Effects of local anesthetics on mechanical characteristics of lipid bilayers and on the ion transport dynamics.

Bilayer lipid membranes (BLM) of various composition were used to study the effects of local anesthetics (LA) carbisocaine and lidocaine on mechanical membrane characteristics and on the transport dynamics of ions across gramicidin D ionic channels. Carbisocaine concentrations of 20 mumols/l-0.1 mmol/l caused a considerable decrease (by 15-40%) in modulus of elasticity E1 in direction perpendicular to membrane surface. The effect of lidocaine was approx. one order of magnitude weaker. LA-induced changes in E1 were shown to depend on both the lipid composition of the membrane and the electrolyte pH. Neutral forms of LA induce marked changes in E1. An analysis of current-voltage (I-V) characteristics of BLM modified by the channel forming agent gramicidin D revealed that carbisocaine significantly affects the superlinear segment of the I-V relationship; this suggests a strong effect on the transport dynamics of ions through the internal channel region. The results of the study suggest that the action of both carbisocaine and lidocaine may be non-specific. The effectivity of the non-specific action of LA is determined by the hydrophobic moiety of the local anesthetic molecule.     

Perturbation effect of the diheptanoyl phosphatidylcholine on rat brain total lipid liposomes. An electron paramagnetic resonance spectroscopy study.

Diheptanoyl phosphatidylcholine (DHPC) was reported to reduce inward sodium current in squid giant axons (Hendry et al., Biophys. J., 47, (1985) 841) and to decrease the frequency of the nicotinic acetylcholine-activated ion channel (Braun and Haydon, Pfügers Arch., 62, (1991) 418). To understand the DHPC effects, an influence of DHPC to increase dynamics/disorder (perturbation effect) in liposomes prepared from rat brain total lipids was studied at the 5th, 12th and 16th carbon membrane depths using the method of EPR spectroscopy of the spin labelled stearic acids and 1-palmitoyl-2-stearoyl phosphatidylcholines. The perturbation efficiency of DHPC in the liposomes was quantitated with the initial slope value of the order parameter S or inner splitting Amin versus DHPC concentration. DHPC at the DHPC/total lipid molar ratio of 1:10, 1:5, 1:3 and 1:2 increased dynamics/disorder of the liposomes. When the perturbation effect, compared at temperature scale, was normalized to 1 at the 5th carbon depth, the relative perturbation effect of DHPC was 1, 4.2, and 6.9 at the 5th, 12th and the 16th carbon depth, respectively, as detected by spin labelled stearic acids. Using the spin labelled lipids the perturbation effect was 1, 1.9 and 2.3, respectively. The differences of the perturbation effect of DHPC at different membrane depths correspond to the published perturbation effect of the local anesthetics lidocaine, tetracaine, dibucaine, heptacaine, IR-9 and carbisocaine on total lipid liposomes. The comparable perturbation properties of DHPC and of the local anesthetics support the hypothesis that the membrane perturbation caused by DHPC may play an important role in its effect on membrane function.     

ATP-dependent interaction of propranolol and local anaesthetic with sarcoplasmic reticulum. Stimulation of Ca2+ efflux.

Preincubation of sarcoplasmic reticulum (SR) with propranolol or tetracaine inhibits Ca2+ accumulation and stimulates ATPase activity by more than 2-fold. This effect is obtained only when the preincubation is carried out in the presence of ATP or other nucleoside triphosphates. The (ATP + drug)-induced inhibition of Ca2+ accumulation is pH-dependent, increasing as the pH rises above 7.5. The presence of micromolar concentrations of Ca2+ or Mg2+ during the preincubation prevents the inhibitory effect of ATP plus drug on Ca2+ accumulation or ATPase activity. The (ATP + drug) modification of SR vesicles resulted in stimulation of a rapid Ca2+ efflux from passively loaded vesicles. The ATP-dependent inhibition of Ca2+ accumulation by the drug is obtained with other local anaesthetics. The drug concentration required for 50% inhibition was 0.15 mM for dibucaine and 0.4 mM for both propranolol and tetracaine, whereas it was 5 mM, 8 mM and greater than 10 mM for lidocaine, benzocaine and procaine respectively. The heavy SR vesicles were only slightly affected by the incubation with propranolol or tetracaine in the presence of ATP, but their sensitivity increased markedly after storage at 0 degrees C for 24-48 h. These results suggest that propranolol and some local anaesthetics, in the presence of ATP, stimulate Ca2+ efflux by modifying a protein factor(s) rather than the phospholipid bilayer.     

