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Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
Na+/H+ exchanger-1 inhibitors decrease myocardial superoxide production via direct mitochondrial action
Carolina D Garciarena1, Claudia I Caldiz, María V Correa
1Centro de Investigaciones Cardiovasces, Facultad de Ciencias Médicas, UNLP 60 y 120, 1900 La Plata, Argentina.
Abstract:
The possibility of a direct mitochondrial action of Na(+)/H(+) exchanger-1 (NHE-1) inhibitors decreasing reactive oxygen species (ROS) production was assessed in cat myocardium. Angiotensin II and endothelin-1 induced an NADPH oxidase (NOX)-dependent increase in anion superoxide (O(2)(-)) production detected by chemiluminescence. Three different NHE-1 inhibitors [cariporide, BIIB-723, and EMD-87580] with no ROS scavenger activity prevented this increase. The mitochondria appeared to be the source of the NOX-dependent ROS released by the "ROS-induced ROS release mechanism" that was blunted by the mitochondrial ATP-sensitive potassium channel blockers 5-hydroxydecanoate and glibenclamide, inhibition of complex I of the electron transport chain with rotenone, and inhibition of the permeability transition pore (MPTP) by cyclosporin A. Cariporide also prevented O(2)(-) production induced by the opening of mK(ATP) with diazoxide. Ca(2+)-induced swelling was evaluated in isolated mitochondria as an indicator of MPTP formation. Cariporide decreased mitochondrial swelling to the same extent as cyclosporin A and bongkrekic acid, confirming its direct mitochondrial action. Increased O(2)(-) production, as expected, stimulated ERK1/2 and p90 ribosomal S6 kinase phosphorylation. This was also prevented by cariporide, giving additional support to the existence of a direct mitochondrial action of NHE-1 inhibitors in preventing ROS release. In conclusion, we report a mitochondrial action of NHE-1 inhibitors that should lead us to revisit or reinterpret previous landmark observations about their beneficial effect in several cardiac diseases, such as ischemia-reperfusion injury and cardiac hypertrophy and failure. Further studies are needed to clarify the precise mechanism and site of action of these drugs in blunting MPTP formation and ROS release.
Insights
Na(+)/H(+) exchanger-1 (NHE-1) inhibitors directly act on mitochondria to reduce reactive oxygen species (ROS) production. This finding impacts understanding of cardiac disease treatments involving NHE-1 inhibition.
Area of Science:
- Cardiovascular Research
- Mitochondrial Physiology
- Pharmacology
Background:
- Reactive oxygen species (ROS) play a role in cardiac dysfunction.
- Na(+)/H(+) exchanger-1 (NHE-1) inhibitors are investigated for cardiovascular benefits.
- The direct mitochondrial effects of NHE-1 inhibitors on ROS production remain unclear.
Purpose of the Study:
- To investigate the direct mitochondrial action of NHE-1 inhibitors on ROS production in cat myocardium.
- To determine if NHE-1 inhibitors can prevent NADPH oxidase (NOX)-dependent ROS generation.
- To elucidate the role of mitochondria in NOX-dependent ROS release and the effect of NHE-1 inhibitors.
Main Methods:
- Assessment of superoxide anion (O(2)(-)) production using chemiluminescence.
- Administration of Angiotensin II and endothelin-1 to induce ROS.
- Treatment with NHE-1 inhibitors (cariporide, BIIB-723, EMD-87580) and mitochondrial modulators.
- Evaluation of mitochondrial swelling and protein phosphorylation (ERK1/2, p90 ribosomal S6 kinase).
Main Results:
- Angiotensin II and endothelin-1 increased NOX-dependent O(2)(-) production.
- NHE-1 inhibitors prevented this increase without scavenging ROS.
- Mitochondria were identified as the source of NOX-dependent ROS via a ROS-induced ROS release mechanism.
- NHE-1 inhibitors, particularly cariporide, directly inhibited mitochondrial swelling and O(2)(-) production, suggesting MPTP pore action.
- Cariporide also prevented downstream signaling pathways (ERK1/2, p90 ribosomal S6 kinase phosphorylation).
Conclusions:
- NHE-1 inhibitors exert a direct mitochondrial effect by reducing ROS production.
- This action involves blunting the ROS-induced ROS release mechanism and potentially modulating the mitochondrial permeability transition pore (MPTP).
- These findings necessitate a re-evaluation of the therapeutic mechanisms of NHE-1 inhibitors in cardiac diseases like ischemia-reperfusion injury and heart failure.
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