Christian Mang

Differential effects of anandamide on acetylcholine release in the guinea‐pig ileum mediated via vanilloid and non‐CB1 cannabinoid receptors

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. Anandamide increased both basal [3H]‐acetylcholine release (pEC50 6.3) and muscle tone (pEC50 6.3). The concentration‐response curves for anandamide were shifted to the right by 1 μM capsazepine (pKB 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 μM). The CB1 and CB2 receptor antagonists, SR141716A (1 μM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide. Anandamide inhibited the electrically‐evoked release of [3H]‐acetylcholine (pEC50 5.8) and contractions (pEC50 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 μM anandamide. The inhibitory effects of anandamide were not changed by either capsazepine (1 μM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 μM). SR141716A (1 μM) produced rightward shifts in the inhibitory concentration‐response curves for anandamide yielding pKB values of 6.6 and 6.2. CP55940 inhibited the evoked [3H]‐acetylcholine release and contractions, and SR141716A (0.1 μM) shifted the concentration‐response curves of CP55940 to the right with pKB values of 8.4 and 8.9. 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.

Antiatherosclerotic Effects of Small-Molecular-Weight Compounds Enhancing Endothelial Nitric-Oxide Synthase (eNOS) Expression and Preventing eNOS Uncoupling

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 Nω-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.

Pentaerythritol Tetranitrate Improves Angiotensin II–Induced Vascular Dysfunction via Induction of Heme Oxygenase-1

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.

Cyclooxygenase 2-selective and nonselective nonsteroidal anti-inflammatory drugs induce oxidative stress by up-regulating vascular NADPH oxidases.

Cyclooxygenase 2-selective inhibitors (coxibs) and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with an increase in cardiovascular events. The current study was designed to test the effect of coxibs and nonselective NSAIDs on vascular superoxide and nitric oxide (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 nonselective NSAIDs diclofenac or naproxen and moderately by rofecoxib or celecoxib in the aorta and heart of SHR. The up-regulation 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. 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 NG-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 nonselective NSAIDs being even more critical than coxibs in this respect.

Modulation of acetylcholine release in the guinea-pig trachea by the nitric oxide donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP)

The effects of the nitric oxide (NO) donor S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) and the NO synthase inhibitor L‐NG‐nitroarginine (L‐NOARG) on the electrically evoked [3H]‐acetylcholine release were studied in an epithelium‐free preparation of guinea‐pig trachea that had been preincubated with [3H]‐choline. SNAP (100 and 300 μM) caused small but significant increases of the electrically evoked [3H]‐acetylcholine release (121±4% and 124±10% of control). Resting outflow of [3H]‐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‐α]quinoxalin‐1‐one (ODQ, 1 μM). The facilitatory effect of SNAP (100 and 300 μM) was reversed into inhibition of release (to 74±4% and to 78±2%) after pretreatment of the trachea with capsaicin (3 μM). ODQ prevented the inhibition. Capsaicin pretreatment alone did not significantly alter the release of [3H]‐acetylcholine. A significant inhibition by SNAP (100 μM) of [3H]‐acetylcholine release (78±3%) was also seen in the presence of the NK2 receptor antagonist SR 48968 (30 nM). L‐NOARG (10 and 100 μM) 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 NK2 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.

Nitrergic modulation of acetylcholine release in the enteric nervous system: differences between guinea-pig and man

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 no 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.