The role of the adenosine triphosphate-sensitive potassium channels in pinacidil-induced vasodilatation of the human saphenous vein in patients with and without type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) increases cardiovascular complications. Diabetic vascular dysfunction is associated with the reduced activity of the different smooth muscle potassium (K+) channels. Thus, the objective of our study was to investigate the role of the adenosine triphosphate (ATP)-sensitive K+ (KATP) channels in the relaxant effect of potassium channel opener, pinacidil on the human saphenous vein (HSV) obtained from the patients with and without T2DM. The rings of HSV without the endothelium, obtained from the patients who had undergone coronary bypass surgery, were mounted in an organ bath system and isometric tension was recorded. The relaxation of HSV, precontracted with phenylephrine, was produced by pinacidil. The expression of KATP subunits (Kir6.1, Kir6.2 and SUR2B) was detected by immunohistochemistry and Western blot. Pinacidil produces comparable effects on HSV in patients with and without T2DM. The suppression of pinacidil effect and its maximal relaxation by glibenclamide, selective blocker of KATP channels, was more pronounced on HSV in patients without T2DM. All three types of KATP subunits are expressed on the smooth muscle cells of HSV. While there are no differences in the expression of Kir6.1 and Kir6.2, the expression of SUR2B is lower in HSV in patients with T2DM. Pinacidil produced comparable KATP-dependent and -independent relaxation of the HSV in patients with/without T2DM. According to the effect of glibenclamide and the applied molecular analysis, presented findings demonstrated that diabetes mellitus was associated with the reduced expression of SUR2B subunit in the vascular smooth muscle of HSV.

Effects of the polyphenol resveratrol on contractility of human term pregnant myometrium.

The ideal agent for prevention and treatment of uterine abnormal contractility has not been found. The polyphenol resveratrol possesses a wide spectrum of pharmacologic properties, but its influence on the contractility of human myometrium is not defined. The present study evaluated the effect of resveratrol on the oxytocin-induced contractions of human term pregnant myometrium in vitro and the contribution of different K(+) channels to resveratrol action. Resveratrol induced a concentration-dependent relaxation of myometrium contractions (pD2 value and maximal responses were 4.52 and 82.25%, respectively). Glibenclamide, a selective blocker of ATP-sensitive (KATP), iberiotoxin, a selective blockers of big-calcium sensitive (BK(Ca)) and 4-aminopiridine, a non-selective blocker of voltage-sensitive (Kv) channels induced a significant shift to the right of the concentration-response curves of resveratrol. Inhibition achieved by 0.1 mM resveratrol was insensitive to all K(+) channel blockers. A K(+) channel opener, pinacidil, inhibited oxytocin-induced contractions of pregnant myometrium with comparable potency and efficacy to resveratrol (pD2 values and maximal relaxation were 4.52 and 83.67%, respectively). Based on K(+) channel opener/blocker affinities, it appears that the inhibitory response of resveratrol involves different myometrial K(+) channels. When applied in high concentrations, resveratrol has an additional K(+)-channel-independent mechanism(s) of action. Furthermore, immunohistochemistry staining and western blot analyses detected the presence and distribution of KATP, BK(Ca) and Kv channel proteins in pregnant myometrium.

Liposomes as carriers of the lipid soluble antioxidant resveratrol: evaluation of amelioration of oxidative stress by additional antioxidant vitamin.

