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The enterovirus genus encompasses many clinically important human pathogens such as poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These viruses are the etiological agents of several human diseases, including hand-foot-and-mouth disease, neonatal sepsis, encephalitis, meningitis, paralysis and respiratory infections. There is an unmet need for antivirals to treat these diseases. The non-structural protein 2C is a AAA+ helicase and plays a key role in viral replication. As such, it is an attractive target for antiviral drug development. Several repurposing screens with FDA-approved drugs have identified 2C-targeting compounds such as fluoxetine and dibucaine, but the molecular basis of 2C inhibition has remained enigmatic. Here we present the 1.5 Å resolution crystal structure of the soluble fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), which reveals a conserved, hydrophobic drug-binding pocket which is distal to the ATP binding site. To decipher the molecular mechanism of inhibition by fluoxetine and other 2C-targeting compounds, we engineered a soluble, hexameric and ATPase competent 2C protein. Using this system, we show that SFX, dibucaine, HBB and guanidine hydrochloride inhibit 2C ATPase activity in a dose-dependent manner. Moreover, using cryo-EM analysis, we demonstrate that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing us to generate the first reconstruction of the oligomeric complex. Taken together, these results provide important structural and mechanistic insights into 2C inhibition and provide a robust engineering strategy which can be used for structural, functional and drug-screening analysis of 2C proteins from current or future enteroviruses.

Background Because ordered membrane domains, called lipid rafts, regulate activation of ion channels related to the nerve pulse, lipids rafts are thought to be a possible target for anesthetic molecules. To understand the mechanism of anesthetic action, we examined influence of representative local anesthetics (LAs); dibucaine, tetracaine, and lidocaine, on raft-like liquid-ordered (Lo)/non-raft-like liquid-disordered (Ld) phase separation. Methods Impact of LAs on the phase separation was observed by fluorescent microscopy. LA-induced perturbation of the Lo and Ld membranes was examined by DPH anisotropy measurements. Incorporation of LAs to the membranes was examined by fluorescent anisotropy of LAs. The biding location of the LAs was indicated by small angle x-ray diffraction (SAXD). Results Fluorescent experiments showed that dibucaine eliminated the phase separation the most effectively, followed by tetracaine and lidocaine. The disruption of the phase separation can be explained by their disordering effects on the Lo membrane. SAXD and other experiments further suggested that dibucaine's most potent perturbation of the Lo membrane is attributable to its deeper immersion and bulky molecular structure. Tetracaine, albeit immersed in the Lo membrane as deeply as dibucaine, less perturbs the Lo membrane probably because of its smaller bulkiness. Lidocaine hardly reaches the hydrophobic region, resulting in the weakest Lo membrane perturbation. Conclusion Dibcaine perturbs the Lo membrane the most effectively, followed by tetracaine and lidocaine. This ranking correlates with their anesthetic potency. General significance This study suggests a possible mechanistic link between anesthetic action and perturbation of lipid rafts.

Mitochondrial membranes are pointed out as the site of cardiotoxic action of local anaesthetics. Its three main phospholipids components are phosphatidylcholine, phosphatidylethanolamine and cardiolipin. Cardiolipins, in eukaryotes, are only found in mitochondria and are essential for the maintenance of its integrity and dynamics. Fluorescence and nuclear magnetic resonance spectroscopy were used to study the interactions of a local anaesthetics, Dibucaine (DBC), with different mitochondrial membrane models constituted by combinations of its three main lipid components in which cardiolipin was a natural extract (CLmix). Both CLmix presence/absence and its percentage in the model membranes were evaluated. Fluorescence spectroscopy showed that DBC lowered the transition temperature of all membrane models understudy. DBC partition showed to be dependent of CLmix presence and phosphatidylethanolamine:CL ratio. Furthermore, the maximum emission wavelength (λmax) exhibited a notorious decreased with increasing phospholipid to DBC ratio, in all the membrane models containing CLmix. Nevertheless, it remained approximately the same in the membrane without CLmix. This indicates a differential membrane localization of the anaesthetics, dependent on the membrane models used. NMR results showed that DBC interaction and location in the membrane models is mainly influenced by CLmix presence, and DBC can significant alter lipid systems properties e.g. percentage and type of lipid phase present. Taken all together it was shown that DBC interaction and location are largely dependent on the membrane model system. Furthermore, DBC is able to produce significant changes in the lipidic systems which might help to explain its high toxicity.

Introduction:This paper presents the development and validation of a novel, fast, sensitive and accurate high performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of dibucaine HCl, fluocortolone pivalate and fluocortolone caproate in pharmaceutical preparations.Experiment:Development of the chromatographic method was based on an experimental design approach. A five-level-three-factor central composite design requiring 20 experiments in this optimization study was performed in order to evaluate the effects of three independent variances including mobile phase ratio, flow rate and amount of acid in the mobile phase.Conclusion:The optimum composition for mobile phase was found as a methanol:water:acetic acid mixture at 71.6 : 26.4 : 2 (v/v/v) ratio and optimum separation was acquired by isocratic elution with a flow rate of 1.3 mL/min. The analytes were detected using a UV detector at 240 nm. The developed method was validated in terms of linearity, precision, accuracy, limit of detection/quantitation and solution stability and successfully applied to the determination of dibucaine HCl, fluocortolone pivalate and fluocortolone caproate in pharmaceutical topical formulations such as suppositories and ointments.