General and local anaesthetics perturb the fusion of phospholipid vesicles

The effects of general and local anaesthetics on Ca2+-induced fusion of negatively charged lipid vesicles have been investigated. Vesicles composed of phosphatidylcholine and phosphatidic acid (2:1 molar ratio) were induced to fuse using 5 mM free Ca2+. Fusion, assessed by an increase in size using gel filtration techniques and confirmed by electron microscopy, displayed a dependence on Ca2+ and Mg2+ concentration and on temperature. The inhalational anaesthetics halothane, methoxyflurane and diethyl ether enhanced fusion as did the uncharged local anaesthetic benzocaine. In contrast, the charged local anaesthetics lignocaine and bupivacaine inhibited the fusion process. It is suggested that the enhancement observed with the inhalational anaesthetics and benzocaine was mediated by an effect on lipid fluidity and the inhibition observed with the charged tertiary amine anaesthetics was due to an antagonism towards Ca2+.     

Correlation of bacterial hyperthermic survival with anaesthetic potency

We have evaluated several local anaesthetics and hypnotics for their relative ability to influence hyperthermic cell killing. Bacterial cell survival following exposure to heat and anaesthetic was used as the assay system. The E. coli bacterium used was the unsaturated fatty acid auxotroph, K1060. It was grown at 37 degrees C in medium supplemented with oleic acid and then exposed to 47 degrees C hyperthermia in the presence of an anaesthetic. The local anaesthetics tested were procaine, lidocaine, tetracaine, and benzocaine, and the general anaesthetics were barbital and pentobarbital. The dose response for each anaesthetic was determined over a five-hour heating period. The anaesthetic concentration required during heating to halve the time for cell killing found with heat alone is 5.9 mM for procaine, 0.8 mM for lidocaine, 0.12 mM for tetracaine, 2.0 mM for benzocaine, 6.7 mM for barbital and 1.2 mM for pentobarbital. There is a direct correlation between equivalent effect doses of the local anaesthetics and published data for the relative potency of the same anaesthetics as determined by respiratory arrest in mice and by myocardial contractile force in dogs. The assay we have described would be a convenient and easy test for the interaction of these drugs with hyperthermia. The use of this interaction with hyperthermia as an adjuvant in combined radiation-hyperthermia therapy should be tested.     

Interaction of benzocaine with model membranes

We measured the absorption properties, water solubility and partition coefficients (P) between n-octanol, egg phosphatidylcholine (EPC) liposomes and erythrocyte ghosts/water for benzocaine (BZC), an ester-type always uncharged local anesthetic. The interaction of BZC with EPC liposomes was followed using Electron Paramagnetic Resonance, with spin labels at different positions in the acyl chain (5, 7, 12, 16-doxylstearic acid methyl ester). Changes in lipid organization upon BZC addition allowed the determination of P values, without phase separation. The effect of BZC in decreasing membrane organization (maximum of 11.6% at approx. 0.8:1 BZC:EPC) was compared to those caused by the local anesthetics tetracaine and lidocaine. Hemolytic tests revealed a biphasic (protective/inductive) concentration-dependent hemolytic effect for BZC upon rat erythrocytes, with an effective BZC:lipid molar ratio in the membrane for protection (RePROT), onset of hemolysis (ReSAT) and 100% membrane solubilization (ReSOL) of 1.0:1, 1.1:1 and 1.3:1, respectively. The results presented here reinforce the importance of considering hydrophobic interactions in the interpretation of the effects of anesthetics on membranes.     

The effect of anaesthetics on protein conformation in membranes as studied by the spin-labelling technique

The effect of general and local anaesthetics was studied on the membrane components (proteins and lipids) of frog sciatic nerve using the spin-labelling technique. A rapid and effective method was developed to incorporate fatty acid labels into the lipid regions and to attach the maleimide spin labels to the thiol sites of the proteins of the membrane. On the basis of the rotational correlation time of the attached maleimide spin labels, at least three different protein domains were identified. One part of the labels has a preferred orientation with respect to the axis of the nerve fibre. The effect of halothane and of local anaesthetics such as lidocaine and tetracaine, which influence primarily the lipid regions of the membrane, is efficiently transferred to the spin-labelled membrane proteins via strong lipid-protein interaction. The results support the concept that the architecture and the physiological activity of the membrane-bound proteins are sensitive to changes in the physical state of membrane lipids.     

Comparison of perturbation effect of propranolol, verapamil, chlorpromazine and carbisocaine on lecithin liposomes and brain total lipid liposomes. An EPR spectroscopy study.

