The dihydropyridine calcium channel blocker benidipine prevents lysophosphatidylcholine-induced endothelial dysfunction in rat aorta

Background  

Lysophosphatidylcholine (LPC), an atherogenic component of oxidized low-density lipoprotein, has been shown to induce the attenuation of endothelium-dependent vascular relaxation. Although benidipine, a dihydropyridine-calcium channel blocker, is known to have endothelial protective effects, the effects of benidipine on LPC-induced endothelial dysfunction remain unknown. We examined the effects of benidipine on the impairment of endothelium-dependent relaxation induced by LPC.     

Nitric oxide increases toxicity of hydrogen peroxide against rat liver endothelial cells and hepatocytes by inhibition of hydrogen peroxide degradation

Nitric oxide (NO) and hydrogen peroxide (H₂O₂) show cooperativity in their cytotoxic action. The present study was performed to decipher the mechanisms underlying this phenomenon. In cultured liver endothelial cells and in cultured, glutathione-depleted hepatocytes, the combined exposure to NO (released by spermine NONOate, 1 mM) and H₂O₂ (released by glucose oxidase) induced cell injury that was far higher than the injury elicited by NO or H₂O₂ alone. In both cell types, the addition of the NO donor increased H₂O₂ steady-state levels, although with different kinetics: in hepatocytes, the increase in H₂O₂ levels was already evident at early time points while in liver endothelial cells it became evident after 2 h of incubation. NO exposure inhibited H₂O₂ degradation, assessed after addition of 50 µM, 200 µM, or 4 mM authentic H₂O₂, significantly in both cell types. However, again, early and delayed inhibition was observed. The late inhibition of H₂O₂ degradation in endothelial cells was paralleled by a decrease in glutathione peroxidase activity. Glutathione peroxidase inactivation was prevented by hypoxia or by ascorbate, suggesting inactivation by reactive nitrogen oxide species (NO_x). Early inhibition of H₂O₂ degradation by NO, in contrast, could be mimicked by the catalase inhibitor azide. Together, these results suggest that the cooperative effect of NO and H₂O₂ is due to inhibition of H₂O₂ degradation by NO, namely to inhibition of catalase by NO itself (predominant in hepatocytes) and/or to inhibition of glutathione peroxidase by NO_x (prevailing in endothelial cells). nitrogen monoxide; catalase; glutathione peroxidase     

Pranidipine,a new 1, 4-dihydropyridine calcium channel blocker,enhances cyclic GMP-independent nitric oxide-induced relaxation of the rat aorta

Pranidipine, a new calcium channel modulator, prolonged endothelium-dependent relaxation induced by acetylcholine in a aortic ring preparation, contracted with prostaglandin F2. This action was not shared by amlodipine. The effect was not modified by indomethacin, suggesting that the action of pranidipine does not involve prostanoid metabolism. N^G-nitro-L-arginine completely prevented the action of Pranidipine. The drug affected neither nitric oxide (NO) synthase activity nor the level of cyclic GMP in the vessel. Pranidipine did not affect the sensitivity of the contractile proteins to calcium. Pranidipine also did not alter cyclic GMP-induced relaxation in a-toxinskinned vascular preparations. Pranidipine also prolonged glyceryl trinitrate-induced relaxation in the endothelium denuded rat aorta. Furthermore, pranidipine enhanced relaxation of the aorta induced by glyceryl trinitrate even in the presence of methylene blue, a guanylyl cyclase inhibitor. This action was not modified by iberiotoxin or by charybdotoxin, two inhibitors of the calciumactivated potassium channel. The results strongly suggest that pranidipine enhances cyclic GMPindependent NO-induced relaxation of smooth muscle by a mechanism other than through NOinduced hyperpolarization. These effects were in direct contrast to amlodipine, another new 1,4dihydropyridine calcium antagonist.     

