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Batrachotoxin blocks saltatory organelle movement in electrically excitable neuroblastoma cells
Brain Research,
‘42 0
Batrachotoxin neuroblastoma
blocks saltatory organelle
211 (1981) 242-247
Elsevier/North-Holland
movement
Biomedical Press
in electrically
excitable
cells
DAVID S. FORMAN
and WILLIAM G. SHAIN, JR.*
Naval Medical Research Institute and Armed Forces Radiobiology Medical Center, Bethesda, Md. 20014 (U.S.A.)
Research
Institute,
National
Naval
(Accepted November 2Oth, 1980) Key
words:
axonal transport - batrachotoxin - neuroblastoma - saltatory organelle movement tetrodotoxin - action potential Na+ channel - video intensification microscopy
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 PM) 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 to; (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 Naf are responsible for the inhibition of fast axonal transport by batrachotoxin.
The fast axonal transport of intra-axonal organelles that can be observed by light microscopy proceeds by a series of saltatory movements*,7,13,16~“3,“*. Saltatory organelle movements (movement in a series of rapid jumps, or saltations, interrupted by pauses and occasional brief reversals of direction) are seen in a variety of other cell typese7. It is not known whether all fast axonal transport, including the transport of organelles that are too small to be directly observed in the light microscope, is also saltatory. If all fast axonal transport and saltatory organelle movements are driven by the same mechanisms, then treatments that alter fast axonal transport should have similar effects on the microscopically visible organelle movements. Several metabolic inhibitors and antimitotic drugs that block fast axonal transport have been shown to also block saltatory movements inside axons 19y23. Another agent that has been reported to inhibit the fast axonal transport of labeled materials is batrachotoxin5,24@. This finding is of special interest because some observations24J5 suggested * Present address: Toxicology Center, Division of Laboratories Department
and Research, New York State of Health, Empire State Plaza, Albany, N.Y. 12201, U.S.A.
243 that this inhibition is independent of the well-established major pharmacological action of batrachotoxin, which is to increase membrane permeability by activating the action potential Naf channels 1~2.We have examined the effect of batrachotoxin on saltatory organelle movements, and found that it does block saltatory organelle movements but that this block is dependent on the presence of action potential Na+ channels. Saltatory organelle movement was studied in neurites and cell bodies of N115 neuroblastoma cells. These cells exhibit a number of neuronal properties including electrical excitabilityll. The cells were grown on glass coverslips in a fully defined, serum-free (N2) mediums; in some instances the cells were stimulated to differentiate morphologically by X-irradiation (1000 rads). The coverslips were transferred to a Dvorak-Stotler observation chamber14 which was continuously perfused with medium at 0.4 ml/h and warmed to 37 “C with an air-stream incubator. Movements of organelles inside the cells were visualized with phase optics, using a Zeiss 63 x/1.4 objective. The images were observed and recorded with a Video Intensification Microscopy system30 consisting of an image-intensifying video camera (RCA TC1030H) and a time-lapse video tape recorder (Panasonic NV-8030). Pharmacological agents (in N2 medium unless otherwise noted) were introduced into the chamber at 0.4 ml/min until 2.0 ml flowed through the 0.2 ml chamber, after which the perfusion rate was returned to 0.4 ml/h. A variety of organelles in the neuroblastoma cells (including large phase-dark organelles, small phase-grey particles, elongated mitochondria, and phase-light vesicles of variable size) all underwent rapid, bidirectional saltatory movements. These movements closely resemble those which have been seen in acutely dissected axons 13J6923, cultured neuron&‘J*, and other types of cell@‘. Batrachotoxin inhibited these movements. In 0.5 PM batrachotoxin movement was noticably reduced by 30 min and had virtually stopped in almost all cells within an hour. At the time when almost all saltatory movement was blocked, membrane ruffling and microspike movements continued, demonstrating that all energy-dependent cell motility was not inhibited. The effects of 60 min in 0.5 ,uM batrachotoxin were reversible in many cells, but with longer incubations the cells eventually died. During the period when saltatory movement was rapidly declining, other changes were seen in some but not all cells, including neurite retraction, swelling of vesicular organelles, Brownian movement, and clumping of organelles in the