Effects of dietary sodium on reactive oxygen species formation and endothelial dysfunction in low-density lipoprotein receptor-deficient mice on high-fat diet

Hypertension and high serum cholesterol level are important risk factors for atherosclerosis and coronary heart disease. In the present study we tested the hypothesis whether high sodium intake, when given in combination with Western type high-fat diet, induces endothelial dysfunction and promotes atherosclerosis. Furthermore, the role and enzyme sources of increased oxidative stress were examined. Low-density lipoprotein receptor-deficient mice (LDLR^−/−) and control C57Bl/6 mice received either high-fat, normal-sodium diet (fat 18% and cholesterol 0.5%; NaCl 0.7%; w/w) or high-fat, high-sodium diet (7% NaCl w/w) for 12 weeks. Superoxide formation was assessed by lucigenin enhanced chemiluminescence, endothelial functions were examined ex vivo, and atherosclerotic lesions from the aorta were assessed by light microscopy. High-fat, high-sodium diet increased systolic blood pressure in LDLR^−/− mice but not in C57Bl/6 mice, whereas it induced cardiac hypertrophy in both mouse strains. Dietary combination of fat and sodium induced endothelial dysfunction in LDLR^−/− mice. Preincubation with a superoxide scavenger Tiron normalized endothelial dysfunction, whereas the hydrogen peroxide scavenger catalase did not alter endothelial function. High sodium intake induced superoxide formation in LDLR^−/− mice on high-fat diet. Stimulation of muscarinic receptors in the endothelial cells by acetylcholine increased superoxide generation, whereas preincubation with the nitric oxide synthase (NOS) inhibitor L-arginine methyl ester or endothelium removal reduced superoxide production. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by apocynin decreased vascular superoxide formation whereas the xanthine oxidase inhibitor oxypurinol did not significantly affect oxidative stress in LDLR^−/− mice. In conclusion, the detrimental effects of dietary sodium on endothelial function and progression of atherosclerosis in LDLR^−/− mice on high-fat diet are mediated by increased ROS formation mainly through uncoupled NOS and NADPH oxidase. The present study also underscores the importance of superoxide and endothelial NOS uncoupling in the pathogenesis of endothelial dysfunction.     

Vascular action of the hypoglycaemic agent gliclazide in diabetic rabbits

Summary ATP-dependent potassium channel blockers used as hypoglycaemic agents may have effects on vascular disease in diabetes mellitus beyond their effect on blood glucose control. This study was designed to determine the effects of treatment with gliclazide on the isolated abdominal aorta of diabetic rabbits in which endothelium-dependent relaxation is impaired by a mechanism involving oxygen-derived free radicals. After induction of diabetes with alloxan, there was no effect of gliclazide (10 mg · kg⁻¹· day⁻¹ orally) on blood glucose or insulin levels over a 6 week period. Hence, this permitted an examination of the vascular effects of gliclazide in diabetic rabbits exclusive of metabolic effects. Acetylcholine- and nitric oxide-induced relaxation in aortae from rabbits treated with or without gliclazide were measured in the absence or presence of the nitric oxide synthase inhibitor, N^G-nitro-l-arginine (l-NAME). Diabetes was associated with significant impairment of acetylcholine-induced endothelium-dependent relaxation of the abdominal aorta which was not significant in diabetic rabbits treated with gliclazide in vivo. Aortae from diabetic rabbits studied in the presence of l-NAME showed an exaggerated contraction to acetylcholine which was prevented in rabbits treated with gliclazide. Gliclazide treatment did not affect the response to acetylcholine of normal rabbit aorta, and gliclazide when added in vitro had no effect on the response of diabetic rabbit aorta, suggesting that the effect of gliclazide was specific to the abnormality arising with diabetes and was not due to an acute effect of the drug. These data indicate that gliclazide, aside from either a direct antioxidant action or an effect on insulin or glucose levels, may ameliorate diabetic endothelial cell dysfunction. [Diabetolgia (1998) 41: 9–15] Received: 31 January 1997 and in final revised form: 25 August 1997     

The oxygen radical generator pyrogallol impairs cardiomyocyte contractile function via a superoxide and p38 MAP kinase-dependent pathway

