Schuhmacher S, Wenzel P, Schulz E, Oelze M, Mang C, Kamuf J, Gori T, Jansen T, Knorr M, Karbach S, Hortmann M, Mäthner F, Bhatnagar A, Förstermann U, Li H, Münzel T, Daiber A (2010)
Pentaerythritol tetranitrate improves angiotensin II-induced vascular dysfunction via induction of heme oxygenase-1.
2010; 55(4): 897-904
The organic nitrate pentaerythritol tetranitrate is devoid of nitrate tolerance, which has been attributed to the induction of the antioxidant enzyme heme oxygenase (HO)-1. With the present study, we tested whether chronic treatment with pentaerythritol tetranitrate can improve angiotensin II-induced vascular oxidative stress and dysfunction. In contrast to isosorbide-5 mononitrate (75 mg/kg per day for 7 days), treatment with pentaerythritol tetranitrate (15 mg/kg per day for 7 days) improved the impaired endothelial and smooth muscle function and normalized vascular and cardiac reactive oxygen species production (mitochondria, NADPH oxidase activity, and uncoupled endothelial NO synthase), as assessed by dihydroethidine staining, lucigenin-enhanced chemiluminescence, and quantification of dihydroethidine oxidation products in angiotensin II (1 mg/kg per day for 7 days)-treated rats. The antioxidant features of pentaerythritol tetranitrate were recapitulated in spontaneously hypertensive rats. In addition to an increase in HO-1 protein expression, pentaerythritol tetranitrate but not isosorbide-5 mononitrate normalized vascular reactive oxygen species formation and augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase I and dihydrofolate reductase in angiotensin II-treated rats, thereby preventing endothelial NO synthase uncoupling. Haploinsufficiency of HO-1 completely abolished the beneficial effects of pentaerythritol tetranitrate in angiotensin II-treated mice, whereas HO-1 induction by hemin (25 mg/kg) mimicked the effect of pentaerythritol tetranitrate. Improvement of vascular function in this particular model of arterial hypertension by pentaerythritol tetranitrate largely depends on the induction of the antioxidant enzyme HO-1 and identifies pentaerythritol tetranitrate, in contrast to isosorbide-5 mononitrate, as an organic nitrate able to improve rather than to worsen endothelial function.
Li H, Hortmann M, Daiber A, Oelze M, Ostad MA, Schwarz PM, Xu H, Xia N, Kleschyov AL, Mang C, Warnholtz A, Munzel T, Forstermann U (2008)
COX2-selective and non-selective NSAIDs induce oxidative stress by upregulating vascular NADPH oxidases.J Pharmacol Exp Ther
2 (COX2)-selective inhibitors (coxibs) and non-selective non-steroidal
anti-inflammatory drugs (NSAIDs) are associated with an increase in
cardiovascular events. The current study was designed to test the
effect of coxibs and non-selective NSAIDs on vascular superoxide and NO
production. mRNA expression of endothelial NO-synthase (eNOS) and of
the vascular NADPH oxidases was studied in spontaneously hypertensive
rats (SHR) and in human endothelial cells. The expression of Nox1,
Nox2, Nox4 and p22phox was increased markedly by the non-selective
NSAIDs diclofenac or naproxen, and moderately by rofecoxib or celecoxib
in the aorta and heart of SHR. The upregulation of NADPH oxidases by
NSAIDs was associated with increased superoxide content in aorta and
heart, which could be prevented by the NADPH oxidase inhibitor
apocynin. NSAIDs reduced plasma nitrite and diminished the
phosphorylation of vasodilator-stimulated phosphoprotein (VASP). This
demonstrates a reduction in vascular NO production. Aortas from
diclofenac-treated SHR showed an enhanced protein nitrotyrosine
accumulation, indicative of vascular peroxynitrite formation.
Peroxynitrite can uncouple oxygen reduction from NO synthesis in eNOS.
