cytochrome-c-t and cariporide

Na+/H+ exchanger isoform 1 (NHE1) is a major acid extrusion mechanism following intracellular acidosis. We hypothesized that stimulation of NHE1 after cerebral ischemia contributes to disruption of Na+ homeostasis and neuronal death. In the present study, expression of NHE1 was detected in cultured mouse cortical neurons. Oxygen and glucose deprivation (OGD) for 3 hours followed by 21 hours of reoxygenation (REOX) led to 68 +/- 10% cell death. Inhibition of NHE1 with the potent inhibitor HOE 642 or genetic ablation of NHE1 reduced OGD-induced cell death by approximately 40% to 50% (p < 0.05). In NHE1 +/+ neurons, OGD/REOX triggered significant increases in Na+ and Ca(i)2+. Genetic ablation of NHE1 and HOE 642 treatment reduced the rise of Na(i)+ by approximately 40% to 50% and abolished the OGD/REOX-mediated Ca2+ accumulation. Moreover, mitochondrial cytochrome C release was significantly attenuated by inhibition of NHE1 activity. These results imply that NHE1 activity disrupts Na+ and Ca2+ homeostasis and contributes to ischemic neuronal damage.

Topics: Animals; Brain Ischemia; Cation Transport Proteins; Cells, Cultured; Cerebral Cortex; Cytochromes c; Guanidines; Membrane Proteins; Mice; Mitochondrial Proteins; Neurons; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sulfones

Although increased Na(+)/H(+) exchanger type-1 (NHE-1) activity has been implicated in the pathogenesis of myocardial infarction, the role of NHE-1 in induction of apoptosis, and the potential mechanisms involved have not been fully characterized. This study tested the hypothesis that NHE-1 activity is involved in hypoxia (H)/re-oxygenation (Re)-induced cardiomyocyte apoptosis by increasing mitochondrial Ca(2+) ([Ca(2+)]m). Primary cultured neonatal rat cardiomyocytes were subjected to 4.5 h of H followed by 12 h of Re. Relative to H alone, the level of X-rhod-1 acetoxymethyl (AM)-labeled [Ca(2+)]m was increased, and the frequency of cell death (propidium iodide (PI) staining) and apoptotic cells (terminal deoxynucleotidyl transferase (TdT)-mediated-UTP nick end labeling [TUNEL]), confirmed by Annexin-V, were augmented at the end of Re, along with appearance of cytosolic cytochrome c, activation of caspase-3, and increased ratio of Bax and Bcl-2. Addition of cariporide (20 micromol/l), a well-known NHE-1 inhibitor, to cultured cells before H significantly reduced [Ca(2+)]m, the number of PI and TUNEL positive cells relative to the levels at end of Re, but did not completely eliminate these changes compared to Sham control. There was a strong trend for attenuation in increased levels of [Ca(2+)]m, and the number of PI and TUNEL positive cells when same dose of cariporide was added only at Re, but the difference in these variables did not reach significance. In contrast, the levels of [Ca(2+)]m and the number of PI and TUNEL positive cells were significantly reduced to a level comparable to Sham control when cariporide (20 micromol/l) was administered before H and during Re, respectively, associated with a reduction in cytosolic cytochrome c, caspase-3 activity and ratio of Bax and Bcl-2. In conclusion, these data suggest that NHE-1 is involved in induction of cardiomyocyte apoptosis during both H and Re through a [Ca(2+)]m-dependent manner, thereby resulting in activation of cytochrome c-caspase-3 signaling pathways.

Topics: Animals; Animals, Newborn; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Caspase 3; Caspases; Cell Hypoxia; Cells, Cultured; Cytochromes c; DNA Fragmentation; Flow Cytometry; Guanidines; In Situ Nick-End Labeling; Mitochondria, Heart; Myocytes, Cardiac; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Signal Transduction; Sodium-Hydrogen Exchangers; Sulfones