Figure 3

AT1 and AT2 receptors in isolated mitochondria (IM) from ventral mesencephalon and the dopaminergic neuron cell line MES 23.5. Effect of oxidative stress and aging. (a–c) Western blot (WB) of whole homogenate (Wh; n=4) and pure IM (Mt; n=4) from the nigral region (a) and MES 23.5 cells (c) showing different compartment markers used to assess the purity of the IM: voltage-dependent anion channel (VDAC) as a mitochondrial marker, tubulin as a cytosol marker and histone deacetylase 2 (HDAC2) as a nuclear marker. Note the higher expression of mitochondrial AT2 compared with AT1 receptors (b). (d)The levels of lactate dehydrogenase specific activity (LDH; micromol of substrate/min/mg; a marker for cytosolic and synaptosomal contamination) of the nigral region and MES 23.5 pure IM (Mt; n=6) were negligible relative to those in Wh. (e and f) WB of IM from the MES 23.5 cells transfected with AT2-YFP (yellow fluorescent protein; n=4) or AT1-EGFP (enhanced green fluorescent protein; n=3) showing the presence of fluorescent-tagged angiotensin receptors in IM in comparison with non-transfected cells (Co, control); 24 h treatment of cells, low doses of MPP⁺ (n=4) or N-acetyl-l-cysteine (NAC; n=3) induced increased expression of mitochondrial AT2 and AT1 receptors, respectively (AT2-YFP+MPP⁺; AT1-EGFP+NAC). (g–i) In young (n=3) and aged (n=4) rats, the expression of AT1 and AT2 receptors was analyzed by RT-PCR in nigral dopaminergic neurons labeled with red retrobeads (RRB) and isolated by laser microdissection (g–h), and by WB of IM from the nigral region of young (n=5–8) and aged rats (n=5-6) (i). Aging induced a significant increase in AT1 expression and a significant decrease in AT2 expression in both dopaminergic neurons and in IM. The results were normalized to the values of young animals. Data are means±S.E.M. *P<0.05 relative to the corresponding controls (Student’s t-test). Scale bars: 50 μm