Accordingly, the eNOS inhibitor N(G)-nitro-L-arginine methyl ester
reduced superoxide content in aortas of NSAID-treated animals,
demonstrating eNOS uncoupling under those conditions. Also in human
endothelial cells, NSAIDs increased Nox2 expression and diminished
production of bioactive NO. In healthy volunteers, NSAID treatment
reduced nitroglycerin-induced, NO-mediated vasodilatation of the
brachial artery. These results indicate that NSAIDs may increase
cardiovascular risk by inducing oxidative stress in the vasculature,
with non-selective NSAIDs being even more critical than coxibs in this
Wohlfart P, Xu H, Endlich
A, Habermeier A, Closs EI, Hübschle T, Mang C,
Strobel H, Suzuki T, Kleinert H, Förstermann U, Ruetten H, Li H (2008)
Antiatherosclerotic effects of small-molecular-weight compounds enhancing endothelial nitric-oxide synthase (eNOS) expression and preventing eNOS uncoupling.
J Pharmacol Exp
Many cardiovascular diseases are associated with reduced levels of bioactive nitric oxide (NO) and an uncoupling of oxygen reduction from NO synthesis in endothelial NO synthase (eNOS uncoupling). In human endothelial EA.hy 926 cells, two small-molecular-weight compounds with related structures, 4-fluoro-N-indan-2-yl-benzamide (CAS no. 291756-32-6; empirical formula C16H14FNO; AVE9488) and 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide (CAS no. 450348-85-3; empirical formula C17H13F2NO3; AVE3085), enhanced eNOS promoter activity in a concentration-dependent manner; with the responsible cis-element localized within the proximal 263 base pairs of the promoter region. RNA interference-mediated knockdown of the transcription factor Sp1 significantly reduced the basal activity of eNOS promoter, but it did not prevent the transcription activation by the compounds. Enhanced transcription of eNOS by AVE9488 in primary human umbilical vein endothelial cells was associated with increased levels of eNOS mRNA and protein expression, as well as increased bradykinin-stimulated NO production. In both wild-type C57BL/6J mice and apolipoprotein E-knockout (apoE-KO) mice, treatment with AVE9488 resulted in enhanced vascular eNOS expression. In apoE-KO mice, but not in eNOS-knockout mice, treatment with AVE9488 reduced cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduced atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice. Aortas from apoE-KO mice showed a significant generation of reactive oxygen species. This was partly prevented by nitric-oxide inhibitor N(omega)-nitro-l-arginine methyl ester, indicating eNOS uncoupling. Treatment of mice with AVE9488 enhanced vascular content of the essential eNOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin and reversed eNOS uncoupling. The combination of an up-regulated eNOS expression and a reversal of eNOS uncoupling is probably responsible for the observed vasoprotective properties of this new type of compounds.
The effects of capsaicin on [(3)H]acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [(3)H]choline. Capsaicin concentration-dependently increased both basal [(3)H]acetylcholine release (pEC(50) 7.0) and muscle tone (pEC(50) 6.1). The facilitatory effects of capsaicin were antagonized by 1 muM capsazepine (pK (B) 7.0 and 7.6), and by the combined blockade of NK(1) and NK(3) tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 muM). This suggests that stimulation by capsaicin of TRPV1 receptors on primary afferent fibres causes a release of tachykinins which, in turn, mediate via NK(1) and NK(3) receptors an increase in acetylcholine release. The capsaicin-induced acetylcholine release was significantly enhanced by the NO synthase inhibitor L-N(G)-nitroarginine (100 muM). This indicates that tachykinins released from sensory neurons also stimulate nitrergic neurons and thus lead, via NO release, to inhibition of acetylcholine release. Capsaicin concentration-dependently reduced the electrically-evoked [(3)H]acetylcholine release (pEC(50) 6.4) and twitch contractions (pEC(50) 5.9). The inhibitory effects were not affected by either capsazepine, NK(1) and NK(3) receptor antagonists, the cannabinoid CB(1) antagonist SR141716A or by L-N(G)-nitroarginine. Desensitization of TRPV1 receptors by a short exposure to 3 muM capsaicin abolished the facilitatory responses to a subsequent administration, but did not modify the inhibitory effects. In summary, capsaicin has a dual effect on cholinergic neurotransmission. The facilitatory effect is indirect and involves tachykinin release and excitation of NK(1) and NK(3) receptors on cholinergic neurons. The inhibition of acetylcholine release may be due to a decrease of Ca(2+) influx into cholinergic neurons.
Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is a powerful vasodilator and possesses vasoprotective effects. Therefore, augmentation of eNOS expression and -activity by pharmacological means could provide protection against cardiovascular disease. However, this concept has been questioned recently, because in several disease models, eNOS upregulation was associated with a dysfunctional enzyme (referred to as eNOS uncoupling). In contrast, the present study demonstrates that an eNOS gene expression-enhancing compound with additional protein kinase C (PKC) inhibitory properties can upregulate eNOS while preserving its enzymatic function. Apolipoprotein E-knockout mice were treated for 7 days with midostaurin (4'-N-benzoyl staurosporine, compound CGP 41251, 50-125 mg/kg/day), a PKC inhibitor previously shown to increase eNOS expression and NO production in cultured human endothelial cells. Midostaurin treatment enhanced eNOS mRNA expression (RNase protection assay) in mouse aorta, kidney, and heart in a dose-dependent fashion. In the dorsal skinfold microcirculation, midostaurin produced an arteriolar vasorelaxation (intravital microscopy), which could be prevented by the NOS inhibitor L-NAME, indicating that the upregulated eNOS remained functional. In organ chamber experiments, the aorta from midostaurin-treated mice showed an enhanced NO-mediated relaxation in response to acetylcholine. Accordingly, serum levels of nitrite/nitrate (NO-Analyzer) were increased, and the production of reactive oxygen species in the aorta (L-012 chemiluminescence) was reduced by midostaurin. Thus, in mice in vivo, midostaurin treatment results in enhanced expression of eNOS with preserved enzyme function and enhanced production of bioactive NO. Given the beneficial effects of endothelial-derived NO, vasoprotective and anti-atherosclerotic effects are likely to ensue.
Stimulation of muscarinic receptors in the urothelium of the pig
bladder causes the release and transfer of a diffusable factor which inhibits contractions of the
underlying detrusor muscle. We investigated whether the contractions of
human detrusor strips elicited by the muscarinic agonist carbachol, by
electrical field stimulation or KCl are affected
by the urothelium.
The effects of the NO donor, SNAP, and of the NO-synthase inhibitor, L-NNA, on release of acetylcholine and contractions were studied in isolated ileum preparations of man and guinea-pig. Strips were incubated with [3H]choline and superfused with a physiological salt solution. Release of [3H]acetylcholine was elicited by electrical stimulation. In the guinea-pig ileum, SNAP (100 µM) increased basal [3H]acetylcholine release and muscle tone, and, in addition, inhibited the electrically-evoked release and contractions. In contrast, SNAP had not effect on basal or evoked [3H]acetylcholine release in the human ileum. L-NNA (300 µM) facilitated the evoked release and contractions of the guinea-pig ileum, but had no effect in the human ileum. It is concluded that endogenous NO exerts a tonic inhibitory effect on cholinergic neurotransmission in the guinea-pig ileum which contributes to the relaxant effect of NO. The results do not indicate that NO has a similar function in the human ileum.
Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor, NG -nitro-L-arginine (L-NNA), and the guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo[4,3-alpha]qui-noxalin-1-one (ODQ), concentration-dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of L-NNA were specifically antagonized by L-arginine. In contrast, L-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor, S-nitroso-N-acetyl-DL-penicillamine, inhibited the electrically evoked release of [3 H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We concluded that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO mediating inhibition of cholinergic neurotransmission in mouse ileum.
The effects of K(ATP) channel blockers (glibenclamide, HMR 1883, HMR 1372) and openers (cromakalim, pinacidil, diazoxide) on the electrically-evoked (5 Hz) release of [(3)H]acetylcholine were studied in isolated guinea-pig atria and myenteric plexus-longitudinal muscle preparations which had been preincubated with [(3)H]choline. Atria: Cromakalim (0.3 microM and 1 microM), pinacidil (10 microM) and diazoxide (30 microM) significantly reduced the stimulation-evoked release of [(3)H]acetylcholine. The inhibition produced by cromakalim and pinacidil was prevented by 1 microM of either HMR 1883, HMR 1372 or glibenclamide. The blockers alone significantly increased the release at concentrations of 30 microM, whereas 1 microM and 10 microM had no effect. Myenteric plexus-longitudinal muscle preparation: The electrically-evoked release of [(3)H]acetylcholine was not affected by K(ATP) channel blockers or openers. In contrast, the contractions of the longitudinal muscle caused by electrical stimulation or by carbachol were strongly inhibited by 1 microM cromakalim which suggests that the relaxant effect of the K(ATP) channel openers is exclusively a direct effect on intestinal smooth muscle.The findings suggest that blockade of activated K(ATP) channels in vagal nerves of guinea-pig atria stimulates acetylcholine release, and that this effect may contribute to the antiarrhythmic actions of K(ATP) channel blockers. By contrast, release of acetylcholine from guinea-pig myenteric plexus is not modulated by K(ATP) channels which suggests heterogeneity of K(ATP) channel distribution in peripheral autonomic nerves.
