Prostaglandin E1 at clinically relevant concentrations inhibits aggregation of platelets under synergic interaction with endothelial cells.

BACKGROUND: The inhibitory effect of prostaglandin E1 (PGE1) on platelet aggregation is considered an important characteristic of this agent. However, the concentration of PGE1 to inhibit aggregation in vitro is higher than those of clinical use (1 ng/ml). To clarify whether PGE1 at clinically relevant concentrations inhibits aggregation under synergic action with endothelial cell-derived factors (nitric oxide and prostacyclin), we evaluated the minimum effective concentration of PGE1 to enhance the anti-aggregating activity of endothelial cells. METHODS: Inhibitory effects of PGE1 and/or the incubation buffer from cultured porcine aortic endothelial (PAE) cells on human platelet aggregation induced by 2 micro g/ml collagen were examined by turbidimetry. RESULTS: PGE1 concentration-dependently (>3 ng/ml) inhibited aggregation: the incubation buffer from PAE cells stimulated by bradykinin also inhibited aggregation. Bradykinin concentration-dependently increased the anti-aggregating activity of the PAE incubation buffer. The half-maximum effective concentration of bradykinin to inhibit aggregation (95.4+/-22.3 nM) was significantly decreased to 10.3+/-2.5 nM by 0.1 ng/ml PGE1 and to 0.9+/-0.5 nM by 1 ng/ml PGE1, respectively. These indicated that PGE1 (=0.1 ng/ml) inhibits aggregation through synergism with endothelial cells. The synergic effect of PGE1 and the anti-aggregating activity of the PAE cells preincubated with 10 micro M indomethacin for 30 min was more potent than that of these cells preincubated with 1 mM NG-nitro-L-arginine methyl ester. This suggested that the interaction of PGE1 with endothelial cell-derived nitric oxide is more powerful than that with endothelial cell-derived prostacyclin. CONCLUSION: Prostaglandin E1 (=0.1 ng/ml) inhibited platelet aggregation under synergic interaction with endothelial cells.

Differential action of spasmolytic vasodilators on platelet aggregation and endothelial cell-dependent anti-aggregation.

BACKGROUND: The perioperative use of spasmolytic vasodilators during reconstructive or vascular surgery is an important therapeutic procedure to prevent vascular spasm. Platelet aggregation at vascular endothelium injured by the surgical manipulation is thought to be associated with the persistency of spasm, although little is known about the effects of these drugs on platelet aggregation and on anti-aggregation provoked by endothelial cells. METHODS: (1) To test the direct effect of vasodilators against platelet aggregation, change in light transmission through platelet-rich plasma (PRP) stimulated with 4 microg/ml collagen was measured in the absence or presence of pentobarbital, papaverine, prostaglandin E1 (PGE1), trinitroglycerin, nitroprusside, nicardipine, and diltiazem. (2) Effects of these drugs on endothelial cell-dependent anti-aggregation were then evaluated. Incubation buffer of cultured porcine aortic endothelial (PAE) cells, which were preincubated with vasodilators for 10 min prior to a 1-min stimulation with 1 microM bradykinin, was transferred to collagen-stimulated PRP. RESULTS: (1) Papaverine and PGE1 directly inhibited platelet aggregation in a concentration-dependent manner. All other drugs failed to inhibit aggregation. (2) Incubation buffer of PAE cells stimulated with bradykinin showed a potent anti-aggregation. Pentobarbital concentration-dependently inhibited the endothelial cell-dependent anti-aggregation. Every other drug did not inhibit the anti-aggregation by PAE cells. CONCLUSION: Because of the direct anti-aggregatory effect without inhibiting endothelial cell-dependent anti-aggregation, we suggested that papaverine and PGE1 were the most promising vasodilators of all drugs examined in this study while further evaluation is required for the clinical relevance of the present study.

Protamine induces elevation of cytosolic free Ca2+ in cultured porcine aortic endothelial cells.

