Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study.

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 µM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

Phentolamine Reverses Epinephrine-Enhanced Skin Antinociception of Dibucaine in Rats.

BACKGROUND: The objective of the experiment was to assess the antinociceptive effect of dibucaine, bupivacaine, and epinephrine. To assess the mechanism of action of the interaction between dibucaine and epinephrine, phentolamine, a nonselective α-adrenergic antagonist, was added to the mixture. METHODS: We assessed sensory blockade with these drugs by injecting 0.6 mL of drug-in-saline in the dorsal thoracolumbar area of rats; pinprick of the "wheal" formed by the injectate was the area targeted for stimulation to elicit a cutaneous trunci muscle reflex. The sensory block of dibucaine was compared with that of bupivacaine or epinephrine. Drug-drug interactions were analyzed by isobologram. Phentolamine was added to investigate the antinociceptive effect of dibucaine coinjected with epinephrine. RESULTS: We demonstrated that dibucaine, epinephrine, and bupivacaine produced dose-dependent skin antinociception. On the median effective dose (ED50) basis, the potency was higher for epinephrine (mean, 0.011 [95% confidence interval {CI}, 0.007-0.015] µmol) than for dibucaine (mean, 0.493 [95% CI, 0.435-0.560] µmol) (P < .01), while there were no significant differences between dibucaine and bupivacaine (mean, 0.450 [95% CI, 0.400-0.505] µmol). On the equipotent basis (75% effective dose, median effective dose, and 25% effective dose), sensory block duration provoked by epinephrine was greater (P < .01) than that provoked by dibucaine or bupivacaine. Coadministration of dibucaine with epinephrine produced a synergistic nociceptive block, whereas phentolamine blocked that synergistic block. CONCLUSIONS: The preclinical data indicated that there is no statistically significant difference between the potency and duration of dibucaine and bupivacaine in this model. Epinephrine synergistically enhances the effects of dibucaine, while phentolamine partially blocked those effects. α-Adrenergic receptors play an important role in controlling synergistic analgesic effect of dibucaine combined with epinephrine.

Discovery of Quinoline Analogues as Potent Antivirals against Enterovirus D68 (EV-D68).

Enterovirus D68 (EV-D68) is an atypical nonpolio enterovirus that mainly infects the respiratory system of humans, leading to moderate-to-severe respiratory diseases. In rare cases, EV-D68 can spread to the central nervous system and cause paralysis in infected patients, especially young children and immunocompromised individuals. There is currently no approved vaccine or antiviral available for the prevention and treatment of EV-D68. In this study, we aimed to improve the antiviral potency and selectivity of a previously reported EV-D68 inhibitor, dibucaine, through structure-activity relationship studies. In total, 60 compounds were synthesized and tested against EV-D68 using the viral cytopathic effect assay. Three compounds 10a, 12a, and 12c were identified to have significantly improved potency (EC50 < 1 µM) and a high selectivity index (>180) compared with dibucaine against five different strains of EV-D68 viruses. These compounds also showed potent antiviral activity in neuronal cells, such as A172 and SH-SY5Y cells, suggesting they might be further developed for the treatment of both respiratory infection as well as neuronal infection.

Subcellular Partitioning and Intramacrophage Selectivity of Antimicrobial Compounds against Mycobacterium tuberculosis.

The efficacy of antimicrobial drugs against Mycobacterium tuberculosis, an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. However, inside infected macrophages, bacteria encounter an environment which differs substantially from broth culture and are subject to important host-dependent pharmacokinetic phenomena which modulate drug activity. Here, we describe how pH-dependent partitioning drives asymmetric antimicrobial drug distribution in M. tuberculosis-infected macrophages. Specifically, weak bases with moderate activity against M. tuberculosis (fluoxetine, sertraline, and dibucaine) were shown to accumulate intracellularly due to differential permeability and relative abundance of their ionized and nonionized forms. Nonprotonatable analogs of the test compounds did not show this effect. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. We further demonstrate that the efficacy of a clinically used compound, clofazimine, is augmented by pH-based partitioning in a macrophage infection model. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis.

Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.

Paraquat Induces Cell Death Through Impairing Mitochondrial Membrane Permeability.