AIM: Resveratrol (RES) is a well-known antioxidant, yet in combination with other antioxidant vitamins, it was found to be more effective than any of these antioxidants alone. Present work aims to compare the antioxidant actions of resveratrol with and without vitamin C following delivery as liposomes tested using chemical and cellular antioxidative test systems. MAIN METHODS: Liposomes were prepared by the thin film hydration method and characterised for percent drug entrapment (PDE), Z-average mean size (nm), polydispersity index (PDI) and zeta potential. Antioxidative capacity was determined by studying the inhibition of AAPH induced luminol enhanced chemiluminescence and inhibition of ROS production in isolated blood leukocytes. Intracellular oxygen-derived radicals were measured using flow cytometry with buffy coats (BC) and human umbilical vein endothelial cells using H2DCF-DA dye. KEY FINDINGS: Particle size varied from 134.2 ± 0.265 nm to 103.3 ± 1.687 nm; PDI ≤ 0.3; zeta potential values were greater than -30 mV and PDE ≥ 80%. Radical scavenging effect was enhanced with liposomal systems; oxidative burst reaction in BC was inhibited by liposomal formulations, with the effect slightly enhanced in presence of vitamin C. Reduction in reactive oxygen species (ROS) production during spontaneous oxidative burst of BC and incubation of HUVECs with H2O2 further intensified the antioxidative effects of pure RES and liposomal formulations. SIGNIFICANCE: The present work clearly shows that the antioxidative effects of resveratrol loaded into liposomes are more pronounced when compared to pure resveratrol. Liposomal resveratrol is even active within the intracellular compartments as RES could effectively quench the intracellular accumulation of ROS.

The effect of resveratrol on contractility of non-pregnant rat uterus: the contribution of K(+) channels.

This study was aimed to evaluate resveratrol (1-100 µM) effect on the spontaneous rhythmic contractions (SRC), oxytocin-induced (0.2 nM, POxC) phasic and tonic (20 nM, TOxC) contractions of isolated rat uterus. The SRC and POxC were more sensitive to resveratrol than TOxC (pD2 values: 4.53 and 4.66 versus 4.06). Different blockers of K(+) channels (glibenclamide, tetraethylamonium, iberiotoxin, 4-aminopyridine) antagonized the response to resveratrol on the SRC and phasic contractions, but did not antagonize the effect of resveratrol on the TOxC. In order to compare the relaxant activities of resveratrol on the TOxC with that of potassium channel openers, a separate experiments with NS 1619, a highly specific big Ca(2+)-sensitive K(+) (BKCa) channels opener and pinacidil, a predominant opener of ATP-sensitive K(+) (KATP) channels were done. NS 1619 (10-100 µM) and pinacidil (10-100 µM) produced more potent inhibition of TOxC than resveratrol (pD2 values were 6.00 and 5.29). Iberiotoxin, a highly selective BKCa channels blocker, antagonized the response to NS 1619 and glibenclamide, a highly selective KATP channels blocker, antagonized the response to pinacidil on the TOxC. To test K(+)- and extracellular Ca(2+)- independent mechanism(s) of resveratrol on TOxC, a K(+)-rich, Ca(2+)-free solution was used. Under this condition, only high concentrations (≥30 µM) of resveratrol inhibited TOxC. Western blots analysis confirmed expression of Kir6.1, Kir6.2, KCa1.1, Kv2.1 and Kv4.2. channel proteins in myometrium. Thus, the effect of resveratrol is dependent on the types of contractions. The inhibitory response of resveratrol on the SRC and phasic contractions involves different myometrial K(+)- channels. When applied in high concentrations, resveratrol has an additional K(+)- channels independent mechanism(s) of action. As the effects of NS 1619, pinacidil and resveratrol on the TOxC are different, we can conclud that resveratrol does not behave as a classical potassium channel opener.

The novel phenylpropiophenone derivates induced relaxation of isolated rat aorta.

