Local anesthetics. Effect of pH on use-dependent block of sodium channels in frog muscle.

Sodium currents were studied under voltage clamp in the presence of neutral, amine, and quaternary local anesthetic compounds. Use-dependent block was observed as a cumulative depression of INa seen with repetitive depolarizing test pulses applied at frequencies of 2-10s-1. With quaternary QX-314, the time constant of use dependence was long, and with neutral benzocaine, very short. With lidocaine and procaine, increasing external pH (pHo) changed the time constant from long to short, but alterations of internal pH have no effect. Inactivation in Na channels was measured by the influence of prepulses on peak INa during test pulses. Single-stimulus inactivation curves were shifted more with lidocaine at high pHo than at low pHo, but inactivation curves measured during pulse trains with any of the drugs and at any pHo were strongly shifted. All measurements show that the drug-receptor reaction was slow for amine drugs at low pHo, as for quaternary drugs at any pHo, and fast for amine drugs at high pHo, as for neutral drugs at any pHo. The major effect of low pHo on amine drugs was to reduce the concentration of drugs in the fiber and to protonate drug molecules on the receptor, thus trapping them in the blocking position for a longer time. Direct effects of pH on the receptor seemed minimal.     

Kinetics of local anesthetic inhibition of neuronal sodium currents. pH and hydrophobicity dependence.

This study assesses the importance of local anesthetic charge and hydrophobicity in determining the rates of binding to and dissociation from neuronal Na channels. Five amide-linked local anesthetics, paired either by similar pKa or hydrophobicity, were chosen for study: lidocaine, two tertiary amine lidocaine homologs, a neutral lidocaine homolog, and bupivacaine. Voltage-clamped nodes of Ranvier from the sciatic nerve of Bufo marinus were exposed to anesthetic externally, and use-dependent ("phasic") block of Na current was observed. Kinetic analysis of binding (blocking) rates was performed using a three parameter, piecewise-exponential binding model. Changes in extracellular pH (pHo) were used to assess the role of drug protonation in determining the rate of onset of, and recovery from, phasic block. For those drugs with pKa's in the range of pHo tested (6.2-10.4), the forward binding rate during a depolarizing pulse increased at higher pH, consistent with an increase in either intracellular or intramembrane concentration of drug. The rate for unbinding during depolarization was independent of pHo. The dissociation rate between pulses also increased at higher pHo. The pHo dependence of the dissociation rate was not consistent with a model in which the cation is trapped relentlessly within a closed channel. Quantitative estimates of dissociation rates show that the cationic form of lidocaine dissociates at a rate of 0.1 s-1 (at 13 degrees C); for neutral lidocaine, the dissociation rate is 7.0 s-1. Furthermore, the apparent pKa of bound local anesthetic was found to be close to the pKa in aqueous solution, but different than the pKa for "free" local anesthetic accessible to the depolarized channel.     

Permeability of axon membranes to local anesthetics

The permeability of the neutral form of tertiary amine local anesthetics across squid axon membranes was studied by utilizing three different experimental methods: (1) narcotic action of axon excitability was measured by monitoring the time derivative of action potential and the results were analyzed in terms of a diffusion reaction equation of local anesthetics to obtain their permeabilities; (2) the influx of local anesthetic into the axon was measured by use of the radioisotope tracer technique; and (3) the desorption rates of the neutral form of local anesthetics from lipid monolayers were measured and the desorption rate was correlated with permeability.The relative permeabilities obtained for procaine, lidocaine and tetracaine by the above three methods were comparable. The order of relative permeabilities was $\text{procaine}\text{>}\text{lidocaine}\text{>}\text{tetracaine}$, and had an inverse correlation with the partition coefficients of anesthetics at oil/water phases. Some discussion concerning the concept of permeability is made when the partition coefficient of a permeant molecule is high.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning alpha-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of "the cation" in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation相似文献   

pH-dependent binding of local anesthetics in single batrachotoxin-activated Na+ channels. Cocaine vs. quaternary compounds.