cell body. These non-specific toxic changes suggest that the block of movement by batrachotoxin is a sequel of a more general derangement of the internal environment of the cell, rather than a specific action on the axonal transport mechanism. The effects of batrachotoxin were dependent on time and concentration. Inhibition of movement began more rapidly in 1.O,uM batrachotoxin than in 0.5 PM, and all movement stopped in many cells within 30 min. In 0.1-0.2 PM batrachotoxin, on the other hand, movement appeared normal in some cells after 60 min although complete inhibition was seen by 3 h. Movement was not inhibited after 3 h in 0.01 ,uM batrachotoxin. This dose-response relationship is similar to that reported for batrachotoxin activation of action potential Na + Phannels in N18 neuroblastoma cellslo. In
244 N18 cells, batrachotoxin
has a half-maximal
effect at a concentration
0.5 ,uM. This suggests that the inhibition consequence of the increase in Na+ permeability
of approximately
of saltatory movement caused by batrachotoxin,
may be a perhaps due
to changes of internal ion concentrations which follow. Although depolarization of the cell membrane would follow an increase in Nat permeability, we think that the inhibition
of saltatory
organelle
movement
is not directly related to membrane
depola-
rization but rather is the result of increased ion permeability. Two observations are consistent with this interpretation. First, fast axonal transport is not inhibited by high external K+ concentrations which depolarize the axonsr2,2*. Second, when growth phase N115 cells are studied, batrachotoxin is still effective. These cells should have a lower resting membrane potential than differentiated cellslr. Several other observations support the hypothesis that the inhibition of organelle movements by batrachotoxin is a result of activation of the action potential Na+ channels : (1) when tetrodotoxin is present (1 .O ,uM), movement continues normally in 0.5 ,uM batrachotoxin for at least 5 h. Tetrodotoxin prevents the effects of batrachotoxin by inhibiting the movements of Nat ions through the batrachotoxin-activated action potential Nat channel@; (2) batrachotoxin (1 .O ,uM) has no effect on saltatory organelle movement in cells that do not exhibit action potential Nam+channels, such as N103as and LB42g neuroblastoma cells, and 3T3 mouse fibroblasts; and (3) if the batrachotoxin-induced increase in permeability is the first step leading to inhibition of saltatory movement, then batrachotoxin should be ineffective in low Nat or Na’free media. When N 115 cells are exposed to batrachotoxin (0.5 PM) in Na+-free, choline-substituted Hanks’ saline, saltatory organelle movements are not inhibited. Batrachotoxin did appear to have some effect on ceils in the choline-substitute medium, since the addition of batrachotoxin was followed by the retraction of neurites in a few cells. Nevertheless, saltatory movements continued for at least 3 h in the cell bodies of these cells, as well as in all of the neurities which did not retract. Although we are unable to explain this apparent effect of batrachotoxin leading to neurite retraction in some cells in the Na+-free, choline-substituted saline, our main finding is that this medium does protect the cells from blockage of saltatory organelle movement. The effects of batrachotoxin were also tested in Hanks’ salines in which Na+ was replaced by sucrose or mannitol. Salines in which all of the NaCl was replaced by isoosmotic concentrations of these sugars (216 mM) could not be used, because saltatory movement ceased in them after 2 h. However, low-Na + salines (13 mM Na+, 194 m M sucrose or mannitol) would support saltatory organelle movement for at least 6 h. Batrachotoxin(0.5pM)did not block saltatoryorganelle movements in these low-Nai salines, and did not induce neurite withdrawal. (In the low-Nat salines there were changes in the shape of the cells, and the cells filled with large pinocytotic vacuoles which, like other vesicular organelles, displayed saltatory movements. However, these morphological changes were not affected by the presence of batrachotoxin.) Our evidence (summarized in Table I) supports the conclusion that batrachotoxin blocks saltatory organelle movement in electrically excitable neuroblastoma cells (and by inference, fast axonal transport in neurons) as a consequence of its activation of the action potential Na+ channels and the subsequent influx of Nat into the cells. A
245 TABLE I Eflect of batrachotoxin Abbreviations:
on saltatory
organelle movement
BTX, batrachotoxin;
TTX, tetrodotoxin.
Cell line
Expresses action potential Na+ channels
Treatment
Saltatory movement
N115 N115 N115 N115 N115 N103 LB4 3T3
+
None BTX BTX + TTX BTX + Na+-free* BTX + 13 mM Na+** BTX BTX BTX
+
+ + + + -
* Choline substituted for Na+.
