Comparison of effects of selected local anesthetics on sodium and potassium channels in mammalian neuron

Effects of procaine, trimecaine, and a new carbanilate local anesthetic, carbizocaine, on early sodium inward current and fast and slow components of potassium outward current in the membrane of the rat dorsal root ganglion neuron were studied using the internal dialysis and potential clamp techniques. All the currents studied were depressed in the presence of the drugs tested. However, for inhibition of the inward current concentrations lower by approximately one to more than two orders were sufficient compared to those required for similar inhibition of the outward currents. Carbizocaine was the most effective, procaine the least effective drug. Almost identical ratios of the negative logarithms of mean effective concentrations for blocking the inward and the outward current respectively, were found for each of the drugs tested. None of the drugs could be characterized as a specific blocker of sodium or potassium channels. It is concluded that the mechanisms of action of these three local anesthetics in all the three types of ion channels studied in the neuronal membrane are very similar regardless of both the type of the chemical bond in the intermediary chain of the molecules (ester, anilide, carbanilate) and the structure of the aromatic moiety, or the absolute potency of the drug.     

Calcium-activated potassium conductance noise in snail neurons

Current fluctuations were measured in small, 3-6 micrometers-diameter patches of soma membrane in bursting neurons of the snail, Helix pomatia. The fluctuations dramatically increased in magnitude with depolarization of the membrane potential under voltage clamp conditions. Two components of conductance noise were identified in the power spectra calculated from the membrane currents. One component had a corner frequency which increased with depolarization. This component was blocked by intracellular injection of TEA and was relatively insensitive to extracellular calcium levels (as long as the total number of effective divalent cations remained constant). It was identified as fluctuations of the voltage-dependent component of delayed outward current. The second component of conductance noise had a corner frequency which decreased with depolarization. It was relatively unaffected by TEA injection and was reversibly blocked by substitution of extracellular calcium with magnesium, cobalt, or nickel. This second component of noise was identified as fluctuations of the calcium-dependent potassium current. The results suggest that the two components of delayed outward current are conducted through physically distinct channels.     

增强UV-B辐射和He-Ne激光对小麦原生质体微管骨架的影响

以小麦叶片原生质体为材料,采用间接免疫荧光定位法标记其微管系统,并利用激光共聚焦扫描显微系统进行观察。研究了低剂量He-Ne激光(5mW.mm-2)、增强UV-B辐射(10.08kJ.m-2.d-1)及二者的复合处理对小麦幼苗叶肉细胞中微管骨架的影响。结果表明,增强UV-B辐射后,小麦叶片细胞中微管骨架发生解聚,呈短棒状或点状分布,微管束弥散且荧光强度减弱;而增强UV-B辐射后再施以He-Ne激光处理,小麦叶肉细胞微管骨架有部分断裂,但较单独UV-B处理组的损伤程度轻,说明低剂量的He-Ne激光可以部分修复增强UV-B辐射对微管骨架的损伤,且对微管的聚合有促进作用。     

GABA-induced potassium channels in cultured neurons

When gamma-aminobutyric acid (GABA) or baclofen were applied to cultured rat hippocampal neurons, single-channel potassium currents appeared after a delay of 30 s or more in patches of membrane on the cell surface isolated from the agonists by the recording pipette. The appearance of currents in patches not exposed to agonist, the delay in their appearance and the suppression of currents in cells pre-incubated with pertussis toxin indicate the involvement of an intracellular second messenger system. The channels were associated with a GABAB receptor rather than a GABAA receptor as they were blocked by baclofen, a GABAB antagonist, but were not affected by bicuculline, a GABAA antagonist. A feature of the single channel currents was their variable amplitude: they had a maximum conductance of ca. 70 pS and displayed many lower conductance states that were integral multiples of 5-6 pS. In several cells exposed to GABA or baclofen, first small currents and then progressively larger currents appeared: current amplitude was a multiple of an elementary current. It is suggested that binding of GABA to GABAB receptors activates a second messenger system causing opening of oligomeric potassium channels.     

Activation and inactivation of single calcium channels in snail neurons

Activation and inactivation properties of Ca currents were investigated by studying the behavior of single Ca channels in snail neurons. The methods described in the previous paper were used. In addition, a zero- phase digital filter has been incorporated to improve the analysis of latencies to first opening, or waiting times. It was found that a decrease in the probability of single channel opening occurred with time. This was especially marked at 29 degrees C and paralleled the inactivation observed in macroscopic currents. The fact that a single channel was observed means that there is a significant amount of reopening from the "inactivated" state. Small depolarizations at 18 degrees C showed little inactivation. From these measurements, histograms of single channel open, closed, and waiting times were analyzed to estimate the rate constants of a three-state model of activation. Two serious discrepancies with the model were found. First, waiting time distributions at -20 mV were slower than those predicted by parameters obtained from an analysis of the single channel closed times. Second, it was shown that the time and the magnitude of the peak of the waiting time histogram were inconsistent with a three-state model. It is concluded that a minimum of four states are involved in activation. Some four-state models may be eliminated from further consideration. However, a comprehensive model of Ca channel kinetics must await further measurements.     

Interaction of local anesthetics with model ion channels

A theoretical study of intermolecular interactions between local anesthetics of the acetanilide series and model ionic channels was performed. A dimer of gramicidine A and melittin were used as models of ionic channels. The geometry of molecules was determined by the method of molecular mechanics. Statistically significant correlation equations were derived, which relate the indices of local anesthetizing activity to the characteristics of intermolecular interactions in the anesthetic-peptide system. A comparative analysis of intermolecular interactions in anesthetic-gramicidine A and anesthetic-melittin systems was carried out.     

Ion conductance and ion selectivity of potassium channels in snail neurones

Summary Delayed potassium channels were studied in internally perfused neurone somata from land snails. Relaxation and fluctuation analysis of this class of ion channels revealed Hodgkin-Huxley type K channels with an average single channel conductance ( _K) of 2.40±0.15 pS. The conductance of open channels is independent of voltage and virtually all K channels seem to be open at maximum K conductance (g _K) of the membrane. Voltage dependent time constants of activation ofg _K, calculated from K current relaxation and from cut-off frequencies of power spectra, are very similar indicating dominant first-order kinetics. Ion selectivity of K channels was studied by ion substitution in the external medium and exhibited the following sequence: T1⁺>K⁺>Rb⁺>Cs⁺>NH ₄ ⁺ >Li⁺>Na⁺. The sequence of the alkali cations does not conform to any of the sequences predicted by Eisenman's theory. However, the data are well accommodated by a new theory assuming a single rate-limiting barrier that governs ion movement through the channel.This paper is dedicated to the memory of Walther Wilbrandt.     

Effect of low-intensity He-Ne laser irradiation on rat mesenteric microcirculation

Changes in microcirculation during low-intensity He-Ne laser irradiation were studied by measurement of blood vessels and lymphangions' diameter in anesthetized rat mesentery using method of videomicroscopy in vivo. We demonstrated that the red coherent light induced a significant increase in contractility of vessels' smooth muscle cells. The flow decreased in the mesenteric microvessels during H2O2-mediated oxidant stress was restored after He-Ne laser irradiation.     

A discrete type of potassium channel conductance in snail neurons

The results of further investigations on a single potential dependent K+ channel are described. It was shown that ionic selectivity of the channel for monovalent ions is too high: Li+, Na+, and Cs+ are practically impermeant ions. Permeability of the channel for Rb+ is approximately 10 times less, and for Tl+ it is 2 times more than permeability for K+. Besides, it was found that open K+ channel has 16 multiple conductance levels.     

Tolbutamide enhances potential-dependent inactivation of calcium channels in snail neurons

A two-microelectrode voltage-clamp method was used to measure a high-threshold calcium current (I_Ca) on isolated snail neurons. Tolbutamide (1–5 mmole/liter) and H-8 (1–30 µmole/liter), inhibitors of kinase A, caused a decrease in the peak amplitude and accelerated I_Ca decay during a depolarizing stimulus. The half-life of the "slow" time constant of I_Ca decay decreased in the presence of tolbutamide, and was two to three times stronger than the half-life of the "fast" time constant. I_Ca inactivation curves plotted in a double-stimulus experiment have shown that after tolbutamide application, I_Ca inactivation elicited by the application of high-amplitude depolarizing pre-stimuli preferentially rises (V_c=+30 to +70 mV). The results suggest that dephosphorylation of Ca²⁺ channels enhances a potential-dependent component of the inactivation process.Scientific-Research Institute of the Brain, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 515–519, September–October, 1991.     

Zinc modulates a-type potassium currents and neuronal excitability in snail neurons

Summary 1. Zinc-induced actions were studied on the A-current and neuronal activity in identified and unidentified nerve cells of the snail,Helix pomatia L., under voltage and current clamp conditions.2. Extracellularly applied Zn²⁺ attenuated the peak amplitude of the A-current in a potential- and dose-dependent way (K _i=1.8 mM at –30 mV,n _H=0.6).3. Attenuation of the A-currents was initiated as Zn²⁺ shifted the potential dependence of both activation and inactivation of the currents toward more positive potential values.4. Zinc concomitantly prolonged the time to peak and decay time constant of the A-currents (K _d=1.7 mM,n _H=1.4) as well.5. Zn²⁺ decreased the resting membrane potential and the spike amplitude and increased the action potential duration and the input resistance of the cells in current clamp experiments.6. A complex action of zinc increased the neuronal excitability, indicating spontaneous and synaptically evoked spike discharges.7. Common and specific zinc binding sites are supposed on vertebrate and invertebrate A-type potassium channel proteins, where binding Zn²⁺ can modulate the gating properties and kinetics of the fast outward potassium currents.     

