Biological functions of entactin

Steady-state and time-resolved fluorescence data are reported for the local anesthetic dibucaine in the absence and presence of phospholipid vesicles. These vesicles were comprised of dimyristylphosphatidyl choline and approximately 10% dimyristylphosphatidyl glycerol. Solute quenching studies show the bound drug to be protected from collision with iodide ion. The fluorescence lifetime of dibucaine is not significantly changed upon binding to vesicles. The fluorescence anisotropy of dibucaine increases upon association with the vesicles. Anisotropy decay measurements show that the rotational correlation time, phi, of bound dibucaine is increased about one hundred fold over that for free dibucaine. This indicates that the rotational motion of bound dibucaine is slowed by its interaction with the phospholipids. However, we find no evidence that the rotational motion of bound dibucaine is anisotropic.     

Normovolaemic haemodilution and hyperoxia have no effect on fractal dimension of regional myocardial perfusion in dogs

The neurotoxicity of dibucaine was compared with that of commercially available local anesthetics in studies using rabbit desheathed cervical vagus nerve preparation. Dibucaine dose-dependently suppressed the evoked action potential of myelinated A beta nerve component and nonmyelinated C nerve component. The potential of A beta nerve component was more strongly suppressed, compared with that of C nerve component. At low concentrations of 0.0001-0.001%, the suppression was reversible and recovery with C nerve component was faster and more complete. At higher concentrations, the suppression was irreversible. The minimum concentrations of irreversible blockade were 0.003% for A beta nerve component and 0.03% for C nerve component. Electron microscopically, marked damages in the myelin layer and intraaxonal structure were observed in nerves treated with 0.03% dibucaine. When the neurotoxic effect of dibucaine was compared, in terms of safety margins (minimum concentration of irreversible blockade/clinically used concentrations), with those of commercially available local anesthetics, the rank order was dibucaine, tetracaine and bupivacaine; dibucaine showing the lowest safety margin.     

Dibucaine interacts differently with membrane and protein in cytochrome c oxidase systems

Dibucaine acts as a weak protonophore in cytochrome c oxidase proteoliposomes. At low concentrations in the presence of permeant anions, it stimulates turnover and collapses enzyme-generated pH gradients. At higher concentrations, dibucaine inhibits activity of cytochrome c oxidase in proteoliposomes and the isolated enzyme. It also induces a red shift in the resting spectrum, indicating a change at the binuclear centre. This spectroscopic effect is kinetically biphasic. Dibucaine inhibits steady-state oxidase activity, but not the rate of the red shift in the cytochrome a3 Soret band during turnover. It reacts faster with the partially reduced state than with resting enzyme. The inhibition is kinetically biphasic with a noncompetitive Ki approximately 0.5 mM. Excess dibucaine effects a maximal turnover decline of 80%. At low ionic strength only the total Vmax is affected; tight binding of cytochrome c and turnover at the "tight" site are unaffected. Dibucaine may bind to an anionic site in a hydrophobic pocket, modifying electron transfer from cytochrome a and CuA to cytochrome a3 - CuB and the oxidized spectrum of the latter centre. Stimulation of turnover in cytochrome c oxidase in proteoliposomes is due to a separate membrane-dependent proton translocation catalysed by dibucaine in the presence of permeant anions.     

Role of metabolism in the biliary excretion of methadone metabolites

1. The effects of six local anaesthetics have been studied on the activities of soluble phospholipases A2 (EC 3.1.1.4) and lysophospholipase (EC 3.1.1.5). 2. Phospholipase A2 activity in human seminal plasma towards sonicated radioactively-labelled phosphatidylethanolamine was slightly stimulated a low and inhibited at high concentrations of all anaesthetic compounds employed. The order of decreasing potency was chlorpromazine, dibucaine, tetracaine, lidocaine, cocaine and procaine. In line with previous findings, the mode of inhibition was seen to be competitive with respect to Ca2+. 3. Phospholipase A2 activity in crude venom of Crotalus adamanteus was not affected or slightly stimulated by local anaesthetics up to 10(-2) M concentrations, when egg yolk was used as substrate. However, with sonicated radioactively-labelled phosphatidylcholine or phosphatidylethanolamine as substrate, stimulation of phospholipase activity was seen with all local anaesthetics up to 10(-2) M, the order of decreasing potency again being chlorpromazine, dibucaine, tetracaine, lidocaine, cocaine and procaine. The mode of stimulation was seen to be un-competitive with respect to substrate and probably independent of any involvement of Ca2+. 4. As in seminal plasma phospholipase A2, the activity in crude Naja naja venom towards sonicated radioactively labelled phosphatidylcholine was stimulated at low and inhibited at high concentrations of dibucaine and chloropromazine, for example. The mode of inhibition was seen to be competitive with respect to Ca2+, whereas stimulation by the anaesthetic drugs was independent of Ca2+. Binding between drug and enzyme was demonstrated by equilibration filtration of purified phospholipase A2 of Naja naja venom through a Sephadex G 25-fine column, previously equilibrated with 0.5 mM radioactively labelled chlorpromazine. 5. Lysophospholipase activity in rat liver cytosol towards radioactively labelled lysophosphatidylcholine was inhibited by all local anaesthetics used; the order of decreasing potency was chlorpromazine, dibucaine, tetracaine, cocaine, lidocaine and procaine. The inhibition was un-competitive with respect to substrate. 6. The inhibitory and stimulatory potencies of the local anaesthetics employed closely parallel their lipid solubilities and anaesthetic potencies.     

