Oxidative stress by monoamine oxidase mediates receptor-independent cardiomyocyte apoptosis by serotonin and postischemic myocardial injury.

BACKGROUND: Serotonin (5-hydroxytryptamine [5-HT]), released by activated platelets during cardiac ischemia, is metabolized by the mitochondrial enzyme monoamine oxidase A (MAO-A). Because hydrogen peroxide is one of the byproducts of 5-HT degradation by MAO-A, we investigated the potential role of reactive oxygen species generated by MAOs in 5-HT-dependent cardiomyocyte death and post-ischemia-reperfusion cardiac damage. METHODS AND RESULTS: Treatment of isolated adult rat cardiomyocytes with 5-HT induced intracellular oxidative stress and cell apoptosis. The apoptotic cascade triggered by 5-HT involves release of cytochrome c, upregulation of proapoptotic Bax protein, and downregulation of antiapoptotic Bcl-2 protein. These effects were prevented by inhibition of amine transporter or MAO, antioxidants, or iron chelation. In contrast, cardiomyocyte apoptosis was only slightly affected by the 5-HT(2B) receptor antagonist SB 206553. In vivo, inhibition of MAO-A largely reduced myocardial ultrastructural damage induced by 30 minutes of ischemia followed by 60 minutes of reperfusion in the rat heart. Cardioprotective effects of MAO inhibitors were associated with the prevention of postischemic oxidative stress, neutrophil accumulation, and mitochondrial-dependent cell death and were not reverted by SB 206553. Administration of MAO-A inhibitors during ischemia was still effective in preventing cardiac damage. CONCLUSIONS: Our results supply the first direct evidence that oxidative stress induced by MAO is responsible for receptor-independent apoptotic effects of 5-HT in cardiomyocytes and postischemic myocardial injury. These findings provide new insight into the mechanisms of 5-HT action in the heart and may constitute the basis for novel therapies.

Dopamine D2-like receptor agonist bromocriptine protects against ischemia/reperfusion injury in rat kidney.

BACKGROUND: Dopamine, via activation of D1-like and D2-like receptors, plays an important role in the regulation of renal sodium excretion. Recently, we demonstrated that dopamine D2-like receptor agonist (bromocriptine) stimulates p44/42 mitogen-activated protein kinases (MAPKs) and Na+,K(+)ATPase (NKA) activity in proximal tubular epithelial cells. Since both these parameters are compromised in ischemia/reperfusion (I/R) injury to the kidney, we investigated whether bromocriptine protects against the injury. METHODS: In this study we used unilateral rat model of renal I/R injury. The Sprague-Dawley rats were divided into vehicle and bromocriptine groups. The vehicle and bromocriptine group was treated with vehicle and bromocriptine (500 microg/kg intravenously), respectively, 15 minutes before the induction of unilateral ischemia followed by 24- or 48-hour reperfusion. At the end of 24 or 48 hours the animals were sacrificed to collect control and ischemic kidney cortices, in which necrosis, apoptosis, NKA activity, NKA alpha1 subunit expression, and p44/42 MAPK phosphorylation were measured. RESULTS: We found extensive necrosis, apoptosis, and decreased NKA activity (with no change in alpha1 subunit) in the ischemic kidney cortex compared to the nonischemic cortex from the vehicle-treated rats as early as 24 hours post-reperfusion. In contrast, I/R injury-induced necrotic, apoptotic, and decrease in NKA activity were absent in the outer cortex of bromocriptine-treated rats after 24 or 48 hours. Interestingly, we detected significantly higher phosphorylation of p44/42 MAPKs in control and ischemic kidneys of bromocriptine-treated rats compared to those of vehicle-treated rats. CONCLUSION: Therefore, bromocriptine, a D1-like receptor agonist, may protect against I/R injury to proximal tubules of the kidney, via p44/42 MAPK activation.

Prevention of apoptotic and necrotic cell death, caspase-3 activation, and renal dysfunction by melatonin after ischemia/reperfusion.

The pineal hormone melatonin has been reported to protect tissue from oxidative damage. This study was designed to determine whether melatonin could prevent cell events leading to tissue injury and renal dysfunction after ischemia/reperfusion (I/R). Using an in vivo rat model of I/R, we show a significant increase in kidney malondialdehyde concentrations, reflecting lipid peroxidation, and cell apoptosis measured by TUNEL staining. This apoptotic cell death was associated with an increase in the activity of the proapoptotic factor caspase-3, determined by fluorometric protease activity assay. Histomorphological analysis of ischemic kidneys revealed that the most extensive tubular necrosis occurred at 24 and 48 h after reperfusion, which correlated with peak elevations in blood urea nitrogen and creatinine. Rat pretreatment with melatonin prevented lipid peroxidation, cell apoptosis, and necrosis and blocked caspase-3 activity. The prevention of tissue injury was associated with the improvement of renal function as shown by the decrease in blood urea nitrogen and creatinine concentrations. The demonstration that melatonin prevents postreperfusion apoptotic and necrotic cell death and improves renal function suggests that melatonin may represent a novel therapeutic approach for prevention of I/R injury.

Activation of pro-apoptotic cascade by dopamine in renal epithelial cells is fully dependent on hydrogen peroxide generation by monoamine oxidases.

