Inhibition of the transthylakoid gradient of electrochemical proton potential by the local anesthetic dibucaine

The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF₀CF₁ ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg²⁺ and strong thylakoid stacking in the presence of only low Mg²⁺ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF₁ and the CF₀CF₁ complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.     

Ion transport processes in corn mitochondria : I. Effect of the local anesthetic dibucaine

The local anesthetic dibucaine inhibited respiration-dependent contraction mediated by the K⁺/H⁺ antiport system of isolated corn mitochondria. Respiration declined concurrently. Nigericin, an exogenous K⁺/H⁺ exchanger, restored ion efflux in dibucaine-blocked corn mitochondria. It was concluded that dibucaine inhibited ion efflux via blockage of the K⁺/H⁺ antiport. Further experiments determined that dibucaine also inhibited proton influx facilitated by protonophores and by the ATPase complex during state III respiration. These results are discussed in relation to the mechanism by which dibucaine inhibits proton translocation across the inner mitochondrial membrane.     

The tertiary amine local anesthetic dibucaine binds to the membrane skeletal protein spectrin

The quinoline-based tertiary amine dibucaine has been shown to bind the membrane skeletal protein spectrin with a dissociation constant of 3.5x10(-5) M at 25 degrees C. Such binding is detected by monitoring the quenching of the tryptophan fluorescence intensity with increasing concentrations of dibucaine only and not with the benzene-based local anesthetics procaine, tetracaine and lidocaine. Binding of dibucaine also indicated changes in the tertiary structure of spectrin indicated by a circular dichroism spectrum in the near-UV region due to absorption of the aromatic side chains. The thermodynamic parameters associated with the binding indicated the interaction of dibucaine and spectrin to be enthalpy-driven and insensitive to an increase in the ionic strength of the buffer.     

Locations of local anesthetic dibucaine in model membranes and the interaction between dibucaine and a Na+ channel inactivation gate peptide as studied by 2H- and 1H-NMR spectroscopies.

To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel inactivation gate peptide have been studied by 2H- and 1H-NMR spectroscopies. The 2H-NMR spectra of dibucaine-d9 and dibucaine-d1, which are deuterated at the butoxy group and at the 3 position in its quinoline ring, respectively, have been observed in multilamellar dispersions of the lipid mixture composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. 2H-NMR spectra of deuterated palmitic acids incorporated, as a probe, into the lipid mixture containing cholesterol have also been observed. An order parameter, SCD, for each carbon segment was calculated from the observed quadrupole splittings. Combining these results, we concluded that first, the butoxy group of dibucaine is penetrating between the acyl chains of lipids in the model membranes, and second, the quinoline ring of dibucaine is located at the polar region of lipids but not at the hydrophobic acyl chain moiety. These results mean that dibucaine is situated in a favorable position that permits it to interact with a cluster of hydrophobic amino acids (Ile-Phe-Met) within the intracellular linker between domains III and IV of Na+ channel protein, which functions as an inactivation gate. To confirm whether the dibucaine molecule at the surface region of lipids can really interact with the hydrophobic amino acids, we synthesized a model peptide that includes the hydrophobic amino acids (Ac-GGQDIFMTEEQK-OH, MP-1), the amino acid sequence of which corresponds to the linker part of rat brain type IIA Na+ channel, and the one in which Phe has been substituted by Gln (MP-2), and measured 1H-NMR spectra in both phosphate buffer and phosphatidylserine liposomes. It was found that the quinoline ring of dibucaine can interact with the aromatic ring of Phe by stacking of the rings; moreover, the interaction can be reinforced by the presence of lipids. In conclusion, we wish to propose that local anesthesia originates from the pi-stacking interaction between aromatic rings of an anesthetic molecule located at the polar headgroup region of the so-called boundary lipids and of the Phe in the intracellular linker between domains III and IV of the Na+ channel protein, prolonging the inactivated state and consequently making it impossible to proceed to the resting state.     