To test the hypothesis that protamine influences calcium movement in endothelial cells, we measured the concentration of intracellular free calcium ([Ca2+]i) in cultured porcine aortic endothelial (PAE) cells in Krebs solution (2.5mM Ca2+, pH 7.4) at 37 degrees C, by fura-2 fluorimetry. The basal [Ca2+]i of PAE cells was 113+/-18 nM (n=6). Protamine increased [Ca2+]i in a concentration-dependent manner (EC50, the concentration having 50% of the maximum effect, 1.4+/-0.3 microg mL(-1), n=6). The response of PAE cells to 100 microg mL(-1) protamine (330+/-80 nM, n=6) was blocked by a Ca2+ chelator, 5 mM glycoletherdiaminetetraacetic acid (EGTA; 131+/-16 nM, n=6), and by a non-selective Ca2+ channel blocker, 3 mM Co2+ (134+/-14 nM, n=6). These results suggest that Ca2+ influx through cell-membrane Ca2+ channels is mainly responsible for the protamine-induced Ca2+ elevation.

Cytosolic Ca2+ movements of endothelial cells exposed to reactive oxygen intermediates: role of hydroxyl radical-mediated redox alteration of cell-membrane Ca2+ channels.

1. The mode of action of reactive oxygen intermediates in cysosolic Ca2+ movements of cultured porcine aortic endothelial cells exposed to xanthine/xanthine oxidase (X/XO) was investigated. 2. Cytosolic Ca2+ movements provoked by X/XO consisted of an initial Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores and a sustained Ca2+ influx through cell-membrane Ca2+ channels. The Ca2+ movements from both sources were inhibited by catalase, cell-membrane permeable iron chelators (o-phenanthroline and deferoxamine), a *OH scavenger (5,5-dimethyl-1-pyrroline-N-oxide), or an anion channel blocker (disodium 4, 4'-diisothiocyano-2, 2'-stilbenedisulphonic acid), suggesting that *O2- influx through anion channels was responsible for the Ca2+ movements, in which *OH generation catalyzed by intracellular transition metals (i.e., Haber-Weiss cycle) was involved. 3. After an initial Ca2+ elevation provoked by X/XO, cytosolic Ca2+ concentration decreased to a level higher than basal levels. Removal of X/XO slightly enhanced the Ca2+ decrease. Extracellular addition of sulphydryl (SH)-reducing agents, dithiothreitol or glutathione, after the removal of X/XO accelerated the decrement. A Ca2+ channel blocker, Ni2+, abolished the sustained increase in Ca2+, suggesting that Ca2+ influx through cell-membrane Ca2+ channels was extracellularly regulated by the redox state of SH-groups. 4. The X/XO-provoked change in cellular respiration was inhibited by Ni2+ or dithiothreitol as well as inhibitors of Haber-Weiss cycle, suggesting that Ca2+ influx was responsible for *OH-mediated cytotoxicity. We concluded that intracellular *OH generation was involved in the Ca2+ movements in endothelial cells exposed to X/XO. Cytosolic Ca2+ elevation was partly responsible for the oxidants-mediated cytotoxicity.

Self-limiting enhancement by nitric oxide of oxygen free radical-induced endothelial cell injury: evidence against the dual action of NO as hydroxyl radical donor/scavenger.

