Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

S-glutathionylation uncouples eNOS and regulates its cellular and vascular function.

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O(2)(•-)), which are key mediators of cellular signalling. In the presence of Ca(2+)/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from l-arginine (l-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH(4)) and l-Arg. In the absence of BH(4), NO synthesis is abrogated and instead O(2)(•-) is generated. While NOS dysfunction occurs in diseases with redox stress, BH(4) repletion only partly restores NOS activity and NOS-dependent vasodilation. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation. Under oxidative stress, S-glutathionylation occurs through thiol-disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione. Cysteine residues are critical for the maintenance of eNOS function; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O(2)(•-) generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O(2)(•-) generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.

Mitochondrial uncoupling does not decrease reactive oxygen species production after ischemia-reperfusion.

Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H(+) leak decreases ROS formation; it has been postulated that increasing H(+) leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H(+) leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H(+) leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H(+) leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H(+) leak. H(+) leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H(+) leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H(+) leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

Fast gated EPR imaging of the beating heart: spatiotemporally resolved 3D imaging of free-radical distribution during the cardiac cycle.

In vivo or ex vivo electron paramagnetic resonance imaging (EPRI) is a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. However, applications of EPRI have been limited by long projection acquisition times and the consequent fact that rapid gated EPRI was not possible. Hence in vivo EPRI typically provided only time-averaged information. In order to achieve direct gated EPRI, a fast EPR acquisition scheme was developed to decrease EPR projection acquisition time down to 10-20 ms, along with corresponding software and instrumentation to achieve fast gated EPRI of the isolated beating heart with submillimeter spatial resolution in as little as 2-3 min. Reconstructed images display temporal and spatial variations of the free-radical distribution, anatomical structure, and contractile function within the rat heart during the cardiac cycle.

The radical trap 5,5-dimethyl-1-pyrroline N-oxide exerts dose-dependent protection against myocardial ischemia-reperfusion injury through preservation of mitochondrial electron transport.

Free radicals are important mediators of myocardial ischemia-reperfusion injury. Nitrone spin traps have been shown to scavenge free radicals. The cardioprotective effect of the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was investigated in an isolated heart model of global ischemia and reperfusion. Rat hearts were perfused and subjected to global ischemia for 30 min followed by reperfusion with four treatment groups of varying DMPO concentration (0.5-10 mM) administered before induction of ischemia. DMPO treatment improved the recovery of left ventricular (LV) function and coronary flow over the 30-min period of reperfusion compared with untreated hearts. Enhanced recovery was observed for all doses studied but was highest with 1 mM treatment with 2.4-fold higher recovery of LV developed pressure and 37% reduction in infarct size. Superoxide was measured by tissue fluorometry using the O(2)* probe hydroethidine. Hearts treated with 1 mM DMPO showed a significant reduction in O(2)* production compared with control hearts both over the first 5 min of ischemia and upon reperfusion after 30 min of global ischemia. Studies of mitochondrial function demonstrated that 1 mM DMPO increased the recovery of function of complexes I, II/III, and IV after 30 min of reperfusion. Immunoblotting with antibodies against complexes I, II, and IV further revealed marked up-regulation of mitochondrial proteins, suggesting that DMPO prevents their ischemic degradation via scavenging oxygen radicals generated during ischemia/reperfusion. Thus, DMPO functions as a protective agent against ischemic and postischemic injury via radical scavenging, conferring robust dose-dependent protection with salvage of mitochondrial function and redox homeostasis.

The functional morphology of the english sparrow cecum.

As birds do not have a urinary bladder, the kidneys and lower gastrointestinal tract must function in concert to maintain fluid and electrolyte homeostasis. In birds, urine is conveyed to the cloaca, and moved by reverse peristalsis into the colon and digestive ceca. Digestive ceca have been well studied for non-passerine birds and have been shown to absorb substrates and water. The ceca of passerine birds have been suggested to be non-functional because of their small size. The present study was undertaken to examine the morphology and cytochemistry of the small ceca of the English sparrow (Passer domesticus). Three-dimensional reconstruction of the ceca from serially sectioned tissue showed these organs to have a central channel with a large number of side channels. Electron micrographs indicated that all of the channels are lined by epithelial cells with a very dense microvillus brush border as well as a region densely packed with mitochondria just below the brush border. Specific staining for Na(+), K(+)-ATPase indicated the enzyme to be localized to the brush border. Quantification of Na(+), K(+)-ATPase activity showed it to be comparable to the coprodeum of domestic fowl. The data suggest that the small ceca of passerine birds may function in fluid and electrolyte homeostasis.