Spread of spinal anesthesia with 3 different hyperbaric solutions used in Japan

We studied the spread of spinal anesthesia with 3 different hyperbaric solutions commercially available in Japan. Percamin-S [0.3% dibucaine in 5% hyperbaric saline] (P), Neo-Percamin.S [0.24% dibucaine with 0.12% T-caine in 9.5% glucose] (N) and 0.5% Tetcaine [tetracaine] in 10% glucose (T) were studied. Two ml of each solution was administered intrathecally using a 25 gauge Quincke needle. Patients (n = 90) were allocated to one of 9 groups receiving 2 ml of P, N or T at L 2-3, L 3-4 or L 4-5 interspace. Both N and T produced significantly higher spread of analgesia than P at any of L 3-4 and L 4-5 interspaces. P and N have the same specific gravity, even though significant differences were found in spread of segmental analgesia. Local anesthesic agents and solvent solutions themselves are considered to influence the spread of spinal anesthesia as the specific gravity of hyperbaric solution does.     

Value of the monitoring of curarisation during prolonged mivacurium induced neuromuscular block

A case of neuromuscular blockade of about 200 min of duration, in a 9-year-old boy from mivacurium 0.15 mg.kg-1 is reported. The diagnosis was delayed, after onset of the first signs of recovery, due to the lack of monitoring of neuromuscular transmission. The neuromuscular blockade was reversed with neostigmine 0.04 mg.kg-1. Complete reversal required fifty minutes. The presence of an abnormal genetic variant of pseudocholinesterases was demonstrated by the measurements of pseudocholinesterase activity and dibucaine number. The importance of monitoring of neuromuscular transmission for diagnosis and treatment of mivacurium-induced neuromuscular blockade is underlined.     

Influence of heart rate, respiration and recipient atrial contraction on pulsed wave transmitral Doppler flow indices in orthotopic heart transplant recipients

Conformational features of charged dibucaine in [2H6]DMSO were elucidated by measuring 13C and 1H spin-lattice relaxation rates and 1H-(1H) and 13C-(1H) nuclear Overhauser effects. The reorientational correlation time of the aromatic moiety was evaluated at 0.16 ns at room temperature and side chains were observed to display segmental motion. Relevant distances were calculated by isolating dipolar interaction terms of 1H-1H or 13C-1H pairs. The 'preferred' conformation in solution was shown to present several analogies, but also some differences, with the structures obtained by solid state experiments, energy calculations and LIS data.     

Human butyrylcholinesterase L330I mutation belongs to a fluoride-resistant gene, by expression in human fetal kidney cells

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.     

A simple quantitative method to determine short chain fatty acid levels in biological fluids

The pseudocholinesterase levels and the nature of the enzyme as shown by the dibucaine number (D.N.) were estimated in 720 controls and 420 lepromatous leprosy patients, and 301 tuberculoid leprosy patients. There was no statistical difference in the esterase levels between leprosy patients and normal controls. But the distribution of D.N. was significantly different in the leprosy patients compared to the normal population studied. The D.N. below 40 indicates the samples with the atypical pseudocholinesterase--the presence of which is genetically determined. The distribution of samples with D.N. below 40 was significantly higher in the lepromatous leprosy patients compared to the normal population or tuberculoid leprosy patients. It is proposed that since there is a greater incidence of the atypical enzyme in lepromatous leprosy cases, the presence of this enzyme or the deficiency of the typical enzyme may make a person more susceptible to leprosy.     

