Drugs were added to the organ chambers in volumes no greater than 0.2 ml. M), an endothelium-dependent vasodilator. The inhibitory effect of melatonin on SNP- and UK14,304-induced relaxations was abolished in the presence of the selective MT2 receptor antagonists 4-phenyl-2-propionamidotetralin (4P-PDOT; 10?7 M) and luzindole (10?7 M). In contrast to melatonin, the selective MT3 receptor agonist 5-methoxycarbonylamino-for 10 min. 3-AP Supernatant was collected, and protein determination was performed using a Bio-Rad Dc Protein assay kit (Bio-Rad Laboratories, Hercules, CA). Aliquots of supernatant containing equal amounts of protein (100 g) were separated on 12% polyacrylamide gel by SDS-polyacrylamide gel electrophoresis, and proteins were electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories). Blots were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS, pH 3-AP 7.4) and incubated overnight at 4C with a primary antibody specific for MT1 receptors, MT2 receptors, or MT3 receptors using a dilution of 1 1:200 (Santa Cruz Biotechnology Inc.). Membranes were washed two times for 15 min using PBS Tween 20 and incubated with a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology Inc.). To ensure equal loading, the blots were analyzed for -tubulin expression using an anti–tubulin antibody (Santa Cruz Biotechnology Inc.). Immunodetection was performed using an enhanced chemiluminescence light detection kit (Thermo Fisher Scientific, Waltham, MA). Immunohistochemistry. Frozen tissue blocks of freshly isolated porcine coronary arteries (= 4) were sectioned at 8 m and mounted onto ProbeOn Plus microscopic slides (Thermo Fisher Scientific). Sections were fixed with ice-cold acetone and air-dried for 30 min at room temperature. Nonspecific antibody binding was blocked with normal donkey serum [1.5% (v/v); Santa Cruz Biotechnology Inc.] in PBS for 1 h at room temperature. Sections were incubated overnight at 4C in PBS containing a primary antibody against MT2 receptors (Santa Cruz Biotechnology Inc.) and/or smooth muscle -actin (Santa Cruz Biotechnology Inc.). For colocalization of the MT2 receptors with smooth muscle -actin, double immunofluorescent staining was performed by incubating the tissue sections with more 3-AP than one primary antibody at the same time. Detection of the primary antibodies against the MT2 receptors and smooth muscle -actin was accomplished using Alexa Fluor 488-conjugated (i.e., rabbit anti-goat IgG; Invitrogen, Carlsbad, CA) and Texas red-conjugated (i.e., goat anti-mouse IgG; Santa Cruz Biotechnology Inc.) secondary antibodies, respectively. Controls were incubated with either nonimmune goat IgG or primary antibody immunoneutralized with a specific blocking peptide (Santa Cruz Biotechnology Inc.) instead of primary antibody. All dilutions and thorough washes between stages were performed using PBS containing Triton X-100 [0.3% (v/v)] unless otherwise stated. Sections were drained by blotting with filter paper, and a drop of mounting medium containing an anti-fade reagent (Vector Laboratories, Burlingame, CA) was added to the slides. The images of the sections Rabbit Polyclonal to Histone H2A were obtained using an Olympus confocal laser-scanning microscope (Olympus, Tokyo, Japan). The images were generated using Olympus FluoView FV300 (v. 4.3) confocal software and Adobe Photoshop 5.5 (Adobe Systems, Mountain View, CA). Organ Chamber Studies. Coronary arterial rings were suspended in water-jacketed organ chambers filled with 25 ml of physiological salt solution, as described previously (Yang et al., 2001). The organ chamber solution was aerated with a mixture of 95% O2/5% CO2, and the temperature was maintained at 37C throughout the experiment. Each ring was suspended by means of two fine stainless steel wire clips passed through the lumen; one clip was anchored inside the organ chamber, and the other was connected to a force transducer (model FT03; Grass Instruments, Quincy, MA). Isometric tension was measured and recorded on a Grass polygraph. The tissues were stretched progressively to the optimal point of their lengthCtension relationship using 20 mM KCl to generate a standard contractile response. After this procedure, the rings were allowed to equilibrate at their optimal length for at least 30 min before exposure to any vasoactive substances. The absence or presence of intact.