Interactions of new lactoglobulin variants with tetracaine: crystallographic studies of ligand binding to lactoglobulin mutants possessing single substitution in the binding pocket.

ß-Lactoglobulin (BLG) like other lipocalins can be modified by mutagenesis to re-direct its ligand binding properties. Local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change BLG ligand preferences. The presented studies are focused on previously described mutants L39Y, I56F, L58F, F105L, and M107L and two new BLG variants, L39K and F105A, and their interactions with local anesthetic drug tetracaine. Binding of tetracaine to BLG mutants was investigated by X-ray crystallography. Structural analysis revealed that for tetracaine binding, the shape of the binding pocket seems to be a more important factor than the substitutions influencing the number of interactions. Analyzed BLG mutants can be classified according to their binding properties to variants: capable of binding tetracaine in the ß-barrel (L58F, M107L); capable of accommodating tetracaine on the protein surface (I56F) and unable to bind tetracaine (F105L). Variants L39K, L39Y, and F105A, had a binding pocket blocked by endogenous fatty acids. The new tetracaine binding site was found in the I56F variant. The site localized on the surface near Arg124 and Trp19 was previously predicted by in silico studies and was confirmed in the crystal structure.

Development of a high-throughput fluorescent no-wash sodium influx assay.

Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495-575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1-1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1-1.7 activator veratridine (EC50 10-29 µM) or the NaV1.1-1.8 inhibitor tetracaine (IC50's 6-66 µM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.

Study of procaine and tetracaine in the lipid bilayer using molecular dynamics simulation.

Despite available experimental results, the molecular mechanism of action of local anesthetics upon the nervous system and contribution of the cell membrane to the process are still controversial. In this work, molecular dynamics simulations were performed to investigate the effect of two clinically used local anesthetics, procaine and tetracaine, on the structure and dynamics of a fully hydrated dimyristoylphosphatidylcholine lipid bilayer. We focused on comparing the main effects of uncharged and charged drugs on various properties of the lipid membrane: mass density distribution, diffusion coefficient, order parameter, radial distribution function, hydrogen bonding, electrostatic potential, headgroup angle, and water dipole orientation. To compare the diffusive nature of anesthetic through the lipid membrane quantitatively, we investigated the hexadecane/water partition coefficient using expanded ensemble simulation. We predicted the permeability coefficient of anesthetics in the following order: uncharged tetracaine > uncharged procaine > charged tetracaine > charged procaine. We also shown that the charged forms of drugs are more potent in hydrogen bonding, disturbing the lipid headgroups, changing the orientation of water dipoles, and increasing the headgroup electrostatic potential more than uncharged drugs, while the uncharged drugs make the lipid diffusion faster and increase the tail order parameter. The results of these simulation studies suggest that the different forms of anesthetics induce different structural modifications in the lipid bilayer, which provides new insights into their molecular mechanism.

Assessing the Potential for Drug-Nanoparticle Surface Interactions To Improve Drug Penetration into the Skin.

There is continued debate as to how nanomaterials enhance the passive diffusion of drugs through the skin. This study examined if drug-nanoparticle surface interactions, which occurred during topical application, had the capability to enhance percutaneous penetration. Atomic force microscopy force adhesion measurements were used to demonstrate that a model drug, tetracaine, strongly adsorbed to the surface of a negatively charged carboxyl-modified polystyrene nanoparticle (NanoPSCOOH) through both its methyl and amine functionalities (up to a 6- and 16-fold greater adhesion force respectively compared with the CH3-CH3 control). These drug-particle adhesion forces were significantly reduced (p < 0.05) to values that were lower than the CH3-CH3 control measurements when tetracaine interacted with a silica nanoparticle (NanoSiO2). This reduction in adhesion was attributed to the lower surface charge of the NanoSiO2 (ca. -23 mV) compared to the NanoPSCOOH (ca. -40 mV), which diminished the electrostatic interactions between positive amine of tetracaine and the negative particle. Mixing NanoPSCOOH with tetracaine on the skin retarded percutaneous drug penetration compared to the control (tetracaine saturated solution without nanoparticles), but the NanoSiO2, which still adsorbed the tetracaine, produced a 3.6-fold enhancement in percutaneous penetration compared to the same control. These data demonstrated the capability of moderate nanoparticle surface interactions that occurred within the application vehicle to promote drug percutaneous penetration.

Investigating the influence of drug aggregation on the percutaneous penetration rate of tetracaine when applying low doses of the agent topically to the skin.

