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.     

Mechanism of local anesthesia: long-range orientation effects

The orientation interaction of a molecule of a local anesthetic with a biomembrane and cell-like liquid was studied, based on the model of adsorption of the anesthetic from a cell-like solution on the surface of a biomembrane for compounds of the trimecaine series. A statistically significant correlation equation was obtained, which relates the minimum blocking concentration to the projection of the dipole moment of the anesthetic on the plane X(1)0X2 of the principal axes of inertia. A model is prosed according to which the "anesthetic-biomembrane" interaction is most effective the molecule of the anesthetic rotates around the axis of the maximum moment of inertia.     

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.     

Differences in the action of Ca2+ ions on a cumulative Na-channel blockade due to tertiary and quaternary amines

In voltage-clamp experiments on frog myelinated fibres it has been established that the increase in Ca2+ concentration from 2 to 20 mM does not effect the use-dependent (cumulative) inhibition of sodium channels (INa) produced by the tertiary local anesthetics (lidocaine, tetracaine, etidocaine) and the quaternary antiarrhythmic drug N-propyl ajmaline (NPA). The NPA-induced inhibition of sodium channels does not undergo any essential changes as the (Ca)0 is raised from 2 to 20 mM. On the contrary, the cumulative blockade produced by the tertiary local anesthetics under such an elevation of the (Ca)0 is sharply reduced. This reduction is caused by the inhibitory action of the (Ca)0 on the local anesthetics-induced transition of sodium channels from the state of rapid to slow inactivation. The (Ca)0 does not affect the interaction of the quaternary NPA with open sodium channels. The data obtained provide evidence in favour of the hypothesis about the existence of the different binding sites responsible for cumulative blockade of the INa induced by tertiary and quaternary amines.     

Differences in the blocking action of benzocaine and amines on batrachotoxin-modified sodium channels of the Ranvier node

Experiments by the voltage clamp method on Ranvier nodes of the frog nerve fiber showed that batrachotoxin sharply reduces the sensitivity of sodium channels to the blocking action of various tertiary (trimecaine, procaine, ajmaline, strychnine) and quaternary (QX-572, N-propylajmaline) amines but has no appreciable effect on blocking of sodium channels by neutral benzocaine. Inhibition of batrachotoxin-modified sodium currents by trimecaine is distinctly time-and potential-dependent in character. None of the amines tested gives rise to frequency-dependent (cumulative) blocking of the modified channels. Unblocking of these channels during rinsing of the node takes place much faster than unblocking of normal channels. The time course of recovery of the normal and modified currents after blocking by benzocaine is about the same. Relations between batrachotoxin "receptors" and the various blocking agents in the sodium channel are discussed.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 636–643, November–December, 1982.     

Modification of cardiac Na+ channels by batrachotoxin: effects on gating, kinetics, and local anesthetic binding.

The purpose of the present study was to examine the characteristics of Na+ channel modification by batrachotoxin (BTX) in cardiac cells, including changes in channel gating and kinetics as well as susceptibility to block by local anesthetic agents. We used the whole cell configuration of the patch clamp technique to measure Na+ current in guinea pig myocytes. Extracellular Na+ concentration and temperature were lowered (5-10 mM, 17 degrees C) in order to maintain good voltage control. Our results demonstrated that 1) BTX modifies cardiac INa, causing a substantial steady-state (noninactivating) component of INa, 2) modification of cardiac Na+ channels by BTX shifts activation to more negative potentials and reduces both maximal gNa and selectivity for Na+; 3) binding of BTX to its receptor in the cardiac Na+ channel reduces the affinity of local anesthetics for their binding site; and 4) BTX-modified channels show use-dependent block by local anesthetics. The reduced blocking potency of local anesthetics for BTX-modified Na+ channels probably results from an allosteric interaction between BTX and local anesthetics for their respective binding sites in the Na+ channel. Our observations that use-dependent block by local anesthetics persists in BTX-modified Na+ channels suggest that this form of extra block can occur in the virtual absence of the inactivated state. Thus, the development of use-dependent block appears to rely primarily on local anesthetic binding to activated Na+ channels under these conditions.     