Oxygen-derived free radicals have been demonstrated to contribute to the pathogenesis of myocardial dysfunction, although the underlying mechanism remains not fully understood. This study was designed to examine the role of the superoxide generator pyrogallol on cardiac contractile function and possible intervention with herbal medicines anisodamine and tetramethylpyrazine (TMP) on pyrogallol-induced cardiac contractile response. Adult rat ventricular myocytes were isolated and stimulated to contract at 0.5 Hz. Mechanical properties were evaluated using an lonOptix system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), and maximal velocity of shortening/relengthening (±dL/dt). A 10-min exposure of pyrogallol (0 to 10⁻² M) did not affect cardiac contractile mechanics. However, longer duration of pyrogallol exposure (1, 3, and 6 h) significantly shortened resting cell length, reduced PS and ±dL/dt, and prolonged TPS and TR₉₀ in time- and concentration-dependent manners. The pyrogallol (10⁻⁴ M with 6-h incubation)-induced mechanical defects were prevented by the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (1 μM) and superoxide dismutase (SOD, 500 U/mL) with the exception that pyrogallol-induced PS depression was unaffected by SOD. Interestingly, incubation of herbal antioxidants anisodamine (10⁻⁷ M) and TMP (10⁻⁷ M) effectively attenuated the pyrogallol-induced cardiac mechanical defects with the exception of PS unaffected by TMP. Our data demonstrate a direct inhibitory effect of pyrogallol on cardiac contraction, probably in a superoxide- and p38 MAP kinase-dependent manner. The antioxidant medicines anisodamine and TMP may be useful in the treatment of oxygen free radical-induced myocardial dysfunction.     

Mechanism of leukotriene-induced contraction of isolated guinea pig tracheal smooth muscle

The mechanical response of guinea pig tracheal smooth muscle to leukotriene (LT) B₄, C₄, D₄ and E₄ was investigated, and the effects of several agents on LT-induced contraction were determined. Agents used in the present study were: verapamil, a Ca-channel blocker; dibutyryl cyclic-AMP (DBcAMP); aminophylline; N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), a calmodulin antagonist; N-(6-aminohexyl)-1-naphthalene-sulfonamide hydrochloride (W-5), resembling W-7 in structure but without calmodulin antagonism. The results were as follows: 1) LTB₄ had a contractile activity, dependent on the [Ca]o concentration; it was suppressed by a [Mg]o increase to 1.2 mM and over. Little effect was observed from other drugs. 2) Contractile activities of LTC₄, D₄ and E₄ were dose-dependent, depending upon [Ca]o as well as LTB₄. 3) Verapamil (5×10⁻⁶−10⁻³ M), DBcAMP (3×10⁻⁶−3×10⁻³ M) and aminophylline (3×10⁻⁸−3×10⁻³ M) suppressed LTs (C₄, D₄, and E₄, 10⁻⁸ M)-induced contraction dose-dependently. However, W-7 and W-5 did not suppress LT-induced contraction. These results suggest that the LTC₄-, D₄-and E₄-induced responses could be explained by the effect of LTC₄, D₄ and E₄ on increasing Ca influx through the plasma membrane.     

Acute effect of smoking on superoxide production by pulmonary alveolar macrophages

We determined the acute effect of smoking on superoxide (O ₂ ⁻ ) production by pulmonary alveolar macrophages (AM) in 32 smokers who had bronchoalveolar lavage both before and after smoking. In 18 subjects, AM were obtained either 10, 30, or 60 minutes after the subjects had smoked 2 cigarettes, while in 14, AM were recovered either at 2, 10, or 60 minutes after they had smoked 4 cigarettes. Prior to smoking, O ₂ ⁻ production by AM was significantly greater in smokers than in 8 control nonsmokers for unstimulated AM, as well as for AM stimulated by zymosan and phorbol myristate acetate. Superoxide production by unstimulated AM increased significantly 1 h after subjects had smoked 2 cigarettes, to 140.8 ± SD 14.7% of levels prior to smoking, but there was no increase in 4 control smokers tested before and 1 h after sham smoking. Superoxide production by unstimulated AM decreased significantly 2 minutes after smoking 4 cigarettes but O ₂ ⁻ production by stimulated AM was not affected. Smoking had no effect on O ₂ ⁻ production by AM under the other conditions tested. We conclude that, in addition to the chronic effect of smoking that increases O ₂ ⁻ production by AM, there may be a stimulating effect of acute smoking, depending on the amount smoked and the time after smoking. The increased O ₂ ⁻ production by AM following smoking may be a factor contributing to inactivation of alpha₁-protease inhibitor. Presented in part at the American Thoracic Society Meetings, Miami, May 1984.     

