Local anesthetics facilitate ion transport across lipid planar bilayer membranes under an electric field: dependence on type of lipid bilayer.

In order to elucidate the role of structural change of lipid membrane bilayer in the mode of action of local anesthetic, we studied the effects of local anesthetics, charged tetracaine and uncharged benzocaine, on ion permeability across various lipid planar bilayers (PC, mixed PC/PS (4/1, mol/mol); mixed PC/PE (1/1, mol/mol); mixed PC/SM (4/1, mol/mol)) under a constant applied voltage. The membrane conductances increased in the order of PC<

A statistical mechanical theory for the adsorption of protein to liposomal membranes.

The observed topology change of spherical lipid vesicles to coffee cups [Saitoh, A. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 1026] was analyzed by a statistical mechanical theory. The topology change was due to the adsorption of talin molecules to the orifices of the coffee cups. The adsorption isotherm of talin between an aqueous solution and the vesicle membrane was analyzed by taking account of the bending energy of the membrane. The equilibrium is determined by the balance of the energy gain for the adsorption of talin to the periphery of the vesicles and the change of the bending energy of the membrane due to the shape change. The observed coexistence of coffee cups and sheet-like vesicles were reproduced. Vesicles with two orifices were also analyzed and theoretically reproduced.

A theory on the instability of tubular membranes to the periodic conformation by laser tweezers.

A mechanical theory to analyze the stability of tubular membranes perturbed by optical tweezers is proposed. I assume that the optical tweezers cause the temporal elevation of hydrostatic pressure inside tubular membranes due to the thermal expansion of solvent water, and I relaxed the conservation of volume per unit length which was strictly maintained in old theories based on the well-known Rayleigh instability. The mechanical energy composed of bending rigidity, interfacial tension, and hydrostatic pressure terms can explain the condition of the previously observed peristaltic and pearling instability. The spontaneous curvature of the membrane is postulated in the theory. The enhanced pressure causes the tubules to enter a peristaltic state with a spinodal line in the phase diagram.

A statistical mechanical analysis of the effect of long-chain alcohols and high pressure upon the phase transition temperature of lipid bilayer membranes.

Long-chain n-alcohols decrease the main phase-transition temperature of lipid vesicle membranes at low concentrations but increase it at high concentrations. The nonlinear phenomenon is unrelated to the interdigitation and is analyzed by assuming that alcohols form solid solutions with solid as well as liquid phases. The biphasic response originates from the balance of the free energy difference of alcohols in the liquid and solid membranes (delta gA) and the alcohol-lipid interaction free energy difference (delta u) between the two phases. When delta gA less than 0 and delta u greater than 0, or delta gA less than delta u less than 0, the transition temperature decreases monotonously according to the increase in the alcohol concentration. When delta gA greater than 0 and delta u less than 0, or delta gA greater than delta u greater than 0, it increases monotonously. Biphasic response occurs with a minimum temperature when delta u greater than delta gA greater than 0, and with a maximum temperature when delta u less than delta gA less than 0. When the alcohol carbon-chain length becomes closer to the lipid carbon-chain length, delta u is equalized by delta gA, and the temperature minimum of the main transition is shifted to extremely low alcohol concentrations. Hence, long-chain alcohols predominantly elevate the main transition temperature and lose their anesthetic potency. High pressure decreased both delta gA and delta u. Presumably, high pressure improves the packing efficiency of liquid membranes and decreases the difference between the solid and liquid membrane properties.

Effect of inhalation anesthetics on swimming activity of artemia salina.

The swimming movement of artemia salina in the artificial sea water was measured by using the video camera system in the absence and presence of anesthetics, i.e. enflurane, halothane, and isoflurane. The movement of artemia looked random at a glance but the obtained distribution curve for the swimming speed was skewed toward the high speed side somewhat resembling a Maxwellian distribution curve seen in the statistics of ideal gases. When anesthetic were added, the distribution curve became sharpened and shifted to the low speed side, which is similar to a behavior of ideal gases when they are cooled down. The mean swimming-speed was decreased eventually leading to an irreversible death with increasing the anesthetic dose. The activity was analyzed by using the hydrodynamic equation. The ED(50), which is a dose that causes a 50% reduction in the activity, of all anesthetics used in this study was quite similar to the MAC values for human. It was also suggested that an interaction between anesthetics and artemia was highly cooperative since the larger Hill coefficients were obtained for all three anesthetics used.

A solid-solution theory of anesthetic interaction with lipid membranes: temperature span of the main phase transition.