Interactions of two local anesthetics, dibucaine and tetracaine have been studied with phospholipid vesicles containing cholesterol and/or monosialogangliosides (GM1) using fluorescence spectroscopy. The fluorescence intensity of tetracaine showed a marked increase with the increasing molar ratio of the phospholipid to tetracaine, while that of dibucaine showed opposite effects. Steady state anisotropy and the wavelength of maximum emission (λmax) decreased with the increasing phospholipids to tetracaine ratio. The extent of such changes in anisotropy and λmax in the presence and absence of two important components of neuronal membranes, cholesterol and GM1 indicated differential membrane localization of the two local anesthetics. To understand the intercellular mode of action of local anesthetics, we have also studied the interactions of dibucaine and tetracaine with brain spectrin which indicate differential spectrin interactions with similar binding strength. Thermodynamic parameters associated with such binding reveal that binding is favored by entropy. Tetracaine brings about distinct structural changes in spectrin compared to dibucaine, as reflected in the tryptophan mean lifetime and far-UV CD spectra. Tetracaine also exhibits a detergent-like property inducing concentration dependent decrease in spectrin anisotropy, further indicating structural changes in brain spectrin with probable implications in its anesthetic potential.

SUMMARY Dibucaine, a local anesthetic, is known to induce flagellar excision in Chlamydomonas reinhardtii. Herein, we investigate whether other local anesthetics have similar effects. Tetracaine, bupivacaine, procaine, and lidocaine also caused flagellar excision, although their potencies were lower than that of dibucaine. Bupivacaine, procaine, and lidocaine induced a morphological change in flagella from a rod-like shape to a disk-like shape before flagellar excision. Except for lidocaine, these local anesthetics caused cell-wall shedding in addition to flagellar excision. The anesthetics in order of their median effective concentration (1-h EC50) for flagellar excision are as follows: dibucaine (1.37 × 10−5 M)

Micelles of a new ABC triblock copolymer, poly(ethylene oxide-b-sodium 2-(acrylamido)-2-methyl-1-propanesulfonate-b-styrene) (PEO-b-PAMPS-b-PS), were prepared in aqueous solutions. The physicochemical properties of the micelle were investigated by combining different techniques: dynamic light scattering, fluorescence spectroscopy, scanning electron microscopy, and transmission electron microscopy (TEM). The micelles have a spherical morphology with a hydrophobic PS core, a hydrophilic and anionic PAMPS shell, and a hydrophilic and nonionic PEO corona as revealed by TEM measurements. The interactions of dibucaine (DC) and dodecyl trimethyl ammonium chloride (DTAC) with PEO-b-PAMPS-b-PS were also studied. The binding of cationic DC and DTAC to the anionic PAMPS shell was confirmed by zeta-potential measurements. It is elucidated that hydrophobic interactions contribute to the binding of DTAC and DC to the polymer, although electrostatic interactions play a primarily important role.

A simple, sensitive, and specific liquid chromatography–mass spectrometry (LC–MS) method for simultaneous determination of dibucaine and naphazoline from serum was developed and validated. The extraction procedure was performed using a monolithic silica spin column. Chromatographic separation of dibucaine and naphazoline was achieved on a C 18 reverse phase column with a mobile phase gradient (mobile phase A: 10 mM ammonium formate and mobile phase B: acetonitrile) at a flow rate of 0.2 mL/min. LC–MS was operated under the selective ion monitoring mode using the electrospray ionization technique in the positive mode. The retention times for naphazoline, dibucaine, and the internal standard (IS) were 6.7, 7.8, and 8.0 min, respectively. A linear graph was obtained for dibucaine and naphazoline with correlation coefficients >0.998 for all analytes by this method. The limit of quantification of dibucaine and naphazoline was 10 and 25 ng/mL, respectively. The mean recoveries were greater than 70%. Both compounds were stable under conditions of short-term storage, long-term storage as well as after freeze–thaw cycles. Monolithic spin column extraction and LC–MS analysis enabled the separation of dibucaine and naphazoline within 20 min.

Summary Estimates of the Michaelis constants for usual5 and ‘atypical’ serum cholinesterase with α-naphthyl acetate as substrate were obtained. Expressed as m-moles α-naphthyl acetate per litre the values were: ‘usual’ enzyme 0-76 + 0-056, ‘atypical’ enzyme 0-58 + 0-057. The inhibition of the activity of the two enzymes by dibucaine, RO 2-0683, and fluoride was also examined using α-naphthyl acetate as substrate. It was found that, in the appropriate concentration range, the ‘usual’ enzyme was inhibited to a much greater degree than the ‘atypical’ enzyme by dibucaine and RO 2-0683. With fluoride, however, the difference, though significant, was much less marked. The findings are compared with similar data obtained when choline esters were used as substrates for the enzymes, and are considered in terms of the hypothesis that a structural difference at the ‘anionic’ site is the cause of the difference in the properties of the two enzymes.

The tetrameric glycoprotein butyrylcholinesterase (BChE; EC 3.1.1.8) is one of two enzymes that hydrolyze choline esters. The controlling gene (BCHE) is comprised of four coding exons and is located on chromosome 3q26. Based on BChE activity measurements in the presence and absence of dibucaine, usual (designated U) and atypical (designated A) gene products have been distinguished. Homozygotes for the A gene product are at risk for prolonged apnea following exposure to the surgical anesthetics succinylcholine or mivacurium. In this report, we detail biochemical and molecular investigations of succinylcholine sensitivity in a prairie Hutterite kindred. Our results establish that BChE activities in the family members are impacted by two distinct BCHE mutations, namely, c.209A>G p. D70G and c.1615G>A p. A539T. However, homozygotes for the c.209A>G mutation (i.e., atypical or A) are the only individuals whose BChE activity could lead to adverse reactions to succinylcholine. Interestingly, haplotype analysis of the chromosomal region containing BCHE indicates that the c.209A>G mutation is carried on a unique haplotype, suggesting that it was likely introduced into the population only once. Conversely, the c.1615G>A mutation is carried on various haplotypes and was likely introduced into the population more than once.