Hydroxyl Radical: A Possible Mediator of Nitroglycerin-Induced Vascular Relaxation

The endothelium-derived relaxing factor, nitric oxide (NO·), is involved in acetylcholine (Ach) and nitroglycerin (NTG)-induced relaxation of vascular smooth muscle. It is known that NO· interacts with the superoxide anion (O₂·^–) at physiological pH and this interaction leads to the generation of hydroxyl radicals (·OH). Hydroxyl radicals are known to induce dilation and act as mediators in acetylcholine (Ach)-induced relaxation of rabbit aortic smooth muscle. Thus, it was hypothesized that ·OH may be involved in NTG-induced relaxation. To test this hypothesis we investigated the effect of NTG on norepinephrine (NE)-precontracted isolated rabbit aortic rings in the absence and/or presence of O₂·^– [superoxide dismutase (SOD)] and ·OH scavengers [dimethylthiourea (DMTU) or mannitol]. Superoxide dismutase, DMTU, and mannitol markedly reduced NTG-induced relaxation of isolated rabbit aortic rings. Superoxide dismutase produced a 32% reduction in NTG-induced vasodilation. Pretreatment with DMTU and/or mannitol reduced NTG-induced relaxation by 56% and 49%, respectively. These results suggest that ·OH may be a mediator involved in NTG-induced vascular relaxation of rabbit aorta.     

Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II

Superoxide anion (O₂⁻) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O₂⁻ is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O₂⁻ production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O₂⁻-generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22^phox, gp91^phox, p47^phox, and p67^phox localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O₂⁻ production. Furthermore, immunodepletion of p67^phox from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67^phox. Further study into the regulation of this adventitial source of O₂⁻ is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.     

Effect of acidosis on contraction, intracellular pH, and calcium in the newborn and adult rabbit aorta

Summary  This study investigated the effect of acidosis on intracellular pH (pH_i), intracellular calcium concentration ([Ca]_i), and vascular contraction in the aorta of the newborn and adult rabbit. Isometric tension, pH_i, and [Ca]_i were measured in an isolated ring preparation. After the vascular contraction was induced with 50 mM KCl, the effect of respiratory acidosis produced by elevation of PCO₂ was studied. Respiratory acidosis caused a transient depression followed by a recovery of contractile tension. The decrease in developed tension was greater in the newborn than in the adult. The decrease in pH_i during acidosis was similar in the two age groups. [Ca]_i increased during acidosis and the increase was greater in the newborn than in the adult. These data show that the vasorelaxant effect of acidosis in the newborn aorta is greater than that in the adult aorta. The greater vasodilation in the newborn cannot be explained by the difference in pH_i or [Ca]_i.     

Relaxant effects of forskolin on guinea pig tracheal smooth muscle

We investigated the relaxant effects of forskolin, a diterpene derivative isolated from the roots ofColeus forskohlii, on guinea pig airway smooth muscle by measuring the isometric tension of tracheal smooth muscle in vitro and transcutaneous Po₂ during the histamine inhalation test (HIT) in vivo. Forskolin (10⁻⁹–10⁻⁵ M) caused dose-dependent relaxant effects on resting tone and on leukotriene C₄ (10⁻⁷ M)-, leukotriene D₄ (10⁻⁷ M)-, and carbachol (3 × 10⁻⁶ M)-induced contraction of tracheal smooth muscle. Moreover, with propranolol pretreatment the relaxant effect of forskolin on tracheal smooth muscle did not change, whereas with the same pretreatment the relaxant effect of isoproterenol diminished. Forskolin (10⁻⁸–10⁻⁶ M) raised tissue cyclic AMP levels dose-dependently in tracheal smooth muscle (6.7–359.9 pmol/mg protein). Forskolin (1 mg/kg) administered subcutaneously raised the respiratory threshold of (RT-histamine in the HIT. The determination of the RT-histamine by measuring tcPo₂ was possible without anesthesia. These results suggest that forskolin relaxes airway smooth muscle in guinea pigs in vitro and in vivo by raising tissue cyclic AMP levels and that its actions are independent ofβ-adrenoceptors.     

