A 2H-NMR study on the interactions of the local anesthetic tetracaine with membranes containing phosphatidylserine

The interaction of the local anesthetic tetracaine with phosphatidylserine-containing model membranes has been studied by 2H-NMR. Charged tetracaine exhibited an unusually large partition coefficient into multilamellar dispersions of phosphatidylserine. The 2H-NMR spectra consisted of a Pake doublet and a narrow line, with the former corresponding to tetracaine in the bilayer and the latter to tetracaine free in solution. A strong pH dependence of the quadrupole splittings indicated different membrane locations for charged and uncharged tetracaine. In equimolar mixtures of phosphatidylserine and phosphatidylcholine the partition coefficients and 2H-NMR spectra were much more like those observed in neat phosphatidylcholine than in neat phosphatidylserine. Dilution studies at pH 5.5 indicated that in phosphatidylserine/phosphatidylcholine mixtures tetracaine experiences a three-site exchange similar to that found earlier for tetracaine in phosphatidylcholine. Tetracaine is in fast exchange between sites weakly bound to membrane and free in solution, and in slow exchange with a strongly bound site in the membrane.     

Interactions of the local anesthetic tetracaine with glyceroglycolipid bilayers: a 2H-NMR study

We have examined the effects of the local anesthetic tetracaine on the orientational and dynamic properties of glycolipid model membranes. We elected to study the interactions of tetracaine with the pure glycolipid 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) and a mixture of beta-DTGL (20 mol%) in dimyristoylphosphatidylcholine (DMPC) by deuterium NMR (2H-NMR) spectroscopy. 2H-NMR spectra of beta-DTGL have been measured as a function of temperature in the presence of both the charged (pH 5.5) and uncharged forms (pH 9.5) of tetracaine. The results indicate that the anesthetic induces the formation of non-lamellar phases. Specifically, the incorporation of uncharged tetracaine results in the formation of a hexagonal phase which is stable from 52 to 60 degrees C. At lower pH, the spectrum at 52 degrees C is very reminescent of that of the beta-glucolipid alone in a bilayer environment, while as the temperature is elevated to 60 degrees C, a transition from a spectrum indicative of axial symmetry to one due to nearly isotropic motion or symmetry occurs, which may result from the formation of a cubic phase. Although it leads to an alteration in the phase behavior, the presence of tetracaine does not induce large changes in the headgroup orientation of beta-DTGL. In contrast to the pure glycolipid situation, the interaction of tetracaine with beta-DTGL (20 mol%) in DMPC does not trigger the formation of non-lamellar phases, but leads to a slight reduction in molecular ordering. The presence of the charged form of the local anesthetic near the aqueous interface of the bilayer appears to induce a small change in the conformation about the C2-C3 bond of the glycerol backbone of beta-DTGL in the mixed lipid system. Thus, the major influence of the local anesthetic on glycolipids is a change in the stability of the lamellar phase, facilitating conversion to phases with hexagonal or isotropic environments for the lipid molecules.     

Influence of the local anesthetic tetracaine on the phase behavior and the thermodynamic properties of phospholipid bilayers.

We investigated the influence of the local anesthetic tetracaine on the thermodynamic properties and the temperature- and pressure-dependent phase behavior of the model biomembrane 1,2-dimyristoyl-sn-glycero-3-phosphocholine by using volumetric measurements at temperatures ranging from 0 degrees to 40 degrees C and at pressures from ambient up to 1000 bar. The pVT measurements were complemented by temperature-dependent differential scanning calorimetric measurements. Information about the influence of different concentrations of the local anesthetic on the thermodynamic changes accompanying the lipid phase transitions, and on the thermal expansion coefficient, the isothermal compressibility, and the volume fluctuations of the lipids in their different phases, could be obtained from these experiments. The incorporation of tetracaine leads to an overall disordering of the membrane, as can be inferred from the depression of the main transition temperature and the reduction of the volume change at the main lipid phase transition. The expansion coefficient alpha p and the isothermal compressibility chi T of the lipid bilayer are enhanced by the addition of tetracaine and strongly enhanced values of alpha p and chi T, and the lipid volume fluctuations are found in the direct neighborhood of the main phase transition region. As tetracaine can be viewed as a model system for amphiphilic molecules, these results also provide insight into the general understanding of the physicochemical action of amphiphilic molecules on membranes. The experimental results are compared with recent theoretical predictions for the phase behavior of anesthetic-lipid systems, and the biological relevance of this study is discussed.     