Spectral analysis of intercycle heart fluctuations in the diethyl-ether-anaesthetized or pithed rat treated with l-hyoscyamine

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.     

Treatment of human pulmonary paragonimiasis with triclabendazole: clinical tolerance and drug efficacy

We report the anesthetic management of a patient with aortitis syndrome using combined spinal and epidural anesthesia. A 28-year-old gravida with aortitis syndrome accompanied by faints was scheduled for an urgent cesarean section. Combined spinal and epidural anesthesia was thought to be better for this case in order to monitor the cerebral circulation by her consciousness level and to reduce the hemodynamic change during surgery as compared to spinal or epidural anesthesia alone. After inserting an epidural catheter at the Th 12/L 1 interspace, spinal anesthesia was performed with 1.5 ml of 0.3% dibucaine at the L 4/L 5 interspace. The level of analgesia was under L 1 with the pinprick method 10 min after the spinal anesthesia. Next, 5 ml of 1.5% mepivacaine was injected through the epidural catheter. The level of analgesia reached to Th 6 without major hemodynamic changes. A healthy 2740 g infant was delivered and she had an uneventful recovery. We conclude that combined spinal and epidural anesthesia is useful in a patient with aortitis syndrome undergoing an urgent cesarean section in order to monitor the cerebral circulation by the consciousness level and to reduce the hemodynamic change.     

An electrophysiological and histological study on the neurotoxicity of lidocaine in excised rabbit cervical vagus nerve

We studied the neurotoxic effect of lidocaine at different concentrations on the desheathed rabbit vagus nerve by measuring the amplitudes of evoked compound action potentials and the histological changes of the nerve by means of the electron microscopy after incubation in lidocaine-Ringer's bicarbonate (RB) solution. The following results were obtained. 1) Minimum concentrations of lidocaine for producing complete conduction block (minimum blocking concentration, MBC) were 0.02% for A beta and A delta fibers, and 0.03% for C fibers. 2) Irreversible conduction blocks of compound action potentials were observed in relation with lidocaine concentrations and the duration of incubation: e.g. 0.5% for 2 hours incubation was equivalent to the block with 1% for 1 hour. 3) Degenerative change of axons was revealed morphologically in the preparations exposed to 2% or a higher concentration of lidocaine. 4) Risk ratio, which means a numerical value calculated as clinical concentration/irreversible concentration with 2 hr exposure, was similar to other local anesthetics except dibucaine HCl, which shows an higher risk ratio. However, it should be noted that lidocaine has an risk of producing irreversible changes in nerve fibers, when applied to the nervous tissue at higher concentrations for longer durations.     

A study of sexual behavior among rural residents of China

Besides the fast tetrodotoxin-sensitive Na+ current, small dorsal root ganglion neurones of rats also possess a slower tetrodotoxin-resistant Na+ current. The blocking effect of commonly used local anaesthetics upon the tetrodotoxin-resistant Na+ current was investigated in the present paper. Dorsal root ganglia were dissected from adult rats and cells were enzymatically isolated. The whole-cell patch clamp technique was then used to measure inward Na+ currents of small dorsal root ganglion neurones. Externally applied local anaesthetics reversibly blocked the tetrodotoxin-resistant Na+ current in a dose-dependent manner. Half-maximal blocking concentrations for tonic block were: lignocaine, 326 microM; prilocaine, 253 microM; mepivacaine, 166 microM; etidocaine, 196 microM bupivacaine, 57 microM procaine, 518 microM benzocaine, 489 microM; tetracaine, 21 microM; and dibucaine, 23 microM. Blocking of the current by lignocaine was independent of temperature. The quaternary lignocaine derivative OX-314 did not have any effect upon the tetrodotoxin-resistant Na+ current when applied externally. High concentrations of tetrodotoxin also blocked the tetrodotoxin-resistant Na+ current with a half-maximal blocking concentration of 115 microM. The block by high tetrodotoxin concentrations did not compete with the lignocaine block, suggesting that there were two independent blocking mechanisms for the two substances. The tetrodotoxin-resistant Na+ currents also showed a marked sensitivity to phasic (use-dependent) block by local anaesthetics.     