Interaction of spin-labeled local anesthetics with the sodium channel of squid axon membranes

The effects of spin-labeled local anesthetics on sodium currents of internally perfused squid axons were studied using the voltage-clamp technique. Internal application (10 mum) of the most potent spin-labeled local anesthetic used in this study produced a small initial block of sodium currents. However, after sixty repetitive pulses (to + 80 mV) given at 1 Hz, the sodium currents were drastically reduced. In addition to this frequency-dependent phenomenon, the anesthetic effect on the sodium currents was also sensitive to the voltage of the pulses. Both the frequency- and voltage-dependent properties remained intact after removal of sodium inactivation with pronase. The recovery of sodium currents from this frequency-dependent anesthetic effect followed a single exponential curve with a surprisingly long time constant of about 10 min. Such a long recovery time, which is longer than any known sodium inactivation process, led us to suggest that the recovery process represents the dissociation of drug molecules from their binding sites. We have also found that increasing hydrophobic character of the homologues series of spin-labeled local anesthetics enhances the frequency- and voltage-dependent block of sodium currents. This effect strongly suggests that hydrophobic interaction is an integral component of the binding site. These probes with their selective effects on the sodium currents, are expected to be highly useful in studying the molecular structure of the sodium channels.     

Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl- aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX- B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX- B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.     

Comparison of the electrophysiological effect of amiodarone, lidocaine and quinidine on rat ventricular cells.

The effects of 20 microM each of amiodarone, lidocaine and quinidine on action potential and membrane currents were studied in rat ventricular cells. At a stimulation frequency of 0.1 Hz, quinidine prolonged the action potential duration (APD50) from 120 +/- 26 to 660 +/- 8 msec and increased the time to peak (Tp) amplitude from 7 +/- 1 msec to 32 +/- 6 msec. Lidocaine shortened APD50 from 123 +/- 15 to 83 +/- 6 msec without altering Tp. Amiodarone changed neither APD50 nor Tp. Voltage clamp study revealed that quinidine inhibited sodium inward current (INa) even when this current was elicited by depolarizing pulses at 0.1 Hz from a holding potential of -90 mV. For amiodarone and lidocaine, the inhibition was observed when INa was elicited from a holding potential of -70 mV. A frequency-dependent inhibition of INa by amiodarone and lidocaine was observed at frequencies higher than 1 Hz. Quinidine showed this inhibition even at 1 Hz. In correlation with the stronger frequency dependent inhibition of INa, a greater delay of the recovery and increase of the non-recovery fraction of INa was induced by quinidine. For lidocaine and amiodarone, only the recovery time constant was delayed. In cells treated with sea anemone toxin (ATX, 0.2 microM), APD50 was prolonged to 4-5 sec in 5 min. Quinidine, but not amiodarone, completely reversed the effect of ATX. Quinidine showed use-dependent inhibition of INa in these ATX-treated cells. Amiodarone, however, did not show this inhibition. It is likely that amiodarone suppresses INa by delaying the recovery of INa instead of blocking the open-state Na(+)-channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of spin-labeled local anesthetics with the sodium channel of squid axon membranes

Summary The effects of spin-labeled local anesthetics on sodium currents of internally perfused squid axons were studied using the voltage-clamp technique. Internal application (10 m) of the most potent spin-labeled local anesthetic used in this study produced a small initial block of sodium currents. However, after sixty repetitive pulses (to +80 mV) given at 1 Hz, the sodium currents were drastically reduced. In addition to this frequency-dependent phenomenon, the anesthetic effect on the sodium currents was also sensitive to the voltage of the pulses. Both the frequency- and voltage-dependent properties remained intact after removal of sodium inactivation with pronase. The recovery of sodium currents from this frequency-dependent anesthetic effect followed a single exponential curve with a surprisingly long time constant of about 10 min. Such a long recovery time, which is longer than any known sodium inactivation process, led us to suggest that the recovery process represents the dissociation of drug molecules from their binding sites. We have also found that increasing hydrophobic character of the homologues series of spin-labeled local anesthetics enhances the frequency- and voltage-dependent block of sodium currents. This effect strongly suggests that hydrophobic interaction is an integral component of the binding site. These probes with their selective effects on the sodium currents, are expected to be highly useful in studying the molecular structure of the sodium channels.     

Combined action of anti-arrhythmia agents

A significant potentiation of antiarrhythmic effect was observed in 121 dogs with arrhythmias 24 and 48 hours after the coronary artery ligation when the following drugs were combined: N-propylajmaline bromide (1A class) and trimecaine (1B class), quinidine (1A class) and trimecaine, N-propylajmaline bromide and anaprilin (2 class). The potentiation is attributed to the different molecular mechanisms of action of the drugs.     