Our aim was to define how different chemical properties of newly developed phenylpropiophenone derivates (PhPds) influenced their potency and efficacy to relax rat aorta. A contribution of ion channels in the PhPds and propafenone mechanism of vasodilatation was tested. PhPds were syntethysed by substitution in the benzyl moiety with -F, -CH3 or -CF3 groups on the ortho or para position. The vasodilatation by PhPds was examined on the rings of rat aorta precontracted with phenylephrine. In order to test involvement of voltage-gated Na+ and K+ channels and L-type Ca2+ channels in a mechanism of action of PhPds, we used their blockers: lidocaine, nifedipine and 4-aminopiridine, respectively. Aorta was more sensitive to 5-ortho-trifluoromethyl derivate than to propafenone and other PhPds. The 5-para-methyl derivate had lower potency and efficacy than propafenone and other PhPds. Lidocaine did not influenced relaxation induced by PhPds, but slightly inhibited the effect of propafenone. The 4-aminopiridine only inhibited relaxation induced by 5-para-methyl derivate. Nifedipine inhibited relaxation of the rat aorta induced by 5-ortho-trifluoromethyl derivate and by propafenone. Introduction of 5-ortho-trifluoromethyl and 5-para-methyl group in the benzyl moiety of propafenone molecule changed its potency, efficacy and mechanism of action in the rat aorta. The 4-aminopiridine- and nifedipine sensitive ion channels are involved in mechanism of action of 5-para-methyl and 5-ortho-trifluoromethyl derivate. The introduction of other tested groups in the benzyl moiety does not affect pharmacological properties of the PhPds in relation to propafenone.

The low-dose combination preparation Vertigoheel activates cyclic nucleotide pathways and stimulates vasorelaxation.

Vertigo of various and often unknown aetiologies has been associated with and attributed to impaired microvascular perfusion in the inner ear or the vertebrobasilar system. Vertigoheel is a low-dose combination preparation of proven value in the symptomatic treatment of vertigo. In the present study we tested the hypothesis that Vertigoheel's anti-vertiginous properties may in part be due to a vasodilatory effect exerted via stimulation of the adenylate and/or guanylate cyclase pathways. Thus, the influence of Vertigoheel or its single constituents on synthesis and degradation of cyclic nucleotides was measured. Furthermore, vessel myography was used to observe the effect of Vertigoheel on the vasoreactivity of rat carotid arteries. Vertigoheel and one of its constituents, Anamirta cocculus, stimulated adenylate cyclase activity, while another constituent, Conium maculatum, inhibited phosphodiesterase 5, suggesting that the individual constituents of Vertigoheel contribute differentially to a synergistic stimulation of cyclic nucleotide signalling pathways. In rat carotid artery rings, Vertigoheel counteracted phenylephrine-induced tonic vasoconstriction. The present data demonstrate a vasorelaxant effect of Vertigoheel that goes along with a synergistic stimulation of cyclic nucleotide pathways and may provide a mechanistic basis for the documented anti-vertiginous effects of this combination preparation.

Effect of an 8-week endurance training program on markers of antioxidant capacity in women.

AIM: The effects of endurance training and of exhaustive treadmill running on low density lipoprotein (LDL) oxidation in women are not clearly established. METHODS: Twenty training and 10 control persons, all not endurance trained, aged 26+/-4 and 23+/-3 years, were recruited for 8 weeks of running training 3x/week 30 min. The susceptibility of LDL to in vitro oxidation, conjugated dienes, malondialdehyde (MDA), nitric oxide (NO) and cholesterol, lipoproteins, triglycerides, apolipoprotein (apo) A-I, apo B and lipoprotein (a) were determined before and after training, at rest and after exhaustive spiroergometric exercise. The training was tailored individually at the speed of the 4 mmol/L lactate threshold. RESULTS: At rest and after treadmill running, training induced an increase in lag-time (P<0.05), a decrease in MDA (P<0.05), and lower values for cholesterol (P<0.001), LDL (P<0.01), triglycerides (P<0.05) and apo B (P<0.001), but no increase for high density lipoprotein (HDL) or apo A-I. Before training, treadmill running induced lower conjugated dienes and malondialdehyde, after training an increase for LDL and decrease for cholesterol and triglycerides, no increase for HDL or apo A-I. In the control group, all parameters remained unchanged, only NO lowered (P<0.01). CONCLUSION: Endurance training in women shows favorable effects on LDL oxidation, cholesterol, LDL-cholesterol, triglycerides and apo B.

