Interaction of articaine hydrochloride with prokaryotic membrane lipids.

OBJECTIVE: Local anesthetics are the most commonly used drugs in dentistry, with a wide range of effects, including antimicrobial activity. High antimicrobial effects have recently been reported on oral microbes from articaine hydrochloride, revealed by the minimum inhibitory concentration and minimal bactericidal concentration. Additionally, articaine has recently been used as an alkaline component in endodontic materials with a proposed antibacterial activity. However, the detailed mechanisms of action have not been discussed. MATERIAL AND METHODS: We determined the Langmuir surface pressure/molecular area isotherms of prokaryotic lipid monolayers, as well as the phospholipid phase transitions, by employing differential scanning calorimetry on unilamellar prokaryotic liposomes (bilayers). RESULTS: Articaine hydrochloride was found to interact with the prokaryotic membrane lipids in both monolayers and bilayers. An increase of the phospholipid molecular area of acidic glycerophospholipids as well as a decrease in phase transition temperature and enthalpy were found with increasing articaine hydrochloride concentration. The thermodynamic changes by adding articaine hydrochloride to prokaryotic membrane lipids are potentially related to the effects observed from antimicrobial peptides resulting from membrane insertion, aggregate composition, pore formation, and lysis. CONCLUSION: Interaction of articaine hydrochloride with prokaryotic membrane lipids is indicated. Hence, further research is necessary to gain insight into where these compounds exert their effects at the molecular level.

The psychotropic drug olanzapine (Zyprexa) increases the area of acid glycerophospholipid monolayers.

The typical antipsychotics chlorpromazine (CPZ) and trifluoperazine (TFP) increase the mean molecular area (mma) of acidic, but not neutral, glycerophospholipids in monolayers at pH 7.36 measured by the Langmuir technique. The atypical antipsychotic olanzapine (OLP(1)) is structurally similar to TFP. We have therefore studied the effects of OLP on glycerophospholipid monolayers and in comparison with CPZ. Olanzapine (10 microM, in subphase, pH 7.36) influenced the isotherms (surface pressure versus mma) in monolayers of the neutral dipalmitoyl phosphatidylcholine (DPPC) and the acidic dipalmitoyl phosphatidylserine (DPPS) or 1-palmitoyl-2-oleoylphosphatidylserine (POPS) in the increasing order of mma: DPPS

alpha-Lactalbumin binding and membrane integrity--effect of charge and degree of unsaturation of glycerophospholipids.

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine alpha-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation-leading to looser packing-promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human alpha-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.

Chlorpromazine interaction with phosphatidylserines: a (13)C and (31)P solid-state NMR study.

Chlorpromazine (CPZ), a cationic, amphiphilic phenothiazine derivative is widely used as an antipsychotic drug because it antagonizes dopaminergic receptors. (13)C and (31)P solid-state NMR techniques were employed on phospholipid bilayers with and without CPZ. Phosphatidylserine from pig brain (PBPS), 1-palmitoyl-2-oleoyl phosphatidylserine (POPS), synthetic 1,2-dipalmitoyl phosphatidylcholine (DPPC) and chlorpromazineHCl were used to make phospholipid bilayers containing two types of phospholipids: DPPC (60%)/PBPS (40%) as well as POPS and PBPS bilayers without and with 10% CPZ. CPZ is found to prefer binding to the phosphate of phosphatidylserine, but also binding to the carboxyl of the serine head group in the DPPC/PBPS/CPZ bilayer is present. (31)P-NMR spectra indicate an effect of acyl chain unsaturation on the anisotropic motion of the charged serine head group. This implies that the serine head group anisotropic motion is restricted by intermolecular rather than intramolecular effects. The degree of phospholipid acyl chain unsaturation determines part of the CPZ bilayer interaction. The PBPS bilayer has the 22:6 acyl chain at 34 mol% and the C(4)?C(5) group of this acyl appears to be a determinant for CPZ bilayer interdigitation.

Interaction of resveratrol with membrane glycerophospholipids in model system in vitro.

