Inhibition Of Fast Axonal Transport Research Articles

Inhibition of fast axonal transport by erythro-9-[3-(2-hydroxynonyl)]adenine.

erythro-9-[3-(2-Hydroxynonyl)]adenine, an inhibitor of protein carboxylmethylation and dynein-ATPase activity, inhibited fast axonal transport in vitro in frog sciatic nerves. Its site of action might be associated with an ATPase on which transport depends, since specific carboxylmethylation inhibitors lacked effects on transport. The levels of high energy phosphates and protein synthesis were unaffected by the drug at a transport-inhibiting concentration, making disturbances due to metabolic effects less likely. An erythro-9-[3-(2-hydroxynonyl)]adenine-sensitive ATPase was looked for in various nerve fractions but has so far not been resolved.

Gelsolin inhibition of fast axonal transport indicates a requirement for actin microfilaments.

The actions of actin-based microfilaments in cell motility suggest a possible role in the mechanism of fast axonal transport, but the pharmacological data evaluating their role in this process are equivocal. Moreover, microfilaments are difficult to preserve and identify in ultrastructural studies, so the organization and function of axonal actin has remained uncertain. We have now evaluated the role of actin microfilaments in intracellular transport of membranous organelles using video-enhanced contrast microscopy and gelsolin to analyse fast axonal transport directly in isolated axoplasm from the squid giant axon. With this preparation it is possible to perfuse axoplasm with large molecules that do not cross the plasmalemma, while controlling cation levels. The 90,000-molecular weight protein gelsolin depolymerizes actin microfilaments in micromolar Ca2+, but not in the absence of Ca2+. Axonal transport of membranous organelles has previously been shown to be unaffected by levels of Ca2+ up to 10 microM. In the presence of EGTA, gelsolin has no effect on the movement of membranous organelles, but in the presence of 10 microM Ca2+ it completely blocks transport of all membranous organelles. No changes in the organization of the axoplasm were detected. These results and results using other probes for actin are consistent with the hypothesis that actin-based microfilaments are involved in the movement of membranous organelles in the axon.

Inhibition of fast axonal transport in vitro by the local anesthetics prilocaine, mepivacaine, and bupivacaine.

The aim of the present study was to establish the concentrations of prilocaine, mepivacaine, and bupivacaine which are effective at blocking fast axonal transport, to determine whether prilocaine and mepivacaine offer a better prospect of dissociating conduction block and transport block in vivo than does lidocaine and whether bupivacaine offers a better prospect than etidocaine in the same context. Fast axonal transport of [3H]leucine-labeled proteins was studied in vitro in bullfrog spinal nerves and quantitated by liquid scintillation counting. Exposure of spinal nerves to 14 mM prilocaine reduced the quantity of 3H-labeled proteins which accumulated at a ligature by 86%, and exposure to 14 mM mepivacaine reduced it by 70%; 10 mM prilocaine reduced this same parameter by 54%, a degree of inhibition close to the 44% reduction caused by 14 mM lidocaine. The D(-) and L(+) stereoisomers of mepivacaine each reduced transport to the ligature by approximately 50% at a concentration of 14 mM. Bupivacaine reduced the accumulation of 3H-labeled proteins at the ligature by 49% at a 10 mM concentration (pH 6.2); its potency is close to that found for etidocaine in a previous study. Since prilocaine and mepivacaine are at least as potent as lidocaine as transport inhibitors and at blocking impulse conduction, these two anesthetics offer no advantage over lidocaine to achieve dissociation of conduction block from transport block in vivo. Bupivacaine appears to offer no advantage over etidocaine in the same context, as the two agents have a similar potency as local anesthetics and a similar potency as inhibitors of fast axonal transport.

Accumulation of [ 3H]fucose-labelled glycoproteins in the Golgi apparatus of dorsal root ganglion neurons during inhibition of fast axonal transport caused by exposure of the ganglion to Co 2+-containing or Ca 2+ -free medium

