Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells.

1. Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve

1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization.     

Lipopolysaccharide decreases bradykinin receptor-induced acidification responses in cultured bovine aortic endothelial cells

The effects of bacterial lipopolysaccharide (Escherichia coli 0127-B8) on bradykinin receptor function in bovine aortic endothelial cells were investigated using a microphysiometer. Bradykinin and Lys(0)-desArg(10)-bradykinin produced concentration-dependent acidification responses with pEC(50) values of 8.87+/-0.20 and 9.78+/-0.08, respectively. These responses were competitively and selectively antagonised by the bradykinin B(2) receptor antagonist, icatibant and the bradykinin B(1) receptor antagonist, desArg(9)-Leu(8)-bradykinin, respectively. The non-peptide bradykinin B(2) receptor antagonist, FR173657 (0.3 and 3 nM), selectively antagonised bradykinin-induced acidification responses, causing rightward shifts of the concentration-response curves to bradykinin, but at the same time, significantly decreasing the maximum response. A preincubation with lipopolysaccharide (0.01 and 0.1 microg/ml) for 24 h caused a significant concentration-dependent decrease in maximal response to bradykinin (27.2+/-1.9 and 9.7+/-0.4% of control) and the bradykinin B(1) receptor agonist, Lys(0)-desArg(10)-bradykinin (59.0+/-7.14 and 25.3+/-7.8% of control), without affecting the EC(50). These results suggest that bradykinin B(1) receptors are constitutively expressed in cultured bovine aortic endothelial cells and that the microphysiometer provides a rapid, sensitive technique to characterise bradykinin receptors and investigate their regulation by cytokines. Interactions between bradykinin receptors and lipopolysaccharide may play a part in the cascade of deleterious effects that occur during septic shock.     

Reduced hyperpolarization in endothelial cells of rabbit aortic valve following chronic nitroglycerine administration

This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the hyperpolarization induced by acetylcholine (ACh) in aortic valve endothelial cells (AVECs) of the rabbit and, if so, whether antioxidant agents can normalize this downregulated hyperpolarization. ACh (0.03-3 microM) induced a hyperpolarization through activations of both apamin- and charybdotoxin-sensitive Ca2+-activated K+ channels (K(Ca)) in rabbit AVECs. The intermediate-conductance K(Ca) channel (IK(Ca)) activator 1-ethyl-2-benzimidazolinone (1-EBIO, 0.3 mM) induced a hyperpolarization of the same magnitude as ACh (3 microM). The ACh-induced hyperpolarization was significantly weaker, although the ACh-induced [Ca2+]i increase was unchanged, in NTG-treated rabbits (versus NTG-untreated control rabbits). The hyperpolarization induced by 1-EBIO was also weaker in NTG-treated rabbits. The reduced ACh-induced hyperpolarization seen in NTG-treated rabbits was not modified by in vitro application of the superoxide scavengers Mn-TBAP, tiron or ascorbate, but it was normalized when ascorbate was coadministered with NTG in vivo. Superoxide production within the endothelial cell (estimated by ethidium fluorescence) was increased in NTG-treated rabbits and this increased production was normalized by in vivo coadministration of ascorbate with the NTG. It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced hyperpolarization in rabbit AVECs, possibly through chronic actions mediated by superoxide.     

Mg2+ and caffeine-induced intracellular Ca2+ release in human vascular endothelial cells.

Interaction of ionized magnesium ([Mg2+]o) and caffeine in regulation of intracellular free calcium concentration ([Ca2+]i) in human aortic endothelial cells was studied using fura-2 and digital imaging microscopy. In 1.2 mM [Mg2+]o, basal [Ca2+]i was 73.7 +/- 22.4 nM, with a heterogeneous distribution within the cells. No significant changes of basal [Ca2+]i were found either when cells were treated with 10 mM caffeine or when [Mg2+]o was lowered from 1.2 mM to 0.3 mM. However, a combined superfusion of the cells with 0.3 mM [Mg2+]o and 10 mM caffeine resulted in a significant elevation of [Ca2+]i to 382.8 +/- 57.1 nM, probably by release of Ca2+ from internal stores, which was attenuated by NiCl2 (1 mM). These results suggest that a Ca(2+)-induced Ca2+ release mechanism is involved in regulation of [Ca2+]i in endothelial cells, which may be either regulated or modulated by Mg2+.     