A simple, rapid, accurate, precise and specific analytical method has been developed, validated and applied for determination of cinchocaine in guinea pig and albino rabbit dorsal skins, after in vivo application of cinchocaine formulations. Extraction was performed using a solvent mixture of ethanol and 0.1 M hydrochloric acid (90:10; v/v). Samples were chromatographed on Spheri-5, RP18 column with a particle size of 5 µm and 220 mm × 4.6 mm i.d. The mobile phase was a mixture of acetonitrile and triethylamine phosphate buffer (pH 2.8; 0.04 M) (60:40, v/v). UV detection was carried out at 247 nm and the run time was 6 min with typical retention time of cinchocaine of 3.63 ± 0.02 min. Specificity was demonstrated, showing that the cinchocaine peak was free of interference from skin endogenous components. The detector response was found to be linear in the concentration range 0.96–56.00 µg/mL with a coefficient of correlation r = 0.99996. The relative standard deviations of within- and between-day analyses were all below 5%. The drug extraction procedure was validated. Satisfactory recoveries with relative standard deviation values below 5% were obtained, indicating efficient quantitative reproducible extraction procedure. Copyright © 2007 John Wiley & Sons, Ltd.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R. I., Epstein P. M., Andrenyak P. M., and Feinstein M. B. (1981) Proc. Natl. Acad. Sci. USA 78, 795–799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.

Although local anesthetics (LA) are considered primarily Na+-channel blockers in the past decade, an alternative action of LA as inhibitors of fusion among compartments of the endocytic/exocytic pathways was described. In epimastigote forms of Trypanosoma cruzi, we observed that 50 mM dibucaine reduced the rates of uptake of bovine serum albumin (BSA) and immunoglobulin to 60% of control values in addition to the delay of exocytosis of cysteine proteases. Fusion among endocytic compartments was not inhibited in the presence of dibucaine because previously labeled reservosomes was loaded with a second label in sequential pulse-chase experiments. However, dibucaine reduced the degradation of BSA-gold complex in the reservosomes, which was not caused either by an inhibition of the whole proteolytic activity of the parasite or by a reduction on the expression levels of cruzipain. The immunocytochemical analysis suggested that the inhibition of the degradation of gold-labeled BSA in reservosomes could be due to a subversion of the regular traffic of proteases toward the reservosomes in dibucaine-treated cells.

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC) containing different mole percents of monosialoganglioside (GM1) has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in the presence of phospholipid vesicles containing various amounts of GM1 yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more hydrophobic and rigid in membranes that contain GM1 than in membranes without it. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed reduced quenching efficiency with the increase in GM1 content of the vesicles, demonstrating lesser accessibility of the iodide quenchers to dibucaine in the presence of GM1, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 and approximately 2.8-3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20-30% lower than in the aqueous medium and showed a definite increase in presence of GM1 from that in the absence of it. All the spectral properties point that dibucaine encountered regions of membrane containing significant amount of GM1 and penetrated deeper in hydrophobic core of the bilayer.

The effects of procaine and some relatedagents on the growth rate of thecyanobacterium Synechococcusleopoliensis were investigated underphotoautotropic growth conditions and thedata compared with those for selectedphytohormones. Low concentrations(0.1–1 μM) of synthetic localanaesthetics (procaine, procainamide,tetracaine, lidocaine, dibucaine) enhancedphotoautotrophic growth. Lidocaine was themost effective growth stimulant, followedin decreasing order by procainamide,tetracaine, dibucaine, and procaine.Increases in growth rate were comparable tothose elicited by similar concentrations ofthe auxins indole-3-acetic acid and2,4-dichlorophenoxyacetic acid and of thecytokinin kinetin. Quantitativestructure-activity relationships betweenthe anaesthetics and phytohormones weresought. Score plots of principal componentsformulated from twelve moleculardescriptors suggest that the proliferativeeffects evoked by the anaesthetics andphytohormones may result from similaraffinities for binding receptor sites onthe cyanobacterial cell surface.Cyanobacterial growth assays couldtherefore be utilised as screens forcompounds with potential applications inagriculture and medicine.evolution

SummaryPhosphatidylserine exposure in platelets is required for normal haemostasis and is also a hallmark of apoptosis. It results from activation of a phospholipid scramblase, which has been shown to be differently stimulated by Ca2+-influx and during apoptosis, thus suggesting that mitochondria may be involved in phosphatidylserine exposure in platelets. It is also well known that local anaesthetics can expose phosphatidylserine in platelets and affect the mitochondrial metabolism in other cells. Thus, the present study was undertaken to evaluate the specific involvement of mitochondria in phosphatidylserine exposure in platelets. For this purpose, we stimulated phosphatidylserine exposure by local anaesthetics and avoided any external Ca2+-influx by performing all experiments in the absence of added Ca2+. We report that phosphatidylserine exposure, induced by the lipophilic local anaesthetics dibucaine and tetracaine, was accompanied by depolarization of the mitochondrial membrane, cytochrome c release, calpain-processing of caspases 9 and 3 to active enzymes, as well as a prolonged increase in both cytosolic and mitochondrial Ca2+ concentrations. In contrast, in the absence of extracellular Ca2+, the Ca2+-ionophore A23187 induced a smaller transient increase in both cytosolic and mitochondrial Ca2+ concentrations, but did not induce any other phenomena, nor phosphatidylserine exposure. However, phosphatidylserine exposure and depolarization induced by dibucaine still occurred in spite of inhibition of intracellular Ca2+ elevation. Thus we conclude that phosphatidylserine exposure in platelets is associated with mitochondrial apoptotic-like events. Therefore, we propose that mitochondria engagement in an apoptotic pathway in platelets could lead to PS exposure without the participation of Ca2+.

