Isolated Unsealed Human Erythrocyte Membranes Research Articles

The acetylcholinesterase (AChE) inhibitor and anti-Alzheimer drug donepezil interacts with human erythrocytes

Donepezil is used to treat symptomatically the Alzheimer's disease (AD). This drug is a specific inhibitor of the enzyme acetylcholinesterase (AChE), whose main physiological function is to hydrolyze the neurotransmitter acetylcholine. The main objective of this work was to study the effect of donepezil on human erythrocytes as AChE is present in its membrane. For this purpose, human erythrocytes and molecular model of its membrane built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were used. The latter correspond to classes of phospholipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively. Our experimental evidences obtained from X-ray diffraction and differential scanning calorimetry (DSC) analysis indicated that donepezil was capable of interacting with both phospholipids. Fluorescence spectroscopy results showed a moderate increase in the fluidity of the hydrophobic tails of DMPC and isolated unsealed human erythrocyte membranes (IUM). On the other hand, results by scanning electron microscopy (SEM) and optical defocusing microscopy (DM) showed that the drug changed the normal biconcave shape of the erythrocytes inducing the formation of stomatocytes (cup-shaped cells). This effect was explained by the incorporation of donepezil molecules into the erythrocyte membrane and interactions with AChE.

Protective effect of epigallocatechin gallate on human erythrocytes.

The interactions and the protective effect of epigallocatechin gallate (EGCG) on human erythrocytes (RBC) and molecular models of its membrane were investigated. The latter consisted of bilayers built- up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. X-ray diffraction and differential scanning calorimetry experiments showed that EGCG induced significant structural and thermotropic perturbations in multilayers and vesicles of DMPC; however, these effects were not observed in DMPE. Fluorescence spectroscopy results revealed that EGCG produced alterations of the molecular dynamics at the level of the hydrophobic-hydrophilic interface in DMPC vesicles, and in isolated unsealed human erythrocyte membranes (IUM). EGCG also induced morphological alterations in RBC from their normal discoid form to echinocytes. These outcomes indicate that EGCG molecules were located in the outer monolayer of the erythrocyte membrane. The assessment of EGCG protective effect demonstrated that it inhibits the morphological alterations and lysis induced by HClO to human erythrocytes. The results obtained from this study suggest that the insertion of EGCG into the outer monolayer of the erythrocyte membrane might prevent the access and deleterious effects of oxidant molecules such as HClO and free radicals into the red cells, protecting them from oxidative damage.

Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes

Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures.

Human erythrocytes and neuroblastoma cells are in vitro affected by sodium orthovanadate

Research on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50μM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25–1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations.

Effects of an antimalarial quinazoline derivative on human erythrocytes and on cell membrane molecular models

Plasmodium, the parasite which causes malaria in humans multiplies in the liver and then infects circulating erythrocytes. Thus, the role of the erythrocyte cell membrane in antimalarial drug activity and resistance has key importance. The effects of the antiplasmodial N6-(4-methoxybenzyl)quinazoline-2,4,6-triamine (M4), and its inclusion complex (M4/HPβCD) with 2-hydroxypropyl-β-cyclodextrin (HPβCD) on human erythrocytes and on cell membrane molecular models are herein reported. This work evidences that M4/HPβCD interacts with red cells as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a 10μM concentration; b) in isolated unsealed human erythrocyte membranes (IUM) a concentration as low as 1μM induced sharp DPH fluorescence anisotropy decrease whereas increasing concentrations produced a monotonically decrease of DPH fluorescence lifetime at 37°C; c) X-ray diffraction studies showed that 200μM induced a complete structural perturbation of dimyristoylphosphatidylcholine (DMPC) bilayers whereas no significant effects were detected in dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of lipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively; d) fluorescence spectroscopy data showed that increasing concentrations of the complex interacted with the deep hydrophobic core of DMPC large unilamellar vesicles (LUV) at 18°C. All these experiments are consistent with the insertion of M4/HPβCD in the outer monolayer of the human erythrocyte membrane; thus, it can be considered a promising and novel antimalarial agent.