Effect of verapamil, propranolol, chlorpromazine and carbisocaine on dynamics and/or order of liposomes (perturbation effect), prepared from different molar ratios of lecithin (PC) and rat brain total lipids (TL) was studied by EPR spectroscopy using spin probes 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The PC liposomes had higher dynamics and/or lower order than the TL liposomes. The perturbation effect of the drugs depended largely on the lipid composition of the liposomes. The drugs at the drug/lipid molar ratios from 0.1 to 1 increased membrane dynamics and/or decreased membrane order. The drugs had the most pronounced perturbation effect in the liposomes prepared from brain total lipids. The effect of the drugs decreased with decreasing the TL/PC ratio in the liposomes and was lowest, almost diminished, in the PC liposomes. Increasing concentration of the drugs decreased the difference between the dynamics and/or order of the PC and TL liposomes and so eliminated the influence of lipid composition on these membrane parameters. The results emphasize the role of lipid composition in studies concerning drug-lipid interactions in model and biological membranes.     

Effects of tertiary amine local anaesthetics on the phase behaviour of the dipalmitoylphosphatidylcholine model membrane. Electron spin resonance tetrametylpiperidinyloxyl partition study

Using the electron spin resonance technique of the spin probe (TEMPO) is shown that local anaesthetics lidocaine, tetracaine, dibucaine, heptacaine and carbizocaine decrease the gel--liquid crystal phase transition temperature and increase the width of the phase transition of the dipalmitoylphosphatidylcholine (DPPC) model membrane. These effects roughly correlate with the efficiencies of the anesthetics to block action potential propagation in the nerves.     

In Vitro Induction of Antibody-Dependent Cytotoxic Macrophages by the Local Anesthetic Lidocaine

Normal macrophages were activated to antibody-dependent cytotoxic effector cells by in vitro treatment with the local anesthetic lidocaine. Experiments on the dose-response and time course of the effect of lidocaine showed that incubation of normal macrophages with 10 mM lidocaine for 10 min at 28 C was enough for induction of antibody-dependent cellular cytotoxicity. The activation by lidocaine was accompanied by enhanced phagocytosis of sheep red blood cells (SRBC) sensitized with anti-SRBC antiserum, but not enhanced ingestion of polystyrene latex particles (PLP). These findings suggest that lidocaine, which has various effects on cell membranes, induces some perturbation of macrophage membranes, resulting in activation of Fc receptor functions in antibody-dependent cytotoxicity and phagocytosis.     

Inhibition of rat liver cholesterol esterase by local anaesthetics.

1. A number of local anaesthetics was shown to inhibit rat liver cholesterol esterase activity towards radioactively labelled cholesterol oleate. The anaesthetics inhibited in the order dibucaine greater than chlorpromazine greater than tetracaine greater than benzocaine greater than procaine greater than lidocaine greater than cocaine. 2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca2+. 3. The inhibition by tetracaine is partially reversed by sodium deoxycholate. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration dependent manner. Only sodium taurocholate stimulates enzymic activity.     

The effects of lidocaine on lymphocyte beta-adrenergic receptor at different pH]

The effects of lidocaine on beta-adrenergic receptor binding and the yield of epinephrine-stimulated cAMP at different pH were measured by radioligand binding assay and radioimmunoassay. The results showed that the binding of H-DHA to the receptor and yield of epinephrine-stimulated cAMP increased with increasing pH (P < 0.001) and lidocaine inhibitory potency also increased with increasing pH (P < 0.001); The results indicate that the response to beta-agonists would increase with pH during cardiopulmonary resuscitation when lidocaine is not present, but it would not increase with pH when lidocaine is present, suggesting that lidocaine may inhibit binding of beta-agonists to receptor by changing lipid fluidity of cell membrane with increasing pH.     

Local anesthetics. Effect of pH on use-dependent block of sodium channels in frog muscle.

Sodium currents were studied under voltage clamp in the presence of neutral, amine, and quaternary local anesthetic compounds. Use-dependent block was observed as a cumulative depression of INa seen with repetitive depolarizing test pulses applied at frequencies of 2-10s-1. With quaternary QX-314, the time constant of use dependence was long, and with neutral benzocaine, very short. With lidocaine and procaine, increasing external pH (pHo) changed the time constant from long to short, but alterations of internal pH have no effect. Inactivation in Na channels was measured by the influence of prepulses on peak INa during test pulses. Single-stimulus inactivation curves were shifted more with lidocaine at high pHo than at low pHo, but inactivation curves measured during pulse trains with any of the drugs and at any pHo were strongly shifted. All measurements show that the drug-receptor reaction was slow for amine drugs at low pHo, as for quaternary drugs at any pHo, and fast for amine drugs at high pHo, as for neutral drugs at any pHo. The major effect of low pHo on amine drugs was to reduce the concentration of drugs in the fiber and to protonate drug molecules on the receptor, thus trapping them in the blocking position for a longer time. Direct effects of pH on the receptor seemed minimal.     