Antioxidative properties of the gastrointestinal phytopharmaceutical remedy STW 5 (Iberogast).

Since inflammation is a common mechanism of many gastrointestinal diseases, reactive oxygen metabolites may play an important role in their pathophysiology. Therefore it is interesting to know, whether phytopharmaceuticals known to modulate gastrointestinal motor function reveal also antioxidative properties. We tested STW 5 (Iberogast), its constituent nine different plant extracts, and some isolated compounds which are present in STW 5 for characterizing their antioxidative and radical quenching activities. The test assays consisted in pure chemical and complex cellular systems in which different types of reactive species were produced. Quantification of the effects was based on chemiluminescence reactions. The results show that all extracts contribute to the effect of the complete remedy STW 5, in the chemical systems in a strongly additive manner, in the cellular systems in a supraadditive manner. The largest contributions resulted from the extracts from peppermint and melissa leaves. Comparison of effects from isolated phytochemical compounds from the extracts with that of the extracts itself shows that usually the extract is more effective than the monosubstance which indicates also the synergism within the whole plant extracts. This means that the plant extracts present in STW 5 provide strong radical quenching activities that could also be involved in the therapeutic gastrointestinal actions.

Intestinal spasmolytic effects of STW 5 (Iberogast) and its components.

Functional gastro-intestinal diseases as the irritable bowel syndrome are very common in the population and are characterized by a broad spectrum of symptoms which mostly are related to spastic or paralytic intestinal function without defined histopathological changes of the tissue. Due to the multifactorial pathogenesis a multifactorial therapy with multi-target action seems to be reasonable. STW 5 (Iberogast), its constituent herbal extracts and some isolated compounds were used in an in vitro model provided by intestinal samples from guinea pig in order to test their activity on histamine-induced contractions and spontaneous motility, respectively. For comparison the known spasmolyticum papaverine was used. The results show that the lytic effect of the phytotherapeuticum on histamine-induced contraction represents additively the actions of the different components and corresponds to approx. 10 microM of papaverine. Spontaneous peristaltic motion was differently modulated by the various constituent extracts. The experiments with silibinin, glycyrrhicine, chelidonine, and protopine showed that the effects of the extracts were not comparable to those of the respective chemical constituents.

Glimepiride reduces mononuclear activation of the redox-sensitive transcription factor nuclear factor-kappa B.