A structure-based approach to nicotinic receptor pharmacology

Infrared difference spectroscopy has been used to examine the structural effects of local anesthetic (LA) binding to the nicotinic acetylcholine receptor (nAChR). Several LAs induce subtle changes in the vibrational spectrum of the nAChR over a range of concentrations consistent with their reported nAChR-binding affinities. At concentrations of the desensitizing LAs prilocaine and lidocaine consistent with their binding to the ion channel pore, the vibrational changes suggest the stabilization of an intermediate conformation that shares structural features in common with both the resting and desensitized states. Higher concentrations of prilocaine and lidocaine, as well as the LA dibucaine, lead to additional binding to the neurotransmitter-binding site, the formation of physical interactions (most notably cation-tyrosine interactions) between LAs and neurotransmitter-binding-site residues, and the subsequent formation of a presumed desensitized nAChR. Although concentrations of the LA tetracaine consistent with binding to the ion channel pore elicit a reversed pattern of spectral changes suggestive of a resting state-like nAChR, higher concentrations also lead to neurotransmitter site binding and desensitization. Our results suggest that LAs stabilize multiple conformations of the nAChR by binding to at least two conformationally sensitive LA-binding sites. The spectra also reveal subtle differences in the strengths of the physical interactions that occur between LAs and binding-site residues. These differences correlate with LA potency at the nAChR.     

The action of local anesthetics on myelin structure and nerve conduction in toad sciatic nerve

X-ray scattering and electrophysiological experiments were performed on toad sciatic nerves in the presence of local anesthetics. In vitro experiments were performed on dissected nerves superfused with Ringer's solutions containing procaine, lidocaine, tetracaine, or dibucaine. In vivo experiments were performed on nerves dissected from animals anesthesized by targeted injections of tetracaine-containing solutions. In all cases the anesthetics were found to have the same effects on the x-ray scattering spectra: the intensity ratio of the even-order to the odd-order reflections increases and the lattice parameter increases. These changes are reversible upon removal of the anesthetic. The magnitude of the structural changes varies with the duration of the superfusion and with the nature and concentration of the anesthetic molecule. A striking quantitative correlation was observed between the structural effects and the potency of the anesthetic. Electron density profiles, which hardly showed any structural alteration of the unit membrane, clearly indicated that the anesthetics have the effect of moving the pairs of membranes apart by increasing the thickness of the cytoplasmic space. Electrophysiological measurements performed on the very samples used in the x-ray scattering experiments showed that the amplitude of the compound action potential is affected earlier than the structure of myelin (as revealed by the x-ray scattering experiments), whereas conduction velocity closely follows the structural alterations.     

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca2+]i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7+/-2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca2+]i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca2+]i increase up to 29.10+/-2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na+-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca2+]i since a significant [Ca2+]i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca2+]i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca2+]i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate.     

Local anesthetics inhibit receptors coupled to phosphoinositide signaling in Xenopus oocytes

Effects and the mechanism of action of quaternary amine local anesthetics on ligand- and voltage-activated ion currents were studied using voltage-clamped ovarian follicles and oocytes from Xenopus laevis. The fast inward and slow outward currents in response to acetylcholine were unaltered by procaine, whereas the oscillatory and smooth inward chloride currents (ICl) were abolished. Potassium currents (IK) elicited by norepinephrine and oscillatory ICl elicited by lysophosphatidic acid were blocked. Procaine caused a noncompetitive inhibition of oscillatory ICl mediated by heterologously expressed neurotransmitter receptors from the rat brain. Threefold differences were found in the procaine sensitivity of the 5-HT2a and 5-HT2c receptors. The rank order of intrinsic inhibitory activity of local anesthetics was: procaine > lidocaine > dibucaine > tetracaine. Extra- or intracellular application of procaine did not alter the Ca2+-activated Cl- current, indicating that neither the endogenous voltage-gated Ca2+ nor the Ca2+-activated Cl- channels account for the inhibition. Procaine caused only a slight reduction in ICl elicited by photolysis of caged inositol 1,4,5-trisphosphate (InsP3) and did not abolish ICl triggered by GTP[gamma-S]-induced direct activation of G proteins. For receptors coupling to the phosphoinositide/Ca2+ signal transduction pathway, the primary and physiologically relevant site of procaine action appears to be on the extracellular surface, upstream from the G protein, presumably on the receptor.     

