Ko Takakura

Modification of α1-adrenoceptors by peroxynitrite as a possible mechanism of systemic hypotension in sepsis

Objective It is well known that nitric oxide synthase is induced by endotoxin or inflammatory cytokines, and consequently large amounts of nitric oxide cause vascular hyporeactivity to vasoconstrictor agents and myocardial dysfunction, hence hypotension. However, there is considerable controversy as to whether these pathologic cardiovascular features are mediated directly by nitric oxide or also through the formation of secondary reaction products such as peroxynitrite (ONOO−1). Our objective was to investigate inhibitory effects of ONOO−1 on &agr;1-adrenoceptors. Design Prospective, controlled, in vitro, laboratory study. Setting Laboratory of a health sciences university. Subjects Chinese hamster ovary cells that expressed the human recombinant &agr;1a-, &agr;1b-, or &agr;1d-adrenoceptors, rat aorta strips. Interventions Binding experiments of [3H]prazosin were done in the Chinese hamster ovary cell membranes pretreated with 100 &mgr;M to 3 mM ONOO−1. Displacement experiments with noradrenaline or 3-nitro-l-tyrosine also were conducted. Mobilization of intracellular Ca2+ evoked by 1 nM to 10 &mgr;M noradrenaline was monitored in a fluorescence spectrophotometer with dual excitation at 340 nm/380 nm and emission at 500 nm in fura-2/AM-loaded Chinese hamster ovary cells. Contractile force produced by noradrenaline was monitored in rat aorta strips that have &agr;1a- and &agr;1d-adrenoceptors, pretreated with 1 mM ONOO−1. Either 0.3 N NaOH or the decomposed ONOO−1 was used as the control. Measurements and Main Results The specific binding of [3H]prazosin to &agr;1a- and &agr;1d-adrenoceptor was inhibited by ONOO−1 in a concentration-dependent manner. We found that 3 mM ONOO−1 decreased maximum binding sites by 40% to 50% in &agr;1a- and &agr;1d-adrenoceptors. Binding affinities for prazosin and noradrenaline were not affected by 1 mM ONOO−1 in all subtypes. We found that 3-nitro-l-tyrosine did not affect the prazosin binding to three adrenoceptor subtypes. Noradrenaline increased intracellular Ca2+ concentration ([Ca2+]i) concentration-dependently, which was inhibited by ONOO−1 in &agr;1a- and &agr;1d-adrenoceptors. ONOO−1 had no effect on &agr;1b-adrenoceptor. Contractile force produced by noradrenaline decreased significantly in aorta strips pretreated with ONOO−1. Conclusion ONOO−1 reduces the binding capacity of &agr;1a- and &agr;1d- but not &agr;1b-adrenoceptors without changing the affinities. Treatment with ONOO−1 attenuates noradrenaline-stimulated increase in [Ca2+]i in &agr;1a- and &agr;1d-adrenoceptors but not in &agr;1b-adrenoceptor. ONOO−1 also weakens noradrenaline-induced contractions in rat aorta that has &agr;1a- and &agr;1d-adrenoceptors. Cardiovascular hyporeactivity to catecholamines in septic shock may be caused in part by the inactivation of &agr;-adrenoceptors by ONOO−1.

Deactivation of Norepinephrine by Peroxynitrite as a New Pathogenesis in the Hypotension of Septic Shock