Vasoconstrictor responses after neo-intima formation and endothelial removal in the rabbit carotid artery.

1. The present study examined the responses of the rabbit carotid artery to five vasoconstrictors after neo-intima formation induced by perivascular collar treatment and evaluated the role of constitutive and inducible nitric oxide (NO) synthase and endothelial cells (ECs). 2. Ring segments of the rabbit carotid artery were mounted in organ chambers for isometric tension recording. Neo-intima-bearing vessels developed less force (Emax) in response to KCl, the thromboxane-mimetic U-46619 and 5-hydroxytryptamine (5-HT), but not to angiotensin I and II. 3. The collar-treatment increased the sensitivity to 5-HT, and decreased the sensitivity to angiotensin II. The sensitivity to U-46619 and angiotensin I remained unchanged. 4. Mechanical removal of ECs and inhibition of NO biosynthesis by NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NOARG) increased the sensitivity to 5-HT in sham and collar-treated segments to the same extent. The effects of collar-treatment and endothelial removal or treatment with inhibitors of NO biosynthesis were additive. Inhibition of NO biosynthesis failed to augment sensitivity to 5-HT after endothelial denudation. L-NOARG increased the force development to KCl in sham and collar-treated segments to the same extent. However, L-NMMA and L-NOARG failed to augment the contractile responses of neo-intima-bearing vessels to 5-HT and KCl after endothelial removal. 5. The responses to angiotensin I were not altered, either by the neo-intima or by endothelial removal. In arteries with a neo-intima the sensitivity to angiotensin II was decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

氧化型低密度脂蛋白诱导主动脉和心内膜内皮细胞凋亡

目的：研究氧化型低密度脂蛋白（ｏｘ－ＬＤＬ）诱导血管和心内膜内皮细胞凋亡。方法：用超速离心法分离健康人血浆低密度脂蛋白（ＬＤＬ），以ＣｕＳＯ４１０μｍｏｌ．Ｌ＾－１氧化，观察ｏｘ－ＬＤＬ对培养新生小牛主动脉内皮细胞及心内膜细胞的损伤作用，琼脂糖凝胶电泳和Ｈｏｅｃｈｓｔ３３２５８荧光密度法定性与定量分析ＤＮＡ降解，结果：ｏｘ－ＬＤＬ诱导血管内皮细胞及以内膜细胞典型凋亡形态学改变，ＤＮＡ降解呈时间和剂     

Effects of sub-chronic oral cyanide on endothelial function in rabbit aortic rings

We have investigated how the endothelium affects vascular responses following sub-chronic low dose cyanide administration. Cyanide exists in low levels in cassava foods, which are widely consumed in tropical Africa. Adult rabbits were administered 0.38 mg/kg per day KCN po for 25 days, and responses of the isolated aortic rings to noradrenaline (NA), calcium chloride (Ca2+) and acetylcholine (ACh) were measured in vitro in the presence and absence of the endothelium. In order to establish that the dose was not toxic, animal weight, some haematological indices, plasma alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured. Results show that endothelium denudation significantly (P <0.05) attenuates NA-induced contraction in rings from cyanide-treated rabbits. There was a similar reduction in response in Ca2+-depleted NA-precontracted endothelium-denuded aortic rings from cyanide-treated rabbits. Endothelium-denuded rings from cyanide-treated rabbits showed significantly (P <0.05) enhanced relaxation to ACh. In rings from control animals, the responses to NA and Ca2+ were not significantly altered, whether in the presence or absence of the endothelium. There were no significant changes in the studied toxicological indices. We conclude that endothelial compromise is necessary for low-dose sub-chronic cyanide-induced to alter vascular reactivity to NA and ACh.     

Stimulation of in vitro angiogenesis by tetrahydrobiopterin in bovine aortic endothelial cells.