Paraquat (PQ) as a Parkinsonian mimetic has been demonstrated to impair dopaminergic (DAergic) neurons and is highly correlated with the etiology of Parkinson's disease (PD) where the death of DAergic neurons has been mainly attributed to impaired mitochondrial functioning. In this study, PQ-induced cytotoxicity focusing on mitochondrial membrane permeability (MMP), which has been implicated to play a part in neurodegeneration, was investigated. Primarily, PQ-induced cytotoxicity and reactive oxygen species (ROS) were inhibited by an inhibitor of NADPH oxidase (NOX), indicating the toxic effect of PQ redox cycling. Further, dibucaine and cyclosporin A which respectively inhibit mitochondrial apoptosis-induced channels (MAC) and mitochondrial permeability transition pores (mPTP) were used and found to prevent PQ-induced mitochondrial dysfunction, such as decreased mitochondrial membrane potential and increased MMP, mitochondrial ROS, and pro-apoptotic factor release. Knockdown of bax and/or bak blocked PQ-induced mitochondrial clusterization of Bax and/or Bak and cytotoxicity, demonstrating the significance of MAC which is composed of Bax and/or Bak. This clusterization coincided with the release of mitochondrial apoptotic factors before there was an increase in inner MMP, indicating that MAC may precede mPTP formation. Besides, NOX inhibitor but not dibucaine attenuated the earlier PQ-induced cytosolic ROS formation or Bax and/or Bak clusterization indicating PQ redox cycling may account for MAC formation. In this model, we have resolved for the first that PQ cytotoxicity through redox cycling may sequentially result in increased outer (MAC) and inner (mPTP) MMP and suggested MMP could be implicated as a therapeutic target in treating neurodegenerative diseases like PD.

[Study of reactive oxygen species on the regulation of platelet apoptosis].

OBJECTIVE: To study the effect of reactive oxygen species (ROS) on the regulation of platelet apoptosis. METHODS: Washed healthy human platelets were pre-incubated with N-caetyl-Lcysteine (NAC), and then stimulated with dibucaine or thrombin. The production of ROS and depolarization of mitochondrial membrane potential (∆ ψm) were detected by flow cytometry. The activation of caspase-3 and expression of Bcl-xL were analyzed by Western blot. RESULTS: (1)The average ROS fluorescence value of NAC+dibucaine group was lower than that of dibucaine group(0.66 ± 0.11 vs 1.06 ± 0.08, P<0.01), while that of NAC+thrombin group was also lower than that of thrombin group(0.45 ± 0.05 vs 0.71 ± 0.11, P=0.001). (2)The percentage of platelets with normal ∆ψm in NAC+Dibucaine group was higher than that of dibucaine group[(86.30 ± 9.37)% vs (13.52 ± 3.01)%, P=0.000], while that of NAC+thrombin group was also higher than that of thrombin group[(93.00 ± 3.03)% vs (76.58 ± 5.28)%, P=0.000]. (3)Fragmentation generated by caspase-3 activation in dibucaine group was much more than that in DMSO control group, while the fragmentation in NAC+dibucaine group was significantly decreased. (4)The expression of anti-apoptosis protein Bcl-xL of NAC+dibucaine group was significantly higher than that of the dibucaine group, while that of NAC+thrombin group was also higher than that of thrombin group. CONCLUSION: Through the regulation of ROS, NAC could inhibit the platelet apoptosis induced by dibucaine or thrombin.

Cytotoxicity and type of cell death induced by local anesthetics in human oral normal and tumor cells.