Mechanisms involved in the contraction of endothelial cells by hydrogen peroxide

The importance of endothelial contraction in the genesis of inflammatory edema has been reported. ROS are metabolites synthesized in pathological conditions in that a significant intravascular fluid leak occurs, such as ischemia-reperfusion. Present experiments were designed to test the hypothesis that ROS, particularly H2O2, may elicit the contraction of endothelial cells, and to explore the mechanisms involved. Bovine aortic endothelial cells incubated with H2O2 showed a significant reduction in planar cell surface area (PCSA), and a significant increase in myosin light chain phosphorylation (MLCP), with a time- and dose-dependent pattern, without any significant toxicity. This effect of H2O2 was not blocked by sulotroban (TxA2 antagonist) or BN 52021 (PAF antagonist). Lanthanum chloride (calcium channel blocker) and EGTA partially inhibited the increase in MLCP induced by H2O2. H7 and staurosporine, PKC inhibitors, and PKC down-regulation (phorbol myristate acetate treatment, 24 h) also blocked H2O2-dependent endothelial contraction, measured as PCSA or MLCP. H2O2 increased the intracellular calcium concentration, an effect blunted by EGTA and lanthanum chloride. H2O2 also increased the phosphorylation of an 80 kD polypeptide, probably MARCKS, a PKC substrate. In summary, the present results demonstrate the ROS-dependent contraction of endothelial cells, an effect that could explain the intravascular fluid leak observed in some pathophysiological situations. Calcium and PKC may be involved in the development of this contraction.     

Ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany. In the present study, to clarify the pharmacological properties of vasodilation produced by GBE, we examined the effect of GBE and quercetin, one of the ingredients in GBE, on the thoracic aorta isolated from Wistar rats. GBE produced a dose-dependent relaxation in the aortic ring precontracted with noradrenaline, and the relaxation was abolished by L-N(G)-nitro arginine methyl ester (L-NAME). Quercetin produced a similar relaxation, which was also abolished by L-NAME. We then examined the effects of GBE and quercetin on the intracellular calcium level ([Ca2+]i) of cultured aortic endothelial cells using a fluorescent confocal microscopic imaging system. Both GBE and quercetin produced significant increases in [Ca2+]i in the endothelial cells. The increase in [Ca2+]i by quercetin (10(-6) M) was abolished by removing the extracellular Ca2+, but was not affected by thapsigargin, a calcium pump inhibitor. These findings suggest that a principal ingredient of GBE producing vasodilation is quercetin, which can activate nitric oxide synthesis and release by increasing [Ca2+]i in vascular endothelial cells.     

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.     

Differential effects of superoxide,hydrogen peroxide,and hydroxyl radical on intracellular calcium in human endothelial cells

Changes in intracellular Ca²⁺ homeostasis are thought to contribute to cell dysfunction in oxidative stress. The hypoxanthine-xanthine oxidase system (X-XO) mobilizes Ca²⁺ from intracellular stores and induces a marked rise in cytosolic calcium in different cell types. To identify the reactive O₂ species involved in the disruption of calcium homeostasis by X-XO, we studied the effect of X-XO on [Ca²⁺]_i by spectrofluorimetry with fura-2 in human umbilical vein endothelial cells (HUVEC). The [Ca²⁺]_i response to X-XO was essentially diminished by superoxide dismutase (SOD) (200 U/ml) and catalase (CAT) (200 U/ml), which scavenge the superoxide anion, O₂^?, or H₂O₂, respectively. The [Ca²⁺]_i increase stimulated by 10 nmol H₂O₂/ml/min, generated from the glucose-glucose oxidase system, or 10 μM H₂O₂, given as bolus, was about a third of that induced by X-XO (10 nmol O₂^?/ml/min) but was comparable to that induced by X-XO in the presence of SOD. The X-XO—stimulated [Ca²⁺]_i increase was significantly reduced by 100 μM o-phenanthroline, which inhibits the iron-catalysed formation of the hydroxyl radical. On the other hand, the [Ca²⁺]_i response to low dose X-XO (1 nmol O₂^?/ml/min) was markedly enhanced in the presence of 1 μM H₂O₂, which itself had no effect on [Ca²⁺]_i. More than 50% of this synergistic effect was prevented by o-phenanthroline. These results indicate that the effect of X-XO on calcium homeostasis appears to result from an interaction of O₂^? and H₂O₂, which could be explained by the formation of the hydroxyl radical. © 1995 Wiley-Liss, Inc.     

