Reinhard Berkels

Aspirin induces nitric oxide release from vascular endothelium: A novel mechanism of action

The study was designed to test the hypothesis that aspirin may stimulate nitric oxide (NO) release from vascular endothelium, a pivotal factor for maintenance of vascular homeostasis. Clinical evidence suggests that low‐dose aspirin may improve vascular endothelial function. Since other cyclooxygenase (COX) inhibitors showed no beneficial vascular effects, aspirin may exhibit a vasculoprotective, COX‐independent mechanism. Luminal NO release was monitored in real time on dissected porcine coronary arteries (PCA) by an amperometric, NO‐selective sensor. Additionally, endothelial NO synthase (eNOS) activity was measured in EA.hy 926 cell homogenates by an L‐[3H]citrulline/L‐[3H]arginine conversion assay. Superoxide scavenging capacity was assessed by lucigenin‐enhanced luminescence. Aspirin induced an immediate concentration‐dependent NO release from PCA with an EC50 of 50 nM and potentiated the NO stimulation by the receptor‐dependent agonist substance P. These effects were independent of an increase in intracellular calcium and could be mimicked by stimulation with acetylating aspirin derivatives. The aspirin metabolite salicylic acid or the reversible cyclooxygenase inhibitor indomethacin failed to modulate NO release. Incubation of soluble eNOS for 15 min with 100 μM aspirin or acetylating aspirin analogues increased the L‐[3H]citrulline yield by 40–80%, while salicylic acid had no effect. Aspirin and salicylic acid showed a similar, but only modest, magnitude and velocity of superoxide scavenging. Our findings demonstrate that therapeutically relevant concentrations of aspirin elicit NO release from vascular endothelium. This effect appears to be due to a direct acetylation of the eNOS protein, but is independent of COX inhibition or inhibition of superoxide‐mediated NO degradation.

Nifedipine Increases Endothelial Nitric Oxide Bioavailability by Antioxidative Mechanisms

Short-term treatment of the endothelium with dihydropyridine calcium antagonists resulted in an increased release in NO that is not due to a modulation of L-type calcium channels, because macrovascular endothelial cells do not express this channel. We investigated whether long-term (48 hours) treatment of porcine endothelial cell cultures with the dihydropyridine calcium antagonist nifedipine resulted in a similar enhanced NO liberation. Regarding to the underlying mechanism, we examined whether (1) nifedipine changed the mRNA and protein levels of the constitutive endothelial NO synthase (NOS) in endothelial cell cultures or (2) nifedipine exerts an NO protective effect via its antioxidative properties, as revealed in a cell culture model and with native endothelium from porcine coronary arteries. Nifedipine induced a significant time- and concentration-dependent increase (132±47%, 1 &mgr;mol/L, 40 minutes’ incubation) in the basal NO liberation (oxyhemoglobin assay). This increased NO release was not due to elevated NOS (type III) mRNA (Northern blot analysis) and protein (Western blot analysis) levels. However, nifedipine (both short- and long-term treatment) significantly reduced the basal and glucose (20 and 30 mmol/L)-stimulated formation of reactive oxygen species (lucigenin assay) of endothelial cell cultures and native cells. We conclude that the calcium antagonist nifedipine enhances the bioavailability of endothelial NO without significantly altering the NOS (type III) mRNA and protein expression, possibly via an antioxidative protection. This increased NO availability may cause part of the vasodilation and might contribute to the antithrombotic, antiproliferative, and antiatherosclerotic effects of dihydropyridine calcium antagonists.