Decrease in maximal sodium permeability and slow sodium inactivation produced by trimecaine in the Ranvier node

The action of trimecaine on sodium permeability of the membrane was studied by the voltage clamp method in single nodes of Ranvier. Like procaine, trimecaine was shown to reduce maximal sodium permeability ( _Na) and to induce slow sodium inactivation. The slow inactivation arising in the presence of trimecaine was qualitatively very similar to that observed during the action of procaine or a high external potassium concentration. Dose-effect curves were obtained for _Na and slow inactivation and they showed that one molecule of trimecaine reacts with one sodium channel in the mechanism of both these effects. The local anesthetic has greater affinity for the receptor responsible for slow inactivation (equilibrium constants about 0.3·10^–3 M for _Na and 0.2·10^–14 M for slow inactivation.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 418–425, July–August, 1976.     

Gating current in the membrane of nodes of Ranvier under conditions of linear alteration of the membrane potential

Asymmetrical displacement currents (Id) in the frog Ranvier node (R. ridibunda) treated with tetrodotoxin and tetraethylammonium were studied by the use of ramp-voltage pulses. In some experiments both ramp- and step-voltage pulses were used. The net Id consists of two components, one of which Id (I) can be blocked by local anesthetic trimecaine, or inactivated with the 10 ms depolarizing prepulse sufficiently large to inactivate the sodium current in the same node (before Na+ removal and TTX application). Parameters of the steady state charge distribution are very close (though not identical) to that of the peak sodium permeability vs. potential relation. The charge carrying Id (I) is estimated as 0.3--0.5 of the net displaced charge. The results suggest that trimecaine- and inactivation-sensitive component of Id may be the true gating current of Na+ channels.     

Reactivation processes of three inward current systems involved in the rising phase of the action potentials in embryonic chick hearts

In embryonic chick hearts during development, there are three inward current systems which are involved in the rising phases of the action potentials (APs): fast INa, slow ICa, and tetrodotoxin-insensitive slow INa. To assess reactivation processes for these three types of inward current channels (fast Na+, slow Ca2+, and slow Na+ channels), diastolic recovery of Vmax was examined in embryonic chick hearts using a paired-pulse protocol. In all cases, the diastolic recoveries were approximated by single exponential functions. The time constants of recovery (tau(V)) and T90% (the diastolic interval which allows 90% recovery of Vmax of the premature AP) were, respectively, 53.1 +/- 5.2 and 61.5 +/- 8.6 ms for Na+-dependent fast AP (n = 10), 376.9 +/- 49.3 and 659.2 +/- 113.1 ms for the Ca2+-dependent slow AP (n = 10), and 40.7 +/- 5.3 and 45.6 +/- 12.0 ms for the Na+-dependent slow AP (n = 10). In the presence of lidocaine, the recovery kinetics also appeared to be single exponentials for diastolic intervals up to 500 ms (fast APs) or 250 ms (slow APs). The reactivation processes for the Na+-dependent fast and slow channels were significantly slowed by 100 microM lidocaine. In addition, in the presence of 100 microM lidocaine, Vmax was depressed in a frequency-dependent manner; the higher the stimulation frequency, the greater the depression. Hence, the fast Na+ channels and the slow Na+ channels had the following similarities: rapid reactivation, reactivation slowed by lidocaine, and frequency-dependent depression in the presence of lidocaine.     

Effects of KC 3791 on sodium and potassium channels in frog node of Ranvier

Effects of a new antiarrhytmic compound KC 3791 on sodium (INa) and potassium (IK) currents were studied in frog myelinated nerve fibres under voltage clamp conditions. When applied externally to the node of Ranvier, KC 3791 (KC) at concentrations of 10(-5)-10(-4) mol.l-1 produced both tonic and cumulative (use-dependent) inhibition of INa. An analysis of the frequency-, voltage- and time dependence of cumulative block by KC suggested that this block resulted from a voltage-dependent interaction of the drug with open Na channels. The progressive decrease in INa during repetitive pulsing was due to accumulation of Na channels in the resting-blocked state: closing of the activation gate after the end of each depolarizing pulse stabilized the KC-"receptor" complex. To unblock these channels a prolonged washing of the node had to be combined with a subsequent repetitive stimulation of the membrane; this suggested that channel could not become cleared of the blocker unless the activation gate has opened. KC also proved to be capable of blocking open K channels at outwardly directed potassium currents (IK). This block increased during membrane depolarization. Unblocking of K channels after the end of a depolarizing pulse proceeded much faster than unblocking of Na channels under identical conditions. Cumulative inhibition of outward IK during high-frequency membrane stimulation was therefore readily reversible upon a decrease in pulsing frequency.     