Understanding the molecular aggregation of therapeutic agents is particularly important when applying low doses of a drug to the surface of the skin. The aim of this study was to understand how the concentration of a drug influenced its molecular aggregation and its subsequent percutaneous penetration after topical application. A model drug tetracaine was shown to form a series of different aggregates across the µM (fluorescence spectroscopy) to mM (light scattering analysis) concentration range. The aggregate formation process was sensitive to the pH of the vehicle in which the drug was dissolved (pH 4, critical aggregation concentration (CAC) - 11 µM; pH 8, CAC - 7 µM) and it appeared to have an impact upon the drug's percutaneous penetration. At pH 4, increasing the concentration of the drug in the donor solution decreased the skin permeability coefficient (Kp) of tetracaine (13.7 ± 4.3 × 10(-3)cm/h to 0.06 ± 0.02 × 10(-3)cm/h), whilst at pH 8, it increased the Kp (29.9 ± 9.9 × 10(-3)cm/h to 75.1 ± 41.7 × 10(-3)cm/h). These data trends were reproduced in a silicone membrane and this supported the notion that the more polar aggregates formed at pH 4 acted to decrease the proportion of species available to pass through the skin, whilst the more hydrophobic aggregates formed in pH 8 increased the membrane diffusing species.

Novel serine-based gemini surfactants as chemical permeation enhancers of local anesthetics: A comprehensive study on structure-activity relationships, molecular dynamics and dermal delivery.

This work aims at studying the efficacy of a series of novel biocompatible, serine-based surfactants as chemical permeation enhancers for two different local anesthetics, tetracaine and ropivacaine, combining an experimental and computational approach. The surfactants consist of gemini molecules structurally related, but with variations in headgroup charge (nonionic vs. cationic) and in the hydrocarbon chain lengths (main and spacer chains). In vitro permeation and molecular dynamics studies combined with cytotoxicity profiles were performed to investigate the permeation of both drugs, probe skin integrity, and rationalize the interactions at molecular level. Results show that these enhancers do not have significant deleterious effects on the skin structure and do not cause relevant changes on cell viability. Permeation across the skin is clearly improved using some of the selected serine-based gemini surfactants, namely the cationic ones with long alkyl chains and shorter spacer. This is noteworthy in the case of ropivacaine hydrochloride, which is not easily administered through the stratum corneum. Molecular dynamics results provide a mechanistic view of the surfactant action on lipid membranes that essentially corroborate the experimental observations. Overall, this study suggests the viability of these serine-based surfactants as suitable and promising delivery agents in pharmaceutical formulations.

Differential interactions of two local anesthetics with phospholipid membrane and nonerythroid spectrin: Localization in presence of cholesterol and ganglioside, GM1.

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.

Interaction of local anesthetics with lipid bilayers investigated by ¹H MAS NMR spectroscopy.

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by ³¹P and ¹H magic-angle spinning (MAS) NMR spectroscopy. The ³¹P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.

Liquid chromatography-tandem mass spectrometry detection of the quaternary ammonium compound mebezonium as an active ingredient in t61.

Quaternary ammonium compounds pose an analytical challenge. Mebezonium, a muscle-relaxing agent contained in veterinary euthanasia solution T61, was analyzed in body fluids, organs, and injection sites of a veterinarian by liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Additionally, embutramide and tetracaine, which are two other active ingredients contained in T61, methadone, xylazine, and analgesics were detected by LC-MS-MS and high-performance liquid chromatography-ultraviolet detection methods. For detection of mebezonium a solid-phase extraction (SPE) combined with ionpairing reagent heptafluorobutyric acid was developed. Separation was achieved on Phenomenex Synergi Hydro RP C(18) column combined with ammonium formate buffer and acetonitrile (pH 3.5). To enrich other drugs, liquid-liquid extraction procedures were used. Most of these drugs were separated on a Restek Allure PFP Propyl column using the mentioned mobile phase. Mebezonium and embutramide were detected in femoral vein serum in concentrations of 10.9 and 2.0 mg/L, respectively. The concentration of xylazine and methadone in serum was 2.0 and 0.4 mg/L, respectively. The LC-MS-MS method with SPE combined with an ion-pairing reagent allowed the quantitation of mebezonium. Methadone was detected in toxic concentrations and was, in combination with xylazine and T61, considered to be the cause of death.

Microcalorimetric and zeta potential study on binding of drugs on liposomes.