Molecular dynamics of the local anesthetic tetracaine in phospholipid vesicles

Upon introduction into phosphatidylcholine vesicles, the 13C magnetic resonance peaks of the aromatic resonances of tetracaine are broadened while the T1 relaxation times show little change. Addition of tetracaine to vesicles containing 30% cholesterol produces a similar broadening in the 13C NMR spectrum of tetracaine. Nuclear magnetic resonance parameters of phosphatidylcholine in vesicles which are unchanged by the addition of equimolar tetracaine include 13C T1 relaxation time and 31P linewidth, T1 relaxation time, and nuclear Overhauser effect enhancement. These results are interpreted as indicating a hydrophobic interaction between hydrocarbon portions of the anesthetic and phospholipid bilayer. The rotational correlation time of tetracaine about its long axis in the vesicles has been calculated from the 13C NMR spin lattice relaxation times to be about 10(-10.3) s and is unchanged by incorporation into the phospholipid bilayer. The positively charged ammonium group of tetracaine interacts with the negatively charged phosphate group of the vesicle lipids. Using shift reagents and 31P NMR, tetracaine has been shown to displace cations from the bilayer surface, and does not undergo fast flip-flop across the vesicle bilayer.     

Effects of the local anesthetic tetracaine on the structural and dynamic properties of lipids in model membranes: a high-pressure Fourier transform infrared study

High-pressure Fourier transform infrared (FT-IR) spectroscopy was used to study the effects of a local anesthetic, tetracaine, on the structural and dynamic properties of lipids in model membranes. The model membrane systems studied were multilamellar aqueous dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC) in the absence and presence of a physiological concentration of cholesterol (30 mol %). The infrared spectra were measured at 28 degrees C in a diamond anvil cell as a function of pressure up to 25 kbar. The results indicate that the effects of tetracaine on the structure of pure DMPC bilayers in the gel state are dependent on the state of charge of the anesthetic. The uncharged tetracaine disorders the lipid acyl chains while the charged form induces the formation of an interdigitated gel phase. The presence of cholesterol in the latter system prevents the formation of the interdigitated phase, whereas in the former system it disorders the lipid acyl chains in the gel state. Moreover, it is shown that the addition of uncharged tetracaine to interdigitated DHPC bilayers does not alter the interdigitated state of the hydrocarbon chains.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Formation of micelles and membrane action of the local anesthetic tetracaine hydrochloride

The formation of micelles of the local anesthetic tetracaine hydrochloride in aqueous phosphate buffer solution of pH 6.5 and ionic strength ($\text{I}$) 0.10 was examined at 22°C by surface tension and using the fluorescent indicators perylene (peri-dinaphthalene) and 8-anilino-1-naphthalene sulfonic acid, sodium salt (ANS). The critical micelle concentration was located at 0.069, 0.071 and 0.063 M by measurements of surface tension, perylene solubilization and enhancement of ANS fluorescence, respectively. In contrast to other cationic surfactants, the anesthetic monomer did not show evidence of forming a fluorescent molecular complex with ANS under the experimental conditions of this study.The formation of micelles by tetracaine-HCl showed a pronounced effect on lipid membranes by inducing an abrupt decrease in the scattered light of egg lecithin liposomes at an anesthetic concentration roughly similar to its critical micelle concentration. This optical behaviour is characteristic of liposome damage and can be interpreted to mean that the lipids become solubilized into tetracaine-HCl micelles.The ability of this local anesthetic to form micelles can be taken as a manifestation of the same hydrophobic forces that lead to partitioning of the drug into membranes.     

Interaction of vasopressin with phosphatidylserine bilayers

Vasopressin causes a decrease in electrical resistance of phosphatidylserine bilayers. The magnitude of the decrease is a function of vasopressin and salt concentrations. The conducting channel is produced probably by aggregation of 4–5 molecules of the hormone.     

Site of action of the local anesthetic tetracaine in a phosphatidylcholine bilayer with incorporated cardiolipin.

Tetracaine (TTC) increases the permeability of phospholipid liposomal membranes to water, and this increase is reduced by the incorporation of cardiolipin into the membranes. We examined the molecular interaction of a phospholipid with the TTC cation in egg-yolk phosphatidylcholine (EyPC) liposomal membranes with incorporated bovine heart cardiolipin (BhCL) by IR spectroscopy and by determination of partitioning and the pKa of membrane-bound TTC. The IR spectra indicated that TTC shifted the stretching band of the BhCL PO2- group, a potential site of hydration in the bilayer, to a lower frequency but did not shift that of EyPC. TTC intercalated into the BhCL bilayer shifted its aromatic C-N stretching band to a lower frequency. One molecule of TTC was found to bind approximately five molecules of EyPC, and the incorporation of negatively charged BhCL into EyPC membranes increased the degree of binding of TTC to the bilayer membranes. The pKa values of TTC bound to membranes were determined as 7.7, 9.4, and 10.2 for EyPC membranes, EyPC membranes containing 50 mol % BhCL, and BhCL membranes, respectively, whereas that in an aqueous 10-mM NaCl solution was 8.5, as it was dependent on the manner of binding. The IR data together with the partitioning and the pKa data suggested differences between the actions of the TTC cation on negatively charged BhCL and on neutrally charged EyPC polar groups in the region close to the aqueous interface of the lipid bilayer.     