Previous in vitro studies have established that Co 2+-containing or Ca 2+-free media interfere with the initiation of the fast axonal transport of proteins. The present study has used light- and electron-microscope radioautography to compare the distribution of [ 3H]fucose-labelled glycoproteins in neuronal cell bodies of control dorsal root ganglia and ganglia incubated for 16–17 h in Ca 2+-free medium or in medium containing 0.18 mM Co 2+. The radioautographic reaction in control cell bodies was diffusely scattered throughout the cytoplasm; grain counts revealed that 22% of the reaction was associated with elements of the Golgi apparatus and 78% was over other organelles and the remainder of the cytoplasm. In most experimental-cell bodies, 78% of the silver grains were clustered over elements of the Golgi complex whereas other organelles and the remainder of the cytoplasm were comparatively much less labelled; structural alterations of the Golgi apparatus were also produced by the modified media. In parallel studies where the radioactivity in nerve trunks and ganglia was measured by liquid scintillation counting, it was found that the Ca 2+-free medium and the Co 2+-containing medium both reduced by approximately 80% the quantity of [ 3H]fucose-labelled glycoproteins which were carried by the fast axonal transport system; they did so without interfering with the incorporation of [ 3H]fucose into glycoproteins. The results indicate that in the presence of Co 2+ or in the absence of Ca 2+ the proteins which are destined for fast axonal transport accumulate at the Golgi apparatus of neuronal cell bodies. These results thus suggest that Ca 2+ is required for proteins to leave the Golgi region in transit to the fast axonal transport system.

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.

On the mechanism of the inhibition of fast axonal transport caused by exposure of frog spinal ganglia to a sodium-deficient medium

Substitution of choline or sucrose for Na + in the medium surrounding frog spinal ganglia causes an inhibition of fast axonal transport in vitro. Since an increased Ca 2+ influx into neurons does occur in these media, the present experiments examined the possibility that the effect on transport is mediated by an increase in intracellular free Ca 2+. The combination of the choline substitution with a 3-fold increase in extracellular Ca 2+ reduced transport of [ 3H]proteins to 75% of control (as compared to the 63% obtained previously with choline alone), thus providing no evidence of an enhancement of choline's effect by a second factor which promotes an increase in intracellular Ca 2+. When Li + was substituted for Na +, the quantity of [ 3H]proteins transported fell to 40% of control despite the fact that the Li + substitution does not modify Ca 2+ fluxes across the plasma membrane in neurons of the frog spinal nerve. The present findings suggest that removal of Na + interferes with fast axonal transport by a mechanism other than an increase in intracellular Ca 2+.

Batrachotoxin blocks saltatory organelle movement in electrically excitable neuroblastoma cells

Batrachotoxin has been reported to inhibit fast axonal transport. We have examined the effect of batrachotoxin on saltatory organelle movements in N115 neuroblastoma cells (a model of fast axonal transport) using time-lapse video intensification microscopy. Batrachotoxin (0.1–1.0 μM) inhibits saltatory organelle movement. Contrary to previously published hypotheses, this inhibition of intra-axonal movement depends upon the action of batrachotoxin on action potential Na + channels. Evidence for this conclusion is: (1) the range of concentrations of batrachotoxin which inhibit saltatory organelle movement is consistent with the dose-response curve for the activation of action potential Na + channels by batrachotoxin in N18 neuroblastoma cells 10; (2) tetrodotoxin, which blocks action potential Na + channels, prevents the inhibition of organelle movements by batrachotoxin; (3) batrachotoxin has no effect on saltatory movement in cells, including some neuroblastoma cell lines, which lack action potential Na + channels; and (4) in Na +-free or low Na + media, batrachotoxin does not block organelle movement. We suggest that changes in internal ion concentrations which follow the influx of Na + are responsible for the inhibition of fast axonal transport by batrachotoxin.

Inhibition of fast axonal transport and microtubule polymerization in vitro by colchicine and colchiceine

The effects of colchicine and colchiceine on fast axonal transport in frog sciatic nerves were studied in vitro. Colchiceine inhibited the transport to about the same extent as colchicine. Preincubation at low temperature potentiated the inhibitory effect of either drug. The polymerization of purified brain tubulin was inhibited by colchiceine at 5-10 times higher concentrations than colchicine. The similarity of the effects obtained with colchicine and colchiceine indicates that both drugs arrest axonal transport by interfering with microtubule function. Colchicine and colchiceine did not affect the levels of high energy phosphates (ATP and CrP) in frog nerves indicating that a reduced energy supply was not responsible for the arrested transport.

Batrachotoxin inhibits axonal transport without affecting membrane potential in single neurons of Aplysia californica.

Intrasomatic injection of 0.25 ng of batrachotoxin (BTX) caused no depolarization of giant cerebral neurons (GCN) of Aplysia californica within 4 hr at 23 degrees C. The amplitude of action potential and the input resistance were slightly and reversibly decreased. The same dose of BTX caused a 68% inhibition of fast axonal transport.

Inhibition of fast axonal transport of [ 3H]protein by cobalt ions

Inhibition of fast axonal transport of [ 3H]protein by cobalt ions