AIMS: Glimepiride has the lowest ratio of insulin release to glucose decrease compared with other sulphonylureas. This prompted us to study in vitro and in vivo in a placebo-controlled study the effect of glimepiride on the redox-sensitive transcription factor nuclear factor-kappa B (NF-kappaB). METHODS: Fifteen patients with type 2 diabetes on glibenclamide with a stable HbA1c over the last 6 months were included. After sampling for determination of baseline values, 10 patients were changed to an equivalent dose of glimepiride, while the placebo group was maintained at glibenclamide plus placebo. The glimepiride dose in these patients was adjusted so that no change in glucose control occurred, allowing for direct comparison. The others were kept on glibenclamide and received additional placebo. After 4 weeks of glimepiride or glibenclamide plus placebo, a second blood sample was taken. Mononuclear cells were isolated and assayed in a tissue-culture-independent electrophoretic mobility shift assay (EMSA)-based detection system for NF-kappaB binding activity, and by Western Blot for nuclear localization of NF-kappaB-p65, the cytoplasmic content of IkappaBalpha and the NF-kappaB-controlled haemoxygenase-1. Glimepiride dose-dependent inhibition of carboxymethyllysin (CML) albumin or tumour necrosis factor alpha (TNFalpha)- and H2O2-induced activation of NF-kappaB binding were determined, using isolated peripheral blood mononuclear cells from healthy volunteers, and transcriptional activity of bovine aortic endothelial cells either left untreated or induced with CML albumin incubated with or without glimepiride. Furthermore, in-vitro studies were implemented to demonstrate radical quenching properties of glimepiride in the cell-free 2,2'-azo-bis(2-aminopropane)-dihydrochloride system. RESULTS: Baseline glucose and HbA1c remained stable in the patients switched from glibenclamide to a corresponding dose of glimepiride or kept on glibenclamide plus placebo. While in the group of patients only taking glibenclamide plus placebo the NF-kappaB binding activity did not change significantly (p = 0.58), the NF-kappaB binding activity in the group of patients taking glimepiride was reduced from 19.3 relative NF-kappaB-p65-equivalents to 15.5 relative NF-kappaB-p65-equivalents (p = 0.04). The nuclear translocation of NF-kappaB-p65 was reduced from 100% at baseline to 58% after 4 weeks (p = 0.04); the cytoplasmic localization of NF-kappaB-p65 increased from 100% to 129% (p = 0.03) and the cytoplasmic content of IkappaBalpha increased from 100% to 109% (p = 0.06). The redox-sensitive haemoxygenase-1 antigen was reduced from 100% to 82% (p = 0.04). To prove directly that glimepiride reduces NF-kappaB activation, we isolated peripheral blood mononuclear cells (PBMC) from healthy volunteers. In vitro, glimepiride reduced TNFalpha-(1 nmol/l) and CML albumin (800 nmol/l)-induced NF-kappaB activation dose dependently, being half maximal at 120 micromol/l. H2O2-mediated NF-kappaB activation was only partially reduced. In addition, glimepiride reduced NF-kappaB-dependent gene expression using a NF-kappaB-driven luciferase reporter system. Finally, a cell-free detection system showed that glimepiride has radical quenching properties. CONCLUSION: Glimepiride can affect the activation of the redox-sensitive transcription factor NF-kappaB in vitro and in vivo.

Intimal plaque development and oxidative stress in cholesterol-induced atherosclerosis in New Zealand rabbits.

Although oxidative stress is well known in atherogenesis, the origin, nature and kinetics of free radicals involved have not been well described till now. Here, we correlated parameters of oxidative stress with cellular components during induction and stabilization of aortic intimal lesions which were induced in rabbits by feeding a cholesterol-enriched diet for 6 weeks and a normal diet for further 68 weeks. Plasma lipids, aortic plaque size and composition (macrophages, smooth muscle cells, oxidized LDL by morphometry), as well as aortic radical production (by luminol-enhanced chemiluminescence and TEMPO-9AC fluorescence) were measured after various time points. The parameters of oxidative stress were correlated with the different cellular components of the aortic plaques. The plaques increased until week 21, no significant regression was found until week 74, plasma cholesterol was maximal at week 6. Macrophages, oxidized LDL and generation of different species of free radicals were increased during plaque development, yet with different time kinetics. Whereas chemiluminescence correlated only weakly with the amount of intimal macrophages, strong correlations were found between TEMPO fluorescence and smooth muscle cells (r = 0.4778, P < 0.001) and between macrophages and oxidized LDL (r = 0.5896, P < 0.0001). Different indicators of oxidative stress were increased during plaque progression and stabilization. However, the various correlations show, that distinct types of reactive species secreted probably from macrophages and smooth muscle cells contribute to oxidative stress in the different phases of plaque development.

Oxidative stress in atherogenesis: Basic mechanisms and problems of therapy with antioxidants.