The effects of internal and external pH on the binding kinetics of local anesthetics (LAs) were studied in single batrachotoxin-activated Na+ channels incorporated into planar bilayers. With internal quaternary QX-314 and RAC421-II drugs, the binding interactions were little affected by either external or internal pH. With tertiary cocaine, the binding kinetics were drastically altered by pH. A decrease in the internal pH from 9.3 to 6.2 decreased the apparent equilibrium dissociation constant (Kd) of internal cocaine by more than 100-fold. This increase in the binding affinity was mostly accounted for by an increase in the apparent cocaine on-rate constant (kon) of approximately 80-fold. The cocaine off-rate constant (koff) was little changed (between 3-4 s-1). These results demonstrate quantitatively that the charged form of cocaine is the active form for BTX-activated Na+ channels. Surprisingly, the apparent pKa of cocaine near its binding site was estimated to be 1.4 units lower than that in bulk solution (7.1 vs. 8.5), indicating that the LA drug encounters a relatively hydrophobic environment. Opposite to the internal pH effect, a decrease of external pH from 8.4 to 6.2 increased the Kd value of internally and externally applied cocaine by approximately 8- and approximately 25-fold, respectively. External pH effect was primarily mediated by modulation of kon; koff was again relatively unaffected. Our findings support a model in which neutral cocaine can readily cross the membrane barrier, but needs to be protonated internally to bind to its binding site.     

Uptake of dibucaine into large unilamellar vesicles in response to a membrane potential

Local amine anesthetics appear to exert their effects in the charged (protonated) form on the cytoplasmic side of excitable membranes. Two features of interest are the mechanism whereby these drugs move across the membrane to the inner monolayer and the actual membrane concentrations achieved. In this work, we have investigated the influence of a K+ diffusion potential, delta psi, on the transmembrane distribution and concentration of the local anesthetic dibucaine employing large unilamellar vesicle systems. It is demonstrated that egg phosphatidylcholine large unilamellar vesicles exhibiting a delta psi (interior negative) actively accumulate dibucaine to achieve high interior concentrations. 31P and 13C nuclear magnetic resonance studies show that the internalized drug is localized to the vesicle inner monolayer, and suggest that the protonated form of the anesthetic is the species that is actively transported. The inner monolayer anesthetic concentrations thus achieved can be an order of magnitude or more larger than predicted on the basis of anesthetic lipid-water partition coefficients. It is suggested that these effects may be related to the mechanisms whereby local anesthetics are localized and concentrated at their sites of action in nerve membranes.     

Local anesthetics: hydrophilic and hydrophobic pathways for the drug- receptor reaction

The properties of Na channels of the node of Ranvier are altered by neutral, amine, and quaternary local anesthetic compounds. The kinetics of the Na currents are governed by a composite of voltage- and time-dependent gating processes with voltage- and time-dependent block of channels by drug. Conventional measurements of steady-state sodium inactivation by use of 50-ms prepulses show a large negative voltage shift of the inactivation curve with neutral benzocaine and with some ionizable amines like lidocaine and tetracaine, but no shift is seen with quaternary OX-572. However, when the experiment is done with repetitive application of a prepulse-testpulse waveform, a shift with the quaternary cations (applied internally) is seen as well. 1-min hyperpolarizations of lidocaine- or tetracaine-treated fibers restore two to four times as many channels to the conducting pool as 50-ms hyperpolarizations. Raising the external Ca++ concentration also has a strong unblocking effect. These manipulations do not relieve block in fibers treated with internal quaternary drugs. The results are interpreted in terms of a single receptor in Na channels for the different drug types. Lipid-soluble drug forms are thought to come and go from the receptor via a hydrophobic region of the membrane, while charged and less lipid-soluble forms pass via a hydrophilic region (the inner channel mouth). The hydrophilic pathway is open only when the gates of the channel are open. Any drug form in the channel increases the probability of closing the inactivation gate which, in effect, is equivalent to a negative shift of the voltage dependence of inactivation.     

Inhibition of sodium currents in frog ranvier node treated with local anesthetics Role of slow sodium inactivation

Effects of different local anesthetics of sodium permeability were studied in single nerve fibres of frog by the method of voltage clamp. Inhibition of sodium current by externally applied tertiary anesthetics, procaine and trimecaine, was the sum of a potentially independent block (reduced $\text{P}{}_{\mathrm{rmNa}}$) and slow sodium inactivation with time constants ranging from tens to hundreds of ms depending on membrane potential (at room temperature). Externally applied uncharged benzocaine produced a potentially independent block only. According to dose-response curves both processes are one-to-one reactions. In the case of trimecaine equilibrium constant the reaction responsible for reduction of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}$ is about 0.3 mM, while that for slow inactivation is more than ten times less (0.02 mM). Increasing pH from 5.6 to 8.5 markedly accelerated the slow inactivation process at all potential values. Divalent cations Ca²⁺ and Ni²⁺ shifted the steady-state slow inactivation curve along the potential axis and simultaneously reduced slow inactivation at the saturation level. Permanently charged quaternary trimecaine was ineffective when applied externally. Internally applied tertiary anesthetics and quaternary trimecaine as well as externally applied quaternary derivative of lidocaine QX-572 produced a progressively irreversible block enhanced by depolarization and inhibition reversibly increased by repetitive short-term depolarization (frequency-dependent inhibition). Inhibition of sodium currents by repetitive stimulation observed also in the case of externally applied tertiary anesthetics is due mainly to slow inactivation. The data suggests the existence of several types of receptor sites through which local anesthetics exert their blocking action on sodium permeability.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation” in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca²⁺, Mg²⁺ and Mn²⁺) and monovalent metal cations (Li⁺, Na⁺, K⁺ and Cs⁺), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.     

Mechanism of penetration and of action of local anesthetics in Escherichia coli cells

Escherichia coli cells were used to study the mechanism of penetration of local anesthetics and the relationship between permeation and functional properties. We show that both the neutral and the protonated form of dibucaine can be accumulated in the cells. Accumulation of the protonated form occurs in response to a transmembrane electrical potential (negative inside) and results in high trapped concentrations (70 mM). Accumulation can lead to an alkalinization of the internal pH. Low concentrations of dibucaine stimulate the respiration, increase the transmembrane electrical potential and raise the accumulation of solutes. Inhibition of these functions occurs at higher concentrations of the drug. Furthermore, the drug concentration required to inhibit these functions is smaller at alkaline external pH than at acidic external pH, suggesting that the inhibition is mainly due to the neutral form of the anesthetics. Other hydrophobic amines also stimulate and inhibit different membrane functions, their efficiency being correlated to their lipophilicity.     

Induction of permeability change and restoration of membrane permeability barrier in transformed cell cultures

Transformed 3T3 cells incubated with ATP at an alkaline pH become permeable to phosphorylated compounds. The increase in membrane permeability can be induced by incubation with ATP at a neutral pH but only if sodium fluoride is present. Fluoride is not necessary for activation of the permeability change in these cultures at the alkaline pH. The effect of fluoride is very rapid, and sodium fluoride by itself does not alter membrane permeability. The alteration of membrane permeability by ATP in 3T6 cells is reversible; the permeability barrier is restored by switching to neutral buffer in the presence or absence of divalent cations. The restoration of the membrane permeability barrier is prevented by fluoride, and by ATP itself; this action of ATP is specific and no other nucleoside triphosphates or chelating agents produce this effect. Untransformed 3T3 cells do not exhibit any appreciable change in permeability as a result of ATP treatment either in the presence or absence of fluoride. These results are consistent with the presence on the transformed cell surface of an ATP-requiring protein kinase and a fluoride-inhibitable protein phosphatase, which would be involved in the control of membrane permeability.     