The localization of the local anesthetic tetracaine in phospholipid vesicles: A fluorescence quenching and resonance energy transfer study

Fluorescence quenching and resonance energy transfer have been used to determine the localization of the local anesthetic tetracaine in vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) as a function of both temperature and ionic strength. The fluorescence behaviour of tetracaine in vesicles can be attributed to its different partition coefficients in acid and basic solution, in gel phase and fluid phase vesicles, respectively. Using both steady-state and time-resolved fluorescence measurements we show that a saturable binding rather than a partitioning model holds for the interaction of tetracaine with gel phase bilayers. The relative quenching efficiencies of the series of n-AS dyes depend on the phase state of the bilayer and suggest a deeper incorporation of tetracaine in fluid phase than in gel phase membranes. Resonance energy transfer measurements support the view that tetracaine is incorporated predominantly in the region of the 9-AS chromophore in DMPC-bilayers.     

The effect of neutral and charged micelles on the acid-base dissociation of the local anesthetic tetracaine

The influence of surfactant micelles on the acid-base dissociation of the charged tertiary amino group of the local anesthetic, tetracaine, has been investigated. From measurements of tetracaine fluorescence as a function of bulk pH, apparent $\text{p}\text{K}$ values of 6.88, 7.58 and 9.92 were found in the presence of cationic, neutral and anionic micelles, respectively, in 10 mM NaCl. These values are considerably displaced with respect to the $\text{p}\text{K}$ in aqueous solution which is 8.26. Such large shifts can be attributed to the effect of the surface polarity and electrical potential on the dissociation behavior of the anesthetic bound to micelles. It can be expected that the acid-base dissociation of a local anesthetic adsorbed to nerve fibers will also be affected by the properties of the membrane surface. Thus, it is suggested that the influence of the interfacial region on the $\text{p}\text{K}$ of surface-bound molecules should not be disregarded when estimating the proportion of charged and uncharged forms of local anesthetics interacting with axonal membranes.     

Multiequilibrium binding of a spin-labeled local anesthetic in phosphatidylcholine bilayers

The equilibria among spin-labeled amine local anesthetic species in dioleoylphosphatidylcholine liposomes at an anesthetic: lipid mole ratio of 1:100 are investigated. Electron spin resonance (ESR) spectra demonstrate that anesthetic mobility within the bilayer is charge-dependent, with the uncharged species the more mobile. Partition coefficient measurements confirm ESR evidence that changes in anesthetic mobility represent anesthetic-phospholipid interaction and not changes in bilayer fluidity. Spin-exchange attenuation experiments show that anesthetics within the bilayer are accessible to the aqueous medium. Dependence of tertiary-amine anesthetic pK on dielectric constant has been used to estimate the interfacial pK. We propose a model of equilibria among species of the tertiary amine anesthetic in the aqueous medium and those intercalated in the bilayer, including a species electrostatically bound to the lipid phosphate. Using experimentally determined equilibrium constants, the model provides the binding constant between the electrostatically bound and unbound cationic anesthetics within the bilayer. The model stimulates the pH dependence of the mobile fraction of total anesthetic population determined by subtraction techniques on experimental ESR spectra.     

Phase separation of cholesterol from phosphatidylserine-cholesterol mixtures in the presence of the local anesthetic tetracaine

Addition of the local anesthetic tetracaine (TTC) to multilamellar dispersions of natural phosphatidylserine (PS) causes changes in the thermotropic properties of the membrane, which can be detected by differential scanning calorimetry, and in the structure of the membrane as detected by X-ray diffraction. At molar ratio [PS]/[TTC]8.5, the melting temperature of the phospholipid shifts downwards by approximately 2.5 °C. The melting endotherm is broadened; however, there is little change in the enthalpy of melting. In ternary mixtures (PS–TTC–cholesterol), the thermotropic changes are enhanced. At [PS]/[TTC]13, the onset of phase separation of cholesterol crystals from PS in the liquid crystalline state occurs at molar fraction cholesterol (X_chol)0.28, marginally smaller than that found in the absence of the anesthetic.     