This study examined whether tetrahydrobiopterin (BH4) stimulates angiogenesis by measuring the formation of tube-like structures in vascular endothelial cells. Bovine aortic endothelial cells that were cultured between two layers of collagen type I gel formed tube-like networks. Addition of BH4 or sepiapterin, a precursor of BH4 synthesis, stimulated the formation of tube-like structures. The sepiapterin-stimulated tube formation was completely inhibited by co-treatment with N-acetylserotonin, an inhibitor of sepiapterin reductase. These findings show that BH4 stimulates in vitro angiogenesis in vascular endothelial cells.     

Endothelium- and sydnonimine-induced responses of native and cultured aortic smooth muscle cells are not impaired by nitroglycerin tolerance

Summary Tolerance to the cyclic GMP-mediated vasodilator action of nitroglycerin develops with prolonged exposure and may be mediated either by formation of less nitric oxide from nitroglycerin or by desensitization of soluble guanylate cyclase to activation with nitric oxide. In the latter case, smooth muscle cells tolerant to nitroglycerin should show cross-tolerance to nitric oxide released from sydnonimines and endothelial cells (endothelium-derived relaxing factor).Therefore cultured smooth muscle cells from rabbit aorta were pretreated for 1 h with vehicle or high concentrations (0.55 mM) of nitroglycerin or the sydnonimine SIN-1. The formation of cyclic GMP induced by subsequent small doses of nitroglycerin, sydnonimine SIN-1 and endothelium-derived relaxing factor (released from cultured endothelial cells) was compared with the changes in activation of soluble guanylate cyclase, cyclic GMP formation and vasodilation in response to the same stimuli in similarly pretreated segments from rabbit thoracic aortae.Both cultured and native smooth muscle cells remained responsive to stimulation with sydnonimine SIN-1 and endothelium-derived relaxing factor after pretreatment with nitroglycerin, vehicle, or sydnonimine SIN-1, even though they were tolerant to nitroglycerin after pretreatment with nitroglycerin. In contrast, activation of soluble guanylate cyclase by nitroglycerin and sydnonimine SIN-1 was attenuated in homogenates of nitrate-tolerant aortae. The findings suggest that nitroglycerin tolerance in intact cells does not involve desensitization of soluble guanylate cyclase, because in intact cells nitrate tolerance can be overcome by direct activators of soluble guanylate cyclase. Send offprint requests to A. Mülsch at the above address     

Palytoxin-induced increase in endothelial Ca2+ concentration in the rabbit aortic valve

Palytoxin (PTX) is one of the most potent toxins isolated from marine coelenterates of the genus Palythoa. It induces depolarization in various types of cells by increasing the permeability for monovalent cations. It has been reported that PTX induces endothelium-dependent relaxation of vascular smooth muscle. In this study, we examined the effect of PTX on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the endothelium of rabbit aortic valves loaded with fluorescent Ca²⁺ indicators, fura-PE3 or fluo-3. PTX (10pM-300nM) irreversibly increased endothelial [Ca²⁺]_i in a concentration-dependent manner. ATP and thapsigargin also increased [Ca²⁺]_i. Imaging of [Ca²⁺]_i with a confocal microscope revealed that PTX increased [Ca²⁺]_i in all endothelial cells studied (n=13). An inorganic Ca²⁺ entry blocker, La³⁺ (30μM), had no effect on the increase in [Ca²⁺]_i induced by PTX whereas it inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. The PTX-induced increase in [Ca²⁺]_i was partially inhibited by ouabain and was abolished by removal of external Ca²⁺ although decrease of Na⁺ concentration in the incubation medium was ineffective. Activation of protein kinase C by 1μM 12-deoxyphorbol 13-isobutyrate or inhibition of phosphatase by 10nM calyculin-A had no effect on the increase in [Ca²⁺]_i induced by PTX, whereas both agents inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. Mn²⁺ influx, measured by the quenching of fura-PE3 fluorescence, was accelerated by ATP or thapsigargin, but not by PTX. These results suggest that PTX increases [Ca²⁺]_i in the endothelium of the rabbit aortic valve by increasing Ca²⁺ influx through a pathway which is different from that activated by ATP or thapsigargin. Received: 28 February 1997     