The dibucaine number (DN) was determined for serum cholinesterase (EC 3.1.1.8, SChE) in plasma samples. The ones with a DN of 79-82 were used, because they had the "usual" SChE variant. The enzyme was assayed colorimetrically by the reaction of 5,5'-dithiobis-[2-nitrobenzoic acid] (DTNB) with the free sulfhydryl groups of thiocholine that were produced by the enzyme reaction with butrylthiocholine (BuTch) or acetylthiocholine (AcTch) substrates, and measured at 412 nm. Dibucaine, a quaternary ammonium compound, inhibited SChE to a minimum within 2 min in a reversible manner. The inhibition was very potent. It had an IC(50) of 5.3 microM with BuTch or 3.8 microM with AcTch. The inhibition was competitive with respect to BuTch with a K(i) of 1.3 microM and a linear-mixed type (competitive/noncompetitive) with respect to AcTch with inhibition constants, K(i) and K(I) of 0.66 and 2.5 microM, respectively. Dibucaine possesses a butoxy side chain that is similar to the butryl group of BuTch and longer by an ethylene group from AcTch. This may account for the difference in inhibition behavior. It may also suggest the existence of an additional binding site, other than the anionic binding site, and of a hydrophobic nature.

Sonic spray ionization (SSI) was compared with atmospheric pressure chemical ionization (APCI) as an interface for liquid chromatography (LC)-mass spectrometry (MS) for the analysis of some local anesthetics. Peaks at [M+H]+ constituted the base peaks for all compounds by both SSI and APCI, except for prilocaine. The sensitivities by SSI for tetracaine, benzoxinate, dibucaine, bupivacaine and mepivacaine were 4–16 times higher than those by APCI; those by SSI for procaine and lidocaine were equivalent to those by APCI. Only for prilocaine was the sensitivity by SSI two times lower than that by APCI. In view of the higher sensitivities obtained for many local anesthetics by SSI, we established a detailed procedure for the assay of these drugs in human plasma and urine by LC-MS with SSI in combination with a diol-bonded silica gel HPLC column that enabled direct injection of crude biological samples without complicated pretreatment. The recoveries, sensitivities, accuracies and precisions were found satisfactory to quantitate them at their therapeutic levels.

Fluorescence emission from three tertiary amine local anesthetics, dibucaine (D), tetracaine (T), and procaine (P) was investigated in the membrane-mimetic environment of AOT/isooctane reverse micelles and water-ethanol mixtures. At room temperature all three show broad UV absorption bands and large Stokes' shifts in emission from aqueous medium. In reverse micelles the emission bands are blue-shifted, with the magnitude of this shift decreasing continuously as the waterto-surfactant molar ratio (w0) of the micelles is increased. The emission intensity of D is much stronger in water and in the interior of micelles than in ethanol, whereas for T and P the emission in water is much weaker than in ethanol and in micelles. Whereas the behavior in micelles is the result of the increased hydrophobicity with respect to bulk water in their interior, that in waterethanol mixtures may be explained on the basis of state reversal of the excited electronic levels of D with increasing hydrogen bonding capability of the solvent mixture. The steady-state anisotropy for both T and P is unusually high in bulk water (∼0.27) and increases with increasing w0 in micelles. However, the rotational correlation time of the anesthetics, as calculated using Perrin's equation, decreases with increasing w0, as expected from physical considerations relating the size of the water pool to the ease of rotational relaxation of probe molecules inside micelles. Together, these results point to a marked difference in emission properties between D on one hand and T and P on the other, arising from differences in electronic characters of their primary emitting chromophores—quinoline for D, para amino benzoate for T and P.

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.

The effect of low concentrations (up to 50 μM) of lipophilic permeant amines on the electron transfer in thylakoid membranes of pea chloroplasts has been investigated. In the presence of heterocyclic amines (9-aminoacridine and neutral red), the electron transfer, initiated from H2O to PS I acceptors, has been shown to be inhibited to a level amounting to less than 50% of control, this taking place for both the basal (at alkaline pH) and the gramicidin-uncoupled transport (at pH 6.5–8.5). Under the same conditions, tertiary amines (dibucaine, tetracaine) cause only a 10–15% inhibition of transport. With all the amines, the rate of electron transport from H2O to DCBQ, PS II acceptor is decreased to 80–90% of control at pH above 8.0, but this effect is completely removed when pH is lowered to 7.7–6.5. In the region of PS I, all the amines accelerate the basal transport, but do not influence the uncoupled electron transfer. A conclusion has been drawn that, parallel with uncoupling, heterocyclic and tertiary amines also cause an inhibition of PS II, appearing at alkaline pH values. Additionally, heterocyclic amines seem to brake electron flow at the level of plastoquinone reduction.

Decreased activity of plasma cholinesterase is responsible for prolonged apnea during anesthesia using neuromuscular blockers such as suxamethonium and mivacurium. More than 20 mutations have been identified so far in the BCHE gene resulting in impaired plasma cholinesterase activity. Biochemical tests are not always able to differentiate between pathological and normal sera; hence in some cases unanticipated complications can still occur during anesthesia even after measurements of enzyme activity and dibucaine numbers within the normal range. Therefore, molecular genetic testing is required for the accurate diagnosis of this deficiency. Here we present a study of plasma cholinesterase activity and BCHE genotyping of patients with a history of prolonged neuromuscular block and most of their pedigrees. All four exons of the BCHE gene were directly sequenced from samples and a number of mutations responsible for the reduction of plasma cholinesterase activity were identified. In most cases the atypical mutation in exon 2 (nt 209A --> G, Asp70 --> Gly) was found together with the K-variant mutation in exon 4 (nt 1615G --> A, Ala539 --> Thr), which is in good agreement with previous data suggesting that these mutations along with two others (at nt -116 and nt 1914) are in linkage disequilibrium.