A laser-temperature-jump method for the study of the rate of transfer of hydrophobic ions and carriers across the interface of thin lipid membranes

The first application of a laser-temperature-jump apparatus for the study of ion transport through planar (artificial) lipid membranes is described. The relaxation of the electric current is detected, either continuously at a constant applied voltage or discontinuously by a series of short voltage pulses. The second technique, a combined voltage- and temperature-jump method, is especially appropriate to investigate the kinetics of the adsorption/desorption process of hydrophobic ions and neutral carriers of cations at the membrane interface and to separate this phenomenon from the diffusion process through the unstirred aqueous layers adjacent to the membrane. The aim is to determine the rate-limiting step of transport. The permeation rate of the hydrophobic anion 2,4,6-trinitrophenolate is limited by the inner membrane barrier. For tetraphenylberate the rate constant of translocation across the inner barrier and that of desorption from the membrane into water are found to be of comparable magnitude. The membrane permeability of the neutral macrocyclic ion carrier enniatin B is strongly interface limited by its comparatively small rate of desorption into water. These results show that the frequently used a priori assumption of partition equilibrium at the membrane interfaces during transport is not justified.     

Steady-state catecholamine distribution in chromaffin granule preparations: a test of the pump-leak hypothesis of general anesthesia

The molecular mechanism of general anesthesia is not understood. Possible modes of action include binding at a protein site, such as a receptor or channel, or physical effects on membrane lipid properties. The pump-leak hypothesis suggests that anesthetics perturb the bilayer of synaptic vesicles, thereby increasing ionic permeability. This results in decay of proton gradients required for transport and accumulation of neurotransmitters. The subsequent loss of neurotransmitters from synaptic vesicles reduces the efficiency of synaptic transmission and results in the anesthetized state. We have determined the effects of general anesthetics on certain parameters of enzyme activity and membrane permeability relevant to the pump-leak hypothesis. We used chromaffin granules as a convenient model system and focused on clinically relevant anesthetic concentrations (ED50), quantitative measurements of permeability changes, and the kinetics of gradient decay. General anesthetics at ED50 have little or no effect on the proton-transport ATPase activity, but do cause modest increments in proton permeability that change the catecholamine distribution in actively pumping chromaffin granule preparations. We found that pH gradients do not collapse entirely under these conditions and that only a fraction of total catecholamine is lost from the chromaffin granules. When total collapse is induced by other means, efflux of catecholamines occurs with a half-time near 30 min. These results suggest that if the pump-leak hypothesis is valid, then very small losses of catecholamines must be sufficient to induce anesthesia. We conclude that the weight of evidence favors other mechanisms, notably direct binding of anesthetics to sensitive proteins.     

Linking solubility and permeability assays for maximum throughput and reproducibility

Solubility and permeability are intimately linked in drug absorption processes. They have, however, been traditionally assayed separately. To support this linkage, a combined solubility/permeability assay was developed for determining absorption properties of chemical entities. First, solubility is determined at 4 pH values by comparing the concentration of a saturated compound solution to its dilute, known concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support parallel artificial membrane permeation assay (PAMPA). The 2 artificial membranes presented here model the gastrointestinal tract and the blood-brain barrier (BBB). Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are 1) reduction of sample usage and preparation time, 2) elimination of interference from compound precipitation in membrane permeability determination, 3) maximization of input concentration to permeability assay for improved reproducibility, and 4) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity.     

The quantitative analysis of three action modes of volatile anesthetics on purple membrane

We quantitatively assessed the spectroscopic changes of purple membrane in relation to the concentrations of a volatile anesthetic. As reported previously, volatile anesthetics show three modes of action on purple membrane. By using an anesthetic for which the concentration in solution could be determined spectroscopically and by applying modified analytical methods regarding the M-intermediate lifetime, we were able to clarify the quantitative relation between anesthetic concentration and each mode of action, a relation which in the past has only been described qualitatively. We also determined through the measurement of transient pH changes with pyranine that the proton pump efficiency per photochemical cycle in an action mode induced with low concentrations of anesthetic does not change from that of the native state. Moreover, we dynamically obtained the individual M-bacteriorhodopsin difference spectrum of each state at room temperature using our flash photolysis system equipped with a wavelength-tunable dye laser. These results demonstrated again that we should clearly distinguish different action modes of anesthetics according to their concentrations.     