Acetylcholine induces Ca-dependent K currents in rabbit endothelial cells

Effects of acetylcholine (ACh) on the membrane potential and current recorded from endothelial cells dispersed from the rabbit aorta were investigated using the patch-clamp technique. ACh hyperpolarized the endothelial cell membrane. Using the whole-cell voltage-clamp procedure, ACh (10(-6) M) induced an outward current, and this current was blocked by atropine (10(-6) M). Application of either pirenzepine (3 x 10(-7) M) or AF-DX 116 (3 x 10(-6) M) slightly inhibited the ACh-induced outward current, and simultaneous application of these two blockers markedly inhibited the outward current. Application of caffeine (2 x 10(-2) M), ryanodine (10(-5) M) or heparin (10(-5) g/ml) reduced the amplitude of the ACh-induced outward current. A single-channel current recording using the patch-clamp technique revealed that ACh opens a Ca-dependent K-channel with a single-channel current conductance of 9 pS. These results indicate that both M1 and M2 receptor subtypes are present in endothelial cells of the rabbit aorta and that ACh activates the Ca-dependent K channel via release of Ca from intracellular store sites. In addition, methylene blue inhibited the ACh-induced outward current from the outside membrane.     

Some new observations on caffeine-induced rhythmic hyperpolarization in frog sympathetic ganglion cells.

Unstimulated bullfrog sympathetic ganglia were studied in vitro by intracellular and extracellular recording methods. In 80% of the cells impaled with K citrate microelectrodes, caffeine caused initial hyperpolarization (ICH) followed by rhythmic membrane hyperpolarization (RMH). Four different patterns of rhythmicity were observed, the most common being a regular beating pattern. RMH frequency depended on both caffeine and Ca2+. Tetraethylammonium reduced RMH amplitude, but did not affect frequency. Caffeine effects on cyclic AMP are not responsible for RMH since neither dibutyryl cyclic AMP nor phosphodiesterase inhibitors elicited RMH. However, the anion in the microelectrode filling solution is critical to both the incidence and amplitude of RMH, the order of effectiveness being: citrate much much greater than glutamate, acetate and chloride. In cells impaled by electrodes filled with K thiocyanate or K iodide, caffeine also caused large amplitude hyperpolarizing oscillations of membrane potential, suggesting that the effectiveness of citrate is not due to Ca2+ chelation. High gain extracellular DC recording revealed no sign of caffeine ICH, RMH or any hyperpolarizing effects. The absence of signs of caffeine hyperpolarization with extracellular recording has several interpretations, and these are discussed.     

Propofol stimulates nitric oxide release from cultured porcine aortic endothelial cells.

Propofol, an intravenous anaesthetic agent, causes marked vasodilatation in vivo. In the present study the effects of propofol on the release of nitric oxide (NO) from vascular endothelial cells was determined in vitro. Application of propofol to co-cultures of porcine aortic endothelial and smooth muscle cells resulted in a rapid increase in cyclic GMP formation. This increase was significantly inhibited following pretreatment of the cells with either NG-nitro-L-arginine (L-NOARG) or in the presence of haemoglobin. When applied to smooth muscle cells alone, propofol did not result in an increase in cyclic GMP levels. These results demonstrate that propofol stimulates the production and release of NO from cultured endothelial cells and suggest that the vasodilatation and hypotension observed when propofol is given in vivo may be due to NO release.     

Mechanism of isoprenaline induced desensitization of rabbit aortic smooth muscle beta-adrenoceptor responses

The effects of short term (1 h) as well as prolonged (16 h) activation of beta-adrenoceptors by isoprenaline on the functioning of the beta-adrenoceptor linked adenylate cyclase system of rabbit aortic smooth muscle cells in the contractile phenotype in primary culture was examined. Smooth muscle cells responded to 50 mumol/l isoprenaline with a rapid increase in intracellular cAMP. Continued exposure of these cells to this concentration of isoprenaline results in a rapid time dependent reduction in maximum beta-adrenoceptor responsiveness. In vitro analyses of adenylate cyclase activities, phosphodiesterase activity and 125I-iodocyanopindolol specific (beta-adrenoceptor) binding sites suggest that the decrease in the cells' ability to increase intracellular cAMP may be due to a reduction in beta-adrenoceptor binding sites. The loss in cell receptor responsiveness and membrane receptor concentration was only very slowly reversible. These results suggest that following prolonged exposure of vascular smooth muscle cells to beta-adrenoceptor agonists, beta-adrenoceptors are rapidly internalized and degraded. This mechanism may account for the haemodynamic tolerance observed to chronic therapy with beta-adrenoceptor agonists.     

