Varied effects of tobacco smoke and e-cigarette vapor suggest that nicotine does not affect endothelium-dependent relaxation and nitric oxide signaling.

Chronic smoking causes dysfunction of vascular endothelial cells, evident as a reduction of flow-mediated dilation in smokers, but the role of nicotine is still controversial. Given the increasing use of e-cigarettes and other nicotine products, it appears essential to clarify this issue. We studied extracts from cigarette smoke (CSE) and vapor from e-cigarettes (EVE) and heated tobacco (HTE) for their effects on vascular relaxation, endothelial nitric oxide signaling, and the activity of soluble guanylyl cyclase. The average nicotine concentrations of CSE, EVE, and HTE were 164, 800, and 85 µM, respectively. At a dilution of 1:3, CSE almost entirely inhibited the relaxation of rat aortas and porcine coronary arteries to acetylcholine and bradykinin, respectively, while undiluted EVE, with a 15-fold higher nicotine concentration, had no significant effect. With about 50% inhibition at 1:2 dilution, the effect of HTE was between CSE and EVE. Neither extract affected endothelium-independent relaxation to an NO donor. At the dilutions tested, CSE was not toxic to cultured endothelial cells but, in contrast to EVE, impaired NO signaling and inhibited NO stimulation of soluble guanylyl cyclase. Our results demonstrate that nicotine does not mediate the impaired endothelium-dependent vascular relaxation caused by smoking.

Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node.

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Adenosine kinase (ADK) inhibition with ABT-702 induces ADK protein degradation and a distinct form of sustained cardioprotection.

Pharmacological inhibition of adenosine kinase (ADK), the major route of myocardial adenosine metabolism, can elicit acute cardioprotection against ischemia-reperfusion (IR) by increasing adenosine signaling. Here, we identified a novel, extended effect of the ADK inhibitor, ABT-702, on cardiac ADK protein longevity and investigated its impact on sustained adenosinergic cardioprotection. We found that ABT-702 treatment significantly reduced cardiac ADK protein content in mice 24-72 h after administration (IP or oral). ABT-702 did not alter ADK mRNA levels, but strongly diminished (ADK-L) isoform protein content through a proteasome-dependent mechanism. Langendorff perfusion experiments revealed that hearts from ABT-702-treated mice maintain higher adenosine release long after ABT-702 tissue elimination, accompanied by increased basal coronary flow (CF) and robust tolerance to IR. Sustained cardioprotection by ABT-702 did not involve increased nitric oxide synthase expression, but was completely dependent upon increased adenosine release in the delayed phase (24 h), as indicated by the loss of cardioprotection and CF increase upon perfusion of adenosine deaminase or adenosine receptor antagonist, 8-phenyltheophylline. Importantly, blocking adenosine receptor activity with theophylline during ABT-702 administration prevented ADK degradation, preserved late cardiac ADK activity, diminished CF increase and abolished delayed cardioprotection, indicating that early adenosine receptor signaling induces late ADK degradation to elicit sustained adenosine release. Together, these results indicate that ABT-702 induces a distinct form of delayed cardioprotection mediated by adenosine receptor-dependent, proteasomal degradation of cardiac ADK and enhanced adenosine signaling in the late phase. These findings suggest ADK protein stability may be pharmacologically targeted to achieve sustained adenosinergic cardioprotection.

The tight junction protein cingulin regulates the vascular response to burn injury in a mouse model.

Edema formation due to the collapse of physiological barriers and the associated delayed healing process is still a central problem in the treatment of burn injuries. In healthy individuals, tight junctions form a barrier to fluid and small molecules. Cingulin is a cytoplasmic component of tight junctions and is involved in the regulation of the paracellular barrier. Endothelial specific cingulin knock-out mice provide new insight into the influence of tight junction proteins on edema formation and angiogenesis during wound healing. Knock-out mice lacking the head domain of cingulin in endothelial cells (CgnΔEC) were created by breeding Cgnfl/fl mice with Tie1-cre mice. Using a no-touch hot air jet a burn trauma was induced on the ear of the mouse. Over a period of 12 days microcirculatory parameters such as edema formation, angiogenesis and leukocyte-endothelial interactions were visualized using intravital fluorescence microscopy. At baseline, CgnΔEC mice surprisingly showed significantly less tracer extravasation compared to Cgnfl/fl littermates, whereas, after burn injury, edema was consistently higher in CgnΔEC mice. Non-perfused area after wounding was increased, but there was no difference in vessel diameters, contraction or dilation of arteries in CgnΔEC mice. Moreover, cingulin knock-out did not cause a difference in leukocyte adhesion after burn injury. In summary, cingulin limits non-perfused area after burn injury and maintains the paracellular barrier of blood vessels. Since edema formation with serious systemic effects is a central problem of burn wounds, understanding the importance of tight junction proteins might help to find new treatment strategies for burn wounds.