Cholinesterases

OBJECTIVE: To review current data on butyrylcholinesterase. DATA SOURCES: Search through Medline data bases of articles in French or English. STUDY SELECTION: Original articles and case reports were selected. Letters to editor were excluded. DATA EXTRACTION: The articles were analyzed in order to obtain current data on biochemical structure, action, major pathological variations, especially with regard to the recent informations obtained by molecular biology concerning the identification of genetic variants. DATA SYNTHESIS: Butyrylcholinesterase must be differentiated from acetylcholinesterase, which cannot hydrolyse succinylcholine. The physiological action of butyrylcholinesterase remains unknown, although it can hydrolyse many drugs. Excluding genetical mutations, several physiopathological situations alter butyryl-cholinesterase activity. Butyrylcholinesterase activity assessment does not allow the diagnosis of genetic variants. Whatever the origin, only deficits of more than 50% modify significantly the metabolism of succinylcholine or mivacurium. The diagnosis of a prolonged neuromuscular blockade is obtained with systematic monitoring of the neuromuscular function in case of administration of mivacurium or succinylcholine. Mivacurium should only be re-injected when one response at train of four is obtained. In case of prolonged neuromuscular blockade, the anticholinesterasic agent should not be administered when no response at train of four is obtained. The biochemical methods using inhibitors (dibucaine, fluoride) of the butyrylcholinesterase and a familial study lead to the diagnosis in most cases because the atypical and fluoride variants are the most frequent. When results are doubtful, genetic molecular methods with the use of PCR and restriction enzymes allow a rapid diagnosis.     

Structure-function relationship studies in human cholinesterases reveal genomic origins for individual variations in cholinergic drug responses

1. Due to their involvement in the termination of neurotransmission at cholinergic synapses and neuromuscular junctions, cholinesterases are the target proteins for numerous drugs of neuro-psychopharmacology importance. 2. In order to perform structure-function relationship studies on human cholinesterases with respect to such drugs, a set of expression vectors was engineered, all of which include cloned cDNA inserts encoding various forms of human acetyl- and butyrylcholinesterase. These vectors were designed to be transcribed in vitro into their corresponding mRNA products which, when microinjected into Xenopus oocytes, are efficiently translated to yield their catalytically active enzymes, each with its distinct substrate specificity and sensitivity to selective inhibitors. 3. A fully automated microtiter plate assay for evaluating the inhibition of said enzymes by tested cholinergic drugs and/or poisons has been developed, in conjunction with computerized data analysis, which offers prediction of such inhibition data on the authentic human enzymes and their natural or mutagenized variants. 4. Thus, it was found that asp70-->gly substitution renders butyrylcholinesterase succinylcholine insensitive and resistant to oxime reactivation while ser 425-->Pro with gly70 gives rise to the "atypical" butyrylcholinesterase phenotype, abolishing dibucaine binding. 5. Furthermore, differences in cholinesterase affinities to physostigmine, ecothiophate and bambuterol were shown in these natural variants. 6. Definition of key residues important for drug interactions may initiate rational design of more specific cholinesterase inhibitors, with fewer side effects. This, in turn, offers therapeutic potential in the treatment of clinical syndromes such as Alzheimer's and Parkinson's disease, glaucoma and myasthenia gravis.     

Biochemical and functional properties of lysine-specific cysteine proteinase (Lys-gingipain) as a virulence factor of Porphyromonas gingivalis in periodontal disease

Intravenous (I.V.) cocaine (0.03-3 mg/kg) produced dose-dependent, rapid, and brief increases in blood pressure (BP) in conscious rats pretreated with the dopamine receptor antagonist, SCH 23390. Monoamine uptake inhibitors structurally analogous to cocaine (cocaethylene, CFT, betaCIT, CPT, (+)-cocaine, norcocaine, and benztropine) also produced this rapid pressor response, whereas structurally unrelated uptake inhibitors with diverse monoamine transporter selectivities (BTCP, indatraline, GBR 12935, mazindol, nomifensine, and zimeldine) either did not produce a rapid pressor response or produced only a small pressor response. At nonconvulsant doses, the sodium channel blockers acetylprocainamide, dibucaine, dyclonine, prilocaine, proparacaine, quinidine, and tetracaine produced a small pressor response or no increase in BP. In rats implanted with telemetric devices, cocaine and its analog, CFT, produced a biphasic pharmacological response that consisted of an initial brief and abrupt behavioral arousal associated with a rapid, large increase in BP followed by prolonged, parallel increases in BP and locomotor activity. Pretreatment with SCH 23390 prevented the prolonged but not the initial rapid and brief pressor and activity responses to both cocaine and CFT administration. The present data suggest that the inhibition of dopamine, norepinephrine, or serotonin transporter functions, either alone or in combination, does not mediate the rapid pressor response to cocaine. The sodium channel-blocking action of cocaine per se does not appear to be involved in the rapid pressor response to cocaine. Finally, the present results confirm previous findings that dopaminergic mechanisms mediate the prolonged increases in BP and locomotor activity produced by cocaine.