Nitric Oxide Formation and Corresponding Relaxation of Porcine Coronary Arteries Induced by Plant Phenols: Essential Structural Features

The high intake of polyphenols is thought to contribute to the beneficial cardiovascular effects of plant-centered diets. A putative mechanism underlying the cardioprotective activity is thought to be a plant phenol–induced increase of nitric oxide formation by the constitutive endothelial nitric oxide synthase. Twenty-eight phenols of different classes commonly occurring in plant foods were examined for their capability of enhancing the endothelial nitric oxide release of isolated porcine coronary arteries by direct real-time measurement of the luminal surface nitric oxide concentration with an amperometric microsensor. Additionally, the relaxing activity of the phenols was measured on porcine coronary rings. Quercetin, myricetin, leucocyanidol, and oligomeric proanthocyanidins induced the highest increases in nitric oxide release (&Dgr;[NO] > 8.5 n M); caffeic acid, fisetin, hyperosid, and isoquercitrin were moderately active (5 n M < &Dgr;[NO] < 8.5 n M); the other phenolic compounds caused only marginal increases of the nitric oxide levels (&Dgr;[NO] < 5 n M). The nitric oxide–stimulating activity of the phenols was uniformly positively correlated with their vasorelaxing activity. However, endothelium-dependent vasorelaxations were limited to phenols inducing nitric oxide elevations > 5 n M (= Km value of the soluble guanylate cyclase). Analysis of structure-activity relations revealed that a high nitric oxide activity was confined to a flavan-moiety with free hydroxyl-residues at C3, C3′, C4′, C5, and C7 and a hydroxyl-, oxo-, or phenolic substituent at C4, whereas the caffeic acid scaffolding emerged as the minimally essential motif for the nitric oxide–dependent vasorelaxation.

Measurement of nitric oxide by reconversion of nitrate/nitrite to NO.

A simple and sensitive method is presented to measure the unstable molecule nitric oxide (NO) by reconversion of nitrate/nitrite to NO. Nitrate and nitrite are the stable degradation products of NO that accumulate in supernatants of biological samples that release nitric oxide. First, nitrate is enzymatically converted to nitrite using nitrate reductase. In a second step, nitrite is reduced to equimolar NO concentrations by an acidic iodide solution and quantified with an amperometric Clark-type electrode. This method provides the ability to assess basal-and agonist-stimulated cumulative NO formation in different biological models and is a sensitive alternative to the widely used Griess assay.

Amlodipine Increases Endothelial Nitric Oxide by Dual Mechanisms

Several experimental and clinical studies have demonstrated the antiatherogenic profile of the long-acting calcium antagonist amlodipine. Given the pivotal role of endothelial (dys)function during atherogenesis, we investigated the influence of amlodipine on endothelial nitric oxide (NO) bioavailability. Acute addition of amlodipine to segments of porcine coronary arteries resulted in a significant increase in NO release which could be blocked by the NO synthase inhibitor L-NMMA (N-monomethylarginine). This effect was mirrored by a rise in intracellular cGMP levels in porcine endothelial cell cultures. Long-term (24 h) treatment of porcine endothelial cell cultures with amlodipine (0.1–10 µmol/l) significantly enhanced the basal NO formation in a concentration-dependent manner which was abrogated in the presence of L-NMMA (0.1 mmol/l). In EA.hy 926 endothelial cells, amlodipine treatment for 24 h significantly increased the endothelial NO synthase protein expression. To evaluate whether the observed increase in NO was additionally due to an antioxidative protection of NO, we examined the influence of amlodipine in different in vitro models. In a cell-free system, amlodipine quenched superoxide anions (hypoxanthine/xanthine oxidase assay) at high concentrations (150 µmol/l). Addition of artificial membrane preparations (dimyristoylphosphatidylcholine) to mimic a physiological environment significantly enhanced this antioxidative effect. In a more physiological model of hyperglycemia (30 mmol/l, 20 min) induced formation of reactive oxygen species from native endothelial cells of porcine coronary arteries, amlodipine concentration dependently attenuated the reactive oxygen species release (>60%; 10 µmol/l). We conclude, that amlodipine increases the endothelial NO bioavailability, firstly via enhanced NO formation and secondly by prolonging the half-life of NO through antioxidative properties. This may result in an improved endothelial function.