Resveratrol is a polyphenol that among other sources occurs in grapes and for this reason, red wines also contain considerable amounts of this compound. Interactions of resveratrol with pure molecular species of phosphatidylcholine (PC), phosphatidylethanoloamine (PE) and phosphatidylserine (PS) were studied with the Langmuir technique on monolayers and with differential scanning calorimetry on unilamellar liposomes. Resveratrol caused a modest increase in the mean molecular area (MMA) of dipalmitoyl-PC (DPCC) and PE (DPPE) monolayers, but profoundly increased the MMA of dipalmitoyl-PS (DPPS). The resveratrol-induced increase in MMA was enlarged in PS species containing stearolyl and oleoyl acyls suggesting that increase in the acyl chain length and unsaturation enhanced the resveratrol-phospholipid interaction. In liposomes resveratrol lowered T(m) (main transition temperature) and increased T(C1/2) (transition interval) in DPPE, DPPS and 1-palmitoyl-2-oleoyl-PS (POPS), suggesting that resveratrol causes an increase in the anisotropy of these liposomes. In DPPE and POPS liposomes resveratrol caused a lowering of ΔH, further substantiating a pure enhancement of anisotropy for these molecular species. However, resveratrol caused a marked increase of ΔH in DPPS liposomes, indicating that, in addition to increase anisotropy, DPPS and resveratrol attracted each other specifically. This study has clearly shown interactions between resveratrol and glycerophospholipids on a molecular level and that these interactions are influenced by the acyl chain length, degree of unsaturation and head group of the lipids.

HAMLET forms annular oligomers when deposited with phospholipid monolayers.

Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH-as observed by liposome content leakage and circular dichroism experiments-and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.

Interaction of ibuprofen with eukaryotic membrane lipids.

With the versatility of the molecular mechanism of amphiphilic drugs there is the possibility that ibuprofen could interact with eukaryotic model membrane lipids. Using the Langmuir technique, we first determined the pressure/molecular area isotherms of glycerophospholipids monolayers at 37 degrees C, and, second, using differential scanning calorimetry (DSC), phase transition parameters in liposomes of the same lipids. Ibuprofen interacted in a concentration-independent manner with monolayers of saturated phosphatidylcholines (PC, i.e. markers of the outer membrane leaflet of eukaryotic cells). Ibuprofen was found to interact with liposomes of saturated and unsaturated phosphatidylcholines and -serines (PS, i.e. markers of the inner membrane leaflet of eukaryotic cells), and saturated ethanolamines (PE, i.e. markers of the inner membrane leaflet of eukaryotic cells). A lowering of the lipid melting temperature (Tm) and a change of enthalpy (deltaH) of the gel to liquid-crystalline phase transitions of liposomes were detected.

Interaction of triclosan with eukaryotic membrane lipids.

The possibility that triclosan and PVM/MA (polyvinylmethyl ether/maleic acid) copolymer, additives to dentrifrices, could interact with eukaryotic membrane lipids was studied by two methods: first, by determining the pressure/molecular area isotherms at 37 degrees C of glycerophospholipid monolayers, using the Langmuir technique; and second, by phase-transition parameters in liposomes of the same lipids, using differential scanning calorimetry (DSC). Triclosan interacted, in a concentration-independent manner, with monolayers of saturated phosphatidylcholines (PC; i.e. markers of the outer membrane leaflet of eukaryotic cells). Triclosan and PVM/MA copolymer mixtures were shown to clearly interact in a concentration-dependent manner with PC. Triclosan was found to interact with liposomes of saturated and unsaturated phosphatidylcholines and phosphatidylserines (PS; i.e. markers of the inner membrane leaflet of eukaryotic cells), and saturated ethanolamines (PE; i.e. markers of the inner membrane leaflet of eukaryotic cells), resulting in a decrease of the lipid melting temperature (Tm). PVM/MA copolymer changed the Tm of PS, PC, and PE in different manners. By adding PVM/MA or triclosan-PVM/MA copolymer mixtures to 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPS) no lipid transitions were detected. A biphasic change of the PC transition temperature resulted when triclosan or triclosan PVM/MA copolymer mixtures were added, indicating domain formation and change of the lipid polymorphism.

The interaction of peripheral proteins and membranes studied with alpha-lactalbumin and phospholipid bilayers of various compositions.

To characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine alpha-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMR-monitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent alpha-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of alpha-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane.