Sun C. 20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca 2ϩ channel via a PKCdependent mechanism in cardiomyocytes. Am J Physiol Heart Circ Physiol 299: H1109 -H1117, 2010. First published July 30, 2010; doi:10.1152/ajpheart.00067.2010.-The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type Ca 2ϩ currents (ICa,L) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10 -100 nM) resulted in a concentration-dependent increase in I Ca,L, and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 M), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on ICa,L. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 M), attenuated the 20-HETE-induced increases in I Ca,L and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca 2ϩ channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases.20-hydroxyeicosatetraenoic acid; L-type calcium channel; protein kinase C; cardiac myocytes; reactive oxygen species 20-HYDROXYEICOSATETRAENOIC ACID (20-HETE) is a lipid metabolite of arachidonic acid that is produced by -hydroxylase enzymes of the cytochrome P-450 (CYP)4A and CYP4F families, which are relatively abundant and exert regulatory functions dependent on the tissue (18). For example, in the kidney, 20-HETE regulates renal functions, such as renal vascular tone, tubuloglomerular feedback, autoregulation of renal blood flow, tubular transport, and mitogenesis (20). In blood vessels, 20-HETE is a potent vasoconstrictor that activates L-type Ca 2ϩ channels and inhibits Ca 2ϩ -sensitive K ϩ channels in vascular smooth muscle cells (28,29). In pulmonary arteries, 20-HETE enhances NADPH oxidase-dependent production of ROS in endothelial cells (21). Thus, it was proposed that 20-HETE plays an important role in the control of apoptosis and angiogensis in vascular endothelial cells in the pulmonary microcirculation (13). Recently, 20-HETE and CYP -hydroxylase were also identified in hearts from the rat and dog (13,26...

Objective 20-Hydroxyeicosatetraenoic acid (20-HETE), a ω-hydroxylation product of arachidonic acid catalyzed by cytochrome P450 4A (CYP4A), may play a role in cardiovascular system. It is well known that CYP450 ω-hydroxylase inhibitors markedly reduced the cardiac ischemia reperfusion injury. However, the direct effect of 20-HETE on cardiomyocytes are still poorly investigated. Here, we studied the effect of 20-HETE on cardiomyocyte apoptosis and the apoptosis-associated signaling pathways. Methods and Results The cardiomyocyte apoptosis was measured by FITC-annexin V/propidium iodide (PI) double staining cytometry, indicating that the percentage of early apoptotic cells increased from 15.6±2.6% to 25.5%±2.5% in control and 20-HETE-treated cells respectively. The mitochondrial membrane potential (ΔΨm) was measured by detecting the ratio of JC-1 green/red emission intensity. A significant decrease in the ratio was observed after treatment with 20-HETE for 24 h in comparison with control group, suggesting the disruptive effect of 20-HETE on mitochondrial ΔΨm. In addition, 20-HETE stimulated caspase-3 activity and Bax mRNA expression in cardiomyocytes. In contrast, the Bcl-2 mRNA levels were significantly decreased by 20-HETE treatment. Conclusion These results demonstrate that 20-HETE induces cardiomyocyte apoptosis by activation of several intrinsic apoptotic pathways. The 20-HETE-induced apoptosis could contribute to the CYP450 ω-hydroxylase-dependent cardiac injure during cardiac ischemia-reperfusion.

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.

Epsilon-toxin (ETX) is produced by types B and D strains of Clostridium perfringens, which cause fatal enterotoxaemia in sheep, goats and cattle. Previous studies showed that only a restricted number of cell lines are sensitive to ETX and ETX-induced hemolysis has not previously been reported. In this study, the hemolytic ability of ETX was examined using erythrocytes from 10 species including murine, rabbit, sheep, monkey and human. We found that ETX caused hemolysis in human erythrocytes (HC50 = 0.2 μM) but not erythrocytes from the other test species. Moreover, the mechanism of ETX-induced hemolysis was further explored. Recent studies showed that some bacterial toxins induce hemolysis through purinergic receptor (P2) activation. Hence, the function of purinergic receptors in ETX-induced hemolysis was tested, and we found that the non-selective P2 receptor antagonists PPADS inhibited ETX-induced lysis of human erythrocytes in a concentration-dependent manner, indicating that ETX-induced hemolysis requires activation of purinergic receptors. P2 receptors comprise seven P2X (P2X1–7) and eight P2Y (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11–P2Y14) receptor subtypes. The pattern of responsiveness to more selective P2-antagonists implies that both P2Y13 and P2X7 receptors are involved in ETX-induced hemolysis in human species. Furthermore, we demonstrated that extracellular ATP is likely not involved in ETX-induced hemolysis and the activation of P2 receptors. These findings clarified the mechanism of ETX-induced hemolysis and provided new insight into the activities and ETX mode of action.

Cardiomyocyte apoptosis is involved in a variety of cardiac stresses, including ischemia-reperfusion injury, heart failure, and cardiomyopathy. Both Angiotensin II (Ang II) and 20-hydroxyeicosatetraenoic acid (20-HETE) induce apoptosis in cardiomyocytes. Here, we examined the relationship between 20-HETE and Ang II in cardiomyocyte apoptosis. Apoptosis was examined using flow cytometry in primary cultured rat cardiomyocytes treated with control, Ang II, and Ang II plus HET0016 (a 20-HETE formation inhibitor). The results demonstrated that the treatment of cardiomyocytes with Ang II or 20-HETE significantly increased the percentage of apoptotic cells and that Ang II-induced apoptosis was markedly attenuated by HET0016 or losartan (an AT1 receptor antagonist). In apoptotic mechanism experiments, Ang II or 20-HETE treatment significantly reduced mitochondrial membrane potential, indicating that a mitochondria-dependent mechanism is involved. Ang II-induced alteration in mitochondrial membrane potential was significantly attenuated by HET0016. Treatment of cardiomyocytes with Ang II also increased superoxide production, and this effect of Ang II was attenuated by HET0016. Treatment of cardiomyocytes with Ang II significantly increased CYP4A1 expression and 20-HETE production, as measured by Western blot, real-time RT-PCR, and mass spectrometric analysis. All results suggest that 20-HETE may play a key role in Ang II-induced apoptosis in cardiomyocytes by a mitochondrial superoxide-dependent pathway.