BACKGROUND: Local anesthetics are often administered to tumors and surrounding tissues during the surgery of the head and neck area, however their effects on oral tissues is not well understood. In the present study, the cytotoxicity of a total of seven local anesthetics towards oral tumor and normal cells was compared. MATERIALS AND METHODS: Tumor-specificity index was determined by the ratio of the mean 50% cytotoxic concentration against normal cells to that for tumor cells. Apoptosis induction was monitored by internucleosomal DNA fragmentation and caspase-3, -8, and - 9 activation. Fine cell structure was observed under transmission electron microscopy. RESULTS: All local anesthetics showed slightly higher cytotoxicity towards oral squamous cell carcinoma (OSCC) cell lines than towards normal oral cells. Dibucaine, with a log p-value of approximately 3, was the most cytotoxic, followed by tetracaine, bupivacaine or ethylaminobenzoate, whereas lidocaine, procaine and mepivacain were much less cytotoxic. When the tumor-specificity was evaluated between OSCC and human skin keratinocytes, the index was 6.6. Dibucaine did not induce apoptosis of OSCC cells. On the other hand, dibucaine did induce mitochondrial injury and swelling, formation of secondary lysosomes, and at high concentrations, rupture of the cell membrane. Autophagy inhibitors did not reduce the cytotoxicity of dibucaine. CONCLUSION: Necrosis may be involved in the induction of antitumor activity by dibucaine.

Examining protection from anoxic depolarization by the drugs dibucaine and carbetapentane using whole cell recording from CA1 neurons.

As an immediate consequence of stroke onset, failure of the Na(+)-K(+)-ATPase pump evokes a propagating anoxic depolarization (AD) across gray matter. Acute neuronal swelling and dendritic beading arise within seconds in the future ischemic core, imaged as changes in light transmittance (ΔLT). AD is itself not a target for drug-based reduction of stroke injury because it is generated in the 1st min of stroke onset. Peri-infarct depolarizations (PIDs) are milder AD-like events that recur during the hours following AD and contribute to infarct expansion. Inhibiting PIDs with drugs could limit expansion. Two types of drugs, "caines" and σ(1)-receptor ligands, have been found to inhibit AD onset (and may also oppose PID initiation), yet their underlying actions have not been examined. Imaging ΔLT in the CA1 region simultaneously with whole cell current-clamp recording from CA1 pyramidal neurons reveal that the elevated LT front and onset of the AD are coincident. Either dibucaine or carbetapentane pretreatment significantly delays AD onset without affecting resting membrane potential or neuronal input resistance. Dibucaine decreases excitability by raising spike threshold and decreasing action potential (AP) frequency, whereas carbetapentane eliminates the fast afterhyperpolarization while accentuating the slow afterhyperpolarization to reduce AP frequency. Orthodromic and antidromic APs are eliminated by dibucaine within 15 min but not by carbetapentane. Thus both drugs reduce cortical excitability at the level of the single pyramidal neuron but through strikingly different mechanisms. In vivo, both drugs would likely inhibit recurring PIDs in the expanding penumbra and so potentially could reduce developing neuronal damage over many hours poststroke when PIDs occur.

Dibucaine mitigates spreading depolarization in human neocortical slices and prevents acute dendritic injury in the ischemic rodent neocortex.

BACKGROUND: Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury. METHODOLOGY/PRINCIPAL FINDINGS: We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model. CONCLUSIONS/SIGNIFICANCE: Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from ischemic injury with an approach that does not rely on the complete abolishment of spreading depolarizations.

A lytic mechanism based on soluble phospholypases A2 (sPLA2) and ß-galactoside specific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractile hemocytes against K562 cell line and mammalian erythrocytes.

Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca²+-dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1-B6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. The B5 activity was blocked by D-galactose, α-lactose, lactulose, LacNAc, thiodigalactoside (TDG), L-fucose, D-mannose, D-glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca²+-dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.

Potent inhibition of anoxic depolarization by the sodium channel blocker dibucaine.

Recurring waves of peri-infarct depolarizations (PIDs) propagate across gray matter in the hours and days following stroke, expanding the primary site of injury. Ischemic depolarization (termed anoxic depolarization or AD in live brain slices) is PID-like but immediately arises in the more metabolically compromised ischemic core. This causes dramatic neuronal and astrocyte swelling and dendritic beading with spine loss within minutes, resulting in acute cell death. AD is evoked in rodent neocortical slices by suppressing the Na(+)/K(+)-ATPase pump with either oxygen/glucose deprivation (OGD) or exposure to ouabain. The process driving AD and PIDs remains poorly understood. Here we show that dibucaine is a potent drug inhibiting AD because of its high binding affinity to the Na(+) channel. Field recording reveals that, when superfused with ouabain (5 min), neocortical slices pretreated with 1 µM dibucaine for 45 min display either no AD or delayed AD onset compared with untreated controls. If ouabain exposure is extended to 10 min, 1 µM dibucaine is still able to delay AD onset by ∼ 60%. Likewise, it delays OGD-evoked AD onset by ∼ 54% but does not depress action potentials (APs) or evoked orthodromic field potentials. Increasing dibucaine to 10 µM inhibits AP firing, gradually putting the slice into a stasis that inhibits AD onset but also renders the slice functionally quiescent. Two-photon microscopy reveals that 10 µM dibucaine pretreatment prevents or helps reverse ouabain-induced structural neuronal damage. Although the therapeutic range of dibucaine is quite narrow, dibucaine-like drugs could prove therapeutically useful in inhibiting PIDs and their resultant neuronal damage.