Regional differences of superoxide dismutase activity enhance the superoxide–induced electrical heterogeneity in rabbit hearts

During myocardial ischemia and the subsequent reperfusion, free radicals are important intermediates of the cellular damage and rhythm disturbances. We examined the effects of superoxide radicals or hydrogen peroxide (H₂O₂) on the action potentials in isolated rabbit Purkinje fibers, atrial muscle and ventricular muscle. Reactive oxygen species (ROS) donors such as adriamycin, xanthine/xanthine oxidase and menadione induced prolongation of APD₉₀ in Purkinje fibers. Menadione (30 µM), the most specific superoxide radical donor, prolonged the action potential duration at 90% repolarization (APD₉₀) by 17% in Purkinje fibers, whereas it shortened the APD by 57% in ventricular muscle, and it did not affect the atrial APD. All these menadione–induced effects were completely blocked by 2,2,6,6–tetramethyl– 1–peperadinyloxy, a superoxide radical scavenger. Superoxide dismutase (SOD) activity was lowest in Purkinje fibers, it was moderate in atrial muscle and highest in ventricular muscle. H₂O₂ shortened the APDs of all three cardiac tissues in a concentration–dependent manner. These results suggest that the different electrical responses to O₂^●– in different cardiac regions may result from the regional differences in the SOD activity, thereby enhancing the regional electrical heterogeneity.Drs. B. H. Choi and K.–Ch. Ha contributed equally to this study.     

The H2O2-Generating Enzyme,Xanthine Oxidase,Decreases Luminal Ca2+ Content of the IP3-Sensitive Ca2+ Store in Vascular Endothelial Cells

Objective: Xanthine oxidase inhibits agonist-stimulated Ca²⁺ signaling in calf pulmonary artery endothelial cells by an H₂O₂-dependent mechanism. We investigated the effect of xanthine oxidase on luminal Ca²⁺ content of the inositol-1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store. Methods: Luminal Ca²⁺ content was estimated from the net release of Ca²⁺ activated by 2,5-di-t-butylhydroquinone (BHQ), an inhibitor of microsomal Ca²⁺ pumps. Results: Initially, xanthine oxidase depleted the IP₃-sensitive Ca²⁺ store of releasable Ca²⁺, but with more prolonged incubation, the enzyme also depleted non-IP₃-sensitive stores. In addition, xanthine oxidase inhibited capacitative Ca²⁺ influx. Similar results were observed when thapsigargin was substituted for BHQ. Conclusions: Depletion of luminal Ca²⁺ content within the IP₃-sensitive Ca²⁺ store contributes to xanthine oxidase inhibition of Ca²⁺ signaling in vascular endothelial cells.     

Deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced vena cava thrombosis

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. Since the role of TAFI in thrombus formation is still controversial in mice, our present study was designed to evaluate mice deficient in TAFI (TAFI^−/−) on FeCl₃-induced vena cava and carotid artery thrombosis. Parallel studies were carried out in wild-type mice using a potato carboxypeptidase inhibitor (PCI), a selective inhibitor of activated TAFI (TAFIa). Significant reduction in thrombus formation was observed in TAFI^−/− mice (n = 8, P < 0.05 compared to wild-type littermates) but not in heterozygous (TAFI^+/−) mice in 3.5% FeCl₃-induced vena cava thrombosis. A similar effect was observed following treatment with 5 mg/kg bolus plus 5 mg/kg/h PCI in the same venous thrombosis model in C57BL/6 mice (n = 8, P < 0.01 compared to vehicle). No compositional difference was observed for the venous thrombi in TAFI^−/− and wild-type littermates with or without PCI treatment using histological assessment. In contrast, neither TAFI deficiency nor treatment with PCI showed antithrombotic efficacy in the 3.5% FeCl₃-induced carotid artery thrombosis model. In a tail transection bleeding time model, both TAFI deficiency and PCI treatment increased bleeding time up to 4.5 and 3.5 times, respectively, over controls (P < 0.05, n = 8). Similar ex vivo fibrinolytic activities were demonstrated for both TAFI deficiency and PCI treatment as enhanced lysis of thrombin-induced plasma clots and lysis of whole blood clot in a thrombelastograph. These data provide direct evidence for the role of TAFIa in vena cava thrombosis without the addition of exogenous thrombolytic in mice. The strong ex vivo fibrinolytic activity of TAFI deficiency or TAFIa inhibition by PCI provides a biomarker of TAFIa inhibition that tracks in vivo antithrombotic efficacy.     