Hydrolysis of phosphatidylcholine liposomes by lysosomal phospholipase A is maximal at the phase transition temperature of the lipid substrate

We have measured the rate of hydrolysis of liposomes made of DL--dipalmitoylphosphatidylcholine (DPPC) and L--dimyristoylphosphatidylcholine by a soluble fraction of highly purified lysosomes isolated from rat liver. Phospholipids are hydrolyzed into lysophospho-lipids and fatty acids at a rate which is maximal near the temperature characteristic of the gel to liquid crystalline phase transition of the lipid bilayer. This strong influence of the physical properties of the substrate on the enzyme activity suggests a structural analogy between the lysosomal phospholipases of the A type (EC 3.1.1.32 and EC 3.1.1.4) and the pancreatic phospholipase A₂.     

Action of phospholipases A2 on phosphatidylcholine bilayers. Effects of the phase transition, bilayer curvature and structural defects

We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.     

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.     

Effect of the local anesthetic dibucaine on the surface membrane in sterol-manipulated Tetrahymena pyriformis studied by freeze-fracture electron microscopy

The effect of the local anesthetic dibucaine on the membrane ultrastructure of sterol-manipulated Tetrahymena pyriformis (NT-1 strain) was studied by freeze-fracture electron microscopy. Dibucaine-treated, ergosterol-replaced Tetrahymena cells had marked alterations in their plasma membranes. IMP-free small depressions (exoplasmic fracture face) and protrusions (protoplasmic fracture face) were formed on the plasma membranes which was in contact with the outer alveolar membrane. In addition, large IMP-free surface "blebs" covered with hexagonally-arranged depressions and protrusions appeared on both the plasma and outer alveolar membranes. These "blebs" were pinched off when the membranes were severely affected. Our previous study (28) demonstrated that the plasma membrane of dibucaine-treated native Tetrahymena cells that contain tetrahymanol showed vertical displacement of its intramembranous particles and that subsequently a smooth, flat surface appeared. Therefore, the structural changes in ergosterol-replaced membranes produced by dibucaine differ strikingly from changes in the native membranes. The remarkable difference in the ultrastructural deformation of the plasma membrane probably is due to a difference in the membrane lipid composition induced by sterol-manipulation.     

Hydrolysis of phosphatidylcholine in phosphatidylcholine-cholate mixtures by porcine pancreatic phospholipase A2

Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.     

Binding of spin-labeled local anesthetics to phosphatidylcholine and phosphatidylserine liposomes

A homologous series of spin-labeled local anesthetics, 2-[N-methyl N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-p-alkoxybenzoates were shown to bind to phosphatidylcholine and phosphatidylserine liposomes. Under similar conditions, 70% of the ethoxy homolog (R2C) of these spin-labeled local anesthetics bound to synthetic dipalmitoyl lecithin while 98% bound to phosphatidylserine liposomes. Five percent of R2C's bound signal could be released by 4 mm calcium from phosphatidylserine liposomes, but calcium had no effect on R2C bound to synthetic lecithin. The butoxy (R4C) and hexyloxy (R6C) homologs bound to phosphatidylcholine in the order R6C > R4C. All of R6C and all of R4C were bound to phosphatidylserine liposomes, while only 90% of R6C bound to synthetic dipalmitoyl lecithin. Calcium was incapable of displacing bound R4C or R6C from either phosphatidylcholine or phosphatidylserine liposomes. The results are discussed in light of anesthetic binding by electrostatic and Van der Waal's forces to phospholipids.     

Effects of the anesthetic dibucaine on the kinetics of the gel-liquid crystalline transition of dipalmitoylphosphatidylcholine multilamellar vesicles.

The effects of the anesthetic dibucaine on the relaxation kinetics of the gel-liquid crystalline transition of dipalmitoylphosphatidylcholine (DC16PC) multilamellar vesicles have been investigated using volume-perturbation calorimetry. The temperature and pressure responses to a periodic volume perturbation were measured in real time. Data collected in the time domain were subsequently converted into and analyzed in the frequency domain using Fourier series representations of the perturbation and response functions. The Laplace transform of the classical Kolmogorov-Avrami kinetic relation was employed to describe the relaxation dynamics in the frequency domain. The relaxation time of anesthetic-lipid mixtures, as a function of the fractional degree of melting, appears to be qualitatively similar to that of pure lipid systems, with a pronounced maximum, tau max, observed at a temperature corresponding to greater than 75% melting. The tau max decreases by a factor of approximately 2 as the nominal anesthetic/lipid mole ratio increases from 0 to 0.013 and exhibits no further change as the nominal anesthetic/lipid mole ratio is increased. However, the fractional dimensionality of the relaxation process decreases monotonically from slightly less than two to approximately one as the anesthetic/lipid mole ratio increases from 0 to 0.027. At higher ratios, the dimensionality appears to be less than one. These results are interpreted in terms of the classical kinetic theory and related to those obtained from Monte Carlo simulations. Specifically, low concentrations of dibucaine appear to reduce the average cluster size and cause the fluctuating lipid clusters to become more ramified. At the highest concentration of dibucaine, where n < 1, the system must be kinetically heterogeneous.     