Background Vascular hyporeactivity to catecholamines limits successful treatment of hypotension in septic shock. Large amounts of nitric oxide (NO) and superoxide anion (O2−1·) are produced in response to bacterial endotoxins and/or inflammatory cytokines. NO reacts with O2−1· to form the potentially toxic NO metabolite, peroxynitrite (ONOO−1). The purpose of this study was to investigate whether ONOO−1 decreases the vasocontractile activity of norepinephrine. Methods Norepinephrine was treated with ONOO−1 or 3-morpholinosydonimine-N-ethyl-carbamine (SIN-1; an ONOO−1 producer) in a 5 × 10−2 m sodium phosphate buffer solution at pH 7.4, and absorbance of the product was measured spectrophotometrically at 295 and 370 nm. Norepinephrine pretreated with ONOO−1 was administered to isolated rat thoracic aortas to observe contractions in functional experiments. The rate constant between norepinephrine and ONOO−1 was determined via a competition assay with cysteine in functional experiments. Norepinephrine pretreated with ONOO−1 was injected intravenously into anesthetized rats to measure blood pressure. Results Norepinephrine pretreated with ONOO−1 was confirmed spectrally as oxidized norepinephrine. Norepinephrine pretreated with ONOO−1 decreased its vasocontractile force in an ONOO−1 (10−6, up to 3 × 10−4 m) concentration–dependent manner (EC50 = 5.1 × 10−5 m). The decrease in its force was lower at pretreatment with ONOO−1 in a lower pH buffer. A rate constant for the ONOO−1–norepinephrine reaction was 6 × 102 m/s. Norepinephrine (10−7 m) incubated with SIN-1 (10−3 m) decreased its vasocontractile force in an incubation time–dependent manner. Administration of norepinephrine pretreated with ONOO−1 to anesthetized rats caused no significant change in arterial blood pressure. Conclusions These results indicate that norepinephrine was oxidized and deactivated by ONOO−1. This deactivation may, at least in part, account for the hyporeactivities of vasocontraction to norepinephrine in septic shock.

Nitric Oxide Produced by Inducible Nitric Oxide Synthase Delays Gastric Emptying in Lipopolysaccharide-treated Rats

Background Endotoxin induces nitric oxide synthase (NOS), resulting in relaxation of gastric smooth muscle. The authors examined the effect of NO produced in response to lipopolysaccharide (LPS) treatment on gastric emptying in rats, and they also examined the effects of a selective inhibitor of inducible NOS (iNOS), aminoguanidine, and a suppressor of iNOS gene expression, dexamethasone. Methods Male Wistar rats weighing 200-250 g were used. LPS-treated rats received LPS (0.2-10 mg/kg) diluted in physiologic saline intraperitoneally. Before and at different intervals up to 8 h after administration of LPS, measurements of gastric emptying were performed in groups of 3-5 rats, by determining the amount of phenol red remaining in the stomach 20 min after intragastric instillation. In additional group of LPS (2 mg/kg)-treated rats, the gastric fundus was isolated 6 h after administration, and the tension changes in response to L-arginine, a substrate for NOS, and electrical transmural stimulation (3 Hz, 5 s) were recorded isometrically. Results (1) Gastric emptying was delayed by pretreatment with LPS in a dose- and time-dependent fashion (reduction from 68 +/- 12% to 22 +/- 7% with a dose of 2 mg/kg for 6 h). Aminoguanidine (50 mg/kg) or dexamethasone (5 mg/kg) partially inhibited the delay (to 39 +/- 4% or to 40 +/- 10%, respectively). (2) L-arginine (0.1 mM) produced a relaxation (28 +/- 2% reduction in active tension) in the gastric fundus strips isolated from LPS-treated rats but not from LPS-untreated rats. The relaxation was inhibited by aminoguanidine (1 mM). In contrast, the relaxation response to the electrical stimulation was not affected by aminoguanidine (0.1-1 mM). Conclusion The present study suggests that NO, probably produced by iNOS, is one of the factors involved in the delay of gastric emptying in the LPS-treated rats and probably in those with sepsis.

Local anesthetics structure-dependently interact with anionic phospholipid membranes to modify the fluidity.