1. The effects of anandamide on [3H]-acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [3H]-choline. 2. Anandamide increased both basal [3H]-acetylcholine release (pEC(50) 6.3) and muscle tone (pEC(50) 6.3). The concentration-response curves for anandamide were shifted to the right by 1 microM capsazepine (pK(B) 7.5 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 microM). The CB1 and CB2 receptor antagonists, SR141716A (1 microM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide. 3. Anandamide inhibited the electrically-evoked release of [3H]-acetylcholine (pEC(50) 5.8) and contractions (pEC(50) 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 microM anandamide. 4. The inhibitory effects of anandamide were not changed by either capsazepine (1 microM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 microM). SR141716A (1 microM) produced rightward shifts in the inhibitory concentration-response curves for anandamide yielding pK(B) values of 6.6 and 6.2. 5. CP55940 inhibited the evoked [3H]-acetylcholine release and contractions, and SR141716A (0.1 microM) shifted the concentration-response curves of CP55940 to the right with pK(B) values of 8.4 and 8.9. 6. The experiments confirm the existence of release-inhibitory CB1 receptors on cholinergic myenteric neurones. We conclude that anandamide inhibits the evoked acetylcholine release via stimulation of a receptor that is different from the CB1 and CB2 receptor. Furthermore, anandamide increases basal acetylcholine release via stimulation of vanilloid receptors located at primary afferent fibres.
The effects of the nitric oxide (NO) donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) and the NO synthase inhibitor L-N(G)-nitroarginine (L-NOARG) on the electrically evoked [(3)H]-acetylcholine release were studied in an epithelium-free preparation of guinea-pig trachea that had been preincubated with [(3)H]-choline. SNAP (100 and 300 microM) caused small but significant increases of the electrically evoked [(3)H]-acetylcholine release (121+/-4% and 124+/-10% of control). Resting outflow of [(3)H]-ACh was not affected by SNAP. The increase by SNAP was abolished by the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo [4,3-alpha]quinoxalin-1-one (ODQ, 1 microM). The facilitatory effect of SNAP (100 and 300 microM) was reversed into inhibition of release (to 74+/-4% and to 78+/-2%) after pretreatment of the trachea with capsaicin (3 microM). ODQ prevented the inhibition. Capsaicin pretreatment alone did not significantly alter the release of [(3)H]-acetylcholine. A significant inhibition by SNAP (100 microM) of [(3)H]-acetylcholine release (78+/-3%) was also seen in the presence of the NK(2) receptor antagonist SR 48968 (30 nM). L-NOARG (10 and 100 microM) significantly enhanced the electrically-evoked smooth muscle contractions, but caused no significant increases of the evoked release from capsaicin pretreated trachea strips. This might indicate that the inhibitory effect of endogenous NO on acetylcholine release is too small to be detected by overflow studies. It is concluded that NO has dual effects on the evoked acetylcholine release. NO enhances release in the absence of modifying drugs, but NO inhibits acetylcholine release after blockade of the NK(2) receptor or after sensory nerve depletion with capsaicin. This suggests that NO and endogenous tachykinins act in series to produce an increase in acetylcholine release.