Modulation of human platelet aggregation by the phosphodiesterase type 5 inhibitor sildenafil

The aim of this study was to investigate if the phospodiesterase type 5 inhibitor sildenafil inhibits collagen-or ADP-induced human platelet aggregation and bleeding time. To investigate this, two studies were designed. In the first, a single oral dose of sildenafil, 100 mg, was administered to healthy men. Bleeding time was determined and agonist (ADP and collagen)-induced platelet aggregation (ex vivo in platelet rich plasma) was measured 0, 1, and 4 h after application. In the second, a single oral dose of sildenafil, 50 mg, was administered and, in addition to the parameters in the first study, we also determined the platelet aggregation after 24 h and measured the effect of a nitric oxide donor (S-nitroso-N-acetylpenicillamine [SNAP]) in combination to mimic a physiologic nitric oxide release from the endothelium. The bleeding time of 1 h after sildenafil medication (100 mg) was significantly prolonged but recovered toward control values after 4 h, whereas application of sildenafil at a lower dose (50 mg) did not alter the bleeding time. Sildenafil (100 and 50 mg) did not inhibit the ADP-induced aggregation, whereas the collagen-induced aggregation (100 mg) was markedly reduced after 1 h and significantly inhibited 4 h after application. This inhibitory effect was overcome by higher concentrations of collagen. SNAP (0.5 &mgr;M) induced an inhibition of platelet aggregation that was potentiated after taking sildenafil (50 mg, 1 and 4 h afterward) and abrogated after 24 h. These data indicates that sildenafil may inhibit collagen-induced platelet aggregation ex vivo. After co-administration of nitric oxide, collagen-and ADP-induced platelet aggregation was significantly inhibited, which may reflect physiologic conditions of an in vivo system.

Dual mode of action of dihydropyridine calcium antagonists: a role for nitric oxide.

Dihydropyridine calcium antagonists have been used for many years in the treatment of angina pectoris and hypertension. According to the common view, their mechanism of action is based on an inhibition of the smooth muscle L-type calcium current, thus decreasing intracellular calcium concentration and inducing smooth muscular relaxation. However, in recent years evidence has accumulated that besides the smooth muscle effects of these agents, their effect on the endothelium must also to be taken into account.It was shown that dihydropyridines can induce the release of nitric oxide (NO) from the vascular endothelium of various vessels and in different species. This was first shown by Günther and colleagues by assaying the methaemoglobin formation in the presence of intact endothelium (in procine coronary arteries) with and without treatment with nitrendipine. These findings were later confirmed by direct measurement of NO or of nitrite production. In addition, in several preparations, including micro- and macrovasculature, the sensitivity of the vasorelaxing effect of the dihydropyridines to inhibitors of NO-synthase, such as L-NG-nitroarginine (LNNA) or L-N-nitro-arginine-methyl-ester ( L-NAME), has been shown. With these studies it became evident that the NO-releasing effect was not unique to nitrendipine but was a group phenomenon shared by the dihydropyridines and several nondihydropyridine calcium antagonists. In addition to their action on vascular endothelium, NO release by nifedipine has also been detected in platelets. There are also studies showing long term effects of calcium antagonists involving NO release.Regarding the underlying mechanism of NO release, nitrendipine was shown, not to decrease but to increase intracellular Ca2+ in cultured endothelial cells. This increase was sensitive to both Ca2+-free extracellular superfusion and to gadolinium, a lanthanide known to inhibit shear-stress activated cation channels. This increase in intracellular calcium can activate endothelial NO-synthase, thus inducing the release of NO.These findings on a dual mode of action, i.e. the direct relaxing effect by inhibition of the smooth muscle L-type calcium current and indirect relaxing effect by release of NO from vascular endothelium may help to understand the beneficial antihypertensive effects of the dihydropyridine calcium antagonists and the preferential effect of certain drugs in certain vascular regions (resistance versus conductive vessels). In addition, NO release from both vascular endothelium and platelets may contribute to the antiatherosclerotic and antithrombotic effects described for certain dihydropyridines.

