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

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Abstract

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 μM) 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 10 mol% cardiolipin, 50 mol% other phospholipids and 40 mol% cholesterol with the potency being bupivacaine  ropivacaine > lidocaine > prilocaine, which agreed with the rank order of cardiotoxicity. Bupivacaine significantly fluidized 2.5–12.5 mol% 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.

Introduction

Local anesthesia is accompanied by the potential risk such as cardiovascular disorders which is a rare but fatal complication. Local anesthetics display cardiotoxic capacities when their concentrations in blood are elevated by an accidental intravenous injection and an absolute overdose. Great concerns over local anesthetic cardiotoxicity include profound bradycardia, arrhythmia, myocardial depression and eventually cardiovascular collapse [1]. While local anesthetics show the pharmacotoxicological diversity, especially the cardiotoxic potency is known to be different between them. Bupivacaine, a long-acting amide anesthetic, has been widely used for cutaneous infiltration, regional nerve block, epidural anesthesia and spinal anesthesia in surgery and obstetrics. However, this agent is more toxic than other commonly used ones. In humans, bupivacaine is noted to exert a cardiotoxic effect at much lower serum levels than those required for other local anesthetics. Serious cardiac arrhythmias develop with the use of bupivacaine, but not with a shorter-acting amide local anesthetic lidocaine [2]. The rank order of cardiotoxic potency has been estimated to be bupivacaine > ropivacaine > lidocaine > prilocaine [3]. However, the detailed mechanism(s) for structure-selective cardiotoxicity is still unclear.

Although the primary target of local anesthetics is referred to as ion channels of cardiomyocytes, the action on other sites has been presumed to contribute to the cardiotoxic discrimination between structurally different drugs [3]. Local anesthetics have the ability to change membrane physicochemical properties such as fluidity or lipid packing order, and their induced membrane fluidization or disordering influences not only directly the functions of biomembranes but also indirectly the activities of membrane-embedded channels, receptors and enzymes through alterations of the membranous lipid environment and the protein conformation [4]. The generation of ionic currents affected by anesthetics also requires the molecular interplay of ion channel proteins and membrane lipids [5]. Since local anesthetics are present in cationic and non-ionic form under physiological conditions, they would show both electrostatic and hydrophobic interaction with cardiomyocyte membranes. Local anesthetics show different potencies to interact with membrane lipid bilayers depending on the lipid composition. In particular, they strongly interact with the membranes consisting of anionic phospholipids [6], [7].

Several cardiotoxic compounds affect the permeability of mitochondrial membranes [8]. While local anesthetics increase membrane permeability by acting on lipid bilayers, such an effect of bupivacaine is associated with the seriousness of cardiotoxicity [9]. Önyüksel et al. [10] presumed membrane cardiolipin to be the possible determinant for local anesthetics to produce their different cardiotoxicity. In their study, bupivacaine, but not lidocaine, increased the permeability of 7.5 mol% cardiolipin-containing liposomes at 200 and 400 μM. However, they found that bupivacaine and lidocaine were inactive at 100 and 400 μM, respectively, despite that these concentrations are higher than their plasma concentrations to produce cardiac collapse and depress myocardial function [11], [12].

Membrane permeability is influenced by the change in membrane fluidity [13]. Local anesthetics induce membrane fluidization which is linked to the mode of pharmacotoxicological action. The aim of this study was to find the drug and membranous system interaction which might be related to the structure-selective cardiotoxicity of local anesthetics. Based on the hypothesis that bupivacaine may fluidize anionic phospholipid membranes more intensively than other local anesthetics at cardiotoxicologically relevant concentrations, we addressed whether local anesthetics interact differently with liposomal and biomimetic membranes containing anionic phospholipids and which specific component(s) is responsible for the structure-selective membrane fluidization.

Section snippets

Materials

Bupivacaine, lidocaine and prilocaine were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ropivacaine was supplied by AstraZeneca (Södertälje, Sweden). Phospholipids: 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), cardiolipin, 1-palmitoyl-2-oleoylphosphatidylserine (POPS), 1-palmitoyl-2-oleoylphosphatidylinositol (POPI), sphingomyelin (SM), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and 1-palmitoyl-2-oleoylphosphatidic acid (POPA)

Results

Local anesthetics interacted with liposomal membranes to increase the fluidity. Their membrane-interacting potencies increased with elevating the membrane content of cardiolipin (Fig. 1A), POPS (Fig. 1B), POPG (Fig. 1C) and POPA (Fig. 1D). Such effects were significantly different between phospholipid species. In comparison of membranes containing 10 mol% anionic phospholipid, the polarization changes (%) by 200 μM bupivacaine were 8.42 ± 0.22 for cardiolipin, 3.01 ± 0.17 for POPS, 3.41 ± 0.15 for POPG

Discussion

The adverse effects of several drugs are pharmacotoxicologically associated with their action on membrane lipid bilayers. Cardiotoxic doxorubicin interacts with anionic phospholipid membranes [21]. The resulting fluidity changes in heart mitochondrial membranes disturb membrane enzymes with the concomitant dysfunction of respiratory responses and the enhancement of hydroxyl radical formation. The amphiphilic property of doxorubicin allows it to interact electrostatically with phospholipid head

Conflict of interest

The authors declare there are no conflicts of interest.

Acknowledgements

The authors thank AstraZeneca for the supply of ropivacaine. This study was supported by a Grant-in-Aid for Scientific Research 20592381 (to H.T.) from the Japan Society for the Promotion of Science.

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