This work elucidates differences in the hemolytic pathway developed by the antipsychotic trifluoperazine (TFP), the local anesthetic dibucaine (DBC) and the antihelminthic praziquantel (PZQ). Their partition coefficients (P) were measured at pH 7.4 between n-octanol, microsomes, liposomes, erythrocyte ghosts and n-octanol/water. The effective drug:lipid molar ratios for the onset of membrane solubilization (ReSAT) and complete hemolysis (ReSOL) were calculated from the experimental P values and compared with a classical surface-active compound treatment Lichtenberg, D. Biochim. Biophys. Acta 821 (1985) 470-478[. The contribution of charged/uncharged forms of TFP and DBC for the hemolytic activity was also analyzed. In all cases the hemolytic phenomena could be related to the monomeric drug insertion into the membrane. Only for TFP at isosmotic condition lysis occurs at concentrations beyond the CMC of the drug, indicating that micellization facilitates TFP hemolytic effect, while DBC and PZQ reach a real membrane saturation at their monomeric form.

Local anaesthetics, in addition to anaesthesia, induce the synthesis of heat shock proteins (HSPs), sensitize cells to hyperthermia, and increase the aggregation of nuclear proteins during heat shock. Anaesthetics are membrane active agents, and anaesthesia appears to be due to altered ion channel activity; however, the direct effect of heat shock is protein denaturation. These observations suggest that local anaesthetics may sensitize cells to hyperthermia by interacting with and destabilizing membrane proteins such that protein denaturation is increased. It is shown, using differential scanning calorimetry (DSC), that the local anaesthetics procaine, lidocaine, tetracaine and dibucaine destabilize the transmembrane domains of the Ca2+ -ATPase of sarcoplasmic reticulum and the band III anion transporter of red blood cells. The transmembrane domain of the Ca2+ -ATPase has a transition temperature (Tm) of denaturation of 61 degrees C which is decreased, for example, to 53 degrees C by 15 mM lidocaine. The degree of destabilization (deltaTm) by each anaesthetic is proportional to the lipid to water partition coefficient, and the increased sensitization by anaesthetics with larger partition coefficients and at higher pH suggests that the uncharged forms of the anaesthetics are responsible for destabilization. A Hill analysis of deltaTm for the Ca2+ -ATPase as a function of the concentration of anaesthetic in water gives dissociation constants (Kd) on the order of 10(-4) M, if binding occurs directly from the aqueous phase. This demonstrates moderate affinity binding. However, dissociation constants of 1-3 M are obtained, if binding occurs through the lipid phase, which demonstrates low affinity binding. Thus, the interaction of local anaesthetics with the Ca2+ -ATPase may be moderately specific or non-specific depending on the mechanism of interaction. The observation that local anaesthetics also destabilize the transmembrane domain of the band III protein of erythrocytes suggests that destabilization of transmembrane proteins is a general property of anaesthetics, which is at least in part a mechanism of sensitization to hyperthermia.

Role of phospholipase A 2 in the cytotoxic effects of oxalate in cultured renal epithelial cells. Background Oxalate, a common constituent of kidney stones, is cytotoxic for renal epithelial cells. Although the exact mechanism of oxalate-induced cell death remains unclear, studies in various cell types, including renal epithelial cells, have implicated phospholipase A 2 (PLA 2 ) as a prominent mediator of cellular injury. Thus, these studies examined the role of PLA 2 in the cytotoxic effects of oxalate. Methods The release of [ 3 H]-arachidonic acid (AA) or [ 3 H]-oleic acid (OA) from prelabeled Madin-Darby canine kidney (MDCK) cells was measured as an index for PLA 2 activity. The cell viability was assessed by the exclusion of ethidium homodimer-1. Results Oxalate exposure (175 to 550 μM free) increased the release of [ 3 H]-AA in MDCK cells but had no effect on the release of [ 3 H]-OA. Oxalate-induced [ 3 H]-AA release was abolished by arachidonyl trifluoromethyl ketone (AACOCF 3 ), a selective inhibitor of cytosolic PLA 2 (cPLA 2 ), but was not affected by selective inhibitors of secretory PLA 2 and calcium-independent PLA 2 . The [ 3 H]-AA release could be demonstrated within 15 minutes after exposure to oxalate, which is considerably earlier than the observed changes in cell viability. Furthermore, AACOCF 3 significantly reduced oxalate toxicity in MDCK cells. Conclusions Oxalate increases AA release from MDCK cells by a process involving cPLA 2 . In addition, based on the evidence obtained using a selective inhibitor of this isoform, it would appear that the activity of this enzyme is responsible, at least in part, for the cytotoxic effects of oxalate. The finding that oxalate can trigger a known lipid-signaling pathway may provide new insight into the initial events in the pathogenesis of nephrolithiasis.

The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variant was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17-20, 24-31 and 34-53 %. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified in terms of enzyme kinetics.

Assays of radiosodium uptake into mouse brain vesicles and the binding of [ 3 H]batrachotoxinin A 20-α-benzoate (BTX-B) were used to compare the actions of six dihydropyrazole (3-aryl-1-arylcarbamoyl-2-pyrazoline) insecticides on mouse brain sodium channels. The relative potencies of the six dihydropyrazoles as inhibitors of either pumiliotoxin B-stimulated sodium uptake measured in the presence of scorpion ( Leiurus quinquestriatus ) venom or veratridine-stimulated sodium uptake were closely correlated with the relative potencies of these compounds as inhibitors of the binding of BTX-B to mouse brain sodium channels. A comparison of the enantiomers of the most potent dihydropyrazole, RH 3421, as inhibitors of radiosodium uptake showed that the (−) enantiomer of RH 3421 was approximately six-fold more potent than the (+) enantiomer. The potencies of these dihydropyrazoles in these assays and the stereoselectivity observed in the action of enantiomers of RH 3421 are in good agreement with available information on insecticidal activity in this group of compounds. Assays of the combined effects of RH 3421 and dibucaine as inhibitors of BTX-B binding revealed mutually competitive interactions between these compounds. This finding is consistent with the existence of a common site of action for dihydropyrazoles and local anesthetics on the sodium channel. The results of these studies provide further evidence for the toxicological relevance of the effects of dihydropyrazoles on sodium channels.