Effects of flavoring compounds used in electronic cigarette refill liquids on endothelial and vascular function.

Electronic cigarette refill liquids are commercially provided with a wide variety of flavoring agents. A recent study suggested that several common flavors may scavenge nitric oxide (NO) and cause endothelial dysfunction. It was the aim of the present study to investigate the effects of these flavors on NO/cyclic GMP-mediated signaling and vascular relaxation. We tested the flavoring agents for effects on Ca2+-induced cGMP accumulation and NO synthase activation in cultured endothelial cells. NO scavenging was studied with NO-activated soluble guanylate cyclase and as NO release from a NO donor, measured with a NO electrode. Blood vessel function was studied with precontracted rat aortic rings in the absence and presence of acetylcholine or a NO donor. Cinnamaldehyde inhibited Ca2+-stimulated endothelial cGMP accumulation and NO synthase activation at ≥0.3 mM. Cinnamaldehyde and diacetyl inhibited NO-activated soluble guanylate cyclase with IC50 values of 0.56 (0.54-0.58) and 0.29 (0.24-0.36) mM, respectively, and caused moderate NO scavenging at 1 mM that was not mediated by superoxide anions. The other compounds did not scavenge NO at 1 mM. None of the flavorings interfered with acetylcholine-induced vascular relaxation, but they caused relaxation of pre-contracted aortas. The most potent compounds were eugenol and cinnamaldehyde with EC50 values of ~0.5 mM. Since the flavors did not affect endothelium-dependent vascular relaxation, NO scavenging by cinnamaldehyde and diacetyl does not result in impaired blood vessel function. Although not studied in vivo, the low potency of the compounds renders it unlikely that the observed effects are relevant to humans inhaling flavored vapor from electronic cigarettes.

Sustained Formation of Nitroglycerin-Derived Nitric Oxide by Aldehyde Dehydrogenase-2 in Vascular Smooth Muscle without Added Reductants: Implications for the Development of Nitrate Tolerance.

According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.

Irreversible Activation and Stabilization of Soluble Guanylate Cyclase by the Protoporphyrin IX Mimetic Cinaciguat.

Belonging to the class of so-called soluble guanylate cyclase (sGC) activators, cinaciguat and BAY 60-2770 are interesting therapeutic tools for the treatment of various cardiovascular pathologies. The drugs are supposed to preferentially stimulate oxidized or heme-depleted, but not native sGC. Since this concept has been challenged by studies demonstrating complete relaxation of nondiseased vessels, this study was designed to reinvestigate the mode of action in greater detail. To this purpose, the effect of cinaciguat was studied on vessel tone of porcine coronary arteries and rat thoracic aortas. Organ bath studies showed that the compound caused time- and concentration-dependent relaxation of precontracted vessels with a maximal effect observed at 90 minutes. The dilatory response was not affected by extensive washout of the drug. Cinaciguat-induced vasodilation was associated with a time- and concentration-dependent increase of cGMP levels. Experiments with purified sGC in the presence of Tween 20 showed that cinaciguat activates the heme-free enzyme in a concentration-dependent manner with an EC50 value of ∼0.2 µM and maximal cGMP formation at 10 µM. By contrast, the effect of cinaciguat on 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one-oxidized (ferric) sGC was moderate, reaching ∼10%-15% of maximal activity. Dilution experiments of cinaciguat/Tween 20-preincubated sGC revealed the irreversible character of the drug. Assuming a sensitive balance between heme-free, ferric, and nitric oxide-sensitive ferrous sGC in cells and tissues, we propose that cinaciguat by virtue of its irreversible mode of action is capable of shifting this equilibrium toward the heme-free apo-sGC species.

Dipeptidyl peptidase-4 independent cardiac dysfunction links saxagliptin to heart failure.

Saxagliptin treatment has been associated with increased rate of hospitalization for heart failure in type 2 diabetic patients, though the underlying mechanism(s) remain elusive. To address this, we assessed the effects of saxagliptin on human atrial trabeculae, guinea pig hearts and cardiomyocytes. We found that the primary target of saxagliptin, dipeptidyl peptidase-4, is absent in cardiomyocytes, yet saxagliptin internalized into cardiomyocytes and impaired cardiac contractility via inhibition of the Ca2+/calmodulin-dependent protein kinase II-phospholamban-sarcoplasmic reticulum Ca2+-ATPase 2a axis and Na+-Ca2+ exchanger function in Ca2+ extrusion. This resulted in reduced sarcoplasmic reticulum Ca2+ content, diastolic Ca2+ overload, systolic dysfunction and impaired contractile force. Furthermore, saxagliptin reduced protein kinase C-mediated delayed rectifier K+ current that prolonged action potential duration and consequently QTc interval. Importantly, saxagliptin aggravated pre-existing cardiac dysfunction induced by ischemia/reperfusion injury. In conclusion, our novel results provide mechanisms for the off-target deleterious effects of saxagliptin on cardiac function and support the outcome of SAVOR-TIMI 53 trial that linked saxagliptin with the risk of heart failure.

TRPC3 contributes to regulation of cardiac contractility and arrhythmogenesis by dynamic interaction with NCX1.

AIM: TRPC3 is a non-selective cation channel, which forms a Ca2+ entry pathway involved in cardiac remodelling. Our aim was to analyse acute electrophysiological and contractile consequences of TRPC3 activation in the heart. METHODS AND RESULTS: We used a murine model of cardiac TRPC3 overexpression and a novel TRPC3 agonist, GSK1702934A, to uncover (patho)physiological functions of TRPC3. GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes. GSK1702934A transiently enhanced contractility and evoked arrhythmias in isolated Langendorff hearts from TRPC3-overexpressing but not wild-type mice. Interestingly, pro-arrhythmic effects outlasted TRPC3 current activation, were prevented by enhanced intracellular Ca2+ buffering, and suppressed by the NCX inhibitor 3',4'-dichlorobenzamil hydrochloride. GSK1702934A substantially promoted NCX currents in TRPC3-overexpressing myocytes. The TRPC3-dependent electrophysiologic, pro-arrhythmic, and inotropic actions of GSK1702934A were mimicked by angiotensin II (AngII). Immunocytochemistry demonstrated colocalization of TRPC3 with NCX1 and disruption of local interaction upon channel activation by either GSK1702934A or AngII. CONCLUSION: Cardiac TRPC3 mediates Ca2+ and Na+ entry in proximity of NCX1, thereby elevating cellular Ca2+ levels and contractility. Excessive activation of TRPC3 is associated with transient cellular Ca2+ overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis. We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli.

Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels.

The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2.

Role of the ubiquitin-proteasome system in cardiac dysfunction of adipose triglyceride lipase-deficient mice.

Systemic deletion of the gene encoding for adipose triglyceride lipase (ATGL) in mice leads to severe cardiac dysfunction due to massive accumulation of neutral lipids in cardiomyocytes. Recently, impaired peroxisome proliferator-activated receptor α (PPARα) signaling has been described to substantially contribute to the observed cardiac phenotype. Disturbances of the ubiquitin-proteasome system (UPS) have been implicated in numerous cardiac diseases including cardiomyopathy, ischemic heart disease, and heart failure. The objective of the present study was to investigate the potential role of UPS in cardiac ATGL deficiency. Our results demonstrate prominent accumulation of ubiquitinated proteins in hearts of ATGL-deficient mice, an effect that was abolished upon cardiomyocyte-directed overexpression of ATGL. In parallel, cardiac protein expression of the ubiquitin-activating enzyme E1a, which catalyzes the first step of the ubiquitination cascade, was significantly upregulated in ATGL-deficient hearts. Dysfunction of the UPS was accompanied by activation of NF-κB signaling. Moreover, the endoplasmic reticulum (ER)-resident chaperon protein disulfide isomerase was significantly upregulated in ATGL-deficient hearts. Chronic treatment of ATGL-deficient mice with the PPARα agonist Wy14,643 improved proteasomal function, prevented NF-κB activation and decreased oxidative stress. In summary, our data point to a hitherto unrecognized link between proteasomal function, PPARα signaling and cardiovascular disease.

Endothelial dysfunction in adipose triglyceride lipase deficiency.

Systemic knockout of adipose triglyceride lipase (ATGL), the pivotal enzyme of triglyceride lipolysis, results in a murine phenotype that is characterized by progredient cardiac steatosis and severe heart failure. Since cardiac and vascular dysfunction have been closely related in numerous studies we investigated endothelium-dependent and -independent vessel function of ATGL knockout mice. Aortic relaxation studies and Langendorff perfusion experiments of isolated hearts showed that ATGL knockout mice suffer from pronounced micro- and macrovascular endothelial dysfunction. Experiments with agonists directly targeting vascular smooth muscle cells revealed the functional integrity of the smooth muscle cell layer. Loss of vascular reactivity was restored ~50% upon treatment of ATGL knockout mice with the PPARα agonist Wy14,643, indicating that this phenomenon is partly a consequence of impaired cardiac contractility. Biochemical analysis revealed that aortic endothelial NO synthase expression and activity were significantly reduced in ATGL deficiency. Enzyme activity was fully restored in ATGL mice treated with the PPARα agonist. Biochemical analysis of perivascular adipose tissue demonstrated that ATGL knockout mice suffer from perivascular inflammatory oxidative stress which occurs independent of cardiac dysfunction and might contribute to vascular defects. Our results reveal a hitherto unrecognized link between disturbed lipid metabolism, obesity and cardiovascular disease.

Cardiac oxidative stress in a mouse model of neutral lipid storage disease.

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4-5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~2.5-fold upregulation of soluble guanylate cyclase activity and a ~2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.

Potent inhibition of aldehyde dehydrogenase-2 by diphenyleneiodonium: focus on nitroglycerin bioactivation.

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI), a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2. DPI (0.3 µM) inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. Denitration and bioactivation of 1-2 µM GTN, assayed as 1,2-glycerol dinitrate formation and activation of purified sGC, respectively, were inhibited by DPI with a half-maximally active concentration of about 0.2 µM in a GTN-competitive manner. Molecular modeling indicated that DPI binds to the catalytic site of ALDH2, and this was confirmed by experiments showing substrate-competitive inhibition of the dehydrogenase and esterase activities of the enzyme. Our data identify ALDH2 as highly sensitive target of DPI and explain inhibition of GTN-induced relaxation by this drug observed previously. In addition, the data provide new evidence for the essential role of ALDH2 in GTN bioactivation and may have implications to other fields of ALDH2 research, such as hepatic ethanol metabolism and cardiac ischemia/reperfusion injury.

Vascular bioactivation of nitroglycerin is catalyzed by cytosolic aldehyde dehydrogenase-2.

RATIONALE: According to general view, aldehyde dehydrogenase-2 (ALDH2) catalyzes the high-affinity pathway of vascular nitroglycerin (GTN) bioactivation in smooth muscle mitochondria. Despite having wide implications to GTN pharmacology and raising many questions that are still unresolved, mitochondrial bioactivation of GTN in blood vessels is still lacking experimental support. OBJECTIVE: In the present study, we investigated whether bioactivation of GTN is affected by the subcellular localization of ALDH2 using immortalized ALDH2-deficient aortic smooth muscle cells and mouse aortas with selective overexpression of the enzyme in either cytosol or mitochondria. METHODS AND RESULTS: Quantitative Western blotting revealed that ALDH2 is mainly cytosolic in mouse aorta and human coronary arteries, with only approximately 15% (mouse) and approximately 5% (human) of the enzyme being localized in mitochondria. Infection of ALDH2-deficient aortic smooth muscle cells or isolated aortas with adenovirus containing ALDH2 cDNA with or without the mitochondrial signal peptide sequence led to selective expression of the protein in mitochondria and cytosol, respectively. Cytosolic overexpression of ALDH2 restored GTN-induced relaxation and GTN denitration to wild-type levels, whereas overexpression in mitochondria (6-fold vs wild-type) had no effect on relaxation. Overexpression of ALDH2 in the cytosol of ALDH2-deficient aortic smooth muscle cells led to a significant increase in GTN denitration and cyclic GMP accumulation, whereas mitochondrial overexpression had no effect. CONCLUSIONS: The data indicate that vascular bioactivation of GTN is catalyzed by cytosolic ALDH2. Mitochondrial GTN metabolism may contribute to oxidative stress-related adverse effects of nitrate therapy and the development of nitrate tolerance.