Dopamine plays a critical role in regulation of different renal functions, including glomerular filtration, renin secretion, and sodium excretion. Recent studies have shown that some of the dopamine effects in the proximal tubule may involve hydrogen peroxide (H(2)O(2)) generation by the catecholamine-degrading enzyme monoamine oxidases (MAO). The present study is an investigation of the potential role of H(2)O(2) generated by MAO during dopamine degradation in apoptosis of proximal tubule cells. Dopamine concentrations between 50 and 200 micro M induced apoptosis of rat proximal tubule and monoamine oxidase B-transfected HEK 293 cells (+73% compared with untreated cells) but not in wild-type HEK 293 cell lacking monoamine oxidases. Apoptosis of proximal tubule cells was preceded by an increase in the ratio of Bax/Bcl2 proteins, the release of mitochondrial cytochrome c, caspase-3 activation, and DNA fragmentation. All these events required dopamine internalization into the cells, its metabolism by MAO, and H(2)O(2) production, as they were prevented by the dopamine uptake inhibitor GBR-12909, the irreversible MAO inhibitor pargyline, or the antioxidant N-acetylcysteine. These results show that, in renal proximal tubule cells, dopamine induces oxidative stress, activation of pro-apoptotic cascade, and cell apoptosis exclusively by mechanisms involving H(2)O(2) production by monoamine oxidases.

Age-dependent increase in hydrogen peroxide production by cardiac monoamine oxidase A in rats.

Oxidative stress is one of the factors involved in age-related impairment of cardiac function. In the present study, we investigated the role of the catecholamine-degrading enzyme monoamine oxidase (MAO) in H(2)O(2) production in the hearts of young, adult, and old rats. MAO-dependent H(2)O(2) production, measured by a chemiluminescence-based assay, increased with age, reaching the maximum in 24-mo-old rats (7.5-fold increase vs. 1-mo-old rats). The following observations indicate that the age-dependent increase in H(2)O(2) generation was fully related to the MAO-A isoform: 1) at all the ages tested, chemiluminescence production was inhibited by the MAO-A inhibitor clorgyline but not by the MAO-B inhibitor RO-19 6327; 2) enzyme assay, Western blot, and semiquantitative RT-PCR analysis showed an age-dependent increase in cardiac MAO-A activity, immunodetection, and mRNA expression, respectively; and 3) the MAO-B isoform was undetectable by enzyme assay and Western blot analysis. These results suggest that MAO-A could be a major source of H(2)O(2) in the aging heart.

Hydrogen peroxide production by monoamine oxidase during ischemia/reperfusion.

Reactive oxygen species have been postulated to play a crucial role in the pathogenesis of renal ischemia-reperfusion injury. However, the intracellular sources of reactive oxygen species during ischemia-reperfusion are still unclear. In the present study, we examined whether catecholamine-degrading enzymes monoamine oxidases contribute to hydrogen peroxide (H(2)O(2)) generation during ischemia-reperfusion using an in vivo rat model of unilateral renal ischemia. The monoamine oxidases were characterized in homogenates of renal cortex by enzyme assay and by Western blot analysis. The monoamine oxidase-dependent H(2)O(2) production was measured by luminol-amplified chemiluminescence assay. Renal monoamine oxidase activity and H(2)O(2) generation by monoamine oxidases were suppressed during ischemia. The monoamine oxidase-dependent H(2)O(2) production was observed during the first 15 min of reperfusion. In addition, enzyme assays showed that monoamine oxidase is also activated in this period. Rat pre-treatment with the irreversible inhibitor of monoamine oxidase, pargyline, prevented H(2)O(2) production. These data suggest that monoamine oxidases are a potential source of H(2)O(2) generation in the early reperfusion following ischemia, which could be involved in renal ischemia-reperfusion injury.

Regulation of JNK/ERK activation, cell apoptosis, and tissue regeneration by monoamine oxidases after renal ischemia-reperfusion.

Reactive oxygen species (ROS) contribute to the ischemia-reperfusion injury. In kidney, the intracellular sources of ROS during ischemia-reperfusion are still unclear. In the present study, we investigated the role of the catecholamine-degrading enzyme monoamine oxidases (MAOs) in hydrogen peroxide (H2O2) generation after reperfusion and their involvement in cell events leading to tissue injury and recovery. In a rat model of renal ischemia-reperfusion, we show concomitant MAO-dependent H2O2 production and lipid peroxidation in the early reperfusion period. Rat pretreatment with the irreversible MAO inhibitor pargyline resulted in the following: i) prevented H2O2 production and lipid peroxidation; ii) decreased tubular cell apoptosis and necrosis, measured by TUNEL staining and histomorphological criteria; and iii) increased tubular cell proliferation as determined by proliferating cell nuclear antigen expression. MAO inhibition also prevented Jun N-terminal kinase phosphorylation and promoted extracellular signal-regulated kinase activation, two mitogen-activated protein kinases described as a part of a "death" and "survival" pathway after ischemia-reperfusion. This work demonstrates the crucial role of MAOs in mediating the production of injurious ROS, which contribute to acute apoptotic and necrotic cell death induced by renal ischemia-reperfusion in vivo. Targeted inhibition of these oxidases could provide a new avenue for therapy to prevent renal damage and promote renal recovery after ischemia-reperfusion.