Biochemical and Molecular Aspects of Vascular Adrenergic Regulation of Blood Pressure in the Elderly
Figure 1
Vascular smooth muscle and chemokine receptor subtype 4 (CXCR4). (a) Aortae from 2-month-old animals () were isolated and mounted on an apparatus to measure vascular reactivity as described [12]. Vessels were exposed to three treatments. The control (CTRL) treatment was that vessels were contracted with phenylephrine (PE) and allowed to stabilize (approximately 10 minutes) that was followed with relaxation stimulated by increasing doses of isoproterenol (ISO). The stromal cell derived factor (SDF1; ligand for the CXCR4 receptor also known as CXCL12) treatment was similar except that after the PE-mediated contraction stabilized, 100 ng/mL SDF1 was added. Following a 5-minute SDF1 incubation, relaxation was stimulated by increasing doses of ISO. SDF1 did not alter the tone produced by PE alone. The AMD-3100 (AMD; a CXCR4 specific antagonist) treatment occurred with a 45 minute, 10 μM AMD incubation preceding the PE-mediated contraction and stabilization (10 minutes), which was followed by relaxation stimulated by increasing doses of ISO. AMD treatment did not effect PE-mediated contraction as compared to CTRL. Also conducted, but not shown, is that addition of AMD blocked the effect of SDF1 thereby implicating a direct effect of CXCR4 on impairing β-AR-mediated vasorelaxation. Data are expressed as percent of PE-induced contraction. The doses for AMD, and SDF1 were used as per LaRocca et al. [50]. (b) Aortic smooth muscle medial layers were homogenized, and membrane-specific fractions were prepared for western blotting as described [51]. Increasing concentrations (5 μg, 10 μg, and 20 μg; as determined with BCA analysis) of total protein extracts were loaded in each lane. A specific antibody for CXCR4 (Abcam; Cambridge, Mass, USA) was used to visualize expression of CXCR4 that is predicted to locate at approximately 39 kilodaltons (kD). Shown also is a size-indicating ladder (Ld).