Bronchodilating effects of the anesthetic ketamine in an in vitro guinea pig preparation

Ketamine, a dissociative anesthetic, is capable of reducing airway resistance and has proved useful in anesthetizing surgical patients with acute or chronic bronchospasm. To determine if ketamine alters smooth muscle tone, the relative responses of large and small airways of the same animal were studied by comparing the pharmacologic reactivity of tracheal smooth muscle strips with that of a specially prepared perfused bronchial tree. Trachealis and bronchial smooth muscle were found to react to methacholine and histamine in a concentration-dependent manner and have similar sensitivities. Ketamine, by itself, in the concentration range 10⁻⁸–10⁻³ M did not alter resting tone as compared to epinephrine, which reduced baseline tone. In tissues precontracted with histamine or methacholine at ED₅₀ doses, ketamine inhibited smooth muscle contraction. In a second series of experiments, the dose-dependent contraction of smooth muscle to histamine and methacholine was reevaluated in the presence of ketamine. Both tissue sensitivity and maximum contractile response to these agonists were reduced by ketamine at 10⁻⁴ M. These data indicate that ketamine alters the in vitro response of guinea pig airways to agonists associated with the asthmatic state. Although ketamine does not reduce airway tone in nonstimulated tissues, its effects on agonist-induced contraction of airway tissues in vitro are consistent with clinical observations that ketamine relieves bronchospasm.     

Effect of PAR2 in regulating TNF-α and NAD(P)H oxidase in coronary arterioles in type 2 diabetic mice

Protease-activated receptor-2 (PAR2) is expressed in endothelial cells and mediates endothelium-dependent vasodilation. We hypothesized that PAR2 regulates tumor necrosis factor-alpha (TNF-α)-induced coronary arteriolar dysfunction in type 2 diabetic (db/db) mice. To test this, coronary arterioles from WT control, db/db, db/db mice treated with PAR2 antagonist FSLLRY–NH₂ (db/db+FSLLRY–NH₂) and db/db mice null for TNF (db^TNF−/db^TNF−) were isolated and pressurized (60 cmH₂O) without flow. Although vasodilation to the endothelium-independent vasodilator sodium nitroprusside (SNP) was not different among WT, db/db, db/db+FSLLRY–NH₂ and db^TNF−/db^TNF−, endothelium-dependent acetylcholine (ACh)- and flow-mediated vasodilation were impaired in db/db mice but were enhanced in db^TNF−/db^TNF− mice and db/db mice treated with PAR2 antagonist. NOS inhibitor N ^G-nitro-l-arginine-methyl ester (l-NAME) significantly reduced ACh-induced dilation in WT, db^TNF−/db^TNF− and db/db+FSLLRY–NH₂, but did not alter the vasodilation in db/db mice. In contrast, cyclooxygenase (COX) inhibitor indomethacin (Indo) did not alter ACh-induced vasodilation in these four groups of mice. PAR2-activating peptide (PAR2-AP, 2-Furoyl-LIGRLO-am)-induced dilation was higher in db/db mice than that in WT, db^TNF−/db^TNF− and db/db mice treated with PAR2 antagonist. These effects were abolished by denudation, or in the presence of l-NAME or Indo. Protein expressions of TNF-α, PAR2, gp91^phox and p47^phox in the heart and isolated coronary arterioles were higher in db/db mice compared to WT mice. Administration of PAR2 antagonist to db/db mice reduced protein expression of TNF-α, gp91^phox and PAR2. Protein expression of gp91^phox and p47^phox was lower in db^TNF−/db^TNF− compared to db/db mice. These results indicate that PAR2 plays a pivotal role in endothelial dysfunction in type 2 diabetes by up-regulating the expression/production of TNF-α and activating NAD(P)H oxidase subunit p47^phox.     