Human cells and cell membrane molecular models are affected in vitro by the nonsteroidal anti-inflammatory drug ibuprofen

This report presents evidence that ibuprofen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a concentration as low as 10μM; b) in isolated unsealed human erythrocyte membranes (IUM) induced mild increase in the water content or in their molecular dynamics at the hydrophobic–hydrophilic interphase, while a corresponding ordering decrease at the deep phospholipids acyl chain level; c) at physiological temperature (37°C), 300μM ibuprofen induced a significant increase in the generalized polarization (GP) of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUV), an indication that ibuprofen molecules locate in the head polar group region of DMPC; d) X-ray diffraction studies showed that ibuprofen concentrations≥300μM induced increasing structural perturbation to DMPC bilayers; e) differential scanning calorimetry (DSC) data showed that ibuprofen was able to alter the cooperativity of DMPC phase transition in a concentration-dependent manner, to destabilize the gel phase and that ibuprofen did not significantly perturb the organization of the lipid hydrocarbon chains. Additionally, the effect on the viability of both human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cells was studied.

Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 μM; (c) X-ray diffraction studies showed that PPA in the 0.1–0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected.

Phospholipid Bilayers as Molecular Models for Drug‐Cell Membrana Interactions.

Since cell membranes are highly complex entities, there has been extensive interest in employing simple models to mimic biological cell membranes. Phospholipids are amphipatic molecules with long hydrophobic acyl chains and zwitterionic polar heads which are assembled into different types of molecular aggregates. The most relevant is the bilayer because of its relation with cell membranes. Among a large variety of relevant biological functions, the cell membrane acts as a diffusion barrier and protects the cell interior. Both structure and functions of the cell membrane are susceptible to alterations as a consequence of interactions with extrinsic chemical species such as therapeutic drugs. On the other hand, pharmacologic activity of a drug frequently involves an intial interaction with cell membranes even if they are not their final targets. With the aim to better understand the molecular mechanisms of the interaction of therapeutic drugs with cell membranes we utilize human erythrocyte membranes and molecular models of red blood cell membranes. Human erythrocytes are an ideal cell system for studying basic drug-membrane interactions, because they are composed of only one membrane and have no internal organelles. On the other hand, although less specialized than many other cell membranes, they carry on enough functions in common with them such as active and passive transport, and the production of ionic and electric gradients, to be considered representative of the plasma membrane in general. Molecular models of cell membranes consist of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers, representative of phospholipid classes located in the outer and inner monolayers of cell membranes, particularly of the human erythrocyte, respectively. The capacity of drugs to interact with membrane bilayers is currently evaluated in our labs by X-ray diffraction on DMPC and DMPE multilayers; DMPC large unilamellar vesicles (LUV) and isolated unsealed human erythrocyte membranes (IUM) are studied by fluorescence spectroscopy, and intact human erythrocytes are observed by scanning electron microscopy (SEM). We have been using these systems and techniques in order to determine the interaction with and the membraneperturbing effects of different compounds of therapeutic interest. As an example of our research, results obtained with the nonsteroidal anti-inflammatory drug diclofenac, the calcium channel blocker verapamil and the neuroleptic chlorpromazine will be presented. Acknowledgements FONDECYT (grant 1090041). L4.2

Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions

Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl3 was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01μM; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5–50μM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 μm–1mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1μM to 100μM.

Structural effects of Zn2+ on cell membranes and molecular models

Zinc is an essential element for nutrition as well as for the proper development and function of brain cells, and its traces are present in a wide range of foods. It is a constituent of many enzyme systems and is an integral part of insulin and of the active site of intracellular enzymes. However, excessive accumulation of zinc or its release from the binding sites may become detrimental for neurons. With the aim to better understand the molecular mechanisms of the interaction of zinc ions with cell membranes, it was incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), cholinergic murine neuroblastoma cells, and molecular models of cell membranes. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, particularly that of human erythrocytes, respectively. The capacity of zinc ions to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, intact human erythrocytes were observed with scanning electron microscopy (SEM), and neuroblastoma cell morphology was observed under inverted microscope. This study presents evidence that 0.1mM Zn and higher concentrations affect cell membrane and molecular models.

Structural effects in vitro of the anti-inflammatory drug diclofenac on human erythrocytes and molecular models of cell membranes

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly on those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated and reported. This report presents the following evidence that diclofenac interacts with red cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that diclofenac interacted with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes it was observed that the drug induced changes different from the normal biconcave morphology of most red blood cells. This is the first time in which structural effects of diclofenac on the human erythrocyte membrane have been described.

