Robert M. Weisbrod

S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide.

Nitric oxide (NO) physiologically stimulates the sarco/endoplasmic reticulum calcium (Ca2+) ATPase (SERCA) to decrease intracellular Ca2+ concentration and relax cardiac, skeletal and vascular smooth muscle. Here, we show that NO-derived peroxynitrite (ONOO−) directly increases SERCA activity by S-glutathiolation and that this modification of SERCA is blocked by irreversible oxidation of the relevant cysteine thiols during atherosclerosis. Purified SERCA was S-glutathiolated by ONOO− and the increase in Ca2+-uptake activity of SERCA reconstituted in phospholipid vesicles required the presence of glutathione. Mutation of the SERCA-reactive Cys674 to serine abolished these effects. Because superoxide scavengers decreased S-glutathiolation of SERCA and arterial relaxation by NO, ONOO− is implicated as the intracellular mediator. NO-dependent relaxation as well as S-glutathiolation and activation of SERCA were decreased by atherosclerosis and Cys674 was found to be oxidized to sulfonic acid. Thus, irreversible oxidation of key thiol(s) in disease impairs NO-induced relaxation by preventing reversible S-glutathiolation and activation of SERCA by NO/ONOO−.

Mechanism of Nitric Oxide–Induced Vasodilatation: Refilling of Intracellular Stores by Sarcoplasmic Reticulum Ca2+ ATPase and Inhibition of Store-Operated Ca2+ Influx

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.

Arterial Stiffening Precedes Systolic Hypertension in Diet-Induced Obesity

Stiffening of conduit arteries is a risk factor for cardiovascular morbidity. Aortic wall stiffening increases pulsatile hemodynamic forces that are detrimental to the microcirculation in highly perfused organs, such as the heart, brain, and kidney. Arterial stiffness is associated with hypertension but presumed to be due to an adaptive response to increased hemodynamic load. In contrast, a recent clinical study found that stiffness precedes and may contribute to the development of hypertension although the mechanisms underlying hypertension are unknown. Here, we report that in a diet-induced model of obesity, arterial stiffness, measured in vivo, develops within 1 month of the initiation of the diet and precedes the development of hypertension by 5 months. Diet-induced obese mice recapitulate the metabolic syndrome and are characterized by inflammation in visceral fat and aorta. Normalization of the metabolic state by weight loss resulted in return of arterial stiffness and blood pressure to normal. Our findings support the hypothesis that arterial stiffness is a cause rather than a consequence of hypertension.

Properties of a Native Cation Channel Activated by Ca2+ Store Depletion in Vascular Smooth Muscle Cells

Depletion of intracellular Ca2+ stores activates capacitative Ca2+influx in smooth muscle cells, but the native store-operated channels that mediate such influx remain unidentified. Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca2+ stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC). Here we characterize these channels in intact cells and present evidence that they belong to the class of store-operated channels, which are activated upon passive depletion of Ca2+ stores. Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca2+ ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches. Channels remained active when inside-out membrane patches were excised from the cells. Excision of membrane patches from resting SMC did not by itself activate the channels. Load-ing SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), which slowly depletes Ca2+ stores without a rise in intracellular Ca2+, activated the same 3-pS channels in cell-attached membrane patches as well as whole cell nonselective cation currents in SMC. TG- and BAPTA-activated 3-pS channels were cation-selective but poorly discriminated among Ca2+, Sr2+, Ba2+, Na+, K+, and Cs+. Open channel probability did not change at negative membrane potentials but increased significantly at high positive potentials. Activation of 3-pS channels did not depend on intracellular Ca2+ concentration. Neither TG nor a variety of second messengers (including Ca2+, InsP3, InsP4, GTPγS, cyclic AMP, cyclic GMP, ATP, and ADP) activated 3-pS channels in inside-out membrane patches. Thus, 3-pS nonselective cation channels are present and activated by TG or BAPTA-induced depletion of intracellular Ca2+ stores in intact SMC. These native store-operated cation channels can account for capacitative Ca2+ influx in SMC and can play an important role in regulation of vascular tone.

