Inhibitors of membrane depolarization regulate acetylcholine receptor synthesis by a calcium-dependent, cyclic nucleotide-independent mechanism

The inhibition of membrane depolarization by tetrodotoxin or the local anesthetic benzocaine elevates the acetylcholine receptor levels in cultured myotubes. The elevated acetylcholine receptor levels are due to increased receptor synthesis rather than to decreased degradation. The effects of tetrodotoxin and benzocaine on acetylcholine receptor levels are not additive, and are not inhibited by exogenously added cyclic GMP analogues or by elevated intracellular levels of cyclic GMP. However, the stimulation of acetylcholine receptor levels by tetrodotoxin or benzocaine is reversed by the addition of the calcium ionophore A23187. In contrast, tetrodotoxin or benzocaine stimulated acetylcholine receptor synthesis beyond the maximal stimulation produced by cholera toxin. These results suggest that the inhibition of membrane depolarization elevates acetylcholine receptor synthesis by a calcium-dependent, cyclic nucleotide-independent mechanism.     

Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier.

The effects of benzocaine (0.5-1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug.     

Haematological evaluation of the anaesthetic benzocaine hydrochloride in the freshwater fish Cyprinus carpio L.

The effects of anaesthetization with different concentrations of benzocaine hydrochloride (BH) and neutralized benzocaine hydrochloride (NBH) were studied on the haematology of Cyprinus carpio. Due to its acidic nature and resultant effects on aquarium water, BH produces haemoconcentration effects with disturbances in acid-base function. The use of NBH, whereby water quality effects were drastically reduced, improved the general haematological profile. In contrast, haemodilution resulted when blood was sampled without the use of an anaesthetic agent.     

Effects of the local anesthetic benzocaine on the human erythrocyte membrane and molecular models

The interaction of the local anesthetic benzocaine with the human erythrocyte membrane and molecular models is described. The latter consisted of isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphospatidylcholine (DMPC), and phospholipid multilayers of DMPC and dimyristoylphospatidyletanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that benzocaine induced the formation of echinocytes. Experiments performed on IUM and DMPC LUV by fluorescence spectroscopy showed that benzocaine interacted with the phospholipid bilayer polar groups and hydrophobic acyl chains. X-ray diffraction analysis of DMPC confirmed these results and showed that benzocaine had no effects on DMPE. The effect on sodium transport was also studied using the isolated toad skin. Electrophysiological measurements indicated a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of benzocaine, reflecting inhibition of active ion transport.     

Anesthesia of fish with benzocaine does not interfere with comet assay results

Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80mg/l for approximately 10min). Furthermore, benzocaine (80-600mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare.     

Local anesthetics QX 572 and benzocaine act at separate sites on the batrachotoxin-activated sodium channel

We have studied the effect of local anesthetics QX 572, which is permanently charged, and benzocaine, which is neutral, on batrachotoxin-activated sodium channels in mouse neuroblastoma N18 cells. The dose-response curves for each drug suggest that QX 752 and benzocaine each act on a single class of binding sites. The dissociation constants are 3.15 X 10(-5) M for QX 572 and 2.65 X 10(-4) M for benzocaine. Equilibrium and kinetic experiments indicate that both drugs are competitive inhibitors of batrachotoxin. When benzocaine and QX 572 are present with batrachotoxin, they are much more effective at inhibiting Na+ flux than would be predicted by a one-site model. Our results indicate that QX 572 and benzocaine bind to separate sites, each of which interacts competitively with batrachotoxin.     

pH-dependent effects of local anaesthetics in perturbing lipid membranes

The effects of local anaesthetics lidocaine, benzocaine, carbisocaine and carbisocaine derivatives, KaQ-7 and Ka-O, in perturbing bovine brain lipid membranes or egg lecithin membranes were compared at pH 6.0; 7.0; and 8.0. The electron spin resonance method with stearic acid labeled at carbon position 16 as the spin probe was employed. The perturbation effects of lidocaine and Ka-O were found to increase with increasing pH of the sample, whereas the effect of carbisocaine decreased with increasing pH. The perturbation effects of benzocaine and KaQ-7 were independent of pH. The pH-dependent perturbation effects of the local anaesthetics tested on lipid membrane fairly corresponded with their pH-dependent potency to block nerve action potentials.     

Binding of benzocaine in batrachotoxin-modified Na+ channels. State- dependent interactions

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX- modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use- dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.     