1. The effects of oxygen free radical scavengers and endothelial cell-derived nitric oxide (EDNO) on the death of porcine cultured aortic endothelial cells exposed to exogenous superoxide-[xanthine (0.4 mM)/xanthine oxidase (0.04 unit ml-1) + diethylenetriaminepentaacetic acid (DTPA, 10 microM)] or hydroxyl radical-generating system(s) [superoxide generating system+ferric iron (Fe3+, 0.1 mM) or peroxynitrite (0-100 microM)] have been evaluated. 2. Spin trapping studies using 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) with electron paramagnetic resonance spectrometry were also conducted to determine qualitatively the oxidant species generated by the oxidant generating systems. 3. Endothelial cell injury provoked by the exogenous superoxide generating system was inhibited by catalase, DTPA and a hydroxyl radical scavenger (dimethyl sulphoxide, DMSO), but not by superoxide dismutase (SOD). Addition of Fe3+ to the superoxide generating system enhanced the cell injury. These suggested that the direct cytotoxicity of exogenous superoxide is limited, and that endogenous transition metal-dependent hydroxyl radical formation is involved in the cell injury. 4. An inhibitor of the constitutive NO-pathway, NG-monomethyl-L-arginine, did not influence cell injury induced by the superoxide generating system, suggesting that basal NO production is not responsible for the cytotoxicity. 5. Stimulation of endothelial cells with bradykinin enhanced cell injury provoked by the exogenous superoxide generating system, but not by the exogenous hydroxyl radical generating system. The enhancement by bradykinin was inhibited by NG-monomethyl-L-arginine and bradykinin B2-receptor antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7] bradykinin, suggesting that an interaction of NO with superoxide is involved in the enhanced cytotoxicity. A possible intermediate of this reaction, peroxynitrite, also caused endothelial cell injury in a concentration-dependent manner. 6. The modulatory effects of NO on hydroxyl radical-like activity (= formaldehyde production) from the superoxide generating system was also evaluated in a cell-free superoxide/NO generating system, consisting of xanthine/xanthine oxidase, DTPA, DMSO, and various amounts of a spontaneous NO generator, sodium nitroprusside (SNP) and were compared with those of Fe3+. At doses up to 10 microM, SNP concentration-dependently increased the formaldehyde production while the higher concentrations of SNP decreased. The maximum amount of formaldehyde produced by SNP was 5 fold less than that produced by Fe3+ (0.1 mM). Peroxynitrite-induced formaldehyde formation was concentration-dependently inhibited by SNP. 7. We conclude that agonist-stimulated but not basal NO production acts as cytotoxic hydroxyl radical donor as well as the endogenous transition metal when endothelial cells are exposed to exogenous superoxide anion, while the modulatory effect of EDNO is limited by a secondary reaction with hydroxyl radicals.

The dehydrofluorinated product of sevoflurane by soda lime reacts with ethanol to produce two products.

Dehydrofluorinated sevoflurane produced by soda lime reacted with methanol to produce methoxylated compounds. It is expected that dehydrofluorinated sevoflurane may react with ethyl alcohol in a patient who has recently ingested alcohol. In a closed vessel system and a model lung with circle absorber, sevoflurane, soda lime and ethanol were reacted at 25 degrees C. The breakdown products of sevoflurane in the gas phase were analyzed by gas chromatography and mass spectrometry. The ethoxylated compounds of dehydrofluorinated sevoflurane [fluoromethyl 2-ethoxy-2,2-difluoro-1-(trifluoromethyl)ethyl ether] and its dehydrofluorinated compound [fluoromethyl 2-ethoxy-2,2-fluoro-1-(trifluoromethyl)vinyl ether] were detected in the mixture of sevoflurane and soda lime in the presence of ethanol. In the closed vessel, the concentrations of these compounds were 69.4 +/- 6.6 ppm and 79.3 +/- 8.8 ppm, respectively, at 120 min in the presence of 11.2 mg/l of ethanol. These concentrations were dependent on the ethanol concentration. These compounds were also detected in the closed anesthetic circuit having a model lung by 180 minutes of circulation of 1.5% sevoflurane and 0.56 mg/l of ethanol with 200 ml/min carbon dioxide gas through soda lime: 24.2 +/- 4.0 ppm and 21.4 +/- 0.9 ppm, respectively. Dehydrofluorinated sevoflurane reacts with ethanol at concentrations that are within the range that may be encountered in a patient who has recently ingested alcohol to produce an ethoxylated compound, and this ethoxylated compound undergoes dehydrofluorination by soda lime.

Inhibitory effect of lidocaine on cultured porcine aortic endothelial cell-dependent antiaggregation of platelets.