BACKGROUND: Oxidative stress is recognized as an essential mechanism of atherogenesis and plaque progression. However, the origin of increased free radical production has not yet been well described. Furthermore, therapy with antioxidants has not shown convincing results. OBJECTIVE: To consider questions concerning the impact of oxidative stress, and the effects and usefulness of antioxidants. ANIMALS AND METHODS: Atherosclerotic plaques were induced in rabbits by feeding them a cholesterol-rich diet (2%) for six weeks. Thereafter a normal diet was given up to 68 weeks. Body weight, food intake, plasma lipid concentration and antioxidative capacity were determined at various time intervals. Aortic plaque size, morphology and radical production were determined in groups of animals killed after six, 14, 21, 29, 40 and 74 weeks, and compared with values in untreated controls. Chemiluminescent methods were used to determine antioxidative capacity of plasma, generation of free radicals and redox reactivity of various antioxidants. RESULTS: Antioxidative capacity, occurrence of modified low density lipoprotein and generation of free radicals indicated oxidative stress during plaque progression; however, they showed different correlations to cellular components of the plaques. Furthermore it was shown that some antioxidants have both anti- and pro-oxidative properties. CONCLUSIONS: Oxidative stress during atherogenesis seems to correlate with different phases of plaque development and can be associated with different types of reactive species. Because plaque remodelling and stabilization may also be a phase of increased free radical generation, therapeutic antioxidants must exert specific and selective activity; in particular, whether their oxidized form acts pro-oxidatively must be determined.

Chronically elevated endothelin-1 concentrations modulate the beta-adrenergic receptor system in vitro and in vivo.

In atherosclerosis and heart failure chronically elevated endothelin-1 (ET-1) plasma concentrations have been found which correlate with an increased mortality. The aim of this study was to determine the effects of chronically elevated ET-1 concentrations in vitro on the expression of the beta-adrenergic receptor (betaAR), the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (G(s alpha)), and to determine betaAR's ability to activate adenylyl cyclase. In order to elucidate the effects of elevated ET-1 concentrations in vivo, male rats were infused with ET-1 and betaAR density was measured. Smooth muscle cells were incubated with ET-1 (10(-7) mol/l) for 6 to 48 h. Densities of betaARs were determined by radioligand binding studies and the G(s alpha) was analyzed by Western blotting. Isoproterenol-mediated adenylyl cyclase activity was measured. Additionally male rats were infused with ET-1 for 3 weeks. In vitro the betaAR density increased by 52% (p < 0.05, n = 5). The G(s alpha) increased to 260%. The isoproterenol-stimulated adenylyl cyclase activity was increased to 228%. In vivo, the pulmonary and myocardial betaAR density was elevated by 43% and 97%, respectively. Chronic ET receptor activation induces a transregulation of betaARs in vitro and in vivo.

Effect of urea and osmotic cell shrinkage on Ca2+ entry and contraction of vascular smooth muscle cells.

The present study was performed to elucidate the effects of urea on vascular smooth muscle cells (SMC). Addition of urea (20, 50, 100 mM) to physiological salt solution blunted the vasoconstrictory effect of phenylephrine (by 17, 25 and 30%, respectively) and of an increased extracellular K+ concentration (by 7, 14 and 19%, respectively) without affecting the basal tone of rabbit arterial rings. According to Fura-2 fluorescence in cultured SMC (A7r5), urea had no effect on basal intracellular calcium activity ([Ca2+]i), but significantly blunted the increase of [Ca2+]i following an increase of extracellular K+. Whole-cell patch-clamp studies revealed that the Ca2+ current through voltage-sensitive Ca2+ channels is significantly inhibited in the presence of urea. As evident from calcein fluorescence, addition of urea leads to sustained cell shrinkage. The effects of urea on vascular tone, [Ca2+]i activity, voltage-gated Ca2+ channels and cell volume are mimicked by addition of raffinose or NaCl. However, the cell shrinkage induced by urea is sustained, whereas the addition of equiosmolar NaCl is only transient and followed by a regulatory cell volume increase. Moreover, hypertonic NaCl increases, whereas urea decreases, the transcription of cell-volume-regulated kinase hsgk. In conclusion, urea leads to sustained shrinkage of vascular smooth muscle cells, which is followed by inhibition of voltage-gated Ca2+ channels, a decrease of [Ca2+]i and thus blunts the vasoconstrictory action of phenylephrine and increased extracellular K+ concentration.

