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.     

The tertiary amine local anesthetic dibucaine binds to the membrane skeletal protein spectrin

The quinoline-based tertiary amine dibucaine has been shown to bind the membrane skeletal protein spectrin with a dissociation constant of 3.5x10(-5) M at 25 degrees C. Such binding is detected by monitoring the quenching of the tryptophan fluorescence intensity with increasing concentrations of dibucaine only and not with the benzene-based local anesthetics procaine, tetracaine and lidocaine. Binding of dibucaine also indicated changes in the tertiary structure of spectrin indicated by a circular dichroism spectrum in the near-UV region due to absorption of the aromatic side chains. The thermodynamic parameters associated with the binding indicated the interaction of dibucaine and spectrin to be enthalpy-driven and insensitive to an increase in the ionic strength of the buffer.     

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.     

Biphasic effect of local anesthetics on the adenosine triphosphate-dependent calcium uptake by lysed brain synaptosomes

Previous studies have shown that an adenosine triphosphate-dependent calcium uptake activity in lysed brain synaptosomes is attributable to the neuronal endoplasmic reticulum elements. The present study has examined the effects of tetracaine, lidocaine, and dibucaine on this calcium uptake process. The adenosine triphosphate-dependent uptake of 45Ca2+ was measured (in the absence and in the presence of drug) by Millipore filtration and liquid scintillation spectrometry. The local anesthetics studied exhibited a biphasic effect on 45Ca2+ uptake by lysed synaptosomes from rat brain cortex. High concentrations (5 mM tetracaine, 50 mM lidocaine, 0.6 mM dibucaine) inhibited the uptake of 45Ca2+; the order of potency for this effect was dibucaine greater than tetracaine greater than lidocaine. Lower concentrations of these local anesthetics produced either no effect on 45Ca2+ uptake (2 mM tetracaine or 30 mM lidocaine) or a stimulation of 45Ca2+ uptake (1 mM tetracaine, 10 mM lidocaine, and 0.3 mM or 0.1 mM dibucaine); the order of potency for stimulation of 45Ca2+ uptake was dibucaine greater than tetracaine greater than lidocaine.     

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.     

Induction of deflagellation by various local anesthetics in Chlamydomonas reinhardtii Dangeard (Chlamydomonadales,Chlorophyceae)

Dibucaine, a local anesthetic, is known to induce flagellar excision in Chlamydomonas reinhardtii. Herein, we investigate whether other local anesthetics have similar effects. Tetracaine, bupivacaine, procaine, and lidocaine also caused flagellar excision, although their potencies were lower than that of dibucaine. Bupivacaine, procaine, and lidocaine induced a morphological change in flagella from a rod‐like shape to a disk‐like shape before flagellar excision. Except for lidocaine, these local anesthetics caused cell‐wall shedding in addition to flagellar excision. The anesthetics in order of their median effective concentration (1‐h EC₅₀) for flagellar excision are as follows: dibucaine (1.37 × 10^?5 M) < tetracaine (3.16 × 10^?5 M) < bupivacaine (4.25 × 10^?4 M) < procaine (2.02 × 10^?3 M) < lidocaine (3.61 × 10^?3 M). In all cases, Ca²⁺ depletion from the solution inhibited flagellar excision. However, Ca²⁺‐channel blockers, IP3 receptor antagonists, and inhibitors of phospholipase C did not prevent excision. We suggest that the local anesthetics induce flagellar excision by increasing the fluidity of the flagellar/cell membrane, thereby allowing extracellular Ca²⁺ to flow into the cell and cause flagellar excision.     

Effects of local anesthetics on guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on beta-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2--20 mM) reduces the responsiveness of adenylate cyclase to (a) guanyl-5'-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5'-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5'-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10(-7) M. Tetracaine also competitively inhibited binding of both the antagonist [3H]dihydroalprenolol and the agonist [3H]hydroxybenzylisoproterenol to beta-adrenergic receptors. However, it was twice as potent in inhibiting [3H]hydroxybenzylisoproterenol as [3H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the beta-adrenergic receptor induced by agonists. Other local anesthetics mimicked the effects of tetracaine on adenylatecyclase and in dissociating high-affinity agonist-receptor complexes. The other of potency for both processes was dibucaine greater than tetracaine greater than bupivacaine greater than lidocaine which agrees with their relative potencies as local anesthetics. By contrast, a different order of potency was observed for competitive inhibition of [3H]dihydroalprenolol binding: dibucaine greater than tetracaine greater than greater than lidocaine greater than bupivacaine.     

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.     

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.     

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.     

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.     

