Inhibition of fast axonal transport in bullfrog nerves by dibenzazepine and dibenzocycloheptadiene calmodulin inhibitors

The effects of the calmodulin inhibitors amitriptyline, desipramine, imipramine, and clomipramine on fast axonal transport, oxidative metabolism, and density of axonal microtubules were measured in bullfrog spinal nerves in vitro. The four drugs tested inhibited the fast orthograde transport of [3H]leucine-labelled proteins and the fast retrograde transport of acetylcholinesterase at a concentration of 0.2 mM. Amitriptyline, desipramine, and imipramine were equipotent inhibitors of transport, and clomipramine was a more potent inhibitor than imipramine. The adenosine triphosphate content of the nerves was reduced by at most 19% by the compounds under study; such a reduction cannot account for the inhibition of fast axonal transport. Desipramine and imipramine had no significant effect on the density of microtubules in unmyelinated axons, whereas amitriptyline only reduced it by 18%; the inhibition of axonal transport by these three drugs can therefore not be explained by microtubule disruption. Clomipramine reduced microtubular density by 40%, and this effect may have contributed to the inhibition of fast axonal transport. The inhibition of fast axonal transport by desipramine, imipramine, and amitriptyline may be related to the inhibition of calmodulin function by these drugs. The similar potency of these three drugs as inhibitors of fast axonal transport goes in parallel with their known similar potency as calmodulin antagonists.     

Inhibition of synaptosomal enzymes by local anesthetics

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na⁺ + K⁺)-ATPase (ouabain-sensitive), Mg²⁺-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPase, for example 7.9 and 10 mM tetracaine for Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F₁-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.     

Inhibition of axonal transport in vitro in frog sciatic nerves by chlorpromazine and lidocaine

Summary The effects of chlorpromazine hydrochloride (CPZ HCl) and prochlorperazin-metansulfonate (PCPZ) on the fast axonal transport of labelled proteins were examined in vitro in a peripheral frog nerve.A 0.1 mM concentration of CPZ HCl and PCPZ reduced the amount of transported proteins by more than 50 per cent. An almost complete block was obtained with a 0.5 mM concentration of these two drugs. The lower concentration hardly affected the protein synthesis. The transport inhibiting effect of 0.1 mM of the drugs was reversible but not that of the higher concentration.The number of microtubuli was strongly decreased and the number of filaments increased at the transport inhibiting concentrations. The ultrastructural changes induced by 0.1 mM of the phenothiazine tranquilizer were largely reversible. The local anesthetics lidocaine (18.3 mM) and tetracaine (3.3 mM) both caused similar changes, i.e. a reduction in the number of microtubuli. No ultrastructural effects were observed after treatment with 1 mM ouabain. These three drugs are known to block the axonal flow in the present system at the above mentioned concentrations.The biochemical and ultrastructural results are discussed in relation to those induced by other drugs affecting axonal transport.The present work was supported by grants from Statens Naturvetenskapliga Forskningsråd (No. 2535-8), C.-B. Nathorsts Vetenskapliga och Allmännyttiga Stiftelser, the Swedish Medical Research Council (B73-12X-2543-05B), H. Hierta's Stiftelse and W. och M. Lundgrens Stiftelse. Thanks are due to Mrs B. Egnér, Mrs E. Fjällstedt, Mrs. E. Norström and Mrs U. Svedin for expert technical assistance.     

Continuation of fast axonal transport in regenerating axons in vitro.

A previous study by McLean and co-workers reported that regenerating axons of the rabbit vagus nerve were unable to sustain axonal transport in vitro for several months after nerve injury. In contrast, we found that sensory axons of the rat sciatic nerve were able to transport 3H-labeled protein into their regenerating portions distal to the site of injury within a week after injury when placed in vitro. Transport in vitro was not significantly less than transport in axons maintained in vivo for the same period. Transport occurred in the medium that was used by the McLean group, but was significantly reduced in calcium-free medium. When axon regeneration was delared, only small amounts of activity were present in the nerve distal to the site of injury, showing that labeled protein normally present in that part of the nerve was associated with axons and was not a result of local precursor uptake by nonneural elements in the sciatic nerve. We were not able to explain the failure of McLean and co-workers to demonstrate transport in vitro in regenerating vagus nerve, but we conclude that there is no general peculiarity of growing axons that makes them unable to sustain transport in vitro.     