Local paclitaxel delivery for the prevention of restenosis: biological effects and efficacy in vivo.

OBJECTIVE: The aim of this study was to evaluate the potential of paclitaxel to prevent restenosis in vivo. BACKGROUND: Paclitaxel (Taxol) is a microtubule-stabilizing compound with potent antitumor activity. It influences the cytoskeleton equilibrium by increasing the assembly of altered microtubules, thereby inducing cellular modifications that result in reduced proliferation, migration and signal transduction. METHODS: Before the in vivo study, delivery efficiency was determined with radiolabeled paclitaxel in porcine hearts. After induction of a defined plaque in the right carotid arteries of 76 New Zealand rabbits by electrical stimulation, 27 animals underwent balloon dilation and subsequent local paclitaxel delivery (10 ml, 10 micromol/liter) with a double-balloon catheter. Twenty-nine animals served as control with angioplasty only, 10 animals underwent local delivery of vehicle only (0.9% NaCl solution) and 10 animals were solely electrostimulated. Vessels were excised one, four, and eight weeks after intervention. RESULTS: The extent of stenosis in paclitaxel-treated animals was significantly reduced compared with balloon-dilated control animals (p = 0.0012, one, four and eight weeks after intervention: 14.6%, 24.6% and 20.5%, vs. 24.9%, 33.8% and 43.1%, respectively). Marked vessel enlargement compared with balloon-dilated control animals could be observed (p = 0.0001, total vessel area after one, four and eight weeks: paclitaxel group: 1.983, 1.700 and 1.602 mm2, control: 1.071, 1.338 and 1.206 mm2, respectively). Tubulin staining and electron microscopy revealed changes in microtubule assembly, which were limited to the intimal area. Vasocontractile function after paclitaxel treatment showed major impairment. CONCLUSIONS: Local delivery of paclitaxel resulted in reduced neointimal stenosis and enlargement in vessel size. Both these effects contribute to a preservation of vessel shape and are likely to be caused by a structural alteration of the cytoskeleton.

Oxidation of plasmalogens produces highly effective modulators of macrophage function.

Model derivatives of plasmalogens and chemically synthesized oxidative degradation products as found e.g. during oxidation of low density lipoproteins show strong effects on phagocytosis induced secretion of reactive oxygen species of macrophages which was measured by luminol-enhanced chemiluminescence. Whereas a plasmalogen epoxide showed enhancing effects in submicromolar range, inhibition was found with higher concentrations as well as with alpha-hydroxyaldehydes. The substances showed only little effects on the non-cellular ROS-dependent chemiluminescence of the reaction between hydrogen peroxide and opsonized zymosan and no cytotoxic effects under the assay conditions used. These results show that oxidative modification and degradation of plasmalogens occuring also under pathophysiological situations in vivo produces effective modulators of macrophage function which could be important; e.g. during inflammation or atherogenesis.

Oxidizability of low density lipoprotein and total antioxidative capacity of plasma are differently altered during induction and regression of hypercholesterolemia in rabbits.

Oxidizability of isolated low density lipoprotein (LDL) and total antioxidative capacity of plasma were measured in rabbits fed for 6 weeks a cholesterol-rich diet and for further 34 weeks a normal diet. Whereas the time to induce copper ion-mediated lipid peroxidation in LDL was prolonged during hypercholesterolemia, total antioxidative capacity as determined by a radical-trapping assay was increased at 6 weeks, but decreased during the time when the plasma cholesterol levels declined slowly to normal. Since aortic plaque progression was continued also during the first 15 weeks of normal diet, increased atherogenicity of hypercholesterolemia might be better reflected by the antioxidant capacity of plasma rather than by oxidation of isolated LDL.

Downregulation of beta-adrenergic receptors by low density lipoproteins and its prevention by beta-adrenergic receptor antagonists.

