The calcium requirement for functional vesicle development and nitrogen fixation by Frankia strains EAN1pec and CpI1

A calcium requirement was shown for both vesicle development and nitrogenase activity by Frankia strains EAN1_pec and CpI1. Washing cells with EGTA or EDTA inhibited both vesicle development and nitrogenase activity. The inhibition of both was reversed by the addition of calcium. A variety of agents known to affect calcium-dependent biological processes, such as a Ca-ATPase inhibitor, Ca-channel blockers, Ca-ionophores, calmodulin antagonists and the local anaesthetics, tetracaine and dibucaine, inhibited nitrogenase activity. Respiratory studies showed that a CN-insensitive respiration process occurred only under nitrogen derepressing conditions. Respiration by NH₄Cl-grown cells was completely inhibited by KCN while N₂-grown cells were inhibited by only 70%. Removal of calcium ions by EGTA or by the addition of dibucaine or tetracaine blocked the CN-insensitive respiration. This CN-insensitive respiration may be involved in protecting nitrogenase inside the vesicles from oxygen.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-( amino-ethyl ether) N,N¹-tetraacetic acid - GI germination inhibitor - MOPS 3-[N-morpholino] propane sulfonic acid - PCMBS p-chloromercuribenzene sulphonate - TMB 8,8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate     

Effects of local anesthetics on phospholipid topology and dopamine uptake and release in rat brain synaptosomes

Summary The effects of local anesthetics on the topology of aminophospholipids and on the release and uptake of dopamine in rat brain synaptosomes have been examined. A metabolically intact preparation of synaptosomes was prepared which maintains aminophospholipid asymmetry and the capacity for sodium-driven uptake and depolarization-dependent release of dopamine. Incubation of synaptosomes with local anesthetics at 37°C induced perturbations in the topology of aminophospholipids as determined by their reactivities to the covalent probe trinitrobenzenesulfonic acid. The reaction of trinitrobenzenesulfonate with phosphatidylethanolamine and phosphatidylserine was inhibited 10–20% by low concentrations of tetracaine (1–100 m) and enhanced by high concentrations (0.3–1.0mm). Other local anesthetics showed a similar biphasic effect with a potency order of dibucaine>tetracaine>lidocaineprocaine. K⁺-stimulated, Ca²⁺-dependent release of [³H]dopamine was inhibited significantly at low concentrations of tetracaine (1–10 m) but enhanced at higher concentrations (0.1–1.0mm). Dibucaine and procaine had a similar biphasic effect on the dopamine release. For each of the local anesthetics tested, the inhibition of the reaction of phosphatidylethanolamine and phosphatidylserine with trinitrobenzenesulfonate occurred at concentrations which were shown also to inhibit the release of [³H]dopamine. Local anesthetics were shown to inhibit uptake of [³H]dopamine with a potency order which reflects their potency in producing anesthesia. The inhibition of dopamine uptake by dibucaine, tetracaine, lidocaine, or procaine was characterized by inhibitory constants (K _I ) of 1.8±0.4 m, 27±5 m, 190 m and 0.5mm, respectively.Abbreviations TNBS 2,4,6-trinitrobenzene sulfonate - PE phosphatidylethanolamine - PS phosphatidylserine - ESR electron spin resonance - TLC thin-layer chromatography - DA dopamine     

Influence of local and neutral anaesthetics on the polymorphic phase preferences of egg yolk phosphatidylethanolamine

(1) The polymorphic phase preferences of egg phosphatidylethanolamine have been examined in the presence of normal alcohols and alkanes of varying chain length, as well as charged amine anaesthetics. (2) It is shown that the charged anaesthetics, ethanol and butanol can stabilize a bilayer arrangement for egg phosphatidylethanolamine. In contrast, longer chain ($\text{C}\text{?6}$) normal alcohols and alkanes induce the hexagonal (H_II) phase. (3) The relative potency of local anaesthetics in vitro (chlorpromazine, dibucaine, tetracaine and procaine) is mirrored by their relative ability to stabilize bilayer structure for hydrated egg phosphatidylethanolamine. Further, the aqueous concentrations of anaesthetic required to affect phospholipid polymorphism is sensitive to the lipid composition. For example, the inclusion of 20 mol% egg phosphatidylserine in egg phosphatidylethanolamine dispersions can reduce the aqueous concentrations of dibucaine required to induce appreciable bilayer stabilization effects from 5.0 mM to 0.5 mM. (4) It is suggested that the ability of amphipatic molecules such as anaesthetics to influence phosphatidylethanolamine polymorphism arises from their molecular shape. The possibility that anaesthetic molecules may exert their effects by virtue of this shape property is raised.     