Concordance of butyrylcholinesterase phenotype with genotype: implications for biochemical reporting.

Butyrylcholinesterase (BChE) metabolizes the paralytic succinylcholine. Extended paralysis occurs in people with inherited BChE variants that may be identified by measuring BChE activity with and without the inhibitor dibucaine to calculate a dibucaine number (DN). Accurate phenotyping requires phenotype-specific BChE and DN reference intervals. We investigated the concordance between the biochemical BChE phenotype and the BCHE genotype to establish interpretive criteria for biochemical results. DNA was extracted from 45 serum specimens for which BChE activity and DN had been determined. The BCHE gene coding region was amplified and sequenced. Phenotype-genotype concordance and discordance occurred in 16 (36%) and 15 (33%) of specimens, respectively. A phenotype could not be assigned for 14 specimens (31%). An incorrectly assigned phenotype did not change the risk of prolonged paralysis or implied a slightly increased risk when there was none. Accurate BChE phenotyping is difficult using only enzyme activity and DN. The combination of biochemistry and BCHE genotype could improve the assessment of patient risk.

Changes in mitochondrial surface charge mediate recruitment of signaling molecules during apoptosis.

Electrostatic interactions with negative lipids contribute to the subcellular localization of polycationic proteins. In situ measurements using cytosolic probes of surface charge indicate that normal mitochondria are not noticeably electronegative. However, during apoptosis mitochondria accrue negative charge and acquire the ability to attract cationic proteins, including K-Ras. The marked increase in the surface charge of mitochondria occurs early in apoptosis, preceding depolarization of their inner membrane, cytochrome c release, and flipping of phosphatidylserine across the plasmalemma. Using novel biosensors, we determined that the increased electronegativity of the mitochondria coincided with and was likely attributable to increased exposure of cardiolipin, which is dianionic. Ectopic (over)expression of cardiolipin-binding proteins precluded the increase in surface charge and inhibited apoptosis, implying that mitochondrial exposure of negatively charged lipids is required for progression of programmed cell death.

Preparation and evaluation of bioadhesive dibucaine gels for enhanced local anesthetic action.

In relieving local pains, dibucaine, one of ester type local anesthetics, has been used. In case of their application such as ointments and creams, it is difficult to expect their effects for a required period of time, because they are easily removed by wetting, movement and contacting. To develop suitable bioadhesive gels, the bioadhesive force of hydroxypropyl cellulose (HPC) was tested using auto-peeling tester. The effect of drug concentration on drug release was studied from the prepared 2% HPC-HF gels using synthetic cellulose membrane at 37 +/- 0.5 degrees C. We investigated the enhancing effects on drug permeation into skins, using some kind of enhancers such as the glycols, the non-ionic surfactants, the fatty acids, and the propylene glycol derivatives. Anesthetic effects of dibucaine gels containing polyoxyethylene 2-oleyl ether were measured by tail flick analgesic meter. The bioadhesive force of various types of HPC such as GF, MF, and HF, was 0.0131, 0.0501, and 0.1346 N, at 2% HPC concentration, respectively. The HPC-HF gels showed the highest bioadhesive force. As the concentration of HPC-HF increased, the drug release increased. As the temperature increased, the drug release increased. Among the enhancers used, polyoxyethylene 2-oleyl ether showed the highest enhancing effects. According to the rat tail flick test, 1% drug gels containing polyoxyethylene 2-oleyl ether showed the prolonged local anesthetic effects. In conclusion, the dibucaine gel containing penetration enhancer and vasoconstrictor showing enhanced local anesthetic action could be developed by using the bioadhesive polymer, HPC.