While bupivacaine is more cardiotoxic than other local anesthetics, the mechanistic background for different toxic effects remains unclear. Several cardiotoxic compounds act on lipid bilayers to change the physicochemical properties of membranes. We comparatively studied the interaction of local anesthetics with lipid membranous systems which might be related to their structure-selective cardiotoxicity. Amide local anesthetics (10-300 microM) were reacted with unilamellar vesicles which were prepared with different phospholipids and cholesterol of varying lipid compositions. They were compared on the potencies to modify membrane fluidity by measuring fluorescence polarization. Local anesthetics interacted with liposomal membranes to increase the fluidity. Increasing anionic phospholipids in membranes enhanced the membrane-fluidizing effects of local anesthetics with the potency being cardiolipin>>phosphatidic acid>phosphatidylglycerol>phosphatidylserine. Cardiolipin was most effective on bupivacaine, followed by ropivacaine. Local anesthetics interacted differently with biomimetic membranes consisting of 10mol% cardiolipin, 50mol% other phospholipids and 40mol% cholesterol with the potency being bupivacaine>>ropivacaine>lidocaine>prilocaine, which agreed with the rank order of cardiotoxicity. Bupivacaine significantly fluidized 2.5-12.5mol% cardiolipin-containing membranes at cardiotoxicologically relevant concentrations. Bupivacaine is considered to affect lipid bilayers by interacting electrostatically with negatively charged cardiolipin head groups and hydrophobically with phospholipid acyl chains. The structure-dependent interaction with lipid membranes containing cardiolipin, which is preferentially localized in cardiomyocyte mitochondrial membranes, may be a mechanistic clue to explain the structure-selective cardiotoxicity of local anesthetics.

Local anesthetic failure associated with inflammation: verification of the acidosis mechanism and the hypothetic participation of inflammatory peroxynitrite

The presence of inflammation decreases local anesthetic efficacy, especially in dental anesthesia. Although inflammatory acidosis is most frequently cited as the cause of such clinical phenomena, this has not been experimentally proved. We verified the acidosis mechanism by studying the drug and membrane lipid interaction under acidic conditions together with proposing an alternative hypothesis. Liposomes and nerve cell model membranes consisting of phospholipids and cholesterol were treated at different pH with lidocaine, prilocaine and bupivacaine (0.05%–0.2%, w/v). Their membrane-interactive potencies were compared by the induced-changes in membrane fluidity. Local anesthetics fluidized phosphatidylcholine membranes with the potency being significantly lower at pH 6.4 than at pH 7.4 (p < 0.01), supporting the acidosis theory. However, they greatly fluidized nerve cell model membranes even at pH 6.4 corresponding to inflamed tissues, challenging the conventional mechanism. Local anesthetics acted on phosphatidylserine liposomes, as well as nerve cell model membranes, at pH 6.4 with almost the same potency as that at pH 7.4, but not on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin liposomes. Since the positively charged anesthetic molecules are able to interact with nerve cell membranes by ion-paring with anionic components like phosphatidylserine, tissue acidosis is not essentially responsible for the local anesthetic failure associated with inflammation. The effects of local anesthetics on nerve cell model membranes were inhibited by treating with peroxynitrite (50 μM), suggesting that inflammatory cells producing peroxynitrite may affect local anesthesia.

Nitric oxide synthase induction and relaxation in lipopolysaccharide-treated gastric fundus muscle of rats

To investigate whether L-arginine/nitric oxide (NO) pathway activated after treatment with lipopolysaccharide (LPS) could relax the gastric fundus smooth muscle, we made functional examinations and measured NO synthase activity by the conversion of radiolabelled L-arginine to L-citrulline in rat gastric fundus strips treated with LPS in vitro. L-arginine caused a relaxation of the mucosa-free gastric fundus strips which had been treated with LPS for 6 h in vitro and then contracted by PGF2alpha beforehand. This relaxation was partially reversed by N(G)-nitro-L-arginine (a nitric oxide synthase inhibitor) or methylene blue (a soluble guanylate cyclase inhibitor). Ca(2+)-independent NO synthase activity was induced after LPS-treatment. Co-incubation with LPS and cycloheximide for 6 h inhibited the relaxation to L-arginine and the induction of NO synthase. On the other hand, Ca(2+)-dependent NO synthase activity was decreased after LPS-treatment. These results strongly suggest that Ca(2+)-independent NO synthase is induced by endotoxin in the gastric fundus muscle, resulting in inhibition of the contractile response.

Interaction of local anaesthetics with lipid membranes under inflammatory acidic conditions.

Abstract.The clinical fact that local anaesthetics do not successfully work in the patients with inflammation has been generally interpreted on the basis of inflamed tissue acidification. In order to verify this hypothesis, the interaction of local anaesthetics with lipid membranes was studied by determining the drug-induced changes of membrane physicochemical property (membrane fluidity) at different pH covering inflammatory acidic conditions. At clinically relevant concentrations, lidocaine, procaine, prilocaine and bupivacaine fluidized 1,2-dipalmitoylphosphatidylcholine membranes with the potency decreased with lowering the pH from 7.9 to 5.9. When treated as the aqueous acidic solution (pH 4.0) similar to marketed injection solutions, lidocaine showed more pronounced pH dependence, so the reduction of its membrane-fluidizing effects at acidic pH theoretically correlated to that of its non-ionized membrane-interactive concentrations. Unlike phosphatidylcholine membranes, however, nerve cell model membranes consisting of different phospholipids and cholesterol were fluidized by lidocaine at pH 6.4–6.9 corresponding to the acidity of inflamed tissues. Cationic lidocaine was effective in fluidizing anionic phosphatidylserine and cardiolipin membranes at pH 6.4, but not zwitterionic phospholipid membranes, whereas it was ineffective on any membranes at pH 2.0 where membrane acidic phospholipids were not ionized. Local anaesthetics are considered to form the ion-pairs specifically with counter-ionic phospholipids and act on the membranes of nerve cells even under inflammatory acidic conditions. The drug and membrane interaction causable in inflamed tissue acidification does not support the conventional theory on the local anaesthetic failure associated with inflammation.

Possible involvement of nitroglycerin converting step in nitroglycerin tolerance.

Nitroglycerin (GTN) produces a dilation of vascular smooth muscle by releasing NO through a putative GTN-converting step. However, the response to GTN is markedly attenuated after prolonged or repeated exposure, resulting in tolerance. We investigated the mechanisms of GTN tolerance, employing exogenous and endogenous NO in rat aorta. In endothelium-denuded rat aortic strips, the GTN-induced relaxation response was attenuated by preceding exposure to either GTN or sodium nitroprusside (SNP). In contrast, the SNP-induced relaxation response was not affected by this protocol of GTN or SNP preexposure. Preincubation of aortic strips with lipopolysaccharide (LPS) +/- L-arginine for 12 h also caused attenuation of GTN-induced responses such as relaxation, cGMP production and nitrite/nitrate formation. The attenuating effect of LPS abolished in aortic strips co-incubated with LPS and cycloheximide or N(G)-nitro-L-arginine. These results suggest that GTN tolerance is predominantly associated with the reduction of NO release from GTN, which is caused through inhibition of a GTN-converting step due to preceding exposure to NO itself.

Protamine sulfate causes endothelium-independent vasorelaxation via inducible nitric oxide synthase pathway.