Organelles in fast axonal transport

The present minireview describes experiments carried out, in short-term crush-operated rat nerves, using immunofluorescence and cytofluorimetric scanning techniques to study endogenous substances in anterograde and retrograde fast axonal transport. Vesicle membrane components p38 (synaptophysin) and SV2 are accumulating on both sides of a crush, but a larger proportion of p38 (about 3/4) than of SV2 (about 1/2) is recycling toward the cell body, compared to the amount carried with anterograde transport. Matrix peptides, such as CGRP, ChRA, VIP, and DBH are recycling to a minor degree, although only 10-20% of surface-associated molecules, such as synapsins and kinesin, appear to recycle. The described methodological approach to study the composition of organelles in fast axonal transport, anterograde as compared to retrograde, is shown to be useful for investigating neurobiological processes. We make use of the "in vivo chromatography" process that the fast axonal transport system constitutes. Only substances that are in some way either stored in, or associated with, transported organelles can be clearly observed to accumulate relative to the crush region. Emphasis in this paper was given to the synapsins, because of diverging results published concerning the degree of affiliation with various neuronal organelles. Our previously published results have indicated that in the living axons the SYN I is affiliated with mainly anterogradely fast transported organelles. Therefore, some preliminary, previously unpublished results on the accumulations of the four different synapsins (SYN Ia, SYN Ib, SYN IIa, and SYN IIb), using antisera specific for each of the four members of the synapsin family, are described. It was found that SYN Ib clearly has a stronger affiliation to anterogradely transported organelles than SYN Ia, and that both SYN IIa and SYN IIb are bound to some degree to transported organelles.     

Depression of fast axonal transport produced by tullidora

The fast axoplasmic transport of labeled proteins was studied in cats showing hindlimb paralysis 4-7 weeks after a single oral dose of tullidora (Karwinskia humboldtiana) toxins. The isotope (3H-leucine) was injected into the spinal ganglion and the contralateral spinal cord of the seventh lumbar segment in order to study transport in sensory and motor fibers. The axoplasmic transport in motor fibers of the sciatic nerve was clearly altered in tullidora-treated cats. The majority of these animals showed a gradual decline of radioactivity from the cord to the periphery instead of the clear-cut wave front always seen in normal cats. An apparent wave was seen in three treated cats but the wave peak was behind the normal position and the slope of the wave front was reduced. While the rate of transport indicated by the farthest extent of the foot of the slope was not in all cases significantly changed, the results all indicated a hindered transport by the reduced slope front in the distal segments of the motor axons. In contrast, the axoplasmic transport appeared normal in the sensory fibers of all but one tullidora-treated cat. Light and electron microscopy of medial gastrocnemius and sural (cutaneous) nerves revealed axonal constrictions and axolemal irregularities associated with organelle retention after tullidora treatment. Also, some mitochondria appeared swollen. These changes were more frequent and intense in the motor nerve fibers than in the cutaneous nerve fibers.     

Inhibition of fast axonal transport in vitro by tetracaine: an increase in potency at alkaline pH, and no change in potency in calcium-depleted nerves

Some of the present in vitro experiments compare the degree of inhibition of fast axonal transport produced by tetracaine at neutral and at alkaline pH. In desheathed spinal nerves from bullfrog, 0.5 mM tetracaine reduced the quantity of [3H]leucine-labeled proteins which were transported to a ligature by 43% at pH 7.2 and by 96% at pH 8.2; separate experiments established that transport was not affected by the pH change in the absence of tetracaine. The relationship between pH and transport-blocking potency of tetracaine (pKa 8.2) is such that the local anesthetic is more potent when more uncharged form of the molecule is present; this may reflect the easier penetration across the axonal plasma membrane by the uncharged form of the tetracaine molecule. The axonal smooth endoplasmic reticulum has been attributed the function of a calcium reservoir, and it appeared possible that local anesthetics could block axonal transport by releasing calcium from this structure. However, the inhibition of transport produced by 1 mM tetracaine (pH 7.1) in sheathed nerves was approximately 80% both in nerves with a lower than normal calcium content (47% of normal) and in nerves with a normal calcium content; this result does not support the hypothesis that inhibition of axonal transport by local anesthetics is mediated by an increase in intracellular free Ca2+, but does not rule out the hypothesis either.     

Inhibition of gramicidin channel activity by local anesthetics.