OBJECTIVE: Vasodilation by beta-adrenergic receptors of smooth muscle cells appears to be impaired early after the onset of hypercholesteremia. The aim of this study was to analyze the modulation of beta-adrenergic receptor density and adenylyl cyclase activity in the presence of moderately elevated concentrations of LDL. The effects of beta 1- and beta 2-adrenergic receptor antagonists on LDL-induced receptor changes were studied. METHODS AND RESULTS: Media explants of porcine coronary arteries were incubated with moderately elevated LDL concentrations (0.7-3.9 mmol/l). The density of beta-adrenergic receptors was determined in plasma membranes using the radioligand [125I]iodocyanopinodolol. LDL (3.9 mmol/l) resulted in a decrease of beta-adrenergic receptor density (control 137 +/- 5 vs. 89 +/- 7 fmol/mg protein, P < 0.01). After removal of LDL and cultivation for an additional 3 days beta-adrenergic receptors increased to 129 +/- 5 fmol/mg. In the presence of the beta 1- or beta 2-adrenergic receptor antagonists the LDL-mediated decrease was inhibited. Addition of metoprolol after 3 days of LDL incubation caused a restoration of receptor density. The basal, isoproterenol- and forskolin-stimulated adenylyl cyclase activities were increased after LDL incubation by 180, 110 or 80%, respectively. CONCLUSION: Moderately elevated LDL levels decreased beta-adrenergic receptor density while adenylyl cyclase activity was simultaneously increased. beta 1- or beta 2-adrenergic receptor antagonists prevented this receptor decrease and might preserve the beta-adrenergic receptor density in the presence of moderately elevated LDL levels.

Inhibition of Fas-induced apoptotic cell death by osmotic cell shrinkage.

Apoptosis is an active physiological mechanism permitting the elimination of cells by triggering an intracellular signalling cascade. Here, we tested whether osmotic alterations of cell volume interfere with apoptotic cell death in Jurkat T-lymphocytes. Apoptotic cell death of Jurkat cells was elicited by activation of the Fas receptor which results in sphingomyelinase stimulation, release of ceramide, activation of Ras, Rac-proteins and formation of O2. Osmotic cell shrinkage inhibited apoptotic cell death induced by the Fas receptor in Jurkat T-lymphocytes. Osmotic cell shrinkage did not interfere with Fas induced activation of the acidic sphingomyelinase or activation of Ras but impaired the formation of O2 suggesting an important function of cell volume in the synthesis of reactive oxygen intermediates upon Fas receptor ligation.

Effects of indapamide on Ca2+ entry into vascular smooth muscle cells.

Part of the antihypertensive action of indapamide has been attributed to a direct inhibitory action on Ca2+ entry into vascular smooth muscle cells. The present study has been performed to identify the possible mechanisms involved. To this end the effect of indapamide on intracellular Ca2+ activity - [Ca2+]i - has been tested under control conditions and under conditions known to increase [Ca2+]i such as osmotic cell swelling (mimicking mechanical stress), depolarization (increase of extracellular K+ concentration) and oxidative stress (H2O2). Indapamide (10 micromol/l) was without effect on control [Ca2+]i, but significantly blunted the increase of [Ca2+]i following potassium-induced depolarization or following osmotic cell swelling. It did not significantly modify the increase of [Ca2+]i induced by H2O2. The effects on cell membrane potential induced by increased [K+], osmotic cell swelling, or H2O2 were not significantly modified by indapamide (10 micromol/l). Voltage-gated Ca2+ currents were not significantly modified by 10 micromol/l indapamide, but were significantly reduced by 100 micromol/l and blunted by 1 mmol/l. In conclusion, indapamide at high concentrations (100 micromol/l) inhibits voltage-gated Ca2+ channels, an effect which blunts the increase of [Ca2+]i during depolarization of the cell membrane at increased extracellular [K+] or osmotic stress. Whether these effects at high concentrations of indapamide are relevant to the antihypertensive action, however, cannot be established from these in vitro studies.