A new look at the hemolytic effect of local anesthetics, considering their real membrane/water partitioning at pH 7.4

The interaction of local anesthetics (LA) with biological and phospholipid bilayers was investigated regarding the contribution of their structure and physicochemical properties to membrane partition and to erythrocyte solubilization. We measured the partition into phospholipid vesicles—at pH 5.0 and 10.5—and the biphasic hemolytic effect on rat erythrocytes of: benzocaine, chloroprocaine, procaine, tetracaine, bupivacaine, mepivacaine, lidocaine, prilocaine, and dibucaine. At pH 7.4, the binding of uncharged and charged LA to the membranes was considered, since it results in an ionization constant (pK_app) different from that observed for the anesthetic in the aqueous phase (pK_w). Even though it occurred at a pH at which there is a predominance of the charged species, hemolysis was greatly influenced by the uncharged species, revealing that the disrupting effect of LA on these membranes is mainly a consequence of hydrophobic interactions. The correlation between the hemolytic activity and the LA potency shows that hemolytic experiments could be used for the prediction of activity in the development of new LA molecules.     

A spin probe study of the effects of chlorpromazine and its derivatives on lipid-protein interactions in synaptosomal membranes.

The electron spin resonance spectra of 16-doxyl stearic acid (16-SA) incorporated into synaptosomes mostly showed a fluid lipid component and a minor motionally-restricted component (MRC) of the molar fraction of 10-20%, measured at 0 degree C. At 10 mmol/l concentration, thioridazine (TRZ), chlorpromazine (CPZ), chlorprothixene (CPT), perphenazine (PFZ) and levopromazine (LPZ) raised the MRC molar fraction in the synaptosomes to 100, 92, 65, 41 and 39%, respectively (as detected by the spin probe at 0 degrees C). At 4% concentration, TRZ, CPZ, CPT, PFZ, and LPZ the respective MRC percentages were 100, 75, 41, 24 and 17%. In synaptosomal membranes, AMRC splitting values of MRC, induced by TRZ and CPZ, were similar to those of the probe in human serum albumin. MRC induced by CPZ and TRZ was constant (+/- 15%) within the temperature range from 0 to 30 degrees C. At drug/lipid ratios > or = 2 : 1, TRZ and CPZ formed rigid complexes with total lipids isolated from the rat brain. The complexes melted upon increasing the temperature of the samples over 10-20 degrees C. The drugs decreased the lipid concentrations in synaptosomes in the order of potency TRZ > CPZ > CPT > PFZ > or = LPZ; this was similar to their effect on MRC increase. The drugs tested increased the membrane dynamics/disordering, and their potency fairly correlated with their MRC increasing effects. It is supposed that the drug-induced 16-SA probe MRC increase in synaptosomes was a result of mainly decreased lipid/protein ratio in the synaptosomal membranes, which in turn probably is connected with perturbation of lipid-protein interactions and/or membrane proteins. The perturbation of lipid-protein interactions and/or membrane proteins may be connected with the drug perturbation effect on the bulk lipid membrane part.     

Disruption of lipid rafts by lidocaine inhibits erythrocyte invasion by Plasmodium falciparum

Membrane lipid rafts have been implicated in erythrocyte invasion process by Plasmodium falciparum. In this study, we examined the effect of lidocaine, a local anesthetic, which disrupts lipid rafts reversibly without affecting membrane cholesterol content on parasite invasion. In the presence of increasing concentrations of lidocaine in the culture medium, parasite invasion was progressively decreased with complete inhibition at 2 mM. Decreased invasion was also seen in erythrocytes pre-treated with lidocaine and cultured in the absence of lidocaine. This inhibitory effect on parasite invasion was reversed following removal of lidocaine from erythrocyte membranes. Our findings show that disruption of lipid rafts in the context of normal cholesterol content markedly inhibits parasite invasion and confirm an important role for lipid rafts in invasion of erythrocytes by P. falciparum.     