Membrane mechanisms of activating depolarized smooth muscle contraction (guinea pig small intestine) during exposure to physiologically active compounds

The experiments were performed on the depolarized smooth muscle of taenia coli with the use of double sucrose-gap arrangement. Muscle contractions were induced by 1) application of hystamin or bradikynin; 2) rectangular long-lasting (10--20 s) pulses of hyperpolarizing current--the strong contraction appeared in response to the switching off the current (off-response). Both the on- and off-responses to the hyperpolarizing current recorded before, during and after hystamine (or bradikynin)-induced contration were, as a rule, very similar. Treatment of smooth muscle with local anaesthetics (procaine, trimecaine, QX-572) removed hystamine- and bradikynin-induced contrations and only decreased off-responses. The analysis of the data obtained suggested the existence of the independent electrically and chemically excitable systems (channels?) OF Ca2+ ion transport in the membrane of smooth muscle cells.     

Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.     

Local anesthetics-induced inhibition of chloroplast electron transport

The effects of local anesthetics on photosynthetic activity of pea chloroplasts were investigated in order to elucidate the role of Ca2+ in photosynthetic electron transport. Dibucaine, benzocaine and tetracaine were found to inhibit the O2-evolving activity. The inhibitory effect decreases in the order dibucaine greater than benzocaine greater than tetracaine greater than trimecaine similarly as does the potency to inhibit propagation of excitation in nerve fibre. As demonstrated in experiments with artificial donors and acceptors, the site of inhibition is the water-splitting site of PSII. The inhibitory power of the anesthetics grows with increasing ionic strength of the incubating mixture (by adding NaCl or MgCl2) and with pH; this is explained by occurrence of the neutral form of amine. At low concentrations the charged anesthetic acts as a protonofore; however, the inactivation of water splitting is not due to the protonophoric effect. The incubation is followed by the disappearance of ESR signal IIs. The role of Ca2+ and Ca2+-binding protein in PSII electron transport and its localization are discussed.     

Local anesthetics: stimulation of incorporation of inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes

Various local anesthetics enhanced the incorporation of [3H]inositol into phosphoinositides in guinea pig cerebral cortical synaptoneurosomes. Dibucaine, QX-572 and dimethisoquin showed maximum stimulation at 100 microM, tetracaine and diphenhydramine at 300 microM, and QX-314 at 1 mM, while quinacrine, lidocaine and cocaine showed no or only slight stimulation. There was no correlation between local anesthetic activity, estimated by inhibition of the 22Na+ flux elicited by the sodium channel activator batrachotoxin, and the potency for stimulation of inositol incorporation. A quaternary, relatively weak, local anesthetic, QX-572, was the most potent agent in stimulation of inositol incorporation, while the next most potent agent was dibucaine, a tertiary, very potent, local anesthetic. Dibucaine did not affect the uptake of [3H]inositol by synaptoneurosomes. The incorporation of [3H]inositol into phosphoinositides was increased in calcium-free buffer. The presence of dibucaine resulted in further stimulation of [3H]inositol incorporation in calcium-free buffer. Although dibucaine and QX-572 markedly stimulated incorporation of [3H]inositol into phosphoinositides, only QX-572 significantly enhanced the incorporation of 32PO4(3-) into phosphoinositides. The results suggest that certain local anesthetics enhance a pathway involving an exchange reaction between inositol and the phosphoinositol ester bond of phosphatidylinositol, but do not markedly affect the de novo pathway of phosphoinositide synthesis.     

The effect of metacaine on the slow variation of peak sodium currents in potential clamped Ranvier nodes following changes in holding potential

The effect of changes in the holding potential on peak sodium currents in isolated myelinated nerve fibres (peak INa) was investigated with the conventional sodium inactivation being kept at h infinity = 1. In Ringer solution no stationary values of peak INa could be obtained over the potential range tested. Near the normal resting potential, ER, peak INa changed with time clearly even after 10 min. Therefore, the individual values of peak INa as normalized by peak INa at ER and corrected for the unevitable run-down of peak INa could not serve as measure for stationary values of any membrane parameter. Under metacaine (1 mmol/l) peak INa changed comparably faster and proved to be less potential dependent as compared to peak INa of the untreated fibre. The effects observed are not necessarily governed by a specific process located inside the nodal membrane.