Bioactivation of pentaerythrityl tetranitrate by mitochondrial aldehyde dehydrogenase.

Mitochondrial aldehyde dehydrogenase (ALDH2) contributes to vascular bioactivation of the antianginal drugs nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN), resulting in cGMP-mediated vasodilation. Although continuous treatment with GTN results in the loss of efficacy that is presumably caused by inactivation of ALDH2, PETN does not induce vascular tolerance. To clarify the mechanisms underlying the distinct pharmacological profiles of GTN and PETN, bioactivation of the nitrates was studied with aortas isolated from ALDH2-deficient and nitrate-tolerant mice, isolated mitochondria, and purified ALDH2. Pharmacological inhibition or gene deletion of ALDH2 attenuated vasodilation to both GTN and PETN to virtually the same degree as long-term treatment with GTN, whereas treatment with PETN did not cause tolerance. Purified ALDH2 catalyzed bioactivation of PETN, assayed as activation of soluble guanylate cyclase (sGC) and formation of nitric oxide (NO). The EC(50) value of PETN for sGC activation was 2.2 ± 0.5 µM. Denitration of PETN to pentaerythrityl trinitrate was catalyzed by ALDH2 with a specific activity of 9.6 ± 0.8 nmol · min(-1) · mg(-1) and a very low apparent affinity of 94.7 ± 7.4 µM. In contrast to GTN, PETN did not cause significant inactivation of ALDH2. Our data suggest that ALDH2 catalyzes bioconversion of PETN in two distinct reactions. Besides the major denitration pathway, which occurs only at high PETN concentrations, a minor high-affinity pathway may reflect vascular bioactivation of the nitrate yielding NO. The very low rate of ALDH2 inactivation, presumably as a result of low affinity of the denitration pathway, may at least partially explain why PETN does not induce vascular tolerance.

Evidence against tetrahydrobiopterin depletion of vascular tissue exposed to nitric oxide/superoxide or nitroglycerin.

Several cardiovascular disorders, including atherosclerosis and tolerance to the antianginal drug nitroglycerin (GTN), may be associated with the generation of superoxide anions, which react with nitric oxide (NO) to yield peroxynitrite. According to a widely held view, oxidation of tetrahydrobiopterin (BH(4)) by peroxynitrite causes uncoupling of endothelial NO synthase (eNOS), resulting in reduced NO bioavailability and endothelial dysfunction under conditions of oxidative stress. In this study we determined the levels of reduced biopterins and endothelial function in cultured cells exposed to peroxynitrite and GTN as well as in blood vessels isolated from GTN-tolerant guinea pigs and rats. BH(4) was rapidly oxidized by peroxynitrite and 3-morpholino sydnonimine (SIN-1) in buffer, but this was prevented by glutathione and not observed in endothelial cells exposed to SIN-1 or GTN. Prolonged treatment of the cells with 0.1 mM GTN caused slow N(G)-nitro-l-arginine-sensitive formation of reactive oxygen species without affecting eNOS activity. Endothelial function and BH(4)/BH(2) levels were identical in blood vessels of control and GTN-tolerant animals. Our results suggest that peroxynitrite-triggered BH(4) oxidation does not occur in endothelial cells or GTN-exposed blood vessels. GTN seems to trigger minor eNOS uncoupling that is unrelated to BH(4) depletion and without observable consequence on eNOS function.

Vascular tolerance to nitroglycerin in ascorbate deficiency.