Comparison of the effects of carbisocaine and other local anesthetics on 32P incorporation into individual and total phospholipids in synaptosomes

The aim of the paper was to study the effect of carbisocaine, a new local anesthetic with high liposolubility on incorporation of 32P into individual and total phospholipids and to compare its effect with that of other local anesthetics (procaine, lidocaine, cinchocaine, heptacaine). Carbisocaine decreased 32P incorporation into neutral phospholipids and increased the incorporation into acid phospholipids, presumably by inhibiting phosphatidate phosphohydrolase, similarly as reported for other anesthetics (Brindley and Bowley 1975). The increased incorporation of 32P into phosphatidylserine induced by carbisocaine suggests that this phospholipid is also synthetised from phosphatidic acid. At low concentrations, the local anesthetics studies were found to increase 32P incorporation into total phospholipids, whereas at high concentrations they reduced 32P incorporation. This biphasic effect is in agreement with the incorporation of 14C from glucose into lipids (Lassánová et al. 1984) and with the effect of cinchocaine on glycerol incorporation into phospholipids (Allan and Michell 1975), suggesting that local anesthetics affect de novo synthesis of phosphatidic acid. Carbisocaine increased 32P incorporation into phospholipids, in concentrations lower by several orders of magnitude as compared to the other local anesthetics studied. A rough correlation was observed between the concentrations at which the local anesthetics showed stimulatory effect on 32P incorporation, and the average effective concentrations of the respective anesthetics. No such correlation could be found for carbisocaine.     

Comparative studies on the membrane actions of depressant drugs: the role of lipophilicity in inhibition of brain sodium and potassium-stimulated ATPase.

Depressant drugs are considered to exert their pharmacological effects as a result of membrane interactions determined by their physico-chemical properties. In this study, a correlation was found between lipid solubility and potency of various local anaesthetics, antihistamines, tricyclic antidepressants and phenothiazine tranquilizers as inhibitors of the Na, K-ATPase activity of a microsomal membrane fraction from bovine brain cortex. Depressant drugs such as chlorpromazine, which have the greatest lipid solubilities, were competitive inhibitors of Na activation but noncompetitive toward K activation, whereas drugs such as tetracaine with lower lipid solubilities were competitive inhibitors of K activation but noncompetitive toward Na activation. Drugs with intermediate lipid solubilities were mixed competitive-noncompetitive inhibitors of both Na and K activation. Both chlorpromazine and tetracaine competitively inhibited cation activation by a heterotropic allosteric mechanism, probably mediated through membrane conformational changes. Whereas quaternization or a decrease in the incubation pH interfered with the ability of chlorpromazine to inhibit Na activation in a competitive fashion, these changes did not affect the ability of tetracaine to compete with K activation. In addition Mn, Ca and phosphatidyl serine were very effective non-competitive antagonists of chlorpromazine inhibition of Na, K-ATPase, whereas these agents competitively antagonized tetracaine inhibition to a lesser extent. These data suggest that the more lipid soluble phenothiazines penetrate into and react in hydrophobic areas of the membrane microenvironment, resulting in a membrane perturbation which interferes with Na activation. On the other hand the less lipid soluble local anaesthetics probably act at superficial sites near the membrane surface, resulting in a different membrane perturbation which interferes with the K activation mechanism. It is suggested that lipid solubility may be a significant factor in determining selectivity in the membrane interactions of various pharmacological agents and hence differences in pharmacological activity among different classes of depressant drugs.     

A study of the mechanism by which some amphiphilic drugs affect human erythrocyte acetylcholinesterase activity.

The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase.     

Effect of charged lidocaine on static and dynamic properties of model bio-membranes

The effect of the charged lidocaine on the structure and dynamics of DMPC/DMPG (mass fraction of 95/5) unilamellar vesicles has been investigated. Changes in membrane organization caused by the presence of lidocaine were detected through small angle neutron scattering experiments. Our results suggest that the presence of lidocaine in the vicinity of the headgroups of lipid membranes leads to an increase of the area per lipid molecule and to a decrease of membrane thickness. Such changes in membrane structure may induce disordering of the tail group. This scenario explains the reduction of the main transition temperature of lipid membranes, as the fraction of lidocaine per lipid molecules increases, which was evident from differential scanning calorimetry results. Furthermore neutron spin echo spectroscopy was used for the dynamics measurements and the results reveal that presence of charged lidocaine increases the bending elasticity of the lipid membranes in the fluid phase and slows the temperature-dependent change of bending elasticity across the main transition temperature.