Anesthetics (or any other small additives) depress the temperature of the main phase transition of phospholipid bilayers. Certain anesthetics widen the temperature span of the transition, whereas others do not. The widening in a first-order phase transition is intriguing. In this report, the effects of additive molecules on the temperature and its span were explained by the solid-solution theory. By assuming coexistence of the liquid-crystal and solid-gel phases of lipid membranes at phase transition, the phase boundary is determined from the distribution of anesthetic molecules between the liquid-crystal membrane versus water and between the solid-gel membrane versus water. The theory shows that when the lipid concentration is large or when the lipid solubility of the drug is large, the width of the transition temperature increases, and vice versa. Highly lipid-soluble molecules, such as long-chain alkanols and volatile anesthetics, increase the width of the transition temperature when the lipid:water ratio is large, whereas highly water-soluble molecules, such as methanol and ethanol, do not. The aqueous phase serves as the reservoir for anesthetics. Depletion of the additive molecules from the aqueous phase is the cause of the widening. When the reservoir capacity is large, the temperature width does not increase. The theory also predicts asymmetry of the specific heat profile at the transition.

Effect of local anesthetics on the electrical characteristics of an excitable model membrane composed of dioleyl phosphate I. Static and steady-state response.

The local anesthetics, tetracaine, procaine and lidocaine, interacted with a negatively charged lipid membrane composed of dioleyl phosphate (DOPH), which exhibited a self-sustained oscillation of the membrane potential. The anesthetics depolarized the membrane potential when present in increasing concentrations, whereas they increased the membrane resistance at low concentrations and decreased it at high concentrations. The above results were analyzed on the basis of electrochemical theory taking into account ion flux across the membrane. The electrical characteristics are affected by both the hydrophobicity and the diffusion constant of local anesthetics within the membrane.

Effect of local anesthetics on the electrical characteristics of an excitable model membrane composed of dioleyl phosphate II. Dynamic response.

The effects of local anesthetics (tetracaine, procaine and lidocaine) on self-sustained electrical oscillations were studied for a lipid membrane comprising dioleyl phosphate (DOPH). This model membrane exhibits oscillation of the membrane potential in a manner similar to that of nerve membranes, i.e., repetitive firing, in the presence of an ion-concentration gradient, on the application of d.c. electric current. Relatively weak anesthetics such as procaine and lidocaine increased the frequency of self-sustained oscillation, and finally induced aperiodic, rapid oscillation. The strong anesthetic tetracaine inhibited oscillation.

Interaction of surfactants with bilayer of negatively charged lipid: effect on gel-to-liquid-crystalline phase transition of dilauroylphosphatidic acid vesicle membrane.

The interaction of surfactants with the vesicle membrane of the negatively charged lipid, dilauroylphosphatidic acid, was investigated through their effect on the gel-to-liquid-crystalline phase transition of the lipid bilayer. Three types of surfactants (anionic, cationic and non-ionic) with different hydrocarbon chain length were examined. (i) Anionic sodium alkylsulfates affected the phase transition temperature, Tm, only weakly. (ii) Non-ionic alkanoyl-N-methylglucamides decreased Tm monotonously with increasing concentration. The depression of Tm induced by these surfactants was analyzed by applying the van't Hoff model for the freezing-point depression, and the partition coefficients of the surfactants between bulk water and lipid membrane were estimated. (iii) Cationic alkyltrimethylammonium bromides affected Tm in a complex manner depending on the hydrocarbon chain length of the surfactants. Octyl-/tetradecyl-trimethylammonium bromide depressed/elevated Tm monotonously with increasing concentration, whereas the change in Tm induced by decyl- and dodecyltrimethylammonium bromides was not monotonous but biphasic. This complex behavior of the phase transition temperature was well explained, based on the statistical mechanical theory presented by Suezaki et al. (Biochim. Biophys. Acta, 818 (1985) 31-37), which takes into account the interaction between surfactant molecules incorporated in the lipid membrane.

Origin of calcium-induced minimum in bulk compressional modulus of lipid membranes. Configurational entropy of adsorbed Ca2+.

Addition of Ca2+ to a dipalmitoylphosphatidylcholine lamellar system decreases the bulk compressional modulus (increases compressibility) of the membrane (S. Aruga, R. Kataoka and S. Mitaku, Biophys. Chem. 21 (1985) 265). The bulk modulus was reported to show a minimum value at 10 mM Ca2+ within the temperature range 20-45 degrees C. In the present report, the occurrence of this minimum in the bulk modulus is explained quantitatively as a result of fluctuation in the number of Ca2+ adsorbed onto the lipid bilayer surface. From this theory, the change in apparent molal volume of Ca2+ upon surface adsorption is estimated to be 5.7 cm3 mol-1, which appears to be a reasonable value. The number of adsorbed Ca2+ at the concentration where the bulk modulus assumes the minimum value is half of the number of allowable adsorption sites on lipid membranes. The configurational entropy of the adsorbed Ca2+ attains a maximum at the minimum point.

