Apelin Reduces Nitric Oxide-Induced Relaxation of Cerebral Arteries by Inhibiting Activation of Large-Conductance, Calcium-Activated K Channels.

Activation of the apelin/APJ receptor signaling system causes endothelium-dependent and nitric oxide (NO)-dependent relaxation in several peripheral arteries. The effects of apelin in cerebral arteries are unknown; however, apelin inhibits voltage-dependent increases in large-conductance, calcium-activated K channel (BKCa) currents in cerebral artery smooth muscle cells. Because NO-induced relaxation of cerebral arteries is mediated, in part, by activation of BKCa channels, the goals of this study were to determine the net effect of apelin in cerebral arteries, as well as test the hypothesis that the actions of apelin in cerebral arteries are secondary to stimulation of APJ receptors. Immunoblot and quantitative reverse transcription polymerase chain reaction analyses detected APJ receptors in cerebral arteries of male Sprague-Dawley rats, and immunofluorescence studies using confocal microscopy confirmed APJ receptor localization in smooth muscle cells. In myograph studies, apelin itself had no direct vasomotor effect but inhibited relaxations to the NO-donor, diethylamine NONOate, and to the endothelium-dependent vasodilator, bradykinin. These effects of apelin were mimicked by the selective BKCa-channel blocker, iberiotoxin, and suppressed by the APJ receptor antagonist, F13A. Apelin also inhibited relaxations evoked by the BKCa-channel openers, NS1619 and BMS 191011, but had no effect on relaxation to levcromakalim, a selective KATP-channel opener. Apelin had no effect on diethylamine NONOate-induced or bradykinin-induced increases in cyclic guanosine monophosphate levels. Patch clamp recordings demonstrated that apelin and iberiotoxin each suppressed the increase in BKCa currents induced by DEA and NS1619 in freshly isolated cerebral artery smooth muscle cells. The results demonstrate that apelin inhibits NO-induced relaxation of cerebral arteries through a mechanism involving activation of APJ receptors and inhibition of BKCa channels in cerebral arterial smooth muscle cells.

ß-adrenergic Receptor-stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 ω-hydroxylase.

Prolonged or excessive ß-adrenergic activation leads to cardiac myocyte loss and heart dysfunction; however, the underlying cellular mechanisms are still unclear. Therefore, we first confirmed the effect of isoproterenol (ISO), a ß-adrenergic receptor agonist, on cardiac toxicity using TUNEL and caspase activity assays in cultured rat cardiomyocytes. ISO treatment significantly increased cardiomyocyte apoptosis. Persistent ISO stimulation of cardiomyocytes also increased the expression of CYP4A3, a major CYP450 ω-hydroxylase that produces 20-hydroxyeicosatetraenoic acid (20-HETE) in a time-dependent manner. Next, we examined the effect of ISO and 20-HETE on cardiomyocyte apoptosis using annexin V and propidium iodide staining. Treatment with either 20-HETE or ISO significantly increased cardiomyocyte apoptosis, and inhibition of 20-HETE production using 17-ODYA, a CYP450 ω-hydroxylase inhibitor, dramatically attenuated ISO-induced cardiomyocyte apoptosis. To determine the apoptotic pathway involved, the mitochondrial membrane potential (ΔΨm) was measured by detecting the ratio of JC-1 green/red emission intensity. The results demonstrated that 17-ODYA significantly abolished ISO-induced disruption of ΔΨm and that 20-HETE alone induced a marked disruptive effect on ΔΨm in cardiomyocytes. In addition, 20-HETE-induced disruption of ΔΨm and apoptosis was significantly attenuated by KN93, a CaMKII inhibitor. Taken together, these results demonstrate that 20-HETE treatment induces significant apoptosis via mitochondrial-dependent pathways, and that inhibition of 20-HETE production using 17-ODYA attenuates ISO-induced cardiomyocyte apoptosis.

20-Hydroxyeicosatetraenoic Acid Is a Key Mediator of Angiotensin II-induced Apoptosis in Cardiac Myocytes.

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.