Absence of a role for superoxide anion, hydrogen peroxide and hydroxyl radical in endothelium-mediated relaxation of rabbit aorta

We tested the hypothesis that the endothelium-dependent relaxation of rabbit thoracic aorta in vitro is mediated by reduced metabolites of oxygen. Helical vascular strips were contracted with either norepinephrine or phenylephrine. Oxygen metabolites, generated by the xanthine oxidase reaction, completely relaxed norepinephrine-induced contractile tone but not tone induced by phenylephrine. A mixture of oxygen metabolite scavengers (superoxide dismutase, catalase and mannitol) eliminated the relaxation induced by the xanthine oxidase products. Acetylcholine caused a dose-dependent and endothelium-dependent relaxation of the strips; this was not inhibited by the presence of the scavengers. We conclude that reduced oxygen metabolites have little direct effect on rabbit aortic smooth muscle in vitro, although they indirectly but specifically relax norepinephrine-induced tone, presumably by oxidation of norepinephrine. Oxygen metabolites do not appear to mediate the endothelium-dependent relaxation response of this tissue to acetylcholine.     

Effects of Recycled Paper Dust Extracts on Isolated Guinea Pig Trachea

The effect of paper dust collected at two different locations in a paper recycling plant (PD1 and PD2) on isolated nonsensitized guinea pig tracheal smooth muscle was studied in vitro. Dust extracts were prepared as a 1:10 w/v aqueous solution. Dose-related contractions of guinea pig tracheal rings were elicited with both PD1 and PD2. Pharmacologic studies were performed with atropine (10⁻⁶ M), indometacin (10⁻⁶ M), pyrilamine (10⁻⁶ M), LY171883 (10⁻⁵ M), nordihydroguaiaretic acid (10⁻⁵ M), and TMB8 (10⁻⁵ M). The possible role of endogenous neuropeptides in this constrictor process was studied by depleting neural mediators with capsaicin (5 × 10⁻⁶ M) before challenge with dust extracts. Constrictor effects were partially inhibited by a wide variety of the mediator blocking agents. The effects of both extracts were almost totally inhibited by the anticholinergic agent atropine, suggesting that a principal pathway mediating this response may involve the parasympathetic nervous system. The intracellular calcium-blocking agent TMB8 also induced a reduction of the contractile responses to PD1 and PD2 concsistent with the well established role of intracellular calcium in smooth muscle constriction. Pretreatment with capsaicin significantly increased the contractile activity of paper dust extracts but only at the higher doses of these extracts. This suggests that the effect of paper dust is not initiated by the release of mediators stored in sensory nerves but that the prerelease of these mediators may enhance the constrictor effects of these dusts. We suggest that paper dust extracts cause dose-related airway smooth muscle constriction possibly associated with the release of cholinergic as well as other mediators. The constrictor effect does not require tissue presensitization or the release of neuropeptides from sensory nerves. Accepted for publication: 21 March 1997     

The Highly Expressed and Inducible Endogenous NAD(P)H:quinone Oxidoreductase 1 in Cardiovascular Cells Acts as a Potential Superoxide Scavenger

It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O₂^•−) though the rate of reaction of O₂^•− with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O₂^•−. We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O₂^•− scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O₂^•−. In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O₂^•− as compared to cytosols from untreated cells. This increased ability to scavenge O₂^•− was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O₂^•− generator further confirmed the ability of NQO1 from HASMCs to scavenge O₂^•−. The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O₂^•− scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O₂^•− scavenger.     

Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice

Aims/hypothesis  We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K⁺ (K_ATP) channels (Kir6.2 ^−/− mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. Methods  Pancreas perfusion experiments were performed using wild-type (Kir6.2 ^+/+ ) or Kir6.2 ^−/− mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2 ^−/− mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K_ATP channels with glibenclamide. Results  The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2 ^−/− pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K_ATP channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2 ^+/+ , as in Kir6.2 ^−/− pancreases. Conclusions/interpretation  Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2 ^−/− beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.     