BACKGROUND: Vascular spasm is a well-known complication during vascular surgery. Topical lidocaine is frequently used to prevent this spasm. However, the effects of lidocaine on the endothelium-dependent antiaggregation are not clear. METHODS: The aggregation of platelet-rich plasma (PRP) obtained from healthy volunteers was measured by the turbidimetric technique at 37 degrees C. (1) Cultured porcine aortic endothelial cells were preincubated with lidocaine (3.7 microM to 37 mM), NG-methyl-L-arginine (300 microM), or indomethacin (10 microM) for 30 min. The preincubation medium was exchanged with a medium containing +/- 1 microM bradykinin for 1-min stimulation of endothelial cells. One hundred microliters of the supernatant was then added to PRP (750 micro1) just after stimulation of PRP with collagen (4 micrograms/ml). (2) Authentic nitric oxide (NO) or prostacyclin (PGI2) was applied to collagen-stimulated PRP with or without lidocaine (100 micrograms/ml). RESULTS: (1) The supernatant from endothelial cells without bradykinin stimulation showed "basal" antiaggregation (13.8 +/- 3.2%; n = 6). Bradykinin enhanced the antiaggregation (100 +/- 0%; n = 6). NG-methyl-L-arginine or indomethacin (antagonists of NO or PGI2) inhibited the bradykinin-evoked antiaggregation (10.3 +/- 2.1% and 13.6 +/- 3.7%, respectively; n = 6). Simultaneous preincubation of both agents completely blocked the effect (-4.2 +/- 2.8%; n = 6). Lidocaine failed to influence basal antiaggregation, but it inhibited bradykinin-stimulated antiaggregation in a concentration-dependent manner (concentration causing 50% inhibition = 108 +/- 41 microM; n = 6). (2) In contrast, lidocaine did not shift the 50% effective concentration of NO (control, 1.3 +/- 0.1 microM vs. lidocaine, 1.6 +/- 0.1 microM) or PGI2 (control, 405 +/- 54 nM vs. lidocaine, 257 +/- 41 nM) for antiaggregation. CONCLUSIONS: Our results suggest that lidocaine has an inhibitory effect on antiaggregation derived from endothelial cells, caused by the inhibition of NO and PGI2 released from endothelial cells.

Inhibitory effect of sevoflurane on nitric oxide release from cultured endothelial cells.

We investigated the effect of sevoflurane (fluoromethyl-2,2,2-trifluoro-1-(trifluoromethyl) ethylether) on intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) release from cultured porcine aortic endothelial cells using fura-2 fluorometry, and direct (ESR spectrometry with NO-trapping by 2-(4-carboxyphenyl)-4,4,5,5-tetramiethylimidazoline-1-oxyl 3-oxide) or indirect (nitrite accumulation measured by Greiss reaction) NO measurement. Sevoflurane alone did not change resting [Ca2+]i, but diminished bradykinin-induced transient increase in [Ca2+]i in a concentration-dependent manner. The inhibitory effect of sevoflurane on bradykinin-induced transient rise in [Ca2+]i was larger than that of a non-selective Ca2+ channel blocker (CO2+). Application of sevoflurane following bradykinin-evoked [Ca2+]i transient diminished [Ca2+]i significantly, while bradykinin B2 receptor antagonist (D-Arg-[Hyp3, Thi5,8, D-Phe7] bradykinin) or CO2+ abolished it. Sevoflurane impaired nitrite accumulation stimulated by bradykinin, and reduced the amount of NO released from endothelial cells. Our results indicate that the negative effect of sevoflurane appears to be due to the inhibition of bradykinin-induced Ca2+ efflux from endoplasmic stores and Ca2+ influx through membrane Ca2+ channels.

Reaction between imidazolineoxil N-oxide (carboxy-PTIO) and nitric oxide released from cultured endothelial cells: quantitative measurement of nitric oxide by ESR spectrometry.

The performance of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide for the monitoring agent of nitric oxide was investigated. The agent (125-500 microM) was mixed with equal volume of nitric oxide solution, and aliquots of the mixture were applied to ESR spectroscopy. ESR spectra of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl, a product of the agent reacted with nitric oxide, were observed. A linear relationship was observed between the amplitude of the signal and concentrations of nitric oxide up to 80 microM. Endothelial cells cultured on microcarriers were packed in a column, perfused with Krebs solutions and the effluent was mixed to the agent. The same ESR spectra were obtained and amplitude of the signal was increased by bradykinin (3-300 nM), decreased by preincubation of NG-monomethyl-L-arginine (3-100 microM) and reversed by following incubation of L-arginine (100 microM).