Ondrias et al. ((1986) Stud. Biophys. 115, 17-22) found that dibucaine, butacaine, and tetracaine reduce the conductance of membranes containing multiple (greater than 10(6)) gramicidin channels. Similar experiments with local anesthetics (LA's) added to the bath while gently stirring showed that the inhibition developed slowly over a time course of 5-10 min. We developed a many (10-20) channel membrane technique which demonstrated that when LA's were added to the bath and the membrane was repeatedly broken and reformed, the channel occurrence frequency declined promptly. In standard single-channel membrane experiments at lower gramicidin densities, the mean single channel conductance and lifetime distributions with LA's present in the bath did not differ from the controls. The predominant channel conductance amplitude was lower by 9.1% than those of controls, but channel amplitude distributions were also modified so that the net reduction in overall population channel conductance was only about 2.0%. Channel currents showed no evidence of flicker blocks. The lifetime histograms of control and LA-exposed channel populations were both satisfactorily fit by a single-exponential function with the same mean. Thus, inhibition is due primarily to a reduction in the frequency of occurrence of conducting channels, implying a reduced concentration of active monomers in the membrane.     

Inhibition of mitogen-induced lymphocyte transformation by local anesthetics.

Chlorpromazine (CPZ) and lidocaine were added to cultures of mouse spleen cells stimulated by concanvalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). Concentrations of CPZ greater than 5 x 10(-6)M and concentrations of lidocaine greater than 2 x 10(-3)M totally inhibited the mitogenic responses to all four mitogens. Minimal inhibitory concentrations of neither drug interferred with cell viability as determined by trypan blue uptake or 51Cr release. The effects were totally reversed by the removal of the drugs from the culture. Addition of the drug at intervals after mitogen exposure demonstrated that the inhibited event occurred relatively soon after exposure to the mitogen. For example, the addition of lidocaine or CPZ more than 24 hr after Con A stimulation had no effect on tritiated thymidine incorporation. Elevated concentrations of cyclic AMP, cyclic GMP (or their derivatives) or calciunown membrane active actions of these drugs and the rapid reversibility of the effect strongly support the idea that the local anesthetics act on the surface membrane of lymphocytes. Binding of radiolabeled Con A or LPS to lymphocyte membranes in the presence of lidocaine or CPZ was not inhibited. The possibility exists that CPZ and lidocaine disorganized cell membranes so as to interfere with the surface membrane elaboration or action of a second messenger, or interfere with cell-cell interactions.     

Dynamics of fast axonal transport.

A phenomenological model of the process of fast axoplasmic transport is presented. The process was conceived of as occurring in two parts: (a) synthesis and storage of material in a cytoplasmic pool; (b) release from the pool and transport distally along the axon. Considering the fate of labeled proteins, the activity at points along the axon relfects events occurring earlier within the pool through the relationship: g(x,t) = const f(t - x/v); where g(x,t) represents axonal activity, f(t) the pool's activity, and v is the transport speed. Using the idea that when there is no further input of radioactivity into the pool its activity declines exponentially due to export of material to the axon. I generalized this concept to the case where activity enters and leaves the pool simultaneously. The model contains two parameters: the relative turnover rate of the pool, alpha, and T, an interval characteristic of the time of synthesis. From this model, the experimental data is unfolded and yields values for these parameters of alpha = 0.004 min-1 and T approximately 60 min.     

Inhibition by local anesthetics and barbiturates of the active transport of H+ in the synaptic vesicle membranes of the brain

The effects of local anesthetics and barbiturates on the ATP-dependent H+ transport in synaptic vesicle membranes from rat brain were studied using a fluorescent probe, acridine orange. Local anesthetics depressed the active H+ transport with the following order of potencies: tetracaine trimecaine lidocaine procaine. Respective IC50 values were 0.07, 0.28, 0.46 and 0.60 mM. The local anesthetics also disrupted the endogenous pH gradient seen in the absence of ATP. Barbiturates inhibited the active H+ transport showing IC50 values in the range of 2-5 mM except for benzobarbital and barbital characterized by IC50 values of 0.5 and 20 mM, respectively. The order of potencies was benzobarbital hexobarbital amobarbital pentobarbital phenobarbital barbital. The endogenous pH gradient was not affected by the barbiturates. The results show that local anesthetics disrupt the H+ transport by acting as permeable weak bases (uncouplers) whereas barbiturates are likely to block and anion channel which maintains electroneutrality of the H+ transport in the membrane of synaptic vesicles.     

Calcium dependent modulation of fast axonal transport

The highly differentiated structure of the neuron poses special problems for the intracellular movement of molecules throughout the cell. Molecular transport distances from the synthesizing neuron cell body along the axon (which has no substantial synthetic capabilities) to the axon terminal are very great. The transported substances, transport support structures, translocator motors, and control elements are currently the focus of intense research. Interruption of this flow of molecules could have disastrous effects upon the cell and ultimately the organism resulting in neuropathological conditions. Calcium plays a critical role in modulating fast-axonal transport (FAT) speeds. Before discussing the effect of calcium on FAT, we summarize our broad perspective on the role of axonal transport in neurologic disease.     

Inhibition of mitochondrial respiration by mepivacaine

In the last years our researches on neurotropic drugs follow our hypothesis that the strong effects on nervous system have always hidden more widespread effects on all tissues and cells. It is often required to employ local anesthetics in practising dentistry and orthodontics, particularly when children have to be treated. We have assayed in vitro one of these dental anesthetics, mepivacaine, on liver rat mitochondria: it depresses the respiration coupled to phosphorylation in mitochondria having a good respiratory control; so respiratory control too is depressed, but P/O ratio is unaffected; also respiration uncoupled by 2.4-dinitrophenol is depressed. Depressing respiration cooperates with anesthesia; unchanging P/O is good for the health of the cells and tissues treated by the mepivacaine.     

Dissociation of the inhibition of fast axonal transport by chlorimipramine from an effect on axonal microtubules

The effect of in vitro exposure of bullfrog spinal nerves to 0.2 mM chlorimipramine on the density of axonal microtubules was studied in an attempt to clarify the mechanism by which chlorimipramine inhibits fast axonal transport. A 17-h exposure to chlorimipramine reduced the density of microtubules in unmyelinated axons by only 18%; this microtubular loss does not reach the upper limit of the range of microtubule reduction associated with inhibition of fast axonal transport. A 23-h exposure to chlorimipramine, which had decreased microtubular density in unmyelinated axons by 40% in a previous study, did not decrease microtubular density in myelinated axons in the present study. These results rule out microtubular destruction as the mechanism responsible for inhibition of fast orthograde axonal transport by chlorimipramine, and greatly reduce the likelihood that microtubular destruction plays a significant role in the inhibition of fast retrograde transport by chlorimipramine.     

A coupled model of fast axonal transport of organelles and slow axonal transport of tau protein

We have developed a model that accounts for the effect of a non-uniform distribution of tau protein along the axon length on fast axonal transport of intracellular organelles. The tau distribution is simulated by using a slow axonal transport model; the numerically predicted tau distributions along the axon length were validated by comparing them with experimentally measured tau distributions reported in the literature. We then developed a fast axonal transport model for organelles that accounts for the reduction of kinesin attachment rate to microtubules by tau. We investigated organelle transport for two situations: (1) a uniform tau distribution and (2) a non-uniform tau distribution predicted by the slow axonal transport model. We found that non-uniform tau distributions observed in healthy axons (an increase in tau concentration towards the axon tip) result in a significant enhancement of organelle transport towards the synapse compared with the uniform tau distribution with the same average amount of tau. This suggests that tau may play the role of being an enhancer of organelle transport.     

Direct inhibition of the actomyosin motility by local anesthetics in vitro.

Using a recently developed in vitro motility assay, we have demonstrated that local anesthetics directly inhibit myosin-based movement of single actin filaments in a reversible dose-dependent manner. This is the first reported account of the actions of local anesthetics on purified proteins at the molecular level. In this study, two tertiary amine local anesthetics, lidocaine and tetracaine, were used. The inhibitory action of the local anesthetics on actomyosin sliding movement was pH dependent; the anesthetics were more potent at higher pH values, and this reaction was accompanied by an increased proportion of the uncharged form of the anesthetics. QX-314, a permanently charged derivative of lidocaine, had no effect on actomyosin sliding movement. These results indicate that the uncharged form of local anesthetics is predominantly responsible for the inhibition of actomyosin sliding movement. The local anesthetics inhibited sliding movement but hardly interfered with the binding of actin filaments to myosin on the surface or with actomyosin ATPase activity at low ionic strength. To characterize the actomyosin interaction in the presence of anesthetics, we measured the binding and breaking force of the actomyosin complex. The binding of actin filaments to myosin on the surface was not affected by lidocaine at low ionic strength. The breaking force, measured using optical tweezers, was approximately 1.5 pN per micron of an actin filament, which was much smaller than in rigor and isometric force. The binding and breaking force greatly decreased with increasing ionic strength, indicating that the remaining interaction is ionic in nature. The result suggests that the binding and ATPase of actomyosin are governed predominantly by ionic interaction, which is hardly affected by anesthetics; whereas the force generation requires hydrophobic interaction, which plays a major part of the strong binding and is blocked by anesthetics, in addition to the ionic interaction.