Effect of tertiary amine local anesthetics on G protein-coupled receptor lateral diffusion and actin cytoskeletal reorganization

Although widely used clinically, the mechanism underlying the action of local anesthetics remains elusive. Direct interaction of anesthetics with membrane proteins and modulation of membrane physical properties by anesthetics are plausible mechanisms proposed, although a combination of these two mechanisms cannot be ruled out. In this context, the role of G protein-coupled receptors (GPCRs) in local anesthetic action is a relatively new area of research. We show here that representative tertiary amine local anesthetics induce a reduction in two-dimensional diffusion coefficient of the serotonin_1A receptor, an important neurotransmitter GPCR. The corresponding change in mobile fraction is varied, with tetracaine exhibiting the maximum reduction in mobile fraction, whereas the change in mobile fraction for other local anesthetics was not appreciable. These results are supported by quantitation of cellular F-actin, using a confocal microscopic approach previously developed by us, which showed that a pronounced increase in F-actin level was induced by tetracaine. These results provide a novel perspective on the action of local anesthetics in terms of GPCR lateral diffusion and actin cytoskeleton reorganization.     

Liquid General Anesthetics Lower Critical Temperatures in Plasma Membrane Vesicles

A large and diverse array of small hydrophobic molecules induce general anesthesia. Their efficacy as anesthetics has been shown to correlate both with their affinity for a hydrophobic environment and with their potency in inhibiting certain ligand-gated ion channels. In this study we explore the effects that n-alcohols and other liquid anesthetics have on the two-dimensional miscibility critical point observed in cell-derived giant plasma membrane vesicles (GPMVs). We show that anesthetics depress the critical temperature (T_c) of these GPMVs without strongly altering the ratio of the two liquid phases found below T_c. The magnitude of this affect is consistent across n-alcohols when their concentration is rescaled by the median anesthetic concentration (AC₅₀) for tadpole anesthesia, but not when plotted against the overall concentration in solution. At AC₅₀ we see a 4°C downward shift in T_c, much larger than is typically seen in the main chain transition at these anesthetic concentrations. GPMV miscibility critical temperatures are also lowered to a similar extent by propofol, phenylethanol, and isopropanol when added at anesthetic concentrations, but not by tetradecanol or 2,6 diterbutylphenol, two structural analogs of general anesthetics that are hydrophobic but have no anesthetic potency. We propose that liquid general anesthetics provide an experimental tool for lowering critical temperatures in plasma membranes of intact cells, which we predict will reduce lipid-mediated heterogeneity in a way that is complimentary to increasing or decreasing cholesterol. Also, several possible implications of our results are discussed in the context of current models of anesthetic action on ligand-gated ion channels.     

Functional asymmetry of the amine transporter from chromaffin granules

The studies presented in this communication describe the existence of a pH-dependent kinetic barrier in amine translocation in membrane vesicles isolated from chromaffin granules from bovine adrenal medulla. This barrier prevents efflux of amines previously accumulated in the membrane vesicles. In these preparations, once the amine is accumulated, there is no further need for ATP. This would suggest a low permeability of the membrane, both to ions and to the amine. In addition, we show that under conditions which thermodynamically favour efflux this is kinetically blocked. This block is due to a rapid protonation of the transporter in the interior of the vesicle. Net efflux can be induced by agents that bring upon an alkalinization of the internal pH such as ammonium salts or nigericin. On the other hand, the rate of exchange with extravesicular substrate is almost identical at the various pH values tested. The physiological implications of this mechanism are discussed.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment.

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.