In this work, isothermal titration calorimetry (ITC) combined with zeta potential measurements was used to study the binding and partitioning of three beta-blockers, alprenolol, labetalol and propranolol, and the local anaesthetic tetracaine into liposomes. The thermodynamic parameters of enthalpy, entropy, the Gibbs energy and the binding constant were determined using the one site model. Furthermore, the binding constants corrected for the electrostatic contribution were used to assess the partition coefficients for the drugs. Also, the effect of the concentration, ionic strength, temperature and membrane curvature on the interaction was included in the evaluation.

A rapid methodology for the characterization of dialkyl tertiary amine-N-oxide metabolites using structurally dependent dissociation pathways and reconstructed ion current chromatograms.

A high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) approach to the characterization of dialkyl tertiary amine-N-oxides is presented. The methodology is based upon forming reconstructed ion current chromatograms (RICCs) of m/z values of product ions known to form through diagnostic losses from dialkyl tertiary amine-N-oxides. The diagnostic losses of N,N-dimethylhydroxylamine and N,N-diethylhydroxylamine were identified through the analysis of a structurally diverse library of compounds by ESI-low-energy collision-induced dissociation (CID)-MS/MS using quadrupole ion trap-mass spectrometry (QIT-MS) and quadrupole time-of-flight-mass spectrometry (QqTOF-MS). The library consisted of dialkyl tertiary amine-containing commercially available pharmaceuticals, along with a number of model, synthetic N-oxides. The loss of the nitrogen-containing group was observed in 89% of the low-energy CID product ion spectra acquired using various collision energies. Further, the resultant product ions, formed through the loss of the nitrogen-containing group, were shown to be unstable because of the observation of second-generation dissociation. These observations regarding gas-phase ion chemistry could be useful to developers of in silico programs for fragmentation prediction by allowing the creation of improved algorithms and models for predicting dissociation. Using the information derived from the library analysis, the characterization methodology was developed and demonstrated using tetracaine. The approach is rapid, MS/MS platform independent, utilizes existing technology, and could be automated. Further, it is definitive and overcomes the limitations of other tools for N-oxide identification by localizing the site of oxidation. Thus, it provides a useful addition to the existing approaches for metabolite identification.

Structural determinants of drugs acting on the Nav1.8 channel.

The aim of this work is to study the role of pore residues on drug binding in the Na(V)1.8 channel. Alanine mutations were made in the S6 segments, chosen on the basis of their roles in other Na(V) subtypes; whole cell patch clamp recordings were made from mammalian ND7/23 cells. Mutations of some residues caused shifts in voltage dependence of activation and inactivation, and gave faster time course of inactivation, indicating that the residues mutated play important roles in both activation and inactivation in the Na(V)1.8 channel. The resting and inactivated state affinities of tetracaine for the channel were reduced by mutations I381A, F1710A, and Y1717A (for the latter only inactivated state affinity was measured), and by mutation F1710A for the Na(V)1.8-selective compound A-803467, showing the involvement of these residues for each compound, respectively. For both compounds, mutation L1410A caused the unexpected appearance of a complete resting block even at extremely low concentrations. Resting block of native channels by compound A-803467 could be partially removed ("disinhibition") by repetitive stimulation or by a test pulse after recovery from inactivation; the magnitude of the latter effect was increased for all the mutants studied. Tetracaine did not show this effect for native channels, but disinhibition was seen particularly for mutants L1410A and F1710A. The data suggest differing, but partially overlapping, areas of binding of A-803467 and tetracaine. Docking of the ligands into a three-dimensional model of the Na(V)1.8 channel gave interesting insight as to how the ligands may interact with pore residues.

Resonance Rayleigh scattering spectra, non-linear scattering spectra of tetracaine hydrochloride-erythrosin system and its analytical application.

The interaction between erythrosine (ET) and tetracaine hydrochloride (TA) was studied by resonance Rayleigh scattering (RRS), frequency doubling scattering (FDS) and second-order scattering (SOS) combining with absorption spectrum. In a weak acidic medium of Britton-Robinson (BR) buffer solution of pH 4.5, erythrosine reacted with tetracaine hydrochloride to form 1:1 ion-association complex. As a result, the new spectra of RRS, SOS and FDS appeared and their intensities enhanced greatly. The maximum peaks of RRS, SOS and FDS were at 342 nm, 680 nm and 380 nm, respectively. The intensities of the three scattering were directly proportional to the concentration of TA in the range of 0.008-4.2 microg mL(-1) for RRS, 0.027-4.2 microg mL(-1) for SOS and 0.041-4.2 microg mL(-1) for FDS. The methods had very high sensitivities and good selectivities, and the detection limits were 0.003 microg mL(-1) for RRS, 0.008 microg mL(-1) for SOS and 0.012 microg mL(-1) for FDS, respectively. Therefore, a new method was developed to determinate trace amounts of TA. The recovery for the determination of TA in blood serum and urine samples was between 97.0% and 103.8%. In this study, mean polarizability was calculated by AM1 quantum chemistry method. In addition, the reasons for the enhancement of scattering spectra and the energy transfer between absorption, fluorescence and RRS were discussed.