Involvement of two-pore channels in hydrogen peroxide-induced increase in the level of calcium ions in the cytoplasm of human umbilical vein endothelial cells

Hydrogen peroxide at concentrations below cytotoxic ones causes an increase in the cytoplasmic calcium concentration in human umbilical vein endothelial cells as a result of calcium release from intracellular stores. Two-pore calcium channel blocker trans-NED19 partially suppresses the increase in the level of calcium ions in the cells in response to the addition of hydrogen peroxide. The staining of endothelial cells with the fluorescent stereoisomer cis-NED19 and LysoTracker confirmed the localization of two-pore calcium channels in lysosomes and endolysosomal vesicles.     

Lymphocyte adhesion-dependent calcium signaling in human endothelial cells

Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3- labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino- phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion- dependent EC signals. mAb studies indicate that the beta 2 integrin- intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1- mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a role in EC alteration by lymphocyte adhesion.     

Acute exercise enhances vasorelaxation by modulating endothelial calcium signaling in rat aortas

The role of endothelial calcium signaling in exercise-enhanced ACh-induced vasorelaxation was examined using male Wistar rats (8~10 wk old) that were divided into control and exercise groups. The exercised animals ran on a treadmill with progressive increments of speed until exhaustion. After decapitation, aortic rings were dissected and loaded with fura 2-AM. After being mounted on a tissue flow chamber, vessels were precontracted with phenylephrine, and ACh-induced endothelial calcium elevation and vasorelaxation were determined simultaneously under an epifluorescence microscope equipped with ratio imaging capability. Our results showed that 1) there was logarithmic correlation between endothelial calcium elevation and vasorelaxation; 2) acute exercise enhanced ACh-induced endothelial calcium elevation and vasorelaxation without altering their relationship; 3) pretreatment with N(omega)-nitro-L-arginine markedly reduced ACh-induced vasorelaxation in both groups but suppressed the calcium response only in the exercise group; and 4) the exercise effect on endothelial calcium elevation was abolished by Ca2+-free buffer or gadolinium. In conclusion, acute exercise increases ACh-induced vasorelaxation by increasing the endothelial calcium influx and the calcium-dependent nitric oxide release.     

Effect of calcium channel modulators on isolated endothelial cells

Indirect evidence, using organic calcium channel modulators suggests that calcium channels exist in endothelial cells. Using freshly prepared and cultured bovine aortic endothelial cells, we have studied the effect of calcium channel modulators on Fura-2 fluorescence and have examined the binding of the dihydropyridine, (+)[3H]PN200-110. In both isolated primary and cultured cells, external calcium (0.5-2 mM) and bradykinin (10(-8) M) increased the intracellular calcium concentration. In cultured cells, the increase in calcium was not significantly attenuated by preincubation with nitrendipine (10(-8) M) or d-cis-diltiazem (10(-6) M). The calcium agonists (-)Bay k8644 and (+)202-791 had no effect on intracellular calcium concentration, but other agonists including ATP (10(-4) M) and thrombin (1.5 micrograms/ml) significantly increased the calcium concentration. Competition binding studies with (+)[3H]PN200-110 indicated specific binding of this ligand with a KD of 57 nM and a Bmax of 2.1 pmol/10(6) cells. While these data do not provide convincing evidence for the existence of calcium channels in cultured or fresh bovine aortic endothelial cells, explanations may yet reconcile our observations with the presence of calcium channels in these cells.     

Antisteroidogenic effects of hydrogen peroxide on rat granulosa cells

Abstract

Rat granulosa cells (GCs) were treated with human chorionic gonadotropin (hCG), 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), forskolin, phorbol 12-myristate 13-acetate (PMA), A23187 or pregnenolone in the absence or presence of hydrogen peroxide (H₂O₂). Different doses of trilostane were applied to GCs treated with steroidogenic precursors, that is, 25-hydroxy-cholesterol (25-OH-C) in the absence or presence of H₂O₂. Results showed that all of the chemicals stimulated the progesterone (PG) release from rat GCs, but the stimulatory effects were inhibited by H₂O₂ dose-dependently. 25-OH-C stimulated the PG release, which was inhibited by H₂O₂ in the presence of trilostane. H₂O₂ attenuated steroidogenic acute regulatory (StAR) protein expression, but did not alter the expression of cytochrome P450 side chain cleavage (P450scc) in Western blotting. This study indicated that H₂O₂ inhibited PG production by GCs via cAMP pathway, protein kinase C (PKC) and the activities of intracellular calcium, P450scc and StAR protein.     