下丘脑神经元钙激活钾通道的整流现象

目的研究SD乳鼠下丘脑神经元中钙激活钾通道的整流现象.方法采用膜片钳内面向外式记录方式.结果记录到一种大电导钙激活钾通道(KCa),在对称140mmol/L[K+]时内向电导为(171±12)pS,不随[Ca2+]变化而改变,而外向电导可受[Ca2+]调控,当[Ca2+]为500μmol/L时,外向电导为(76±14)pS.[Ca2+]越大,整流现象越明显,Mg2+对这种KCa的整流作用不明显.结论下丘脑神经元中KCa具有Ca2+依赖性整流现象,它可能与神经元的兴奋性和稳定性有关.     

Calcium channels of neurons and mechanism of action of low intensity 337 nm laser irradiation

The effect of low intensity laser irradiation upon calcium current of Helix neurons was studied by means of intracellular dialysis technique under voltage clamp conditions. It was found that irradiation with the rate dose 0.4 J/cm2 decreased calcium currents irreversibly without changing the channels voltage-dependence. The molecule of carotenoid is assumed to be the photosensitive gating particle of the calcium channel.     

Permeation and interaction of divalent cations in calcium channels of snail neurons

We have studied the current-carrying ability and blocking action of various divalent cations in the Ca channel of Lymnaea stagnalis neurons. Changing the concentration or species of the permeant divalent cation shifts the voltage dependence of activation of the Ca channel current in a manner that is consistent with the action of the divalent cation on an external surface potential. Increasing the concentration of the permeant cation from 1 to 30 mM produces a twofold increase in the maximum Ca current and a fourfold increase in the maximum Ba current; the maximum Ba current is twice the size of the maximum Ca current for 10 mM bulk concentration. Correcting for the changing surface potential seen by the gating mechanism, the current-concentration relation is almost linear for Ba2+, and shows only moderate saturation for Ca2+; also, Ca2+, Ba2+, and Sr2+ are found to pass through the channel almost equally well. These conclusions are obtained for either of two assumptions: that the mouth of the channel sees (a) all or (b) none of the surface potential seen by the gating mechanism. Cd2+ blocks Lymnaea and Helix Ca channels at concentrations 200 times smaller than those required for Co2+ or Ni2+. Ca2+ competes with Cd2+ for the blocking site; Ba2+ binds less strongly than Ca2+ to this site. Mixtures of Ca2+ and Ba2+ produce an anomalous mole fraction effect on the Ca channel current. After correction for the changing surface potential (using either assumption), the anomalous mole fraction effect is even more prominent, which suggests that Ba2+ blocks Ca current more than Ca2+ blocks Ba current.     

Influence of liposomal local anesthetics on platelet aggregation in vitro

We assessed the effect of local anesthetics (LA) from different families such as esters (benzocaine), linear aminoamides (lidocaine) and cyclic aminoamides (bupivacaine) on the platelet aggregation induced by ADP. Liposomal formulations of the three LA, prepared with egg phosphatidylcholine:cholesterol alpha-tocopherol, were also tested. The three LA were able to inhibit platelet aggregation induced by ADP, in the following order: bupivacaine > lidocaine > benzocaine. After encapsulation into liposomes the inhibitory effect increased for all anesthetics studied, showing that aggregation tests could be used to assess the toxicity of new drug formulations.     

The effect of irradiation by a He-Ne laser and phytohemagglutinin on lymphocyte mitochondria

Electron-microscopic morphometry has been applied to study mitochondria on ultrathin sections of lymphocytes from human peripheral blood. It has been shown that the stimulation of lymphocytes by the mitogen phytohemagglutinin (PHA) 1 h causes increases in the quantity of mitochondria per cellular section (17%) as well as in the total area of mitochondria per cell section (35%), i.e. an increase in mitochondrial mass. Taking into account known facts about growth and division of mitochondria in late phases of cellular cycle, one can suppose that described above changes in mitochondria during G0----G1 transition under action of PHA belong to an early phase of biogenesis of mitochondria. In the contrary, irradiation of lymphocytes with He-Ne-laser (lambda = 632.8 nm) in dose 56 J/m2 which does not cause the G0----C1 transition, results in the increase in the number of mitochondria per cellular section (20%) but not increase in the total area of mitochondria per cell section. The last finding indicates to some modification of space configuration of the mitochondria without any changes in their mass. The increase in the quantity of mitochondria per cellular section after the irradiation could be related with the increase in electrochemical proton gradient and in phosphorylating activity of mitochondria. He-Ne-laser radiation as well as mitogen PHA cause some deaggregation of mitochondria (this is more pronounced in case of PHA) which may be related to their functional activation.