High-pressure 31P NMR study of dipalmitoylphosphatidylcholine bilayers.

High-pressure 31P NMR was used for the first time to investigate the effects of pressure on the structure and dynamics of the phosphocholine headgroup in pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar aqueous dispersions and in DPPC bilayers containing the positively charged form of the local anesthetic tetracaine (TTC). The 31P chemical shift anisotropies, delta sigma, and the 31P spin-lattice relaxation times, T1, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for both pure DPPC and DPPC/TTC bilayers. This pressure range permitted us to explore the rich phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as gel I (P beta'), gel II (L beta'), gel III, gel IV, gel X, and the interdigitated, Gi, gel phase. For pure DPPC bilayers, pressure had an ordering effect on the phospholipid headgroup within the same phase and induced an interdigitated Gi gel phase which was formed between the gel I (P beta') and gel II (L beta') phases. The 31P spin-lattice relaxation time measurements showed that the main phase transition (LC to gel I) was accompanied by the transition between the fast and slow correlation time regimes. Axially symmetric 31P NMR lineshapes were observed at pressures up to approximately 3 kbar but changed to characteristic axially asymmetric rigid lattice lineshapes at higher pressures (3.1-5.1 kbar).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine. Spin label and titration studies

Electrometric titrations and spin label data demonstrate changes in the experimentally determined apparent pK of an ionizable drug in the presence of membranes. This effect is attributed to the difference in partition coefficients for the charged and uncharged forms of the drug. Investigation of the binding of a local anesthetic, tetracaine, to egg phosphatidylcholine membranes indicates that the drug apparent pK decreases in the presence of membranes, the decrease being a function of membrane concentration. The agreement between titration and spin label studies is very good and could be simulated by calculating membrane-bound and free populations of charged and uncharged tetracaine from the independently-measured partition coefficients for the two forms.     

Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine spin label and titration studies

Electrometric titrations and spin label data demonstrate changes in the experimentally determined apparent pK of an ionizable drug in the presence of membranes. This effect is attributed to the difference in partition coefficients for the charged and uncharged forms of the drug. Investigation of the binding of a local anesthetic, tetracaine, to egg phosphatidylcholine membranes indicates that the drug apparent pK decreases in the presence of membranes, the decrease being a function of membrane concentration. The agreement between titration and spin label studies is very good and could be simulated by calculating membrane-bound and free populations of charged and uncharged tetracaine from the independently-measured partition coefficients for the two forms.     

Molecular simulation study of phospholipid bilayers and insights of the interactions with disaccharides

Molecular simulations of hydrated dipalmitoylphosphatidylcholine lipid bilayers have been performed for temperatures in the range of 250-450 K. The area per headgroup increases with temperature from 58 to 77 A(2). Other properties such as hydration number, alkyl tail order parameter, diffusion coefficients, and radial distribution functions exhibit a clear dependence on temperature. Simulations of bilayers have also been performed in the presence of two disaccharides, namely trehalose and sucrose, at concentrations of up to 18 wt % (lipid-free basis). The simulated area per headgroup of the bilayer is not affected by the presence of the disaccharides, suggesting that the overall structure of the bilayer remains undisturbed. The results of simulations reveal that the interaction of disaccharide molecules with the bilayer occurs at the surface of the bilayer, and it is governed by the formation of multiple hydrogen bonds to specific groups of the lipid. Disaccharide molecules are observed to adopt specific conformations to fit onto the surface topology of the bilayer, often interacting with up to three different lipids simultaneously. At high disaccharide concentrations, the results of simulations indicate that disaccharides can serve as an effective replacement for water under anhydrous conditions, which helps explain their effectiveness as lyophilization agents for liposomes and cells.     

Structure stability of lytic peptides during their interactions with lipid bilayers.

In this work, molecular dynamics simulations were used to examine the consequences of a variety of analogs of cecropin A on lipid bilayers. Analog sequences were constructed by replacing either the N- or C-terminal helix with the other helix in native or reverse sequence order, by making palindromic peptides based on both the N- and C-terminal helices, and by deleting the hinge region. The structure of the peptides was monitored throughout the simulation. The hinge region appeared not to assist in maintaining helical structure but help in motion flexibility. In general, the N-terminal helix of peptides was less stable than the C-terminal one during the interaction with anionic lipid bilayers. Sequences with hydrophobic helices tended to regain helical structure after an initial loss while sequences with amphipathic helices were less able to do this. The results suggests that hydrophobic design peptides have a high structural stability in an anionic membrane and are the candidates for experimental investigation.