Maki Mizogami

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.

Membrane interactivity of charged local anesthetic derivative and stereoselectivity in membrane interaction of local anesthetic enantiomers.

With respect to the membrane lipid theory as a molecular mechanism for local anesthetics, two critical subjects, the negligible effects of charged drugs when applied extracellularly and the stereoselective effects of enantiomers, were verified by paying particular attention to membrane components, phospholipids with the anionic property, and cholesterol with several chiral carbons. The membrane interactivities of structurally-different anesthetics were determined by their induced fluidity changes of liposomal membranes. Lidocaine (3.0 μmol/mL) fluidized phosphatidylcholine membranes, but not its quaternary derivative QX-314 (3.0 μmol/mL). Similarly to the mother molecule lidocaine, however, QX-314 fluidized phosphatidylserine-containing nerve cell model membranes and acidic phospholipids-constituting membranes depending on the acidity of membrane lipids. Positively charged local anesthetics are able to act on lipid bilayers by ion-pairing with anionic (acidic) phospholipids. Bupivacaine (0.75 mol/mL) and ropivacaine (0.75 and 1.0 μmol/mL) fluidized nerve cell model membranes with the potency being S(−)-enantiomer < racemate < R(+)-enantiomer (P < 0.01, vs antipode and racemate) and cardiac cell model membranes with the potency being S(−)-ropivacaine < S(−)-bupivacaine < R(+)-bupivacaine (P < 0.01). However, their membrane effects were not different when removing cholesterol from the model membranes. Stereoselectivity is producible by cholesterol which increases the chirality of lipid bilayers and enables to discriminate anesthetic enantiomers. The membrane lipid interaction should be reevaluated as the mode of action of local anesthetics.

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.

Membrane effects of ropivacaine compared with those of bupivacaine and mepivacaine

We compared the effects of ropivacaine, bupivacaine and mepivacaine on membrane lipids in an attempt to determine the anaesthetic mechanism of ropivacaine with structure‐dependent potency. The membrane effects were determined by measuring anaesthetic‐induced changes in the phase transition temperature and the fluorescence polarization of liposomal membranes prepared with cholesterol and phosphatidylcholine. Bupivacaine, ropivacaine and mepivacaine depressed the membrane lipid phase transition and decreased the polarization of liposomal membranes at 0.0625–1.0 mg/mL, indicating that these anaesthetics fluidize membranes at concentrations lower than those in clinical use. Ropivacaine and bupivacaine were effective in fluidizing the membrane core rather than the membrane surface, whereas mepivacaine was a membrane fluidizer acting equally on both regions. In the comparison of membrane fluidization at an equimolar concentration (3.0 mmol/L), ropivacaine was found to be less potent than bupivacaine and more potent than mepivacaine. This membrane‐fluidizing potency was also consistent with the hydrophobic properties of these substances evaluated by reversed‐phase chromatography. Structure‐dependent membrane fluidization associating with hydrophobicity appears to underlie the local anaesthetic effect of ropivacaine as well as those of bupivacaine and mepivacaine.

Stereostructure-based differences in the interactions of cardiotoxic local anesthetics with cholesterol-containing biomimetic membranes

Amide-type pipecoloxylidide local anesthetics, bupivacaine, and ropivacaine, show cardiotoxic effects with the potency depending on stereostructures. Cardiotoxic drugs not only bind to cardiomyocyte membrane channels to block them but also modify the physicochemical property of membrane lipid bilayers in which channels are embedded. The opposite configurations allow enantiomers to be discriminated by their enantiospecific interactions with another chiral molecule in membranes. We compared the interactions of local anesthetic stereoisomers with biomimetic membranes consisting of chiral lipid components, the differences of which might be indicative of the drug design for reducing cardiotoxicity. Fluorescent probe-labeled biomimetic membranes were prepared with cardiolipin and cholesterol of varying compositions and different phospholipids. Local anesthetics were reacted with the membrane preparations at a cardiotoxically relevant concentration of 200 μM. The potencies to interact with biomimetic membranes and change their fluidity were compared by measuring fluorescence polarization. All local anesthetics acted on lipid bilayers to increase membrane fluidity. Chiral cardiolipin was ineffective in discriminating S(-)-enantiomers from their antipodes. On the other hand, cholesterol produced the enantiospecific membrane interactions of bupivacaine and ropivacaine with increasing its composition in membranes. In 40 mol% and more cholesterol-containing membranes, the membrane-interacting potency was S(-)-bupivacaine<racemic bupivacaine<R(+)-bupivacaine, and S(-)-ropivacaine<R(+)-ropivacaine. Ropivacaine (S(-)-enantiomer), levobupivacaine (S(-)-enantiomeric), and bupivacaine (racemic) interacted with biomimetic membranes in increasing order of intensity. The rank order of membrane interactivity agreed with that of known cardiotoxicity. The stereoselective membrane interactions determined by cholesterol with higher chirality appears to be associated with the stereoselective cardiotoxic effects of local anesthetics. The stereostructure and membrane interactivity relationship supports the clinical use and development of S(-)-enantiomers to decrease the adverse effects of pipecoloxylidide local anesthetics on the cardiovascular system.

Comparative study on determination of antioxidant and membrane activities of propofol and its related compounds

Certain anesthetics have been suggested to protect against the pathological states associated with oxidative stress. We compared the antioxidant and membrane activities of propofol (2,6-diisopropylphenol) and its related compounds to address the structure-activity relationship especially in a lipid membrane phase. They were studied for the effects on 1,1-diphenyl-2-picrylhydrazyl radicals, nitro blue tetrazolium reduction by superoxide anions and membrane lipid peroxidation by peroxynitrite, and also for the induced changes in membrane fluidity of liposomes. 2-Isopropylphenols scavenged free radicals with the potency being propofol>2,5-diisopropylphenol>2-isopropylphenol>2,4-diisopropylphenol, but not 3- and 4-isopropylphenols and 1,3- and 1,4-diisopropylbenzenes. The tested compounds showed no significant superoxide dismutase-like effects. Propofol inhibited membrane lipid peroxidation more intensively than 2,5-diisopropylphenol, 2,4-diisopropylphenol and 2-isopropylphenol. Despite structurally resembling antioxidant alpha-tocopherol, 2,6-dimethylphenol was less potent than propofol. Propofol produced 50% inhibition of the lipid peroxidation in unsaturated phosphatidylcholine liposomal membranes and cell-mimetic membranes at 4.0 and 10.1 microM, respectively. Propofol and 2-alkylphenolic compounds interacted with membranes to increase their fluidity with the potency correlating with lipid peroxidation inhibiting activity. The 2-isopropylphenol structure is a requisite for both lipid peroxidation inhibition and membrane fluidity modification. The structure-specific membrane interactivity appears to be one of possible antioxidant mechanisms for propofol.

Interaction of Local Anesthetics with Biomembranes Consisting of Phospholipids and Cholesterol: Mechanistic and Clinical Implications for Anesthetic and Cardiotoxic Effects

Despite a long history in medical and dental application, the molecular mechanism and precise site of action are still arguable for local anesthetics. Their effects are considered to be induced by acting on functional proteins, on membrane lipids, or on both. Local anesthetics primarily interact with sodium channels embedded in cell membranes to reduce the excitability of nerve cells and cardiomyocytes or produce a malfunction of the cardiovascular system. However, the membrane protein-interacting theory cannot explain all of the pharmacological and toxicological features of local anesthetics. The administered drug molecules must diffuse through the lipid barriers of nerve sheaths and penetrate into or across the lipid bilayers of cell membranes to reach the acting site on transmembrane proteins. Amphiphilic local anesthetics interact hydrophobically and electrostatically with lipid bilayers and modify their physicochemical property, with the direct inhibition of membrane functions, and with the resultant alteration of the membrane lipid environments surrounding transmembrane proteins and the subsequent protein conformational change, leading to the inhibition of channel functions. We review recent studies on the interaction of local anesthetics with biomembranes consisting of phospholipids and cholesterol. Understanding the membrane interactivity of local anesthetics would provide novel insights into their anesthetic and cardiotoxic effects.

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.