Inhibition of synaptosomal enzymes by local anesthetics

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na⁺ + K⁺)-ATPase (ouabain-sensitive), Mg²⁺-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPase, for example 7.9 and 10 mM tetracaine for Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F₁-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.     

Further studies on F1-ATPase inhibition by local anesthetics

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F₁ extracted from these particles. All of these agents cause inhibition of ATPase in F₁ as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F₁. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F₁ complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.     

On the mechanism of inhibition of the bovine heart F1-ATPase by local anesthetics

The rate of inactivation of the mitochondrial F₁-ATPase by dicyclohexylcarbodiimide is slowed by concentrations of chlorpromazine, dibucaine, or tetracaine which have been shown by others (B. Chazotte, G. Vanderkooi, and D. Chignell (1982)$\text{Biochim. Biophys. Acta}\text{680}$, 310–316) to inhibit the hydrolytic reaction catalyzed by the enzyme. The order of effectiveness of the drugs as protectors of the enzyme against inactivation by dicyclohexylcarbodiimide is: chlorpromazine > dibucaine > tetracaine. Examination of the steady state kinetics showed the chlorpromazine inhibits the ATPase competitively at concentrations up to 18.5 μM while complex kinetic behavior is exhibited at chlorpromazine concentrations from 25–50 μM. These results suggest that the drugs inhibit the F₁-ATPase by interacting with the catalytic site of the enzyme and not by promoting its dissociation.     

Simultaneous determination of local anesthetics including ester-type anesthetics in human plasma and urine by gas chromatography–mass spectrometry with solid-phase extraction

The present study describes the simultaneous determination of seven different kinds of local anesthetics and one metabolite by GC–MS with solid-state extraction: Mepivacaine, propitocaine, lidocaine, procaine (an ester-type local anesthetics), cocaine, tetracaine (an ester-type local anesthetics), dibucaine (Dib) and monoethylglycinexylidide (a metabolite of lidocaine) were clearly separated from each other and simultaneously determined by GC–MS using a DB-1 open tubular column. Their recoveries ranged from 73–95% at the target concentrations of 1.00, 10.0 and 100 μg/ml in plasma, urine and water. Coefficients of variation of the recoveries ranged from 2.3–13.1% at these concentrations. The quantitation limits of the method were approximately 100 ng/ml for monoethylglycinexylidide, propitocaine, procaine, cocaine, tetracaine and dibucaine, and 50 ng/ml for lidocaine and mepivacaine. This method was applied to specimens of patients who had been treated with drip infusion of lidocaine, and revealed that simultaneous determination of lidocaine and monoethylglycinexylidide in the blood and urine was possible.     

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.     

Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

We have studied the effects of local anesthetics (dibucaine, tetracaine, lidocaine, and procaine) on calcium fluxes through the plasma membrane of synaptosomes. All these local anesthetics inhibit the ATP-dependent calcium uptake by inverted plasma membrane vesicles at concentrations close to those that promote an effective blockade of the action potential. The values obtained for the K0.5 of inhibition of calcium uptake are the following: 23 microM (dibucaine), 0.44 mM (lidocaine), 1.5 mM (procaine), and 0.8 mM (tetracaine). There is a good correlation between these K0.5 values and the concentrations of the local anesthetics that inhibit the Ca2(+)-dependent Mg2(+)-ATPase of these membranes. In addition, except for procaine, these local anesthetics stimulate severalfold the Ca2+ outflow via the Na+/Ca2+ exchange in these membranes. This effect, however, is observed at concentrations slightly higher than those that effectively inhibit the ATP-dependent Ca2+ uptake, e.g., 80-700 microM dibucaine, 2-10 mM lidocaine, and 1-3 mM tetracaine. The results suggest that the Ca2+ buffering of neuronal cytosol is altered by these anesthetics at pharmacological concentrations.     

