U46619

ADMA, BK and L-citrulline were purchased from Sigma-Aldrich (St. Louis, MO, USA). U46619 was from Cayman Chemical (Ann Arbor, Mi, USA). Stock solutions of U46619 and BK were kept at –20 °C until required.

The reagents that we used are given below: SQ29548 and U46619 (Cayman chemical, Ann Arbor, MI, USA); rat anti-mouse CD324 (Biolegend, San Diego, CA, USA); rat anti mouse CD41 (AbD Serotec, Oxford, UK); rabbit anti-thromboxane synthase antibody (Abcam, Cambridge, UK); goat anti-VE-cadherin antibody (Santa Cruz, Dallas, Texas, USA); rhodamine-phalloidin (Life Technologies, Carlsbad, CA, USA); Escherichia coli lipopolysaccharide O55:B5, 66,100 Da FITC-dextran, and 70,000 Da FITC-dextran (Sigma-Aldrich, St. Louis, MO, USA); Ozagrel (LKT laboratories, Inc., St. Paul, MN, USA); Y27632 (Merck Biosciences, Darmstadt, Germany); LaCl₃ (Nakarai tesque, Kyoto, Japan); and histamine dihydrochloride (Wako, Osaka, Japan).

The composition of the KRB buffer was (in mM): NaCl 118.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.0, KCl 4.7, CaCl 2.5 and glucose 5.5. Solutions containing different concentrations of K⁺ were prepared by replacing part of the NaCl of the KRB buffer by an equimolar amount of KCl. U46619 was obtained from Cayman Chemical (Ann Arbor, MI). The other drugs were from Sigma (St. Louis, MO). All drugs were dissolved initially in distilled deionized water (except U46619, A23187 and BRL 37344 in DMSO and forskolin and indomethacin in ethanol) to prepare adequate stock solutions and further dilutions were also made in deionized water. The final bath concentration of DMSO or ethanol did not exceed 0.1 % and had no effect on mechanical activity.

U46619, SQ29548, and 2-APB were purchased from Cayman Chemical (Ann Arbor, MI). Hanks balanced salt solution (HBSS) and Fura-2 AM were obtained from Invitrogen (Carlsbad, CA). Enzymes for cardiomyocyte digestion were purchased from Worthington Biochemical (Lakewood, NJ). Total RNA Isolation kits were purchased from IBI Scientific (Peosta, IA), and the real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was performed using a TaqMan RNA-to-CT 1 step kit and primers and probes from ABI (Carlsbad, CA). Primary antibody for TXA2R was purchased from Abcam (Cambridge, MA). Gentamicin and fetal bovine serum was obtained from Sigma-Aldrich (St. Louis, MO). DeadEnd Fluorometeric TUNEL stain was purchased from Promega (Madison, WI). All remaining reagents were purchased from Fisher Scientific.

L-NAME, ACh, PE, AA, and the non-selective COX inhibitor indomethacin were purchased from Sigma (St Louis, MO, USA). The TP agonist U46619, PGI₂, PGF_2α, PGE₂, and PGD₂, the TP antagonist SQ29548, the IP antagonist CAY10441, the EP3 antagonist L798106, and the EP1 antagonist, SC19220 were bought from Cayman Chemical (Ann Arbor, MI, USA). The composition of physiological salt solution (PSS; pH 7.4 with 95%O₂–5% CO₂) was as follows (in mM): NaCl 123, KCl 4.7, NaHCO₃ 15.5, KH₂PO₄ 1.2, MgCl₂ 1.2, CaCl₂ 1.25, and D-glucose 11.5. The 60 mM K⁺ -PSS (K⁺) was prepared by replacing an equal molar of NaCl with KCl.
L-NAME, PE, AA, and ACh were dissolved in distilled water (purged with N₂ for dissolving AA), while PGI₂ was dissolved in carbonate buffer (50 mM, pH 10.0). PGF_2α, PGE₂, PGD₂, CAY10441, SQ29548, L798106, and indomethacin were dissolved in dimethyl sulfoxide (DMSO). The final ratio of a solvent (distilled water, carbonate buffer, or DMSO) to working PSS was 0.5/1,000, which doesn’t alter the final pH value of the working buffer (pH 7.4). The concentration of an inhibitor or antagonist used was based on previous reports, which would selectively inhibit the effect of its intended target27 (link)54 (link)55 (link).

After pigs had been monitored for 20 h following I/R, they were sacrificed by inducing cardiac arrest via application of high doses of propofol (200 mg) and potassium chloride (40 mmol) intravenously. Next, the eyes were enucleated and transferred into ice-cold Krebs–Henseleit buffer of the following ionic composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11 glucose. After opening the eye globe, the retina was carefully isolated as described previously [34 (link)]. Retinal arterioles of the first order were then isolated and cleaned from surrounding retinal tissue by Vannas scissors and fine-point tweezers. Vascular measurements were conducted after cannulation of blood vessels onto two micropipettes as described previously [53 (link),54 (link)]. Only when the luminal arteriole diameter decreased by at least 30% in response to 100 mM KCl, the vessel was used for experiments. Concentration–response curves were started after development of basal tone, which was achieved after an equilibration time of 45 min. A myogenic tone of 30%–50% of the initial diameter was defined as preconstricted for the following concentration–response curves to vasodilators. If not achieved, the thromboxane mimetic, U46619 (Cayman Chemical, Ann Arbor, MI, U.S.), was titrated into the circulating Krebs–Henseleit buffer to achieve proper preconstriction.