Aldose reductase inhibition restores endothelial cell function in diabetic rabbit aorta

A possible relationship between increased aldose reductase activity and abnormal endothelium-dependent relaxation was examined in aorta from al-loxan-induced diabetic rabbits. Isolated aorta of diabetic rabbits, contracted submaximally with phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine or adenosine diphosphate compared to those from normal rabbits. Basal and acetylcholine-stimulated levels of cyclic GMP and the relaxations in response to an endothelium-independent vasodilator, sodium nitroprusside, were not significantly different between diabetic and normal rabbits, indicating that nitric oxide release and action on the vascular smooth muscle were unchanged. The release of thromboxane A2 from diabetic vessels was increased, as previously demonstrated. Treatment with an aldose reductase inhibitor, zopolrestat, normalized the elevated red blood cell sorbitol levels in diabetic rabbits. Zopolrestat also restored the abnormal acetylcholine- and adenosine diphosphate-induced relaxations of the aorta. The aldose reductase inhibitor had no effect on the levels of cyclic GMP or on the increased release of thromboxane A2 in diabetic aorta. These findings suggest that increased activity of the aldose reductase pathway in hyperglycemia is responsible for the abnormal endothelium-dependent relaxation in diabetic blood vessels. Significant alterations in endothelial production of neither nitric oxide nor vasoconstrictor prostanoids could be directly implicated in the improvement caused by the drug, suggesting another mechanism of action.

The Thromboxane A2 Receptor Antagonist S18886 Prevents Enhanced Atherogenesis Caused by Diabetes Mellitus

Background— S18886 is an orally active thromboxane A2 (TXA2) receptor (TP) antagonist in clinical development for use in secondary prevention of thrombotic events in cardiovascular disease. We previously showed that S18886 inhibits atherosclerosis in apolipoprotein E–deficient (apoE−/−) mice by a mechanism independent of platelet-derived TXA2. Atherosclerosis is accelerated by diabetes and is associated with increased TXA2 and other eicosanoids that stimulate TP. The purpose of this study was to determine whether S18886 lessens the enhanced atherogenesis in diabetic apoE−/− mice. Methods and Results— Diabetes mellitus was induced in apoE−/− mice with streptozotocin and was treated or not with S18886 (5 mg · kg−1 · d−1). After 6 weeks, aortic lesion area was increased >4-fold by diabetes in apoE−/− mice, associated with similar increases in serum glucose and cholesterol. S18886 largely prevented the diabetes-related increase in lesion area without affecting the hyperglycemia or hypercholesterolemia. S18886 prevented deterioration of endothelial function and endothelial nitric oxide synthase expression, as well as increases in intimal markers of inflammation associated with diabetes. In human aortic endothelial cells in culture, S18886 also prevented the induction of vascular cell adhesion molecule-1 and prevented the decrease in endothelial nitric oxide synthase expression caused by high glucose. Conclusions— The TP antagonist inhibits inflammation and accelerated atherogenesis caused by diabetes, most likely by counteracting effects on endothelial function and adhesion molecule expression of eicosanoids stimulated by the diabetic milieu.

The Polyphenols Resveratrol and S17834 Prevent the Structural and Functional Sequelae of Diet-Induced Metabolic Heart Disease in Mice

Background— Diet-induced obesity is associated with metabolic heart disease characterized by left ventricular hypertrophy and diastolic dysfunction. Polyphenols such as resveratrol and the synthetic flavonoid derivative S17834 exert beneficial systemic and cardiovascular effects in a variety of settings including diabetes mellitus and chronic hemodynamic overload. Methods and Results— We characterized the structural and functional features of a mouse model of diet-induced metabolic syndrome and used the model to test the hypothesis that the polyphenols prevent myocardial hypertrophy and diastolic dysfunction. Male C57BL/6J mice were fed a normal diet or a diet high in fat and sugar (HFHS) with or without concomitant treatment with S17834 or resveratrol for up to 8 months. HFHS diet–fed mice developed progressive left ventricular hypertrophy and diastolic dysfunction with preservation of systolic function in association with myocyte hypertrophy and interstitial fibrosis. In HFHS diet–fed mice, there was increased myocardial oxidative stress with evidence of oxidant-mediated protein modification via tyrosine nitration and 4-OH-2-nonenol adduction. HFHS diet–fed mice also exhibited increases in plasma fasting glucose, insulin, and homeostasis model assessment of insulin resistance indicative of insulin resistance. Treatment with S17834 or resveratrol prevented left ventricular hypertrophy and diastolic dysfunction. For S17834, these beneficial effects were associated with decreases in oxidant-mediated protein modifications and hyperinsulinemia and increased plasma adiponectin. Conclusions— Resveratrol and S17834 administered concurrently with a HFHS diet prevent the development of left ventricular hypertrophy, interstitial fibrosis, and diastolic dysfunction. Multiple mechanisms may contribute to the beneficial effects of the polyphenols, including a reduction in myocardial oxidative stress and related protein modifications, amelioration of insulin resistance, and increased plasma adiponectin. The polyphenols resveratrol and S17834 may be of value in the prevention of diet-induced metabolic heart disease.