Influence of anaesthetics on the movement of the mobile charges in the algal cell membrane of Valonia utricularis

Voltage relaxation studies in the presence of anaesthetics were performed on cells of the giant marine alga Valonia utricularis using intracellular microelectrodes. From the decay of the initial membrane voltage which can be described by two relaxation processes the conclusion can be drawn that protein-linked, mobile charges are present which are probably involved in turgor-pressure-dependent potassium transport (Büchner, K.-H., Rosenheck, K. and Zimmermann, U. (1985) J. Membrane Biol. 88, 131-137). The anaesthetics halothane and chloroform were found to affect reversibly, procaine and tetracaine irreversibly the translocation rate k of the mobile charges at concentrations which were equal to (for halothane and chloroform) or significantly below (for procaine and tetracaine) clinical and nerve blocking levels. The concentration of the mobile charges Nt as well as the specific membrane resistance Rm and the specific membrane capacitance Cm remained unchanged in these concentration ranges. The data suggest a specific interaction of anaesthetics with specialized target sites of a transport protein to which the mobile charges are coupled.     

Locations and dynamical perturbations for lipids of cationic forms of procaine, tetracaine, and dibucaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy

Locations and dynamical perturbations for lipids of local anesthetics (procaine . HCl, tetracaine . HCl, and dibucaine . HCl) in sonicated egg yolk phosphatidylcholine (PC) vesicles have been studied by 1H-1H nuclear Overhauser effect (NOE) measurements. It was found that tetracaine and dibucaine bind much strongly to the neutral lipids than does procaine and that their mobilities are lowered to such an extent that spin diffusion is transmitted (i.e., omega 2 tau c2 much greater than 1). The intermolecular NOEs between drugs and PC were more effective in the case of dibucaine than with tetracaine, indicating that dibucaine binds to the lipids more strongly than tetracaine; this order agrees well with that of anesthetic potency. However, it was only tetracaine that gave any appreciable dynamical perturbation to the PC vesicles when they were monitored by the extent of transfer of the negative NOE from alpha-methylene protons to choline methyls, olefinic methines, acyl methylenes and terminal methyl protons. This finding was interpreted as being due to the differences in the locations of these drugs in small unilamellar vesicles: (1) procaine interacts with lipids very weakly at the outer surface of the vesicles; (2) tetracaine binds to the lipids both at the outer and inner halves of the bilayer, inserting its rod-like molecule in a forest of acyl chains of PC; (3) dibucaine binds tightly to the polar head-group of PC, which resides only at the outer half of the bilayer vesicles. It was concluded that the relative order of anesthetic potency within these drugs can be correlated not with the ability to affect membrane fluidity but with the ability to bind to lipids at the polar head-group of the bilayer vesicles.     

Further studies on F1-ATPase inhibition by local anesthetics

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F1 extracted from these particles. All of these agents cause inhibition of ATPase in F1 as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F1. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F1 complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.     

Local anesthetic inhibition of pancreatic phospholipase A2 action on lecithin monolayers.

Using quantitative data previously reported for the penetration of local anesthetics into lecithin monolayers, the effects of surface and subphase concentrations of anesthetics on the inhibition of pancreatic phospholipase A2 action on didecanoyl phosphatidylcholine monolayers was investigated. Inhibition as a function of subphase concentration of anesthetic was in the order: dibucaine greater than tetracaine greater than butacaine greater than lidocaine = procaine. Inhibition as a function of surface concentration showed no obvious correlation; procaine inhibited at a very low surface concentration, followed by lidocaine at a somewhat higher concentration, and tetracaine, butacaine and dibucaine only at rather high concentrations. Ultraviolet difference spectroscopy indicated an interaction between lidocaine and enzyme in the subphase. Fluorescence studies showed that lidocaine is a competitive inhibitor of enzyme-lipid interface interaction. It is proposed that the more surface-active anesthetics inhibit by surface effects while the less surface-active anesthetics (lidocaine and procaine) inhibit by interaction with the enzyme in the subphase, which prevents enzyme penetration at the monolayer interface.     