The localisation of the extraneuronal monoamine transporter (EMT) in rat brain.

The extraneuronal monoamine transporter plays an important role in the inactivation of monoamine transmitters. A basal extraneuronal tissue expression of this transporter has been reported, but it is also expressed in CNS glia. As little is known about the expression pattern and the function of the extraneuronal monoamine transporter in the brain, we performed a detailed investigation. Firstly, a northern blot analysis of different rat organs revealed that the transporter is strongly expressed in placenta, lung and heart and less prominently in the whole brain, brain stem, intestine, testis, epididymis, stomach, kidney and skeletal muscle. It was not expressed in cerebellum, liver and embryo. Using an in situ hybridization to the rat brain, we detected a marked and highly confined expression of the extraneuronal monoamine transporter in the area postrema, but in no other brain areas. These findings were confirmed by polyclonal antibodies against rat extraneuronal monoamine transporter showing an intensive signal in the area postrema, although a few cells in the cerebellum and the brain stem also showed a signal. Additionally, a partly overlapping expression pattern of the monoamine oxidase‐B was detected. Summarizing, we firstly describe a marked and highly confined expression of the extraneuronal monoamine transporter in the rat area postrema by in situ hybridisation which may play a role in physiological functions of this circumventricular organ such as emesis, food intake and the regulation of cardiovascular functions.

Vascular protective effects of dihydropyridine calcium antagonists. Involvement of endothelial nitric oxide.

Dihydropyridine calcium antagonists play an important role in the treatment of hypertension and angina pectoris. They lower blood pressure by a well-characterized mechanism of blocking L-type calcium channels in smooth muscle cells. Additionally, there is growing evidence that dihydropyridines also modulate endothelial functions by other mechanisms, since macrovascular endothelial cells do not express L-type calcium channels. A number of studies have demonstrated that dihydropyridine calcium antagonists enhance bioavailability of endothelial nitric oxide (NO). Endothelium-derived NO plays a pivotal role in the regulation of vasorelaxation, leukocyte adhesion and platelet aggregation and an impaired NO release is associated with the genesis and progression of atherosclerotic diseases. This review summarizes results from experimental findings that dihydropyridine calcium antagonists increase endothelial NO formation as well as studies which demonstrate these effects in vivo both in animals and humans. Moreover, the influence of dihydropyridine calcium antagonists on the progression of atherosclerosis is discussed. These pleiotropic effects of dihydropyridine calcium antagonists may underlie or contribute to antiatherosclerotic effects of this substance class.

Switched single-electrode voltage-clamp amplifiers allow precise measurement of gap junction conductance

Measurement of gap junction conductance ( g j) with patch-clamp amplifiers can, due to series resistance problems, be subject to considerable errors when large currents are measured. Formulas developed to correct for these errors unfortunately depend on exact estimates of series resistance, which are not always easy to obtain. Discontinuous single-electrode voltage-clamp amplifiers (DSEVCs) were shown to overcome series resistance problems in single whole cell recording. With the use of two synchronized DSEVCs, the simulated g j in a model circuit can be measured with a maximum error of <5% in all recording situations investigated (series resistance, 5-47 MΩ; membrane resistance, 20-1,000 MΩ; g j, 1-100 nS). At a very low g j of 100 pS, the error sometimes exceeded 5% (maximum of 15%), but the error was always <5% when membrane resistance was >100 MΩ. The precision of the measurements is independent of series resistance, membrane resistance, and g j. Consequently, it is possible to calculate g j directly from Ohms law, i.e., without using correction formulas. Our results suggest that DSEVCs should be used to measure g j if large currents must be recorded, i.e., if cells are well coupled or if membrane resistance is low.