Expression of Melatonin Receptor 1 in Rat Mesenteric Artery and Perivascular Adipose Tissue and Vasoactive Action of Melatonin

Fig. 1. The MT1, but not MT2, gene is expressed in mesenteric artery (MA) and perivascular adipose tissue (PVAT) derived from MA (MA-PVAT). a RT-PCR analysis of MT1 mRNA expression in MA, MA-PVAT, and eye. Total RNA isolated from rat tissues was subjected to RT-PCR using specific primers for MT1 (316 bp) cDNA. Non-reversely transcribed samples (-RT, RNA instead of cDNA) and negative controls (PCR-neg.co, water instead of cDNA; RT-neg.co, water instead of RNA) are also shown. b RT-PCR analysis of MT2 mRNA expression in MA, PVAT derived from MA (MA-PVAT), and eye. Total RNA isolated from rat tissues was subjected to RT-PCR using specific primers for MT2 (390 bp; nested PCR, upper panel) and β-actin (496 bp; ACTB, lower panel) cDNAs. Two individual samples per tissue are shown. c Relative gene expression levels of MT1 in rat MA (n = 6) and PVAT derived from MA (MA-PVAT, n = 5). The experiments were carried out in technical triplicates for each sample. The distribution of data is displayed by box plots. The box represents the range from the first to third quartiles; the band near the middle of the box is the median, and the lines above and below the box indicate the locations of the minimum…

Fig. 2. MA and MA-PVAT express MT1. a Rat cerebellum (Cb) and eye were used to validate the antibody. Total tissue lysates (25 µg/lane) were probed with anti-MT1 antibody (left panel) or anti-MT1 antibody in the presence of the specific blocking peptide (right panel) by Western blot. b Total tissue lysates of rat Cb (20 µg/lane), MA (75 µg/lane), and MA-PVAT (75 µg/lane) were probed with anti-MT1 antibody. Arrows point at the two major bands of approximately 40 kDa and 50 kDa, respectively, detected in the tissues

Fig. 3. MT1 protein localization in smooth muscle layer of rat MA and associated PVAT. Tissue Sects. (4 μm) were stained with anti-MT1 antibody and a corresponding AlexaFluor647-conjugated secondary antibody (white staining). Nuclei were stained with DAPI (blue staining). a MA and associated PVAT stained for MT1. b Transmission light micrograph corresponding to (a). c Significantly reduced MT1 staining in MA and associated PVAT after pre-incubation of the anti-MT1 antibody with the corresponding blocking peptide. d Transmission light micrograph corresponding to (c). Arrows indicate adipocytes (examples), and arrowheads point to smooth muscle cells. Bar is 100 µm

Fig. 4. Frequency–response curves to transmural electrical stimulation in rat superior MA with intact PVAT (+ PVAT) and removed PVAT (- PVAT), before and after their incubation with melatonin (MEL). Values represent the mean ± SEM of 9 rats. *p < 0.05 arterial preparations with PVAT intact vs. PVAT removed (before melatonin incubation); +p < 0.05 before vs. after melatonin incubation (arteries without PVAT)