Single-wall carbon nanotubes induce oxidative stress in rat aortic endothelial cells

Oxidative stress is a major factor contributing to endothelial cell damage. Single-wall carbon nanotubes (SWCNTs) have oxidative properties; however, the oxidative effects of SWCNTs on endothelial cells are not fully understood. In the present study, we investigated the effects of oxidative stress induced by SWCNTs on rat aortic endothelial cells (RAECs). Various markers of cellular damage were assessed, such as biochemical and ES immunity indexes, and DNA and protein damage. Our findings suggest that RAEC endured oxidative damage following SWCNT exposure. Specifically, after SWCNTs exposure, non-enzymatic antioxidant glutathione was activated prior to superoxide dismutase activation in order to defend against oxidative stress. Additionally, it was found that as SWCNT concentration increased, so did the stress protein, heme oxygenase-1 (HO-1), expression levels. These changes may induce RAEC damage, and result in many serious diseases.     

Single-wall carbon nanotubes induce oxidative stress in rat aortic endothelial cells

Oxidative stress is a major factor contributing to endothelial cell damage. Single-wall carbon nanotubes (SWCNTs) have oxidative properties; however, the oxidative effects of SWCNTs on endothelial cells are not fully understood. In the present study, we investigated the effects of oxidative stress induced by SWCNTs on rat aortic endothelial cells (RAECs). Various markers of cellular damage were assessed, such as biochemical and ES immunity indexes, and DNA and protein damage. Our findings suggest that RAEC endured oxidative damage following SWCNT exposure. Specifically, after SWCNTs exposure, non-enzymatic antioxidant glutathione was activated prior to superoxide dismutase activation in order to defend against oxidative stress. Additionally, it was found that as SWCNT concentration increased, so did the stress protein, heme oxygenase-1 (HO-1), expression levels. These changes may induce RAEC damage, and result in many serious diseases.     

Kynurenic acid production in cultured bovine aortic endothelial cells. Homocysteine is a potent inhibitor

Kynurenic acid (KYNA) is a broad-spectrum antagonist at all subtypes of ionotropic glutamate receptors, but is preferentially active at the strychnine-insensitive glycine allosteric site of the N-methyl-d-aspartate (NMDA) receptor and is also a non-competitive antagonist at the alpha7 nicotinic receptor. KYNA occurs in the CNS, urine, serum and amniotic fluid. Whilst it possesses anticonvulsant and neuroprotective properties in the brain, its role in the periphery, however, is unknown. In this study we demonstrated the presence of kynurenine aminotransferase (KAT) I and II in the cytoplasm of bovine aortic endothelial cells (BAEC). BAEC incubated in the presence of the KYNA precursor l-kynurenine synthesized KYNA concentration- and time-dependently. KYNA production was inhibited by the aminotransferase inhibitor aminooxyacetic acid but was not affected by a depolarising concentration of K⁺ or by 4-aminopyridine. The glutamate agonists l-aspartate and l-glutamate depressed KYNA production significantly. The selective ionotropic glutamate receptor agonists -amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropionic acid (AMPA) and NMDA were ineffective in this respect. d,l-Homocysteine and l-homocysteine sulphinic acid lowered KYNA production in BAEC. Further investigations are needed to assess the role and importance of KYNA in vessels and peripheral tissues.     

Pharmacological modulation of 86Rb efflux from aortic endothelial cells

The ATP-induced efflux of 86Rb from prelabelled bovine aortic endothelial cells was inhibited by quinine (50 microM) but not by a tetraethylammonium (5 mM) or apamin (50 nM). Neither sulfonylureas nor pinacidil had a significant effect on the rate of 86Rb efflux from the endothelial cells. These data are consistent with the presence of intermediate conductance Ca2(+)-activated K+ channels in endothelial cells. ATP-dependent K+ channels, sensitive to sulfonylureas and pinacidil, could not be detected.