L-arginine (Sigma, A5006) as these concentrations of these compounds were previously shown to have significant effects on RBC deformability^{19 (link)}. After one hour the samples were placed on ice and scanned in the ektacytometer.