RATIONALE: A quantitative technique was used to compare the pharmacological potency in healthy volunteers of angiotensin II receptor antagonists (AIIA): candesartan cilexetil, losartan, irbesartan, valsartan, and telmisartan. METHODS: In a randomised, double-blind, parallel-group (4x12 subjects) study, single oral doses of candesartan cilexetil 4, 8 and 16 mg, losartan potassium 25, 50 and 100 mg, valsartan 40, 80 and 160 mg, and irbesartan 75, 150 and 300 mg were administered on three consecutive days. Telmisartan 20, 40 and 80 mg was similarly evaluated in 12 volunteers in an open amendment. Angiotensin II (Ang II) antagonistic effects were determined in vivo from rightward shifts in Ang II dose-response curves for diastolic blood pressure (BP) and dose ratios were calculated. Apparent K(i)-doses, i.e. doses (in mg) required to induce a two-fold shift in Ang II dose-response curves (equivalent to approx. 50% blockade of receptors) were determined, using Schild regression analysis. RESULTS: All treatments dose-dependently attenuated increases in diastolic BP induced by infusion of exogenous Ang II. Candesartan cilexetil appeared to have a more pronounced increase in effect following cumulative dosing. At 24 hours, apparent K(i)-doses were: candesartan cilexetil 6 mg, irbesartan 123 mg, valsartan 93.5 mg, and telmisartan 54 mg. It was not possible to determine an apparent K(i)-dose for losartan at 24 hours. CONCLUSION: Consistent with results from experimental pharmacology, candesartan cilexetil displayed the highest pharmacological potency (i.e. antagonistic activity per mg substance) of the AIIAs tested. Apparent K(i)-doses at 24 hours were within the dose range recommended for clinical use in patients with hypertension.
OBJECTIVES: To compare the angiotensin II antagonistic properties of the usual recommended oral starting doses of various angiotensin II receptor antagonists-150 mg irbesartan, 80 mg valsartan, and 50 mg losartan-in humans. SUBJECTS AND METHODS: Eighteen healthy men were enrolled in a double-blind, randomized crossover study. Angiotensin II dose-effect curves of diastolic blood pressure and radioreceptor assay were performed before and up to 47 hours after single and multiple doses of the antagonists. The rightward shift of the angiotensin II dose-effect curves (dose ratio-1) assessed the antagonistic effects in vivo. The degree of receptor occupancy in plasma was detected by a rat lung radioreceptor assay ex vivo in vitro. RESULTS: All of the drugs clearly showed antagonistic effects to angiotensin II in vivo (dose ratio-1) and in vitro (radioreceptor assay). Within the given doses the dose ratio-1 for irbesartan was greater than for valsartan and losartan after single and repetitive dosing, reaching statistical significance at various time points up to 36 hours versus valsartan and up to 47 hours versus losartan. The apparent half-lives of the decay of the effects were approximately 8 hours for valsartan and losartan, whereas 15 to 18 hours were obtained with irbesartan. These findings were supported by the radioreceptor assay data: the percentage of receptor occupancy for irbesartan was significantly greater than for valsartan and losartan up to 47 hours. CONCLUSION: Angiotensin II antagonistic effects of irbesartan, valsartan, and losartan were compared. Irbesartan showed the slowest decay and longest duration of its antagonistic effects. With the recommended initial doses used in this study, the following rank order of antagonistic intensity was obtained: irbesartan > valsartan > losartan. The findings of this study, specifically the longer-lasting effects of irbesartan, may have clinical implications.
Alveolar macrophages (AM) located on the alveolar surface are directly exposed to air pollutants. We evaluated the effect of exposure to SO2 on the oxidative metabolism of AM and peripheral blood mononuclear cells (PBMNC) by measuring the spontaneous and stimulated reactive oxygen-intermediates (ROI) release. AM or PBMNC were placed on a polycarbonate membrane, which was in contact with the surface of a reservoir filled with RPMI 1640 allowing the cells to be supplied with nutrients by capillarity. For SO2 exposure times of 10, 20 and 30 minutes and concentrations of 2.5, 7.5 and 12.5 ppm were chosen. A 10-minute SO2-exposure up to 12.5 ppm induced a dose dependent maximal 3.6 fold increase of spontaneous ROI-production (r = 0.876; p < 0.005). A 30-minute exposure of 12.5 ppm SO2 exhibited a cytotoxic effect inducing the death of 62 +/- 9% of AM and caused a 63% decrease of ROI-release compared to 2.5 ppm SO2-exposure under identical conditions (r = -0.96; p < 0.005). These experiments demonstrate that AM and PBMNC are activated by SO2 and that concentrations in the range of 12.5 ppm SO2 are toxic and induce a decrease in ROI-release after 30 minutes exposure of these cells.