Efficacy of Tricaine Methanesulfonate (MS-222) as an Anesthetic Agent for Blocking Sensory-Motor Responses in Xenopus laevis Tadpoles

Anesthetics are drugs that reversibly relieve pain, decrease body movements and suppress neuronal activity. Most drugs only cover one of these effects; for instance, analgesics relieve pain but fail to block primary fiber responses to noxious stimuli. Alternately, paralytic drugs block synaptic transmission at neuromuscular junctions, thereby effectively paralyzing skeletal muscles. Thus, both analgesics and paralytics each accomplish one effect, but fail to singularly account for all three. Tricaine methanesulfonate (MS-222) is structurally similar to benzocaine, a typical anesthetic for anamniote vertebrates, but contains a sulfate moiety rendering this drug more hydrophilic. MS-222 is used as anesthetic in poikilothermic animals such as fish and amphibians. However, it is often argued that MS-222 is only a hypnotic drug and its ability to block neural activity has been questioned. This prompted us to evaluate the potency and dynamics of MS-222-induced effects on neuronal firing of sensory and motor nerves alongside a defined motor behavior in semi-intact in vitro preparations of Xenopus laevis tadpoles. Electrophysiological recordings of extraocular motor discharge and both spontaneous and evoked mechanosensory nerve activity were measured before, during and after administration of MS-222, then compared to benzocaine and a known paralytic, pancuronium. Both MS-222 and benzocaine, but not pancuronium caused a dose-dependent, reversible blockade of extraocular motor and sensory nerve activity. These results indicate that MS-222 as benzocaine blocks the activity of both sensory and motor nerves compatible with the mechanistic action of effective anesthetics, indicating that both caine-derivates are effective as single-drug anesthetics for surgical interventions in anamniotes.     

Metamorphosis and the steady state anesthetic concentrations of tricaine,benzocaine and ethanol

In the classical tadpole assay employed for Meyer-Overton type correlations, tricaine and benzocaine are 7–8 times more potent than n-alkanols of equivalent lipid solubility. Median anesthetic concentrations for loss of the righting reflex, AC₅₀(RR)s, were 0.165 and 0.103 mM for tricaine and benzocaine, and log Ps (octanol: water) were 1.81 and 1.98, respectively. Tadpoles receiving a half AC₅₀(RR) each of tricaine and ethanol showed less than additive effects, suggesting a substantial difference in their mechanism(s) of action. AC₅₀(RR)s for both tricaine and benzocaine increased two fold coincident with metamorphosis, reflecting a change from an equilibrium to a nonequilibrium steady state.     

Differential effects of anesthetics on electrical properties of the rainbow trout (Oncorhynchus mykiss) heart

We compared electrocardiographic signals in hatchery-reared, non-spinally-transected, immature rainbow trout (Oncorhynchus mykiss Walbaum) under clove oil (25 ppm), tricaine methanesulfonate (tricaine, 60 ppm), and benzocaine (108 ppm) general anesthesia (35 min, 14 degrees C). For all 3 anesthetics, the mean heart rate (HR) and QRS amplitude did not differ, and QRS duration and QT interval were independent of HR. Heart rate variability (HRV) was significantly (4-fold, P=0.032) higher under benzocaine than under clove oil and tricaine, but did not differ between clove oil and tricaine. QRS duration differed between groups (P<0.001, F=121); benzocaine anesthesia resulted in longer QRS complexes compared to clove oil (P<0.001) and tricaine (P<0.001) anesthesia, and QRS complexes under clove oil were longer than those under tricaine (P<0.001). High HRV and QRS amplitude variation with benzocaine were associated with HR oscillations as anesthetic exposure time increased, and suggest benzocaine toxicity which may influence cardiac function studies. Similar clove oil and tricaine ECG patterns suggest comparable autonomic effects, and maintenance of myocardial excitability. Given its low cost, ease of use, and similar ECG profiles to tricaine, clove oil is a viable alternative for studies of cardiac function in anesthetized rainbow trout.     

In vivo effects of the anesthetic,benzocaine, on liver microsomal cytochrome P450 and mixed-function oxidase activities of gilthead seabream (Sparus aurata)

Gilthead seabreams were exposed to benzocaine, 4-aminobenzoic acid ethyl ester, 57 mg/l in sea water for 3 min, daily, for 2 or 3 consecutive days. The fish were killed 20 hr after the last treatment. Benzocaine treatment for 2 or 3 days resulted in 57% and 67% inhibition of liver microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase activities,respectively. The total cytochrome P450 content of fish liver microsomes was unaltered following the 2-day benzocaine treatment. However, additional 3 min benzocaine treatment on day 3 reduced cytochrome P450 level by 50%. Benzocaine produced type II difference spectra with rabbit liver microsomes. Difference spectra of fish liver microsomes elicited by benzocaine were complex. The position of peak and intensity were greatly influenced by the concentration of benzocaine.     

THE USE OF BENZOCAINE HYDROCHLORIDE AS FISH TRANQUILLIZER AND ANAESTHETIC IN SALINE WATERS

SUMMARY

The anaesthetic effects of various concentrations of benzocaine hydrochloride were tested on Liza macrolepis and Sarotherodor mossambicus in sea water and diluted sea water, respectively. Induction time for anaesthesia was negatively correlated with increasing anaesthetic concentrations in L. macrolepis.

In S. mossambicus, however, operculum clamping appeared to be responsible-f or induction times increasing with increased anaesthetic concentration.

The tranquillizing effects of low concentrations of benzocaine hydrochloride on L. macrolepis was also studied.     