This study concerns measurements and interpretations of the trans-thylakoid membrane pH gradient, ΔpH, and xanthophyll cycle-dependent energy dissipation in Photosystem II (PS II). Compared and contrasted are the concentration-dependent inhibitory effects and interactions between two lipophilic tertiary amines, namely, 9-aminoacridine the ΔpH indicator and dibucaine a local anesthetic reported to inhibit both the ΔpH and xanthophyll cycle deepoxidation. Chlorophyll a fluorescence monitored both electron transport efficiency and xanthophyll cycle-dependent energy dissipation, high-performance liquid chromatography monitored deepoxidase and chloroplast ATPase activities and steady-state fluorescence monitored various activities of the amines in solution. Low concentrations (up to 2 μM) of both 9-aminoacridine and dibucaine showed similar fluorescence properties and ΔpH-dependent uptake into thylakoids. Importantly both amines exhibited mutually competitive inhibitory effects with respect to this ΔpH-dependent uptake and fluorescence quenching. The fluorescence yields of both compounds in aqueous solution were strongly quenched by sodium ascorbate, a necessary cofactor for in vitro deepoxidation. Both compounds similarly inhibited several light induced activities including deepoxidation, photosynthetic electron transport and PS II energy dissipation. However, for all these activities 9-aminoacridine was 2 to 5 times more potent. Importantly, 9-aminoacridine inhibited deepoxidation with an I50≈1 μM, a concentration far below that which inhibits the ΔpH, ATP synthesis/hydrolysis or electron transport. The inhibitory effects of both compounds on PS II energy dissipation were exerted at 3 to 5 times lower concentration if added before as opposed to after a saturating level of deepoxidation. This result confirms the important role for deepoxidation in mediating PS II energy dissipation. Compared to 9-aminoacridine and in contrast to similar effects on the light-induced activities, dibucaine exhibited significantly different inhibitory effects on ATPase activity and ATPase mediated PS II energy dissipation. However, we conclude from the more potent inhibition by 9-aminoacridine and the similar inhibitory patterns of all the light-induced activities that neither 9-aminoacridine nor dibucaine possess unique capacities to neutralize the light-mediated ΔpH. DCMU–3-(3,4-dichlorophenyl)-1,1-dimethylurea; DTT–dithiothreitol; fx–fractional intensity of fluorescence lifetime component x; F(′)m–maximal PS II Chl a fluorescence intensity with all QA reduced in the absence (presence) of thylakoid membrane energization; Fo–minimal PS II Chl a fluorescence intensity with all QA oxi dized; Fs–steady state PS II Chl a fluorescence; HPLC–high performance liquid chromatography; I(o)–intensity of fluorescence in the presence (absence) of quencher; Ka–association constant between Z (and A) and protonated PS II units; LA–local anesthetic; NaAsc–sodium ascorbate; NR–neutral red; PAM–pulse-amplitude modulation fluorometer; PFD–photon-flux density, μmols photons m-2 s-1; PS I–Photosystem I; PS II–Photosystem II; [PS II-+]–concentration of PS II units with inactive/deprotonated (active/protonated ) xanthophyll binding sites; [PS IItot]–total concentration of PS II units; [PS II+-Z]–concentration of PS II units with Z or A bound; Q–fraction of fluorescence intensity that is quenched; Qmax–fraction of fluorescence intensity that is quenched under control conditions; QA–primary quinone electron acceptor of PS II; V–violaxanthin; Z–zeaxanthin; 9AA–9-aminoacridine; ΔpH–trans-thylakoid membrane proton gradient; τf–lifetime of Chl a fluorescence

We noticed a Japanese male showed low serum butyrylcholinesterase(BCHE) activity on health examination. The phenotyping analysis revealed a reduced dibucaine number (DN) and an especially low fluoride number (FN), similar to an FS phenotype. A homozygous missense mutation, a T to A transversion at nucleotide 988, was identified in his BCHE gene. This mutation resulted in the replacement of leucine by isoleucine at codon 330 (L330I). DN and FN of recombinant BCHE(L330I) secreted by human fetal kidney cells were compared to recombinant wild-type(usual gene)BCHE and normal serum BCHE. These results showed this amino acid substitution of BCHE, Leu330 to Ile, really caused the abnormal DN and FN. We conclude that the BCHE L330I mutation is a fluoride-resistant gene, a Japanese type fluoride-resistant gene.

1 Glutamate receptor activation has been previously shown to result in mitochondrial depolarization and activation of the mitochondrial permeability transition pore in cultured neurones. In this study, we characterized the effects of two putative permeability transition inhibitors, namely trifluoperazine and dibucaine, on mitochondrial depolarization in rat intact, cultured forebrain neurones. 2 Permeability transition was monitored by following mitochondrial depolarization in neurones loaded with the mitochondrial membrane potential-sensitive fluorescent indicator, JC-1. Trifluoperazine (10–20 μM) and dibucaine (50–100 μM) inhibited or delayed the onset of glutamate-induced permeability transition. 3 We also investigated the effects of trifluoperazine and dibucaine on neuronal recovery from glutamate-induced Ca2+ loads. Trifluoperazine affected Ca2+ recovery in a manner similar to the mitochondrial Na+/Ca2+ exchange inhibitor, CGP-37157, while dibucaine had no apparent effect on Ca2+ recovery. Therefore, inhibition of permeability transition does not appear to be involved in Ca2+ recovery from glutamate-induced Ca2+ loads. 4 Trifluoperazine and dibucaine did not inhibit [3H]-dizocilpine binding at the concentrations that prevented mitochondrial depolarization. 5 These studies suggest that trifluoperazine and dibucaine inhibit permeability transition in intact neurones. Trifluoperazine also appears to inhibit mitochondrial Na+/Ca2+ exchange. These drugs should prove to be valuable tools in the further study of the role of mitochondrial permeability transition in glutamate-induced neuronal death. British Journal of Pharmacology (1997) 122, 803–808; doi:10.1038/sj.bjp.0701442