Taurodeoxycholate Modulates Apical Cl<Superscript>−</Superscript>/OH<Superscript>−</Superscript> Exchange Activity in Caco2 Cells

Bile acid malabsorption has been shown to be associated with diarrhea in cases such as ileal resection Crohn’s disease of the ileum, and radiation enteritis. The mechanisms of bile acid-induced diarrhea are not fully understood. Although the induction of colonic chloride secretion in response to bile acids has been extensively investigated, to date the direct effect of bile acids on intestinal chloride absorption has not been well defined. Therefore, the current studies were undertaken to investigate the effect of bile acids on the apical Cl⁻/OH⁻ exchange process utilizing Caco2 monolayers as an in vitro cellular model. Cl⁻/OH⁻ exchange activity was measured as DIDS-sensitive pH gradient-driven ³⁶Cl uptake. The results are summarized as follows: (i) short-term exposure (20 min) of Caco2 cells to taurodeoxycholate (TDC; 200 μM) and glycochenodeoxycholate (GCDC; 200 μM) acids significantly inhibited apical Cl⁻/OH⁻ exchange (by ∼60–70%); (ii) the Ca²⁺ chelator BAPTA-AM blocked the inhibition by TDC; (iii) the reduction in Cl⁻/OH⁻ exchange by TDC was reversed by the PKC inhibitor, chelerythrine chloride; (iv) functional and inhibitor studies indicated that TDC induced inhibition of Cl⁻/OH⁻ exchange was mediated via the activation of the PKCβI isoform; (v) the effect of TDC on apical Cl⁻/OH⁻ exchange was completely blocked by the PI3 kinase inhibitor LY294002 (5 μM); and (vi) the PKA inhibitor, RpcAMP, had no effect on TDC induced inhibition of Cl⁻/OH⁻ exchange. In conclusion, our studies provide direct evidence for inhibition of human intestinal apical Cl⁻/OH⁻ exchange activity by bile acids via Ca²⁺-, PI3 kinase-, and PKCβI-dependent pathways in Caco2 cells.     

Arrhythmogenic peroxynitrite-induced alterations in mammalian heart contractility and its prevention with quercetin-filled liposomes

The aim of the present study was to evaluate the effects of quercetin-filled phosphatidylcholine liposomes (PCLs) on peroxynitrite (ONOO⁻)-induced cardiac arrhythmias. Experiments were done using different experimental models, including isolated rat papillary muscle, Langendorff perfused rat hearts, and anesthetized animals. Being exogenously applied in a concentration greater than 50 μM, ONOO⁻, caused inhibition of isometric twitch amplitude in isolated papillary muscles and led to an appearance of arrhythmias. Decomposed ONOO⁻ had no similar effects and reversibly increased twitch amplitude. Authentic nitric oxide (NO, 100 μM) did not produce arrhythmias and had no significant effect on twitch amplitude. Verapamil and ruthenium red were with-out effect on ONOO⁻-induced arrhythmias, whereas tetrodotoxin and nicorandil effectively prevented arrhythmias development. Ouabain increased the arrhythmogenic effect of ONOO⁻. ONOO⁻ significantly decreased coronary perfusion pressure (CPP) and mean left-ventricular pressure (MLVP) in the Langendorff perfused rat heart and produced severe arrhythmias. Authentic nitric oxide (NO) decreased CPP and MLVP insignificantly and resulted in a low incidence of arrhythmias. The NO donor SIN-1 in doses greater than 50 μM led to the appearance of low-incidence arrhythmias in anesthetized rats. Intraventricular injection of ONOO⁻ promotes the appearance of a high incidence of arrhythmias in anesthetized rats and decreased MLVP. PCLs filled with the antioxidant quercetin restored normal cardiac contractility in both isolated tissues and anesthetizes animals. In conclusion, we hypothesized that ONOO⁻, but not its decomposed products, can initiate membrane lipid peroxidation and damage the phospholipid environment of ionic channels in myocardial cell plasma membranes inducing abnormal cardiac action potentials, arrhythmogenesis, and contractile dysfunction. Quercetin-filled PCL provide reliable protection against peroxynitrite-induced myocardial injury in isolated cardiac tissues and anesthetized animals primarily as a result of the decomposition of endogenously formed ONOO⁻.