ACCUMULATION OF ADENOSINE 3'',5''-MONOPHOSPHATE IN BRAIN SLICES: INTERACTION OF LOCAL ANAESTHETICS AND DEPOLARIZING AGENTS

Abstract— Membrane depolarizing agents such as veratridine, ouabain and high concentrations of potassium ions elicit a remarkable accumulation of cyclic AMP in brain slices incubated in vitro , and this accumulation, but not that elicited by biogenic amines, is prevented by a membrane stabilizer, cocaine. The effect of various local anaesthetics (compounds which are known to stabilize the membrane of peripheral sensory nerves) on the accumulation of cyclic AMP elicited by depolarizing agents in incubated slices of guinea pig brain has now been examined. At optimal concentrations the anaesthetics inhibited by more than 95 per cent the accumulation of cyclic AMP elicited with veratridine, ouabain, and high concentrations of potassium ions. The order of the inhibitory potency vs. veratridine was: dibucaine (ED₅₀= 9.5 ± 10⁻⁶ M) > tetracaine > cocaine (ED₅₀= 1·3 ± 10⁻⁴ M) > lidocaine > procaine (ED₅₀= 1.7 ± 10⁻³M). This order is consistent with the order of their local anaesthetic potency, but is not consonant with the order of the relative toxicity of these agents when used as spinal anaesthetics.     

Effect of local anesthetics on stearoyl-CoA desaturase of Tetrahymena microsomes

The effect of local anesthetics on the stearoyl-CoA desaturase activity was studied using Tetrahymena microsomal preparation. Dibucaine, tetracaine, and propranolol, a beta-blocking agent, nonspecifically inhibited the activities of NADPH-ferrihemoprotein reductase as well as of stearoyl-CoA desaturase and the terminal component, but lidocaine and procaine had no effect on these activities. The inhibitory potency was decreased in the order of dibucaine greater than propranolol greater than tetracaine much greater than lidocaine = procaine. According to the double-reciprocal plots of stearoyl-CoA desaturase, the inhibition by dibucaine appeared to be noncompetitive with respect to stearoyl-CoA as substrate. However, the activity of NADH-ferricyanide reductase was not significantly affected by concentrations of propranolol and tetracaine lower than 10mM, but by dibucaine. The terminal component, cyanide-sensitive factor, was most sensitive to local anesthetics among the microsomal electron transport components, suggesting a rate-limiting enzyme.     

Pharmacological evidence for cell surface control of oral regeneration in Stentor coeruleus

This study suggests that membrane perturbations can affect oral morphogenesis in Stentor, possibly by a mechanism involving calcium ions. Exposure of regenerating Stentor to micromolar concentrations of the membrane active local anesthetics dibucaine, tetracaine, or procaine greatly delayed the progress of oral regeneration. In the case of tetracaine and dibucaine the greatest delays were observed in the early stages of regeneration prior to stage 4, when the majority of essential synthetic activity is occurring. The effects of dibucaine were generally readily reversible upon removal of the cells from the drug, with some residual effects occurring at higher dibucaine concentrations. Regenerating cells in the presence of dibucaine and excess extracellular calcium were not delayed, suggesting that the effects of dibucaine were reversible by calcium ions. The effects of tetracaine were not reversible by calcium ions, however. Exposure of regenerating cells to medium either lacking in, or containing an excess of, extracellular calcium had no effect on the time required to complete oral regeneration. The plant lectin, phytohemagglutinin, can also delay oral regeneration. The possible implications of these findings on the control of oral regeneration are discussed.     

ATP-dependent interaction of propranolol and local anaesthetic with sarcoplasmic reticulum. Stimulation of Ca2+ efflux.