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.     

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.     

Solubilizing effects caused by the nonionic surfactant dodecylmaltoside in phosphatidylcholine liposomes.

The interaction of the nonionic surfactant dodecylmaltoside (DM) with phosphatidylcholine liposomes was investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein released from the interior of vesicles and bilayer solubilization as a decrease in the static light scattered by liposome suspensions. This surfactant showed higher capacity to saturate and solubilize PC liposomes and greater affinity with these structures than those reported for the octyl glucoside. At subsolubilizing level an initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the effective molar ratio of surfactant to phospholipid in bilayers (Re) rose. However, at solubilizing level a direct dependence was established between both parameters. A direct correlation took place in the initial interaction steps (Re up to 0.28) between the growth of vesicles, their fluidity, and Re. A similar direct dependence was established during solubilization (Re range from 0.9 to 1.7) between the decrease in both the surfactant-PC aggregate size, the light scattering of the system, and Re (composition of aggregates). The fact that the free DM concentration at subsolubilizing and solubilizing levels showed values lower than and similar to its critical micelle concentration indicates that permeability alterations and solubilization were determined, respectively, by the action of surfactant monomer and by the formation of mixed micelles.     

Partition of piperidinoethylesters of 2-alkyloxyphenylcarbamic acid in unilamellar phosphatidylcholine liposomes.

Molar partition coefficients for amphiphilic N-[2-(2-alkyl-oxyphenyl-carbamoyloxy)-ethyl]-piperidinium chlorides (PAA) between small unilamellar egg yolk phosphatidyl choline liposomes and saline, as determined by ultraviolet difference spectroscopy at 22 degrees C, pH 5-6, v = 34640 cm-1, and at 100 mumol/l PAA concentration, were 149, 1990, and 7474 for PAA with 5, 7, and 9 carbon atoms in the alkyloxy substituent, respectively. At the PAA concentration used, the cut-off in biological activities of PAAs with long alkyloxy substituents could not be caused by the self-association of PAA molecules in the aqueous phase.     

Action of phospholipases on the phosphatidylcholine exchange protein from beef liver.

The phospholipases A2, C and D have been used to investigate the localization of phosphatidylcholine in the phosphatidylcholine exchange protein from beef liver. The rate of enzymatic hydrolysis of the protein-bound phosphatidylcholine was found to be very low. Addition of deoxycholate, isobutanol or dioxane to the native protein, under conditions where delipidation did not occur, greatly enhanced the hydrolytic action of the phospholipases. From these results it is concluded that phosphatidylcholine may be buried in the protein molecule.     

Hydrolysis of N-acylated glycerophospholipids by phospholipases A2 and D: a method of identification and analysis

We have previously identified N-acylethanolamine phospholipids in infarcted dog heart and in normal fish brain by chemical and enzymatic degradation. We now report that hydrolysis with phospholipase D from Streptomyces chromofuscus removes N-acylethanolamine from N-acylethanolamine phospholipids and lyso N-acylethanolamine phospholipids, or N-acylserine from lyso N-acylserine phospholipids. At acidic pH, a phosphatase present in the phospholipase D preparation further hydrolyzes the resulting phosphatidic acid (PA) or lyso-PA to diacyl- or monoacylglycerol. Because N-acylserine phospholipids are a poor substrate for the phospholipase D, pretreatment with phospholipase A2 (Trimeresurus flavoviridis venom) is used to remove the 2-O-acyl group. Thus, both types of N-acylated phospholipids can be analyzed by consecutive phospholipase A2 and phospholipase D treatment. Reaction products, i.e., free fatty acids, monoacylglycerols and N-acylethanolamine or N-acylserine, are separable by thin-layer chromatography. Both N-acyl components can be further characterized by conversion to the t-butyldimethylsilyl derivatives. The method was used to identify and analyze the N-acylserine phospholipids of bovine brain.