Objectif Le mecanisme precis de ľhypotension generale souvent observee avec ľusage de protamine n’est pas clair. Il a ete demontre que la protamine stimule la liberation ďoxyde nitrique (NO) a partir de la NO synthase (NOS) de ľendothelium, mais ľassociation avec la NOS inductible (NOSi) est inconnue malgre ľinduction de NOSi par les lipopolysaccharides (LPS) et/ou les cytokines inflammatoires lors de la circulation extracorporelle (CEC). Notre etude veut determiner si la protamine stimule la liberation de NO a partir de la NOSi induite par les LPS.PurposeThe precise mechanism of systemic hypotension frequently observed with the use of protamine is unclear. Although it has been reported that protamine stimulates the release of nitric oxide (NO) from endothelium NO synthase (eNOS), the association with inducible NOS (iNOS) remains unknown, despite the induction of iNOS by lipopolysaccharides (LPS) and/or inflammatory cytokines during cardiopulmonary bypass (CPB). The purpose of this study was to determine whether protamine stimulates the release of NO from iNOS induced by LPS.MethodsWe performed prospective and controlled functional examinations with isolated endothelium-denuded thoracic aortas from 21 male Wister rats. Aortic strips were mounted in Krebs solution and treated with LPS (1 µg·mL-1) for six hours to induce iNOS. Changes in tension caused by L-arginine (a substrate of NOS), protamine or a heparin-protamine complex (heparin: protamine = 1 unit: 10 µg) were measured in strips pre-contracted by phenylephrine.ResultsNo drug relaxed the strips before LPS-treatment, but each drug relaxed the strips in a dose-dependent manner after LPS-treatment (P < 0.05). Aminoguanidine (an iNOS inhibitor) and methylene blue (a guanylyl cyclase inhibitor) inhibited the relaxations.ConclusionThese results indicate that protamine and the heparin-protamine complex stimulated the release of NO from iNOS. As iNOS is induced during CPB, protamine or a heparin-protamine complex might cause systemic hypotension, at least in part, by stimulating iNOS.RésuméMéthodeNous avons réalisé des examens fonctionnels prospectifs et contrôlés ďaortes thoraciques sans endothélium prélevées chez 21 rats mâles Wister. Les bandes aortiques ont été montées dans des solutions de Krebs et traitées avec des LPS (1 µg·mL-1) pendant six heures pour induire la NOSi. Les modifications de la tension causées par la L-arginine (un substrat de la NOS), la protamine ou un complexe ďhéparine-protamine (héparine: protamine = 1 unité 10 µg) ont été mesurées dans les bandes précontractées par la phényléphrine.ObjectifLe mécanisme précis de ľhypotension générale souvent observée avec ľusage de protamine n’est pas clair. Il a été démontré que la protamine stimule la libération ďoxyde nitrique (NO) à partir de la NO synthase (NOS) de ľendothélium, mais ľassociation avec la NOS inductible (NOSi) est inconnue malgré ľinduction de NOSi par les lipopolysaccharides (LPS) et/ou les cytokines inflammatoires lors de la circulation extracorporelle (CEC). Notre étude veut déterminer si la protamine stimule la libération de NO à partir de la NOSi induite par les LPS.RésultatsAucun médicament n’a détendu les bandes avant le traitement aux LPS, mais chaque médicament les a détendues en fonction de la dose après le traitement aux LPS (P < 0,05). Ľaminoguanidine (un inhibiteur de NOSi) et le bleu de méthylène (un inhibiteur de la guanylyl cyclase) ont inhibé les relâchements.ConclusionLa protamine et le complexe ďhéparine-protamine ont stimulé la libération de NO à partir de NOSi. La NOSi étant induite pendant la CEC, la protamine ou un complexe ďhéparine-protamine peuvent, en partie, causer une hypotension générale en stimulant la NOSi.

Stereospecific interaction of bupivacaine enantiomers with lipid membranes.

Background and Objectives: S(−)‐Bupivacaine has the pharmacotoxicological advantage over its antipode and racemate. The interaction with lipid membranes was compared between S(−)‐, R(+)‐ and racemic bupivacaine. Methods: The bupivacaine‐induced changes in membrane property were determined by turbidity and fluorescence polarization measurements of membrane preparations to which bupivacaine stereoisomers of 1.0‐5.0 mmol/L were applied. Liposomal membranes were made of 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine without or with cholesterol (5 to 15 mol%), and nerve cell model membranes of 55 mol% different phospholipids and 45 mol% cholesterol. The purity and hydrophobic interaction of bupivacaine were analyzed by reversed‐phase high‐performance liquid chromatography. Results: Both S(−)‐ and R(+)‐bupivacaine were not different in lowering the phase transition temperature of membrane 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine. S(−)‐, R(+)‐ and racemic bupivacaine disordered 100 mol% 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine liposomal membranes, although the potency was indistinguishable between stereoisomers. By adding cholesterol to membranes, however, the membrane‐disordering effects showed stereostructure‐specificity that was enhanced with increasing the cholesterol content (0 to 15 mol%). The enantio‐differentiating effects resulted from neither impurities in enantiomers nor hydrophobic interaction with phosphatidylcholine acyl chains. Bupivacaine disordered nerve cell model membranes with the potency being S(−)‐enantiomer < racemate < R(+)‐enantiomer, which resembled their relative stereopotency in nerve and cardiac channel inhibition. Membrane‐disordering stereospecificity disappeared in the membranes without containing cholesterol. Conclusions: Bupivacaine stereostructure‐specifically interacts with membranes containing cholesterol, which is consistent with the clinical features of S(−)‐bupivacaine. Membrane cholesterol appears to increase the chirality of lipid bilayers and enable them to interact with S(−)‐, racemic and R(+)‐bupivacaine differently.