Phospholipids Bilayers as Molecular Models for Drug‐ Membrane Interactions. The Case of the Antiepileptics Phenytoin and Carbamazepine

AbstractWith the aim to better understand the molecular mechanisms of the interaction of phenytoin and carbamazepine with cell membranes we utilized a well‐established model consisting in intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of its membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidyl‐ethanolamine (DMPE), representative of phospholipid classes respectively located in the outer and inner monolayers of erythrocytes and other cell membranes. This report presents the following evidence that phenytoin and carbamazepine interact with membrane phospholipids: a) X‐ray diffraction and fluorescence spectroscopy showed that both drugs preferentially interacted with DMPC; b) in IUM, the drugs induced a disordering effect on the polar head groups and acyl chains of the eryhrocyte membrane lipid bilayers; c) electron microscopy observations of human erythrocytes showed the echinocyte formation, an effect due to phenytoin and carbamazepine insertion in the outer monolayer of the red cell membrane.

Cr(III) exerts stronger structural effects than Cr(VI) on the human erythrocyte membrane and molecular models

Chromium exists in many oxidation states, of which only the hexavalent Cr(VI) and the trivalent Cr(III) ions are stable under environmental conditions. It is generally reported that Cr(VI) is highly toxic while Cr(III) is relatively innocuous, although others have reported just the opposite. On the other hand, despite the many studies on chromium toxicity, and particularly after the knowledge that Cr(VI) anions readily enter the erythrocytes where they are reduced to Cr(III), there are practically no reports on the structural effects induced by chromium compounds on the erythrocyte membrane. With the aim to better understand the molecular mechanisms of the interaction of Cr(III) and Cr(VI) with cell membranes, CrCl3, and K2CrO4 were incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of the erythrocyte membrane. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylcholine (DMPE), phospholipid classes present in the outer and inner monolayers of the erythrocyte membrane, respectively. The capacity of Cr(III) and Cr(VI) to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed with scanning electron microscopy (SEM). In all these systems, it was found that Cr(III) induced considerably higher structural perturbations than Cr(VI).

A study of the perturbation effects of the local anesthetic procaine on human erythrocyte and model membranes and of modifications of the sodium transport in toad skin

The interaction of the local anesthetic procaine with human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), isolated toad skins, and molecular models is described. The latter consisted of phospholipid multilayers built-up of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that procaine induced the formation of stomatocytes. Experiments performed on IUM at 37 °C by fluorescence spectroscopy showed that procaine interacted with the phospholipid bilayer polar groups but not with the hydrophobic acyl chains. X-ray diffraction indicated that procaine perturbed DMPC structure to a higher extent when compared with DMPE, its polar head region being more affected. Electrophysiological measurements disclosed a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of procaine to isolated toad skin, reflecting inhibition of active ion transport.

Structural effects of titanium citrate on the human erythrocyte membrane

The structural effects of titanium citrate on the human erythrocyte membrane were studied through its interaction with intact erythrocytes and isolated unsealed human erythrocyte membranes (IUM). The studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Titanium citrate induced shape changes in erythrocytes, which were damaged and ruptured leaving empty and retracted membranes. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar head group and the acyl chain packing arrangements of the membrane phospholipid bilayer. Titanium citrate also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that titanium citrate induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC, while the effects on DMPE bilayers were negligible. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles. All these findings indicate that the structural perturbations induced by titanium to human erythrocytes can be extended to other cells, thereby affecting their functions.

The Antiepileptic Drug Diphenylhydantoin Affects the Structure of the Human Erythrocyte Membrane

Phenytoin (diphenylhydantoin) is an antiepileptic agent effective against all types of partial and tonic-clonic seizures. Phenytoin limits the repetitive firing of action potentials evoked by a sustained depolarization of mouse spinal cord neurons maintained in vitro. This effect is mediated by a slowing of the rate of recovery of voltage activated Na+ channels from inactivation. For this reasons it was thought of interest to study the binding affinities of phenytoin with cell membranes and their perturbing effects upon membrane structures. The effects of phenytoin on the human erythrocyte membrane and molecular models have been investigated in the present work. This report presents the following evidence that phenytoin interacts with cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that phenytoin perturbed a class of lipids found in the outer moiety of cell membranes; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the polar head groups and acyl chains of the erythrocyte membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes the formation of echinocytes was observed, due to the insertion of phenytoin in the outer monolayer of the red cell membrane. This is the first time that an effect of phenytoin on the red cell shape is described. However, the effects of the drug were observed at concentrations higher than those currently found in plasma when phenytoin is therapeutically administered.