Local Anesthetics Inhibit the Ca2+,Mg2+-ATPase Activity of Rat Brain Synaptosomes

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

The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria

The ATP/ADP-antiporter inhibitors and the substrate ADP suppress the uncoupling effect induced by low (10-20 microM) concentrations of palmitate in mitochondria from skeletal muscle and liver. The inhibitors and ADP are found to (a) inhibit the palmitate-stimulated respiration in the controlled state and (b) increase the membrane potential lowered by palmitate. The degree of efficiency decreases in the order: carboxyatractylate (CAtr) greater than ADP greater than bongkrekic acid, atractylate. GDP is ineffective, Mg.ADP is of much smaller effect, whereas ATP is effective at much higher concentration than is ADP. Inhibitor concentrations, which maximally suppress the palmitate-stimulated respiration, correspond to those needed for arresting the state 3 respiration. The extent of the CAtr-sensitive stimulation of respiration by palmitate has been found to decrease with an increase in palmitate concentration. Stimulation of the controlled respiration by p-trifluoromethoxycarbonylcyanide phenylhydrozone (FCCP) and gramicidin D at any concentrations of these uncouplers is CAtr-insensitive, whereas that caused by a low concentrations of 2,4-dinitrophenol and dodecyl sulfate is inhibited by CAtr. The above effect of palmitate develops immediately after addition of the fatty acid. It is resistant to EGTA as well as to inhibitors of phospholipase (nupercain) and of lipid peroxidation (ionol). Moreover, palmitate accelerates spontaneous release of the respiratory control, developing in rat liver mitochondria under certain conditions. This effect takes several minutes, being sensitive to EGTA, nupercain and ionol. Like the fast uncoupling, this slow effect is inhibited by ADP but CAtr and atractylate are stimulatory rather than inhibitory. In artificial planar phospholipid membrane, palmitate does not increase the membrane conductance, FCCP increases it strongly and dinitrophenol only slightly. In cytochrome oxidase proteoliposomes, FCCP, gramicidin and dinitrophenol (less effectively) lower, whereas palmitate enhances the cytochrome-oxidase-generated membrane potential. In this system, monensin substitutes for palmitate. It is concluded that the ATP/ADP antiporter is somehow involved in the uncoupling effect caused by low concentrations of palmitate and, partially, of dinitrophenol, whereas uncoupling produced by FCCP and gramicidin is due to their action on the phospholipid part of the mitochondrial membrane. A possible mechanism of this effect is discussed.     

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.     

Studies on the mechanism of action of local anesthetics on proton translocating ATPase from Mycobacterium phlei

We have measured the inhibitory potencies of local anesthetics (procaine, lidocaine, tetracaine and dibucaine) on ATP-mediated H+-translocation, Ca2+-transport and ATPase activity in membrane vesicles from Mycobacterium phlei. Procaine and lidocaine up to 1 mM concentration did not inhibit ATP-dependent H+-translocation, Ca2+-transport and ATPase activity. However, tetracaine and dibucaine at 0.2 mM concentration caused dissipation of the proton gradient, measured by the reversal of the quenching of fluorescence of quinacrine, and inhibition of active Ca2+-transport. Tetracaine (1 mM) inhibited membrane-bound ATPase activity without affecting solubilized F1-ATPase activity. Studies show that these local anesthetics do not prevent the inactivation of F0-F1 ATPase by dicyclohexylcarbodiimide (DCCD). Binding of [14C]DCCD to F0-proteolipid component remained unchanged in the presence of tetracaine indicating that DCCD and tetracaine do not share common binding sites on the F0-proteolipid sector. The inhibition of H+-translocation and membrane-bound ATPase activity by tetracaine was substantially additive in the presence of vanadate.     

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.     

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.     

Interaction of the local anesthetics dibucaine and tetracaine with sarcoplasmic reticulum membranes. Differential scanning calorimetry and fluorescence studies

The local anesthetics dibucaine and tetracaine inhibit the (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum [DeBoland, A. R., Jilka, R. L., & Martonosi, A. N. (1975) J. Biol. Chem. 250, 7501-7510; Suko, J., Winkler, F., Scharinger, B., & Hellmann, G. (1976) Biochim. Biophys. Acta 443, 571-586]. We have carried out differential scanning calorimetry and fluorescence measurements to study the interaction of these drugs with sarcoplasmic reticulum membranes and with purified (Ca2+ + Mg2+)-ATPase. The temperature range of denaturation of the (Ca2+ + Mg2+)-ATPase in the sarcoplasmic reticulum membrane, determined from our scanning calorimetry experiments, is ca. 45-55 degrees C and for the purified enzyme ca. 40-50 degrees C. Millimolar concentrations of dibucaine and tetracaine, and ethanol at concentrations higher than 1% v/v, lower a few degrees (degrees C) the denaturation temperature of the (Ca2+ + Mg2+)-ATPase. Other local anesthetics reported to have no effect on the ATPase activity, such as lidocaine and procaine, did not significantly alter the differential scanning calorimetry pattern of these membranes up to a concentration of 10 mM. The order parameter of the sarcoplasmic reticulum membranes, calculated from measurements of the polarization of the fluorescence of diphenylhexatriene, is not significantly altered at the local anesthetic concentrations that shift the denaturation temperature of the (Ca2+ + Mg2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation by local anesthetics of the metabolism of acidic phospholipids in the rat pineal gland

The efficacy of five local anesthetics in causing stimulation of phospholipid metabolism in rat pineal gland $\text{in vitro}$ paralleled their anesthetic potency and decreased in the order: dibucaine, tetracaine, cocaine, procaine, lidocaine. When stimulation occurred, the patterns of labeling resembled that produced by propranolol, a β-adrenergic receptor blocking agent with local anesthetic activity. Isotope incorporation into phosphatidylglycerol and CDP-diglyceride was markedly enhanced and increases of labeling of phosphatidic acid and phosphatidylinositol were also seen. At concentrations of 1–10 mM, propranolol and local anesthetics inhibited labeling of phosphatidylcholine and phosphatidylethanolamine by more than 90% and incorporation of ³²P_i into other phospholipids to a smaller extent.