Upregulation of Nox4 by TGFβ1 Oxidizes SERCA and Inhibits NO in Arterial Smooth Muscle of the Prediabetic Zucker Rat

Rationale: Vascular smooth muscle cell (SMC) migration is an important pathological process in several vascular occlusive diseases, including atherosclerosis and restenosis, both of which are accelerated by diabetes mellitus. Objective: To determine the mechanisms of abnormal vascular SMC migration in type 2 diabetes, the obese Zucker rat (ZO), a model of obesity and insulin resistance, was studied. Methods and Results: In culture, ZO aortic SMCs showed a significant increase in Nox4 mRNA and protein levels compared with the control lean Zucker rat (ZL). The sarco-/endoplasmic reticulum Ca2+ ATPase (SERCA) nitrotyrosine-294,295 and cysteine-674 (C674)-SO3H were increased in ZO SMCs, indicating oxidant stress. Unlike ZL SMC, nitric oxide (NO) failed to inhibit serum-induced SMC migration in ZO. Transfection of Nox4 small interference RNA or overexpression of SERCA2b wild type, but not C674S mutant SERCA, restored the response to NO. Knockdown of Nox4 also decreased SERCA oxidation in ZO SMCs. In addition, transforming growth factor-&bgr;1 via Smad2 was necessary and sufficient to upregulate Nox4, oxidize SERCA, and block the antimigratory action of NO in ZO SMCs. Corresponding to the results in cultured SMCs, immunohistochemistry confirmed that Nox4 and SERCA C674-SO3H were significantly increased in ZO aorta. After common carotid artery injury, knockdown of Nox4 by adenoviral Nox4 short hairpin RNA decreased Nox4 and SERCA C674-SO3H staining and significantly decreased injury-induced neointima. Conclusion: These studies indicate that the upregulation of Nox4 by transforming growth factor-&bgr;1 in ZO SMCs is responsible for the impaired response to NO by a mechanism involving the oxidation of SERCA C674. Knockdown of Nox4 inhibits oxidation of SERCA, as well as neointima formation, after ZO common carotid artery injury.

Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide

The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10−7 M), NO (10−10–10−6 M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura‐2 fluorimetry. Zaprinast (10−4 M), increased NO‐stimulated levels of cyclic GMP by 3–20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. The guanylyl cyclase inhibitor, H‐(1,2,4) oxadiazolo(4,3‐a) quinoxallin‐1‐one (ODQ, 10−5 M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp‐8‐Br‐cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10−10–10−8 M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10−7–10−6 M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10−7–10−5 M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp‐8‐Br‐cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP‐independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.

Reduced Responsiveness of Hypercholesterolemic Rabbit Aortic Smooth Muscle Cells to Nitric Oxide

The response to nitric oxide of intracellular free Ca2+ levels, measured by fura 2 fluorimetry, and cyclic GMP, measured by RIA, was evaluated on smooth muscle cells of the thoracic aorta in primary culture from normal and cholesterol-fed rabbits. Relaxation to acetylcholine and nitric oxide was also determined in isolated rings of aorta. After 10 weeks of high-cholesterol diet, the intact aorta relaxed less to both acetylcholine and nitric oxide. In cultured cells from hypercholesterolemic rabbits, intracellular Ca2+ oscillated, and the mean Ca2+ levels were approximately twofold greater than in normal aortic cells. Nitric oxide failed to affect basal Ca2+ in either cell type. The peak and sustained rise in intracellular Ca2+ induced by angiotensin II (10(-7) mol/L) were similar in the two cell types. However, nitric oxide (10(-10) to 10(-6) mol/L) decreased the sustained Ca2+ levels to a significantly smaller extent in cells from cholesterol-fed rabbits. In addition, in cells from hypercholesterolemic rabbits, nitric oxide added before angiotensin II inhibited to a smaller degree the transient increase in intracellular free Ca2+ caused by angiotensin II in the nominal absence of extracellular Ca2+, as well as the increase in Ca2+ associated with the addition of extracellular Ca2+. Measurements of fura 2 quenching caused by Mn2+ influx confirmed that nitric oxide inhibited the entry of extracellular divalent cations significantly less in cells from hypercholesterolemic rabbits. Basal levels of cyclic GMP were significantly less than normal, and nitric oxide increased levels of cyclic GMP to a significantly smaller degree in cells from cholesterol-fed rabbits. These data indicate a substantial resistance to nitric oxide action in aortic smooth muscle cells of cholesterol-fed rabbits. This observation is consistent with the notion that resistance of smooth muscle cells to nitric oxide contributes to abnormal endothelium-dependent vasodilation during hypercholesterolemia and can play a role in the pathogenesis of atherosclerosis.