AIMS: Nitroglycerin (GTN) acts through release of a nitric oxide (NO)-related activator of soluble guanylate cyclase in vascular smooth muscle. Besides enzymatic GTN bioactivation catalysed by aldehyde dehydrogenase, non-enzymatic reaction of GTN with ascorbate also results in the formation of a bioactive product. Using an established guinea pig model of ascorbate deficiency, we investigated whether endogenous ascorbate contributes to GTN-induced vasodilation. METHODS AND RESULTS: Guinea pigs were fed either standard or ascorbate-free diet for 2 or 4 weeks prior to measuring the GTN response of aortic rings and isolated hearts. The effects of ascorbate on GTN metabolism were studied with purified mitochondrial aldehyde dehydrogenase (ALDH2) and isolated mitochondria. Ascorbate deprivation led to severe scorbutic symptoms and loss of body weight, but had no (2 weeks) or only slight (4 weeks) effects on aortic relaxations to a direct NO donor. The EC(50) of GTN was increased from 0.058 +/- 0.018 to 0.46 +/- 0.066 and 5.5 +/- 0.9 microM after 2 and 4 weeks of ascorbate-free diet, respectively. Similarly, coronary vasodilation to GTN was severely impaired in ascorbate deficiency. The potency of GTN was reduced to a similar extent by ALDH inhibitors in control and ascorbate-deficient blood vessels. Up to 10 mM ascorbate had no effect on GTN metabolism catalysed by purified ALDH2 or liver mitochondria isolated from ascorbate-deficient guinea pigs. CONCLUSION: Our results indicate that prolonged ascorbate deficiency causes tolerance to GTN without affecting NO/cyclic GMP-mediated vasorelaxation.

Tissue-toxic effects of phosphatidylcholine/deoxycholate after subcutaneous injection for fat dissolution in rats and a human volunteer.

BACKGROUND: The safety of the lipodissolution procedure for the cosmetic treatment of fat is unknown. OBJECTIVES: The objective was to determine the subcutaneous tissue effects of phosphatidylcholine solubilized with deoxycholate (PC/DC) in rats and a human volunteer. METHODS: Rats were treated subcutaneously three times with 50, 300, or 600 microL of PC/DC formula on the abdomen in a chronic study (30 days). A human volunteer undergoing elective liposuction was similarly treated. Cell membrane lysis, cell viability, and histologic status were determined on fresh biopsies of subcutaneous fat from the injection sites. RESULTS: PC/DC dose-dependently reduced membrane integrity and cell viability. Histologic alterations induced by PC/DC included fibroplasia, bandlike fibrosis in the region of the cutaneous muscle, and partial muscle loss. The highest dose caused widespread fat necrosis, fat cyst formation, and necrotic changes of the walls of small blood vessels. Histologic sections of subcutaneous tissue from the human volunteer showed dose-dependent panniculitis, fat cysts, and vessel necrosis. DC (2.5%), tested for comparison in the rat, exerted membrane and histologic effects similar to those of PC/DC. Solvent controls caused negligible alterations. CONCLUSIONS: Injection lipolysis with PC/DC causes tissue fibrosis and necrosis of adipose and vascular tissues in rat and man, making the long-term safety of PC/DC for nonsurgical treatment of subcutaneous fat deposits uncertain.

Role of myocardial nitric oxide in diabetic ischemia-reperfusion dysfunction: studies in mice with myocyte-specific overexpression of endothelial nitric-oxide synthase.

We investigated the role of nitric oxide (NO) in myocardial ischemia-reperfusion injury of diabetic mice with myocyte-specific overexpression of endothelial NO synthase (NOS). Four weeks after diabetes induction with streptozotocin (blood glucose approximately 29 mM), isolated isovolumic heart function and cellular NO metabolites in response to brief normothermic ischemia-reperfusion were determined. Under normoxic conditions transgenic (TG) hearts from nondiabetic and diabetic animals generated less left-ventricular developed pressure compared with wild-type (WT) control hearts, and this abnormality was unaffected by NOS inhibition. During ischemia, the rise in end-diastolic pressure was less in the TG than WT group of nondiabetic hearts, whereas the transgene had no effect in the diabetic group. Similarly, the transgene also improved reperfusion systolic and diastolic function in nondiabetic but not in diabetic hearts. NOS inhibition worsened reperfusion function in diabetic hearts. Postischemic nitrite and cGMP formation were higher in nondiabetic TG than WT hearts, but in diabetic hearts cGMP was no longer elevated. The formation of reactive oxygen species (superoxide and peroxynitrite) during early reperfusion, measured by electron spin resonance spectroscopy, was similar in nondiabetic WT and TG hearts, but it was significantly higher in diabetic TG hearts. Stimulating endogenous NO production with 10 microM bradykinin more strongly reduced myocardial O(2) consumption in diabetic TG than diabetic WT hearts perfused in normoxia, whereas there was no difference after ischemia-reperfusion. Thus, providing additional endogenous NO is sufficient to protect nondiabetic hearts against ischemia-induced injury, but for a similar protection in diabetic hearts, effective scavenging of reactive oxygen species is also important.