Preincubation of sarcoplasmic reticulum (SR) with propranolol or tetracaine inhibits Ca2+ accumulation and stimulates ATPase activity by more than 2-fold. This effect is obtained only when the preincubation is carried out in the presence of ATP or other nucleoside triphosphates. The (ATP + drug)-induced inhibition of Ca2+ accumulation is pH-dependent, increasing as the pH rises above 7.5. The presence of micromolar concentrations of Ca2+ or Mg2+ during the preincubation prevents the inhibitory effect of ATP plus drug on Ca2+ accumulation or ATPase activity. The (ATP + drug) modification of SR vesicles resulted in stimulation of a rapid Ca2+ efflux from passively loaded vesicles. The ATP-dependent inhibition of Ca2+ accumulation by the drug is obtained with other local anaesthetics. The drug concentration required for 50% inhibition was 0.15 mM for dibucaine and 0.4 mM for both propranolol and tetracaine, whereas it was 5 mM, 8 mM and greater than 10 mM for lidocaine, benzocaine and procaine respectively. The heavy SR vesicles were only slightly affected by the incubation with propranolol or tetracaine in the presence of ATP, but their sensitivity increased markedly after storage at 0 degrees C for 24-48 h. These results suggest that propranolol and some local anaesthetics, in the presence of ATP, stimulate Ca2+ efflux by modifying a protein factor(s) rather than the phospholipid bilayer.     

The penetration of local anesthetics into phosphatidylcholine monolayers.

The penetration of tetracaine into monolayers of phosphatidylcholine and trioctanoin at different surface pressures, and the penetration of dibucaine, tetracaine, butacaine, lidocaine, and procaine into monolayers of didecanoylphosphatidylcholine at II = 10 mN/m was determined by the use of a modified Gibbs adsorption equation. These data were shown to fit a geometric model and compared favorably with data determined by a method based on the geometric model. The penetration of tetracaine into phosphatidylcholine monolayers was pressure dependent. At II = 10 mN/m, the local anesthetics penetrate into a phosphatidycholine monolayer in the order: dibucaine greater than tetracaine greater than butacaine greater than lidocaine greater than procaine. This correlates with their potencies in blocking nerve conduction and inhibiting phospholipase A2.     

Interaction of local anesthetics with lipid bilayers investigated by (1)H 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 (31)P and (1)H magic-angle spinning (MAS) NMR spectroscopy. The (31)P 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.     

A comparative study of the effects of procaine, lidocaine, tetracaine and dibucaine on the functions and ultrastructure of isolated rat liver mitochondria

The effects of procaine, lidocaine, tetracaine and dibucaine (10(-5) - 10(-2) M) were tested on isolated rat liver mitochondria by measurements of the respiratory rates and of the membrane potential and by electron microscopy. A general concentration-dependent stimulation of the basal state (respiration before ADP addition) was observed for all local anesthetics studied. Up to the concentration of 10(-3) M, the order of stimulation was: procaine less than lidocaine less than dibucaine less than tetracaine. However, with the exception of dibucaine, which inhibited state-3 respiration (ADP present) in a strictly concentration-dependent manner, the other drugs had a biphasic effect: slight stimulation of state 3 at low and moderate concentrations (less than or equal to 10(-3) M) and inhibition at higher concentrations. Nevertheless, due to a stronger stimulation of the basal state, the acceptor control ratio decreases progressively (uncoupling effect) as the concentration of the drugs increases. The only exception to this observation is procaine in the range of 10(-5) - 10(-4) M, where the stimulation of the two respiration states (although small) is approximately equal and thus the uncoupling effect is absent or negligible. Membrane potential recordings suggested that membrane integrity and phosphorylation capacity were negatively affected at high drug concentrations (greater than 10(-3) M), especially in the case of tetracaine and dibucaine, when 5 x 10(-3) M even produced the collapse of the membrane potential and complete loss of the phosphorylation ability. Electron microscopy confirmed these effects, showing an abundance of either swollen or supercondensed mitochondria, with many membrane ruptures. The action mechanisms of the tertiary amines studied are discussed in terms of interaction of drug with the lipid bilayer and with the membrane proteins. It is concluded that both the inhibitory and the uncoupling effects are dependent, in the first place, on the degree of hydrophobicity of each local anesthetic.