The role of calpain in the regulation of ADAM17-dependent GPIbalpha ectodomain shedding.

There are evidence that both a disintegrin and metalloproteinase 17 (ADAM17) and calpain are involved in platelet glycoprotein (GP)Ibalpha ectodomain cleavage. However, the relationship between the two enzymes in the shedding process remains unclear. Here we show that calcium ionophore A23187- and alpha-thrombin-induced GPIbalpha shedding is completely inhibited by the metalloproteinase inhibitor GM6001, whereas it is only partially inhibited by calpain inhibitors. Calpain activator dibucaine-induced GPIbalpha shedding was completely inhibited by both metalloproteinase and calpain inhibitors. On the other hand, calpain inhibitors did not inhibit GPIbalpha shedding induced by the reagents that specifically activate ADAM17. Furthermore, A23187-induced GPIbalpha shedding in Chinese hamster ovary cells expressing wild-type or mutant GPIb-IX was also partially inhibited by calpain inhibitors and almost completely inhibited by GM6001. Therefore, these data indicate that calpain plays an important role in the regulation of ADAM17-dependent GPIbalpha ectodomain shedding in both platelets and nucleated cells.

Ethanol aggravates itch-related scratching in hairless mice developing atopic dermatitis.

In patients with atopic dermatitis, alcoholic beverages can sometimes trigger or enhance itching. We have previously reported that HR-1 hairless mice fed a commercial special diet, HR-AD, but not a normal diet, develop atopic dermatitis-like skin inflammation with prolonged spontaneous scratching, and that skin barrier dysfunction is involved in the basal scratching. In the present study, the effects of ethanol on itch-related scratching were examined in this mouse model. When ethanol (30%, 10 ml/kg) was given orally to HR-AD-fed mice, scratching with long duration was further markedly increased, while oral ethanol administration had little effect on the scratching response in normal diet-fed mice. The scratching response after oral ethanol administration in HR-AD-fed mice (ethanol-induced scratching) was attenuated by antagonism of the mu-opioid receptor or local skin anesthesia, as in human itching. Ethanol-induced scratching was also suppressed by improvement of skin barrier function by an application of petrolatum ointment, while ethanol administration itself did not affect the function. This suggests that ethanol indirectly aggravates the basal scratching. Although antagonism of the transient receptor potential vanilloid-1 did not affect ethanol-induced scratching, blockade of ethanol actions in the central nervous system (CNS), including gamma-aminobutyric acid type A receptor antagonism and N-methyl-d-aspartate receptor activation, inhibited it. Taken together, the present study demonstrates that orally administered ethanol markedly aggravates itch-related scratching in HR-AD-fed mice developing atopic dermatitis, and suggests that the CNS depressant actions of ethanol play an important role in the aggravation.

Dibucaine effects on structural and elastic properties of lipid bilayers.

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPC) were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes.

Amphiphilic effects of dibucaine HCl on rotational mobility of n-(9-anthroyloxy)stearic acid in neuronal and model membranes.

We studied dibucaine's effects on specific locations of n-(9-anthroyloxy)palmitic acid or stearic acid (n-AS) within phospholipids of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV) and model membranes. Giant unilamellar vesicles (GUVs) were prepared with total lipids (SPMVTL) and mixture of several phospholipids (SPMVPL) extracted from SPMV. Dibucaine.HCl increased rotational mobility (increased disordering) of hydrocarbon interior, but it decreased mobility (increased ordering) of membrane interface, in both native and model membranes. The degree of rotational mobility in accordance with the carbon atom numbers of phospholipids comprising neuronal and model membranes was in the order at the 16, 12, 9, 6 and 2 position of aliphatic chain present in phospholipids. The sensitivity of increasing or decreasing effect of rotational mobility of hydrocarbon interior or surface region by dibucaine.HCl differed depending on the neuronal and model membranes in the descending order of SPMV, SPMVPL and SPMVTL.

Molecular basis of succinylcholine sensitivity in a prairie Hutterite kindred and genetic characterization of the region containing the BCHE gene.

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.