Effects of the local anesthetic benzocaine on the human erythrocyte membrane and molecular models

The interaction of the local anesthetic benzocaine with the human erythrocyte membrane and molecular models is described. The latter consisted of isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphospatidylcholine (DMPC), and phospholipid multilayers of DMPC and dimyristoylphospatidyletanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that benzocaine induced the formation of echinocytes. Experiments performed on IUM and DMPC LUV by fluorescence spectroscopy showed that benzocaine interacted with the phospholipid bilayer polar groups and hydrophobic acyl chains. X-ray diffraction analysis of DMPC confirmed these results and showed that benzocaine had no effects on DMPE. The effect on sodium transport was also studied using the isolated toad skin. Electrophysiological measurements indicated a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of benzocaine, reflecting inhibition of active ion transport.

The Local Anesthetic Proparacaine Modifies Sodium Transport in Toad Skin and Perturbs the Structures of Model and Cell Membranes

Experimental results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of proparacaine to isolated toad skin, which may reflect an inhibition of the active transport of ions. This finding was explained on the basis of the results obtained from membrane models incubated with proparacaine. These consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), phospholipid multilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively, and in large unilamellar vesicles (LUV) of DMPC X-ray diffraction showed that proparacaine interaction with DMPC and DMPE bilayers perturbed both structures, especially DMPC. This result, confirmed by fluorescence spectroscopy of DMPC LUV at 18 degrees C, demonstrated that the local anesthetic (LA) could interact with the lipid moiety of cell membranes. However, effects observed by scanning electron microscopy (SEM) of human erythrocytes and by fluorescence spectroscopy of IUM might also imply proparacaine-protein interactions. Thus, the LA may alter epitheial sodium channels through interaction with the lipid matrix and with channel protein residues.

Structural Effects of the Local Anesthetic Bupivacaine Hydrochloride on the Human Erythrocyte Membrane and Molecular Models

ABSTRACT The interaction of the local anesthetic bupivacaine with the human erythrocyte membrane and molecular models is described. The latter consisted of isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC), and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy revealed that bupivacaine induced erythrocyte spheroechinocytosis. According to the bilayer couple hypothesis, this result implied that bupivacaine inserted in the outer monolayer of the erythrocyte membrane. Experiments performed on IUM and DMPC LUV by fluorescence spectroscopy and X-ray diffraction on DMPC and DMPE multilayers confirmed this result. Changes in the molecular organization of membranes alter lipid–protein interactions and induce functional perturbation of membrane proteins such as Na + channels. Since local anesthetics may control the influx of Na + into the human erythrocyte, in order to relate the structural perturbations induced by bupivacaine in these systems to Na + transport, the interaction of this anesthetic with isolated toad skin was also studied. Electrophysiological measurements indicated a significant decrease in the potential difference and in the short-circuit current of the skin after the application of the anesthetic, reflecting inhibition of the active transport of ions. These results suggest that bupivacaine-induced conformational changes of the lipid molecules alter the lipid–protein boundaries of the outer moiety of the erythrocyte membrane, thus interfering with the function of neighboring sodium channels.

Dibucaine-induced modification of sodium transport in toad skin and of model membrane structures.

The interaction of the local anesthetic dibucaine with the isolated toad skin and membrane models is described. The latter consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of dibucaine in toad skin, which may be interpreted as reflecting inhibition of the active transport of ions. This finding might be explained on the basis of the results obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, dibucaine induced structural perturbations in IUM, DMPC LUV and phospholipid multilayers. Scanning electron microscopy revealed that dibucaine induced erythrocyte stomatocytosis. According to the bilayer couple hypothesis an echinocytic type of shape change would have been expected given the preferential interaction of dibucaine with DMPC. Although it is still premature to define the molecular mechanism of action of dibucaine, the experimental results confirm the important role played by the phospholipid bilayers in the association of the anesthetic with cell membranes.