Regions necessary for pH-dependent assembly of gizzard myosin rod.

Aggregated and disaggregated forms of gizzard myosin rod and its fragments in various concentrations of NaCl (0-0.30 M) at various pH (7.4-8.6) were distinguished from each other by their permeability through a Sepharose 4B column. The rod existed in three forms, namely: large aggregates impermeable to the column, small aggregates eluted at the void volume of the column and a disaggregated monomer which penetrated the column. The relative proportions of the three forms varied depending on the salt concentration and pH. The monomeric rod was detected in NaCl solutions above 0.20 M and its relative proportion at 0.25 M NaCl was larger than those of the small and large aggregates. The small aggregates of the rod were predominant at below 0.05 M NaCl and, upon decrease in pH from 8.6 to 7.4, these small aggregates in NaCl solutions between 0.10 M and 0.15 M were replaced by the large aggregates. Light meromyosin, which corresponded to the C-terminal two-thirds of the rod, existed exclusively as large aggregates in NaCl solutions below 0.15 M; increase of NaCl concentration to above 0.20 M resulted in the formation of its monomer, instead of the large aggregates. In contrast to the rod, no small aggregated form of the light meromyosin was detected. Truncated light meromyosin which had lost a small segment from either the C-terminal or N-terminal of light meromyosin was eluted only as a monomer in any NaCl concentration at any pH. It may be deduced from the above results that a small segment in the light meromyosin is requisite for the assembly of both rod and light meromyosin in the NaCl solutions below 0.15 M and that the relative proportion of small and large aggregates of the rod is determined in a pH-dependent manner by the subfragment 2 segment, the N-terminal third of the rod.

Influence of metal ions and a local anesthetic on the conformation of the choline group of phosphatidylcholine bilayers studied by Raman spectroscopy.

A Raman band assigned to the 'totally' symmetric stretching vibration of the choline C-N bonds is relatively strong and sensitive to the conformation of the choline backbone (Akutsu, H. (1981) Biochemistry 20, 7359-7366). By monitoring this Raman band, the influence of Eu3+, La3+, Ca2+ and a local anesthetic, dibucaine, on the conformation of the choline group was examined for the bilayers of dipalmitoylphosphatidylcholine and those of deuterated one at the choline methyl group (-N(C2H3)3). NMR lanthanide-shift studies proposed that the interaction with metal ions induces a conformational change from the gauche to the trans form in the O-C-C-N+ backbone of the choline group. However, present Raman work clearly showed that neither metal ions nor anesthetics induce such a conformational change. Therefore, a structural change in the polar group detected by 2H-NMR on addition of metal ions should not include a significant conformational change in the choline group as well. Deuterated phosphatidylcholine used here was proved to be more suitable for the direct detection of the amount of the trans conformation by Raman spectroscopy than the nondeuterated one. The spectra of the deuterated compound in the gel and liquid-crystalline states confirmed that the trans conformation of the choline group does not appear at all in both states.

Molecular origin of biphasic response of main phase-transition temperature of phospholipid membranes to long-chain alcohols.

A statistical mechanical theory is proposed which explains the molecular mechanism of the nonlinear response of the phase-transition temperature of phospholipid vesicle membranes to added 1-alkanols. By assuming that the free energy of transfer of 1-alkanols from the aqueous phase to the membrane and the interaction energy between 1-alkanol molecules are linear functions of alkanol alkyl chain-length, the nonlinear behavior is explained in the Bragg-Williams approximation. For dipalmitoylphosphatidylcholine vesicle membranes, the theory reveals a larger free energy of transfer of 1-alkanols from the aqueous phase to the solid-gel membrane than to the liquid-crystalline membrane when the number of carbon atoms of 1-alkanol exceeds 12. When the intermolecular interaction force between 1-alkanol molecules residing in the gel phase is stronger than the interaction force between those residing in the liquid-crystalline phase, the ligand effect is to tighten the lipid matrix structure, causing the transition temperature to rise. The interaction force is a quadratic function of 1-alkanol concentration; hence, the response of the transition temperature to the 1-alkanol concentration is nonlinear. At low concentrations of the long-chain 1-alkanols that predominantly elevate the transition temperature, this intermolecular interaction force is negligible. In this case, the entropic effect of the incorporated ligand molecules, which loosens the lipid matrix, predominates, and the transition temperature decreases. The biphasic action of long-chain 1-alkanols originates from the balance of these two opposing effects: entropy and intermolecular interaction.

Atypical Langmuir adsorption of inhalation anesthetics on phospholipid monolayer at various compressional states: difference between alkane-type and ether-type anesthetics.

