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Epoxyeicosatrienoic acids (EETs) are endothelium-derived arachidonic acid metabolites of cytochrome P450. They dilate coronary arteries, open K+ channels, and hyperpolarize vascular smooth muscles. However, the mechanisms of these smooth muscle actions remain unknown. This study examined the effects of EETs on the large-conductance Ca(2+)-activated K+ channel (KCa) in smooth muscle cells of small bovine coronary arteries. In cell-attached patch-clamp experiments, 11,12-EET produced a 0.5- to 10-fold increase in the activity of the KCa channels when added in concentrations of 1, 10, and 100 nmol/L. In the inside-out excised membrane patch mode, 11,12-EET was without effect on the activity of the KCa channel unless GTP (0.5 mmol/L) or GTP and ATP (1 mmol/L) were added to the bath solution. In the presence of GTP and ATP, the increase in the KCa channel activity with 11,12-EET in inside-out patches was comparable to that in cell-attached patches. This effect of 11,12-EET in inside-out patches was blocked by the addition of GDP-beta-S (100 mumol/L). In outside-out patches, 11,12-EET also increased the KCa channel activity when GTP and ATP were added to the pipette solution. The addition of a specific anti-Gs alpha antibody (100 nmol/L) in the pipette solution completely blocked the activation of the KCa channels induced by 11,12-EET. An anti-G beta gamma or anti-Gi alpha antibody was without effect. We conclude that 11,12-EET activates the KCa channels by a Gs alpha-mediated mechanism. This mechanism contributes to the effects of EETs as endothelium-derived hyperpolarizing factors to hyperpolarize and relax arterial smooth muscle.

Docosahexaenoic acid, a major -3 fatty acid in human brain, synapses, retina, and other neural tissues, displays beneficial actions in neuronal development, cancer, and inflammatory diseases by mechanisms that remain to be elucidated. In this study we found, using lipid mediator informatics employing liquid chromatography-tandem mass spectrometry, that (10,17S)-docosatriene/neuroprotectin D1, now termed protectin D1 (PD1), is generated from docosahexaenoic acid by T helper type 2-skewed peripheral blood mononuclear cells in a lipoxygenase-dependent manner. PD1 blocked T cell migration in vivo, inhibited tumor necrosis factor ␣ and interferon-␥ secretion, and promoted apoptosis mediated by raft clustering. These results demonstrated novel anti-inflammatory roles for PD1 in regulating events associated with inflammation and resolution.

Background/Aim: Plasma trimethylamine-N-oxide (TMAO), a product of intestinal microbial metabolism of dietary phosphatidylcholine has been recently associated with atherosclerosis and increased risk of cardiovascular diseases (CVD) in rodents and humans. However, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. The present study tested whether TMAO induces NLRP3 inflammasome formation and activation and thereby contributes to endothelial injury initiating atherogenesis. Methods: Inflammasome formation and activation was determined by confocal microscopy, caspase-1 activity was measured by colorimetric assay, IL-1β production was measured using ELISA, cell permeability was determined by microplate reader and ZO-1 expression was determined by western blot analysis and confocal microscopy. In in vivo experiments, TMAO was infused by osmotic pump implantation. Results: TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase-1, caspase-1 activity, IL-1β production, cell permeability in carotid artery endothelial cells (CAECs) compared to control cells. Pretreatment with caspase-1 inhibitor, WEHD or Nlrp3 siRNA abolished the TMAO-induced inflammasome formation, activation and cell permeability in these cells. In addition, we explored the mechanisms by which TMAO activates NLRP3 inflammasomes. TMAO-induced the activation of NLRP3 inflammasomes was associated with both redox regulation and lysosomal dysfunction. In animal experiments, direct infusion of TMAO in mice with partially ligated carotid artery were found to have increased NLRP3 inflammasome formation and IL-1β production in the intima of wild type mice. Conclusion: The formation and activation of NLRP3 inflammasomes by TMAO may be an important initiating mechanism to turn on the endothelial inflammatory response leading to endothelial dysfunction.

Hydrogen sulfide (H 2 S), a novel endogenous gaseous bioactive substance, has recently been implicated in the regulation of cardiovascular and neuronal functions. However, its role in the control of renal function is unknown. In the present study, incubation of renal tissue homogenates with L-cysteine (L-Cys) (as a substrate) produced H 2 S in a concentration-dependent manner. This H 2 S production was completely abolished by inhibition of both cystathionine ␤-synthetase (CBS) and cystathionine ␥-lyase (CGL), two major enzymes for the production of H 2 S, using amino-oxyacetic acid (AOAA), an inhibitor of CBS, and propargylglycine (PPG), an inhibitor of CGL. However, inhibition of CBS or CGL alone induced a small decrease in H 2 S production. In anesthetized Sprague-Dawley rats, intrarenal arterial infusion of an H 2 S donor (NaHS) increased renal blood flow, glomerular filtration rate (GFR), urinary sodium (U Na ⅐V), and potassium (U K ⅐V) excretion. Consistently, infusion of both AOAA and PPG to inhibit the endogenous H 2 S production decreased GFR, U Na ⅐V, and U K ⅐V, and either one of these inhibitors alone had no significant effect on renal functions. Infusion of L-Cys into renal artery to increase the endogenous H 2 S production also increased GFR, U Na ⅐V, and U K ⅐V, which was blocked by AOAA plus PPG. It was shown that H 2 S had both vascular and tubular effects and that the tubular effect of H 2 S might be through inhibition of Na ϩ /K ϩ /2Cl Ϫ cotransporter and Na ϩ /K ϩ /ATPase activity. These results suggest that H 2 S participates in the control of renal function and increases urinary sodium excretion via both vascular and tubular actions in the kidney.

Abstract-Mitochondrial ATP-sensitive potassium (mitoK ATP ) channels have been suggested as triggers and end effectors in myocardial ischemic preconditioning. However, the intracellular mechanism regulating mitoK ATP channels remains unclear. In the present study, mitoK ATP channels from bovine ventricular myocardium were reconstituted using planar lipid bilayers, and the effect of superoxide (O 2 Ϫ·) on the activity of these reconstituted channels was examined. After incorporation, a potassium-selective current was recorded. The mean conductance of this current was 56 pS at 150 mmol/L KCl, which was substantially inhibited by 1 mmol/L MgATP. 5-Hydroxydecanoate (5-HD, 10 to 100 mol/L), a selective mitoK ATP antagonist, reduced the open state probability (NPo) of these channels in a concentration-dependent manner, whereas diazoxide (10 mol/L), a selective mitoK ATP agonist, significantly increased channel activity. HMR-1098 (100 mol/L), a selective sarcolemmal K ATP antagonist, had no effect on the activity of reconstituted channels. Addition of xanthine/xanthine oxidase (100 mol/L per 0.038 U/mL), an O 2 Ϫ· -generating system, resulted in a marked activation of mitoK ATP channels; the NPo of the channels was increased from 0.60Ϯ0.10 to 1.94Ϯ0.02. This O 2 Ϫ· -induced channel activation was completely abolished by pretreatment with 5-HD (100 mol/L) or a sulfhydryl alkylating compound, N-ethylmaleimide (2 mmol/L). It is concluded that myocardial mitoK ATP channels can be reconstituted into lipid bilayers and that O 2 Ϫ· activates these channels. The effect of O 2 Ϫ· may be associated with its direct action on the sulfhydryl groups of the channel protein. Key Words: ATP-sensitive K ϩ channel Ⅲ mitochondria Ⅲ superoxide Ⅲ heart Ⅲ channel reconstitution S ingle or multiple brief periods of myocardial ischemia confer protection against infarction produced by a subsequent prolonged ischemia, a phenomenon termed ischemic preconditioning (IPC). 1 Although a variety of intracellular signaling pathways and molecules have been implicated in the protective effect of IPC, there is substantial evidence suggesting that the adenosine 5Ј-triphosphate-sensitive potassium (K ATP ) channel is an important component of this phenomenon and may serve as a distal effector in this process. 2 Cardiac myocytes contain 2 distinct forms of K ATP channels, as follows: the sarcolemmal K ATP (sarcK ATP ) channel 3 and the mitochondrial ATP-sensitive potassium (mitoK ATP ) channel. 4 The mitoK ATP channels are present in the mitochondrial inner membrane and modulate mitochondrial function by regulating mitochondrial membrane potential, ion homeostasis, and matrix volume. 5 Recent studies suggest that mitoK ATP channels more importantly contribute to IPCinduced cardioprotection compared with sarcK ATP channels. 6,7 It has been demonstrated that diazoxide, a selective mitoK ATP agonist, 8 mimicked the beneficial effect of IPC to reduce infarct size or cell killing of cardiomyocytes and that sodium 5-hydroxydecanoate (5-HD), a selective mitoK ATP a...

This study examined the role of acid sphingomyelinase (ASM) and its redox amplification in mediating the formation of lipid raft (LR) redox signaling platforms in coronary arterial endothelial cells (CAECs). Using small interference RNA (siRNA) of ASM, Fas ligand (FasL)-induced increase in ASM activity, production of ceramide, and LR clustering in CAECs were blocked, and clustered Fas was also substantially reduced in detergent-resistant membrane fractions of CAECs. LR clustering, gp91(phox) aggregation, and p47(phox) translocation to the LR clusters induced by FasL were also blocked in ASM-siRNA transfected CAECs. Corresponding to this reduction of LR clustering with NAD(P)H oxidase subunits in ASM-siRNA transfected CAECs, superoxide (O(2)(-*)) production was significantly decreased as measured by either ESR or fluorescent spectrometry. Interestingly, superoxide dismutase (SOD) not only scavenged (O(2)(-*)), but also markedly attenuated LR clustering. Xanthine/xanthine oxidase, an exogenous (O(2)(-*)) generating system, dramatically increased ASM activity and LR clustering in EC membrane and enhanced FasL-induced LR clustering, which were blocked by SOD. These results suggest that that ASM activates LR clustering to form redox signaling platforms, where (O(2)(-*)) production enhances ASM activity, and thereby results in a forwarding amplification of LR and redox signaling. This ASM-mediated feedforwarding mechanism may be critical for an efficient transmembrane signaling through LRs.

These results indicate that Hcys activates NADH/NADPH oxidase by stimulating de novo ceramide synthesis, and subsequently enhancing Rac GTPase activity in rat MG cells. This ceramide-Rac GTPase signaling pathway may mediate Hcys-induced oxidative stress in these glomerular cells.