Effects of benzocaine and its isomers on sodium permeability and steady state sodium inactivation in the myelinated nerve, obtained by an improved dissection technique

The blocking effects of benzocaine and its isomers (1 mmol/l) on sodium currents in myelinated nerve fibres were tested. As far as the so-called fast sodium inactivation is concerned, benzocaine shifted the h alpha-curve in negative direction to a stronger extent than did its isomers, while the potency of the isomers did not differ significantly from each other. The drug-induced reductions of maximum sodium permeability PNa were tested at constant test pulses at h alpha = 1. In this kind of experiments all the three isomers had the same potencies. The findings could not be correlated to the lipid solubilities of the drugs as measured by the corresponding octanol/water partition coefficients. In addition, efforts were undertaken to minimize any noxious pull during the isolation of the axon. Some consequences of the improvements introduced are discussed in terms of the reliability of ionic current measurements in Ranvier nodes.     

The uptake of the anaesthetic benzocaine hydrochloride by the gills and the skin of three freshwater fish species

The uptake of benzocaine hydrochloride and neutralized benzocaine hydrochloride by the skin and the gills of Cyprinus carpio, Oreochromis mossambicus and Salmo gairdneri were studied. The differences observed can mainly be ascribed to degree of ionization and the lipid solubility of the anaesthetic.     

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.     

General and local anaesthetics perturb the fusion of phospholipid vesicles

The effects of general and local anaesthetics on Ca2+-induced fusion of negatively charged lipid vesicles have been investigated. Vesicles composed of phosphatidylcholine and phosphatidic acid (2:1 molar ratio) were induced to fuse using 5 mM free Ca2+. Fusion, assessed by an increase in size using gel filtration techniques and confirmed by electron microscopy, displayed a dependence on Ca2+ and Mg2+ concentration and on temperature. The inhalational anaesthetics halothane, methoxyflurane and diethyl ether enhanced fusion as did the uncharged local anaesthetic benzocaine. In contrast, the charged local anaesthetics lignocaine and bupivacaine inhibited the fusion process. It is suggested that the enhancement observed with the inhalational anaesthetics and benzocaine was mediated by an effect on lipid fluidity and the inhibition observed with the charged tertiary amine anaesthetics was due to an antagonism towards Ca2+.     

Use-dependent inhibition of Na+ currents by benzocaine homologs.

Most local anesthetics (LAs) elicit use-dependent inhibition of Na+ currents when excitable membranes are stimulated repetitively. One exception to this rule is benzocaine, a neutral LA that fails to produce appreciable use-dependent inhibition. In this study, we have examined the use-dependent phenomenon of three benzocaine homologs: ethyl 4-diethylaminobenzoate, ethyl 4-ethoxybenzoate, and ethyl 4-hydroxybenzoate. Ethyl 4-hydroxybenzoate at 1 mM, like benzocaine, elicited little use-dependent inhibition of Na+ currents, whereas ethyl 4-diethylaminobenzoate at 0.15 mM and ethyl 4-ethoxybenzoate at 0.5 mM elicited substantial use-dependent inhibition--up to 55% of peak Na+ currents were inhibited by repetitive depolarizations at 5 Hz. Each of these compounds produced significant tonic block of Na+ currents at rest and shifted the steady-state inactivation curve (h infinity) toward the hyperpolarizing direction. Kinetic analyses showed that the decaying phase of Na+ currents during a depolarizing pulse was significantly accelerated by all drugs, thus suggesting that these drugs also block the activated channel. The recovery time course for the use-dependent inhibition of Na+ currents was relatively slow, with time constants of 6.8 and 4.4 s for ethyl 4-diethylaminobenzoate and ethyl 4-ethoxybenzoate, respectively. We conclude that benzocaine and 4-hydroxybenzoate interact with the open and inactivated channels during repetitive pulses, but during the interpulse the complex dissociates too fast to accumulate sufficient use-dependent block of Na+ currents. In contrast, ethyl 4-diethylaminobenzoate and ethyl 4-ethoxybenzoate dissociate slowly from their binding site and consequently elicit significant use-dependent block. A common LA binding site suffices to explain the presence and absence of use-dependent block by benzocaine homologs during repetitive pulses.     

Comparative study of the action of procaine and benzocaine on normal and aconitine-modified sodium channels]

Ionic currents of normal and aconitine modified sodium channels of the Ranvier node membrane were measured under voltage clamp conditions. The experiments with local anesthetics in the external Ringer solution have showed that dissociation constant (Kdis) of normal channel-anesthetic complex for procaine is 0.27 + 0.03 mM, and for benzocaine is 0.68 +/- 0.04 mM. With aconitine modified channels, Kdis increases and becomes 1.32 +/- 0.5 mM and 1.52 +/- 0.3 mM for procaine and benzocaine, respectively. It is ascertained that the development of aconitine effect is inhibited by neutral benzocaine to a lesser extent than by procaine. It is shown that the aconitine effect cannot be reversed by a high concentration of anesthetic. Hence, it appears that aconitine and anesthetic receptors do not coincide.