Access and binding of local anesthetics in the closed sodium channel.

Local anesthetics (LAs) are known to bind Na+ channels in the closed, open, and inactivated states and reach their binding sites via extracellular and intracellular access pathways. Despite intensive studies, no atomic-scale theory is available to explain the diverse experimental data on the LA actions. Here we attempt to contribute to this theory by simulating access and binding of LAs in the KcsA-based homology model of the closed Na+ channel. We used Monte Carlo minimizations to model the channel with representative local anesthetics N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium (QX-314), cocaine, and tetracaine. We found the nucleophilic central cavity to be a common binding region for the ammonium group of LAs, whose aromatic group can extend either along the pore axis (vertical binding mode) or to the III/IV domain interface (horizontal binding mode). The vertical mode was earlier predicted for the open channel, but only the horizontal mode is consistent with mutational data on the closed-channel block. To explore hypothetical access pathways of the permanently charged QX-314, we pulled the ligand via the selectivity filter, the closed activation gate, and the III/IV domain interface. Only the last pathway, which leads to the horizontal binding mode, did not impose steric obstacles. The LA ammonium group mobility within the central cavity was more restricted in the vertical mode than in the horizontal mode. Therefore, occupation of the selectivity-filter DEKA locus by a Na+ ion destabilizes the vertical mode, thus favoring the horizontal mode. LA binding in the closed channel requires the resident Na+ ion to leave the nucleophilic central cavity through the selectivity filter, whereas the LA egress should be coupled with reoccupation of the cavity by Na+. This hypothesis on the coupled movement of Na+ and LA in the closed channel explains seemingly contradictory data on how the outer-pore mutations as well as tetrodotoxin and micro-conotoxin binding affect the ingress and egress of LAs.

The effects of prehydration on the properties of cerebrospinal fluid and the spread of isobaric spinal anesthetic drug.

BACKGROUND: In a two-part clinical study, we investigated the effect of the administration of fluids "prehydration" on the physical properties of cerebrospinal fluid (CSF) and intrathecal spread of local anesthetics. METHODS: First, in the clinical spinal anesthesia study, 68 patients were allocated randomly into the prehydration or nonprehydration groups. One group was prehydrated with 10 mL/kg of lactated Ringer's solution, and spinal anesthesia was performed with 12 mg of 0.5% isobaric tetracaine in all patients at the lumbar level. The arterial blood pressure, heart rate, and sensory block level were assessed. Second, in a magnetic resonance image study, 24 male volunteers were enrolled. CSF motion variables were measured after infusion of 10 mL/kg of lactated Ringer's solution to examine the net flow and volume displacement of the CSF at the L2-3 disk level. RESULTS: In the clinical study, there were no significant differences in arterial blood pressure, heart rate, and median peak sensory block level between the two groups, but the median time to reach peak sensory block level (26.4 +/- 15.7 vs 16.5 +/- 9.2 min, P < 0.05) was longer in group P. In posthydration magnetic resonance images, the CSF regurgitant fraction (caudal flow) was significantly increased after hydration, but the stroke volume, absolute stroke volume, mean flux, stroke distance, and mean velocity in the cranial direction were significantly decreased. CONCLUSIONS: Rapid crystalloid prehydration can affect CSF flow in the lumbar region, reducing cephalic spread of 0.5% isobaric tetracaine and delaying the time to reach the peak sensory level.

Lipid composition alters drug action at the nicotinic acetylcholine receptor.

We tested the hypothesis that membrane lipid composition influences drug action at membrane proteins by studying local anesthetic action at the nicotinic acetylcholine receptor (nAChR). Infrared difference spectra show that concentrations of tetracaine consistent with binding to the ion channel (<50 microM) stabilize a resting-like state when the nAChR is reconstituted into phosphatidylcholine membranes containing the anionic lipid, phosphatidic acid, but have no effect on the nAChR reconstituted into membranes lacking phosphatidic acid, either in the presence or absence of cholesterol. Concentrations of tetracaine above 200 microM lead to neurotransmitter site binding in all membranes. In the presence of phosphatidic acid, cholesterol, or both, neurotransmitter site binding leads to the formation of quaternary amine-aromatic interactions between tetracaine and binding site tyrosine/tryptophan residues and the stabilization of a desensitized state. One interpretation suggested by lipid partitioning studies is that phosphatidic acid enhances tetracaine action at the channel pore by increasing the partitioning of tetracaine into the lipid bilayer, thereby enhancing access to the transmembrane pore. However, subtle membrane-dependent variations in the vibrations of tyrosine and tryptophan residues, and agonist analog binding studies indicate that the structures of the agonist-bound neurotransmitter sites of the nAChR in membranes lacking both phosphatidic acid and cholesterol differ from the structures of the agonist-desensitized neurotransmitter sites in the presence of both lipids. Lipid action at the nAChR thus involves more than a simple modulation of the equilibrium between resting and desensitized states.

Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization.

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.

Photo-induced covalent attachment of agonists as a tool to study allosteric mechanisms of nicotinic acetylcholine receptors.

Muscular and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels and contain either two or five binding sites for acetylcholine (ACh). Binding of ACh molecules on the nAChR will trigger the fast opening of the channel and subsequent slow desensitization process. Neuronal alpha7 nicotinic receptors are made up of five identical subunits and possess five binding sites for ACh; this raises the question of how many sites must be occupied before channel opening. However, the effect of each ligand binding on gating is difficult to assess because of the reversible aspect of ligand binding at each site. One solution is to photochemically tether agonists to their binding sites. Such methodology has been applied elegantly and successfully on the homotetrameric cyclic-nucleotide-gated channels to evaluate the functional effects of each ligand binding on gating (Ruiz and Karpen, 1997). We therefore decided to develop a similar approach on Torpedo and neuronal alpha7 nAChRs with the photoactivatable agonist AC5 to investigate the effect of binding site occupancy on allosteric transitions of the receptor. In the dark, AC5 (see structure below) evokes robust currents on oocytes expressing Torpedo nAChR, displaying maximal amplitude comparable to ACh, with EC50 = 1.2 microM (Mourot et al., 2005). When the voltage-clamp oocyte was exposed to UV light in the presence of 30 microM AC5 for 50 s, there was a prolonged activation of the Torpedo nAChR, not reversible by washing, but inhibited by the noncompetitive blockers tetracaine and proadifen (see structure below). Both UV light and AC5 are required for this effect. However, further studies are required to determine whether the gradual decrease of the inward current reflects a slow desensitization process. AC5 is thus a potent photoactivatable agonist of the nAChR, which is able, upon UV irradiation, to incorporate covalently into the ACh-binding sites and to prolong activation of the nAChR. By extending this methodology to patch-clamp experiments, we will be able to incorporate one or several AC5s covalently into the muscular and neuronal nAChR at the single-channel level. Such study will help us understand the observed cooperative effect of gating and will contribute decisively to the controversial concerted vs sequential models for nAChR allosteric transitions.

Blood concentrations of tetracaine and its metabolite following spinal anesthesia.

Blood concentrations of tetracaine and its metabolite, p-butylaminobenzoic acid, were measured after spinal anesthesia with tetracaine which had been administered to patients under going orthopedic surgery. Tetracaine, an ester anesthetic, was given to 10 patients, the dose was 8-14mg, and blood samples were collected 1, 2 and 6h after the injection of tetracaine. We used gas chromatography/mass spectrometry for purposes of analysis. Tetracaine was not detected in any blood sample, but the metabolite was detected in each sample with the mean concentrations of 126.5, 97.9 and 43.3ng/ml at 1, 2 and 6h, respectively. This data will be useful in determination of the cause of death after spinal anesthesia with tetracaine.

Distribution of tetracaine and its metabolite in rabbits after high versus normal spinal anesthesia.

High spinal anesthesia is one cause of sudden death associated with the spinal anesthesia. We did animal experiments to verify high spinal anesthesia by analyzing tetracaine and its metabolite, p-butylaminobenzoic acid in tissue samples. Tetracaine (0.25% in 10% glucose solution) 0.21-0.28 mg/kg was administered to two groups of rabbits to induce high and normal spinal anesthesia. Tetracaine and the metabolite in rabbit tissues were analyzed by gas chromatography-mass spectrometry, as a free base for tetracaine and as tert-butyldimethylsilyl derivative for the metabolite. In the group given high spinal anesthesia, levels of the metabolite in the brain stem were higher than in the cerebrum, cerebellum and whole blood. On the other hand, in the group given normal spinal anesthesia, the opposite results were obtained. Therefore, high spinal anesthesia induced by tetracaine can be diagnosed by comparing the concentrations of metabolite in whole blood, cerebrum, cerebellum and brain stem.