Antisteroidogenic effects of hydrogen peroxide on rat granulosa cells

Rat granulosa cells (GCs) were treated with human chorionic gonadotropin (hCG), 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), forskolin, phorbol 12-myristate 13-acetate (PMA), A23187 or pregnenolone in the absence or presence of hydrogen peroxide (H(2)O(2)). Different doses of trilostane were applied to GCs treated with steroidogenic precursors, that is, 25-hydroxy-cholesterol (25-OH-C) in the absence or presence of H(2)O(2). Results showed that all of the chemicals stimulated the progesterone (PG) release from rat GCs, but the stimulatory effects were inhibited by H(2)O(2) dose-dependently. 25-OH-C stimulated the PG release, which was inhibited by H(2)O(2) in the presence of trilostane. H(2)O(2) attenuated steroidogenic acute regulatory (StAR) protein expression, but did not alter the expression of cytochrome P450 side chain cleavage (P450scc) in Western blotting. This study indicated that H(2)O(2) inhibited PG production by GCs via cAMP pathway, protein kinase C (PKC) and the activities of intracellular calcium, P450scc and StAR protein.     

Effect of hydrogen peroxide on calcium homeostasis in smooth muscle cells.

One of the major biological targets of free radical oxidations, prone, for anatomical reasons, to oxidative challenges, is the cardiovascular system. In the present paper the effect of hydrogen peroxide on intracellular ionized calcium ([Ca2+]i) homeostasis in smooth muscle cells (SMC) is studied, the major aim of the study being a better understanding of the protective effect of antioxidants and Ca2+ channel blockers. The exposure of SMC to 300 microM H2O2 induced a rapid increase of [Ca2+]i, followed by a decrease to a new constant level, higher than the basal before the oxidative challenge. When incubation medium was Ca2+ free, the pattern of [Ca2+]i change was different. The rapid increase was still observed, but it was followed by a rapid decrease to a level only slightly above the basal before the oxidative challenge. The involvement of intracellular Ca2+ stores was tested by using vasopressin, a hormone able to induce discharge of inositol 1,4,5-triphosphate-sensitive Ca2+ stores. When H2O2 was added after vasopressin no [Ca2+]i increase was observed. Treatment of cells, in which the stable increase of [Ca2+]i was induced by H2O2, with disulfide reducing compounds, induced a progressive decrease of [Ca2+]i toward the level observed before the oxidative challenge. Calcium channel blockers and antioxidants, on the other hand, effectively prevented the stabilization of [Ca2+]i at the high steady-state, after the internal Ca2+ release phase. Dihydropyridine Ca2+ channel blockers were by far more active than verapamil and among those the most active was lacidipine. Also the antioxidants trolox and N,N'-diphenyl-1,4-phenylenediamine both prevented the [Ca2+]i unbalance. These results suggest that Ca+ channel blockers and antioxidants, although inactive on oxidative stress-induced Ca2+ release from intracellular stores, prevent the increased influx apparently related to a membrane thiol oxidation.     

Inhibition of leukotriene actions by the calcium channel blocker, nisoldipine

Nisoldipine, a calcium channel blocker having a highly potent effect on vascular smooth muscle relative to cardiac muscle, was tested to determine its anti-leukotriene properties. Nisoldipine, at concentrations from 1 to 300 ng/ml, significantly attenuated the vasoconstrictor effects of both LTC4 and LTD4 in isolated perfused cat coronary arteries and in isolated Langendorff perfused cat hearts. In isolated perfused coronary arteries, nisoldipine exerted a greater percentage inhibition of LTC4- and LTD4-induced constriction than of the constriction induced by the thromboxane analog, carbocyclic thromboxane A2 (CTA2). In isolated cat lung fragments, higher concentrations of nisoldipine were required to inhibit leukotriene formation (i.e., 10-200 microM). These concentrations of nisoldipine markedly inhibited the formation of the chemotactic leukotriene (LTB4) as well as the peptide leukotrienes (LTC4 and LTD4) stimulated by A-23187. Both types of leukotrienes were inhibited to a comparable degree. Thus, nisoldipine has significant anti-leukotriene actions. At normally employed concentrations, nisoldipine inhibits leukotriene actions on vascular smooth muscle, and at higher concentrations, it inhibits leukotriene formation.     

Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide

Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.