Peroxide formation has been implicated in impairment of motor function by cyanide which occurs in both animals and man. The present study employs the neuronal model, rat pheochromocytoma (PC12) cells to evaluate peroxidation as a toxic mechanism of cyanide. Confocal imaging shows that peroxides form within a few seconds in cell cytoplasm after cyanide exposure and continue to accumulate over a period of several minutes. Peroxide generation by cyanide is decreased to about 50% by phospholipase A2 inhibitors indicating involvement of arachidonic acid in the oxidative process. Also antioxidant defense enzymes (CuZn superoxide dismutase and especially catalase) in PC12 cells are inhibited by cyanide. It appears that peroxide accumulation after cyanide treatment involves both inhibition of breakdown and increased production. Furthermore, both peroxide accumulation and cell death induced by cyanide in PC12 cells are blocked by an antioxidant (ascorbate). These data support the hypothesis that the cytotoxic action of cyanide is related in part to an oxidative process.

An unstable variant of human butyrylcholinesterase (BChE) is described in four apparently unrelated individuals sensitive to succinylcholine. Sequencing of genomic DNA revealed a single nucleotide substitution which results in the replacement of amino acid residue Gly115 by Asp. This variant can be recognized by its increased instability under extremes of temperature such as heating and also freezing and thawing, both in homozygous and heterozygous states. When in heterozygous combination with the Atypical variant, it produces dibucaine and fluoride numbers which are intermediary between those of Atypical homozygotes and heterozygotes. After repeated freezing and thawing, however, these values approach those of homozygous Atypical plasma. Measurement of activity and immunoreactive BChE protein in plasma of individuals representing different combinations of this allele indicated that the presence of the Usual or Atypical enzymes seems to partially protect this variant from denaturation in vivo. Phenotyping fresh serum or plasma samples, before they are frozen, is critical for the identification of this, and possibly some other, unstable variants.

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.

The dynamics of structural changes in pea chloroplasts in the presence of 25–50 μM dibucaine or tetracaine has been examined using electron microscopy. The light-induced uptake of anesthetic cations by thylakoids is attended by the appearance of local fusions of stroma-exposed thylakoid membranes. The first membrane protrusions and interthylakoid contacts are observed after 4 s illumination and they become numerous by 10 s. As a result, a network of anastomoses is formed which is maintained during at least 10 min. These effects are reversible in the dark and can be reproduced several times. The formation of membrane fusions is inhibited by the addition of protonophore. It is supposed that the energy-dependent uptake of protonated anesthetics by thylakoids leads to an increase in positive surface charge and thus a lateral pressure on the inner side of the thylakoid membrane. The appearance of membrane protrusions (crinkles) having the positive curvature of their inner surface may be considered as a way of compensating for lateral pressure. Presumably, anastomoses result from the fusion of crinkles to adjacent thylakoids.

The effects of both neutral and anionic lipids on the structure of the nicotinic acetylcholine receptor (nAChR) have been probed using infrared difference spectroscopy. The difference between infrared spectra of the nAChR recorded using the attenuated total reflectance technique in the presence and absence of the neurotransmitter analog, carbamylcholine, exhibits a complex pattern of positive and negative bands that provides a spectral map of the structural changes that occur in the nAChR upon ligand binding and subsequent desensitization. This spectral map is essentially identical in difference spectra recorded from native, native alkaline-extracted, and affinity-purified nAChR reconstituted into either soybean asolectin or egg phosphatidylcholine membranes containing both neutral and anionic lipids. This result suggests both a similar structure of the nAChR and a similar resting to desensitized conformational change in each membrane environment. In contrast, difference spectra recorded from the nAChR reconstituted into egg phosphatidylcholine membranes lacking neutral and/or anionic lipids all exhibit an essentially identical pattern of band intensity variations, which is similar to the pattern of variations observed in difference spectra recorded in the continuous presence of the desensitizing local anesthetic, dibucaine. The difference spectra suggest that the main effect of both neutral and anionic lipids in a reconstituted egg phosphatidylcholine membrane is to help stabilize the nAChR in a resting conformation. In the absence of neutral and/or anionic lipids, the nAChR is converted into an alternate conformation that appears to be analogous to the local anesthetic-induced desensitized state. Significantly, the proportion of receptors found in the resting versus the putative desensitized state appears to be dependent upon the final lipid composition of the reconstituted membrane. A lipid-dependent modulation of the equilibrium between a channel-active resting and channel-inactive desensitized state may account for the modulations of nAChR activity that are observed in different lipid membranes.

A recently completed model of Ca concentration and movements in the cardiac cell diadic cleft space predicts that removal or neutralization of inner sarcolemmal (SL) leaflet anionic Ca-binding sites at the sarcolemmal border of this space will greatly diminish Na/Ca exchange-mediated Ca efflux. The present study tests this prediction using the local anesthetic dibucaine as a probe. It is shown, in isolated SL, that dibucaine competitively displaces Ca specifically from anionic phospholipid headgroups. Dibucaine also displaces Ca from the SL when applied to intact cells. It does not affect the content or release of Ca from sarcoplasmic reticulum (SR) in these cells. This eliminates a primary effect on SR Ca as a contributing factor to dibucaine's effect on Na/Ca exchange-mediated Ca efflux. Measurement of this efflux from whole cells shows a highly significant reduction of 58% (p < 0.001) by 0.5 mM dibucaine. The inhibiting effect of dibucaine on Na/Ca exchange-mediated Ca efflux can be significantly reversed by augmentation of Ca release from SR by caffeine at the time of activation of Na/Ca exchange. This supports the contention that the dibucaine-SL interaction is a competitive one vis-a-vis Ca. The results are supportive of the model in which inner SL leaflet Ca-binding sites account for the delay of Ca diffusion from the diadic cleft, thereby prolonging the time for which [Ca] remains elevated in the cleft. The prolonged increased [Ca] significantly enhances the ability of Na/Ca exchange to remove Ca from the cell during the excitation-contraction cycle.

