Figure 1
(A) Quantificative RT-PCR analysis of OX1R and OX2R in the rat hypothalamus (hypo) and rat heart (***P<0.001 for heart compared with hypothalamus). (B) Immunohistochemical analysis of rat heart ventricular sections (n=3) for OX1R (I) and OX2R (II). Magnification ×400. Negative control (III) confirmed the specificity of the immunostaining. (C) Immunohistochemical analysis of OX1R (I) and OXR2 (II) in rat cardiomyocytes and negative control (III). Identical results obtained from four independent experiments; measuring at least 50 cells. (D) Expression of OX1R and OX2R in rat heart compartments (n=3). Lane 1 cDNA from LV; Lane 2 cDNA from left auricle; Lane 3 cDNA from septum; Lane 4 cDNA from RV; Lane 5 cDNA from right auricle; and Lane 6 is DNA marker. (E) Single-cell RT-PCR in cardiomyocytes (n=6): Lane-1: β-Actin; Lane-2: OX1R; Lane-3: negative control; Lane-4: OX2R. (F) Expression of OX1R and OX2R in different human cardiac compartments: Lane-1: aorta; Lane-2: apex of the heart; Lane-3: LA; Lane-4: right atrium (RA); Lane-5: right auricle; Lane-6: left auricle; Lane-7: LV; Lane-8: RV; Lane-9: interventricular septum; and Lane-10: atrioventricular (AV) node. (G) Expression of prepro-orexin in rat heart chambers (n=3). Prepro-orexin is expressed as a 302-bp PCR product in the LV (lane 1), LA (lane 2), interventricular septum (lane 3), RV (lane 4), and RA (lane 5). M = Marker; DNA ladder. (H,I) Prepro-orexin (PPO) and the cleaved peptides OR-A and OR-B were detected using Western blotting, with molecular weights of 15 kDa for PPO, 3.5 and 2.9 kDa for OR-A (H) and OR-B (I), respectively; Lane 1: rat heart lysates; and Lane 2: rat hypothalamus lysates (n=3). (J) Immunohistochemical analysis for OR-A on rat thymus section (I) and rat spleen (II). (K) Immunohistochemical analysis of rat heart ventricular sections (n=3) for OR-A (I) and OR-B (II) and negative control (III). Magnification ×400. (L) Rat cardiomyocyte immunofluorescent analysis of OR-A (I), OR-B (II) and negative control (III). Identical results obtained from four independent experiments, measuring at least 50 cells.
Expression of orexin-1 (OX1R) and orexin-2 (OX2R) receptors in the rat and human heart

(A) Quantificative RT-PCR analysis of OX1R and OX2R in the rat hypothalamus (hypo) and rat heart (***P<0.001 for heart compared with hypothalamus). (B) Immunohistochemical analysis of rat heart ventricular sections (n=3) for OX1R (I) and OX2R (II). Magnification ×400. Negative control (III) confirmed the specificity of the immunostaining. (C) Immunohistochemical analysis of OX1R (I) and OXR2 (II) in rat cardiomyocytes and negative control (III). Identical results obtained from four independent experiments; measuring at least 50 cells. (D) Expression of OX1R and OX2R in rat heart compartments (n=3). Lane 1 cDNA from LV; Lane 2 cDNA from left auricle; Lane 3 cDNA from septum; Lane 4 cDNA from RV; Lane 5 cDNA from right auricle; and Lane 6 is DNA marker. (E) Single-cell RT-PCR in cardiomyocytes (n=6): Lane-1: β-Actin; Lane-2: OX1R; Lane-3: negative control; Lane-4: OX2R. (F) Expression of OX1R and OX2R in different human cardiac compartments: Lane-1: aorta; Lane-2: apex of the heart; Lane-3: LA; Lane-4: right atrium (RA); Lane-5: right auricle; Lane-6: left auricle; Lane-7: LV; Lane-8: RV; Lane-9: interventricular septum; and Lane-10: atrioventricular (AV) node. (G) Expression of prepro-orexin in rat heart chambers (n=3). Prepro-orexin is expressed as a 302-bp PCR product in the LV (lane 1), LA (lane 2), interventricular septum (lane 3), RV (lane 4), and RA (lane 5). M = Marker; DNA ladder. (H,I) Prepro-orexin (PPO) and the cleaved peptides OR-A and OR-B were detected using Western blotting, with molecular weights of 15 kDa for PPO, 3.5 and 2.9 kDa for OR-A (H) and OR-B (I), respectively; Lane 1: rat heart lysates; and Lane 2: rat hypothalamus lysates (n=3). (J) Immunohistochemical analysis for OR-A on rat thymus section (I) and rat spleen (II). (K) Immunohistochemical analysis of rat heart ventricular sections (n=3) for OR-A (I) and OR-B (II) and negative control (III). Magnification ×400. (L) Rat cardiomyocyte immunofluorescent analysis of OR-A (I), OR-B (II) and negative control (III). Identical results obtained from four independent experiments, measuring at least 50 cells.

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