Adsorption of chloroform, halothane, enflurane and diethyl ether on the air/water interface was compared with adsorption on the dipalmitoylphosphatidylcholine monolayer, spread on the air/water interface, at four compressional states; 88.5, 77.0, 66.5 and 50.5 A2 surface area per phosphatidylcholine molecule. Anesthetics were administered from the gas phase. The affinities of these agents to the phosphatidylcholine monolayer varied according to the state of the monolayer. Chloroform and halothane showed a stronger affinity to the highly compressed phosphatidylcholine monolayer (50.5 A2) than to the expanded monolayer (88.5 A2) or to the air/water interface without the monolayer. Diethyl ether behaved in reverse; a stronger affinity to the expanded monolayer was exhibited than to the compressed monolayer. Enflurane showed the highest affinity to the intermediately compressed monolayer (77.0 A2). The adsorption isotherm of anesthetics to the monolayer was characterized by atypical Langmuir-type, in which available number of binding sites changed when anesthetics were adsorbed. The mode of adsorption onto the monolayer was dissimilar to adsorption onto air/water interface, where adsorption followed the Gibbs surface excess. A theory is presented to explain the above differences. The adsorbed anesthetic molecules do not stick to phosphatidylcholine molecules but penetrate into the monolayer lattice and occupy the phosphatidylcholine sites at the interface. Quantitative agreement between the theory and the experimental data was excellent. For the monolayer at 50.5 A2 compression, the changes in the transfer free energy accompanying the anesthetic adsorption from the gas phase to the monolayer were in the order of chloroform greater than halothane greater than enflurane greater than diethyl ether, in agreement with the clinical potencies.

Anesthetic-protein interaction. Random versus helix polylysine monolayers and interaction with 1-alkanols.

Penetration of 1-alkanols into monolayers of hydrophobic polypeptides, poly(epsilon-benzyloxycarbonyl-L-lysine) and poly(epsilon-benzyloxycarbonyl-DL-lysine), was compared with their adsorption on the air/water interface in the absence of monolayers. The polypeptide prepared from L-lysine is generally considered to be in the alpha-helical form whereas DL-copolymer polypeptide contains random-coiled portions due to the structural incompatibility between the two isomers. The free energy of adsorption of 1-alkanols on the air/water interface at dilute concentrations was -0.68 kcal X mol-1 per methylene group and 0.15 kcal X mol-1 for the hydroxyl group at 25 degrees C. In the close-packed state, the surface area occupied by each molecule of 1-alkanols of varying carbon chain-lengths showed nearly a constant value of about 27.2 A2, indicating perpendicular orientation of the alkanol molecules at the interface. About 75% of the water surface was covered by 1-butanol in this close-packed state. The mode of adsorption of 1-alkanols on the vacant air/water interface followed the Gibbs surface excess while the mode on the polypeptide membranes followed the Langmuir adsorption isotherm, indicating that the latter is characterized by the presence of a finite number of binding sites. The free energies of adsorption of 1-alkanols on the L-polymer monolayers were more negative than those on the vacant air/water interface and less negative than those on the DL-copolymer monolayers. Thus, the affinity of 1-alkanols to the interface was in the order of vacant air/water interface less than L-polymer less than DL-copolymer. The difference between the air/water interface and L-polymer was about 0.54 kcal X mol-1 and that between L-polymer and DL-copolymer was 0.17 kcal X mol-1 at 25 degrees C: the adsorption of 1-alkanols to the DL-copolymer was favored compared to the L-polymer. The polar moieties of the backbone of the DL-copolymer may be exposed to the aqueous phase at the disordered portion. Dipole interaction between this portion and 1-alkanol molecules may account for the enhanced adsorption of the alkanols to the DL-copolymer.

Anesthetics and high-pressure interaction upon elastic properties of a polymer membrane.

Anesthetics expand cell membranes, and high pressures (about 10-15 MPa) antagonize the anesthetic action. It is also known that inhalation anesthetics expand elastomer membranes. The mechanism of pressure antagonism of anesthetic action on membrane expansion was investigated in the present study with Silastic membranes. Halothane increased the length of Silastic membrane (0.14% per kPa), with an accompanying decrease of Young's modulus (3.7.10(5) Newton/m2 per kPa). High pressure decreased the length of the membrane and increased Young's modulus. The magnitudes of the pressure effect on the length and Young's modulus of the Silastic membrane in the presence of the anesthetic were not identical with those observed in the absence of the anesthetic. In the presence of halothane at pressures common to clinical applications, the bulk modulus of the membrane decreased about 4.6-4.0%. These results suggest that the effects of pressure and anesthetic upon the elastomer may not be completely independent of each other.