Effects of basic glycoside antibiotic aculeximycin (ACM) on the oxidative phosphorylation of rat-liver mitochondria were examined. ACM was shown to be a potent uncoupler of the oxidative phosphorylation. To cause the same extent of respiration release, higher concentration of ACM was required in phosphate (Pi)-free medium than in Pi medium. During the uncoupling caused by ACM in Pi medium, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, indicating that ACM remarkably enhances permeability of the inner mitochondrial membrane. The Pi uptake via Pi/H+ symporter was shown to play an important, but not essential, role in the uncoupling by ACM, indicating the increase in membrane permeability is mostly due to acceleration of Pi/H+ influx through Pi/H+ symporter activated by ACM. ACM is the first naturally occurring antibiotic, to our knowledge, which activates Pi/H+ symporter. However, since the inhibition of Pi/H+ symporter by N-ethylmaleimide did not completely abolish the uncoupling activity of ACM, and ACM induced the uncoupling even in Pi-free medium, an increase in the membrane permeability for other ions, such as Na+ and K+, due to a different action mechanism has also to be considered. On the other hand, positively charged amine local anesthetics, like dibucaine, prevented the uncoupling activity by ACM in both Pi and Pi-free medium. The uncoupling activity of N-diacetylated ACM lacking free amino groups was ca. 1/120th that of ACM, indicating that positively charged amino groups are important for the uncoupling activity. It is suggested that some specific interactions between positively charged amino groups of ACM and the binding site, which is probably negatively charged, are triggers that affect the permeability of the inner mitochondrial membrane. Amine local anesthetics may mask the negative charge of the binding site, thereby interfering with ACM binding.

The polarographic behaviour of dibucaine was studied using various electrochemical techniques. In acidic media, a non-reversible diffusion-controlled reduction process involving two electrons takes place. The linear relationship between peak current intensity and dibucaine concentration allowed the differential-pulse polarographic determination of dibucaine over a wide concentration range, from 10–6 to 10–4 mol l–1 at pH 2.6, with a relative standard deviation of 1.9%(nine determinations at the 10–5 mol l–1 level). A simple extraction procedure was employed for the determination of the drug in pharmaceutical formulations and the validity of the method was confirmed by parallel determinations using the official method of the US Pharmacopeia (HPLC).

The purpose of these experiments was to determine the reversibility of α-chaconine and α-solanine inhibition of human plasma butyrylcholinesterase (BuChE). For the substrate α-naphthylacetate, optimal assay conditions were 0.50 M sodium phosphate buffer and a substrate concentration of 3–5 × 10 −4 M . Dibucaine (1 × 10 −5 M ) indicated the usual phenotype for all subjects; α-chaconine and α-solanine at 2.88 × 10 −6 M inhibited BuChE about 70 and 50%, respectively. One- and 24-hr incubations at 1 × 10 −5 M with α-chaconine, α-solanine, paraoxon, eserine, and ethanol yielded reversible inhibition with dilution except for paraoxon. Twenty-four-hour dialyses of incubations showed no inhibition except for paraoxon. PAGE enzyme activity gels of 1- and 24-hr incubations also showed no inhibition except for paraoxon. α-Chaconine and α-solanine are reversible inhibitors of human butyrylcholinesterase. At estimated tissue levels, α-chaconine, α-solanine, and solanidine inhibited BuChE 10–86%. In assays which combined α-chaconine, α-solanine, and solanidine, inhibition of BuChE was less than additive. No inhibition of albumin α-naphthylacetate esterase (an arylesterase) was noted with any inhibitor. The importance of these data to adverse toxicological effects of potato alkaloids is discussed..

A direct colorimetric method was described for the rapid, sensitive and accurate determination of dibucaine, lidocaine, bupivacaine, procaine and tetracaine in pharmaceutical preparations. The method involves the use of haematoxylin reagent in the presence of boric acid to give a reddish-violet chromogen (λmax = 555 nm). Beer's law was obeyed in the range from 2–60 μg/ml. No interference was observed from the commonly present additives or agents in pharmaceutical formulations.

Lophotoxin and the bipinnatins are members of the lophotoxin family of marine neurotoxins, which covalently react with Tyr190 in the alpha-subunits of the nicotinic acetylcholine receptor. Bipinnatin-A, -B, and -C are protoxins that have been shown to spontaneously convert from inactive to active toxins during preincubation in buffer. However, in this report, we show that preincubation of lophotoxin did not result in an increase in the subsequent rate of irreversible inhibition of nicotinic receptors. Thus, unlike the bipinnatins, lophotoxin does not appear to be an inactive protoxin. Lophotoxin preferentially inhibited one of the two acetylcholine-binding sites on the receptor, and this preference resulted from both a higher reversible affinity and a faster rate of irreversible inhibition at this site. Association of 125I-alpha-bungarotoxin in the presence of lophotoxin was analyzed to obtain the apparent reversible association and dissociation rate constants for lophotoxin. The apparent association rate constant of lophotoxin was approximately 10(6)-fold slower than expected for a diffusion-limited interaction, indicating that lophotoxin is a slow binding irreversible inhibitor. The kinetic constants that describe the interaction of lophotoxin with the receptor did not change in the presence of dibucaine, suggesting that, unlike agonists, the slow apparent association of lophotoxin does not result from a slow transition of the receptor to a desensitized conformation.