+ + + + + +
** Sucrose or mannitol substituted for Naf.
similar mechanism probably accounts for the block of axonal transport by veratridiners which affects neurons by a mechanism similar to that of batrachotoxinrs. Two previous publications suggested that batrachotoxin might inhibit fast axonal transport by a mechanism independent of the activation of Na+ action potential channels by the toxin; those results can now also be reinterpreted in terms of an action on Na+ channels : (I) although most of their evidence was compatible with a primary action on the sodium channels, Ochs and Worth25 found that fast transport in sensory axons of cat sciatic nerve in vitro was blocked by batrachotoxin even in a sodium-free medium. However, this result was apparently due to retention of sodium by the nerve sheath, since in a desheathed nerve, batrachotoxin does not block fast transport in a sodium-free mediumis (R. M. Worth and S. Ochs, manuscript in preparation); and (2) Kumara-Siri reported that intracellular injections of batrachotoxin into the soma of giant cerebral neurons of Aplysia californica would block the fast axonal transport of serotonin but did not significantly alter either antidromically elicited action potentials, input resistance, or membrane potential; however, there was a transient decrease in action potential amplitude and input resistance. These observed effects of batrachotoxin may be due to the particular properties of many Aplysia neurons. In those neurons, there are two action potential mechanisms: one is tetrodotoxinsensitive and Na+ is the current-carrying ion; the other is cobalt-sensitive and Casf is the current-carrying ion 9317. Furthermore, it has been observed that these two mechanisms are unequally distributed on a given neuron, with the Ca2+ mechanism being predominantly located in the cell body 213s. There is, however, sufficient density of Ca2+ action potential current in axons to permit antidromic activation of tetrodotoxin-insensitive action potentialsso. Thus, one possible interpretation of Kumara-Siri’s observations that would be compatible with our results is as follows: the injected batrachotoxin may transiently affect the Naf action potential channels, resulting in the observed decrease in the action potential amplitude and input
246 resistance
of the cell soma. There may be no effect on the resting membrane
because of the relative numbers (pump) sites in these neurons. channels,
of action potential
Na+ channels
and Nat-K+
However, the axon, with a greater predominance
would be more sensitive to batrachotoxin.
The duration
potential ATPase of Na+
and the extent of the
effects of batrachotoxin in the axon may not be evident in recordings made in the neuronal soma because of the relatively small size of the axon compared to the large somaao. Thus, the reduction in fast axonal transport of serotonin might still result from batrachotoxin activation of axonal action potential Na-* channels and the resulting
alterations
in intra-axonal
ion distributions,
Our finding that batrachotoxin blocks saltatory organelle movement in electrically excitable cells is further evidence for our hypothesis that the mechanisms which drive fast axonal transport
and saltatory
organelle
movement
are the same. Although
our results show that an increase in permeability to Nat- is a necessary initial event in the inhibition of saltatory organelle movement by batrachotoxin, they do not identify which changes in internal ionic concentration are responsible for the block. Increase in Caa+ are all possible internal Na+, loss of internal K +, or an increase in internal candidates. Naval Medical Research and Development Command, Research Task ZF58.524.013.1023. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. We wish to thank Brenda Bolden for expert technical assistance with the cell culture, and David K. Wood, Donna Mitchell and David Weisman for assistance with the time-lapse video microscopy. Batrachotoxin was a generous gift from Dr. John W. Daly. We wish to thank Dr. William A. Catterall for the LB 4 cells and Dr. Katherine Holmes for the 3T3 cells. We also wish to thank Drs. Daly, Catterall, and Sidney Ochs for helpful discussion.
1 Albuquerque, E. X. and Daly, J. W., Batrachotoxin, a selective probe for channels modulating sodium conductances in electrogenic membranes. Jn P. Cuatrecasas (Ed.), The Specificity and Action of Animal, Bacterial and Plant Toxins (Receptors and Recognition, Series B) Vol. I, Chapman and Hall, London, 1977, pp. 297-338. 2 Albuquerque, E. X., Daly, J. W. and Witkop, B. Batrachotoxin: chemistry and pharmacology, Science, 172 (1972) 995-1002. 3 Albuquerque, E. X., Sasa, M. and Sarvey, J. M., Batrachotoxin has no effect on the electrogenic membranes of lobster and crayfish muscles, Lz$ Sci., I I (1972) 357-363. 4 Berlinrood, M., McGee-Russell, S. M. and Allen, R. D., Patterns of particle movement in nerve fibers in vitro-an analysis by photokymography and microscopy, J. Cell&-i., 11(1972) 875-886. 5 Boegman, R. J. and Albuquerque, E. X., Axonal transport in rats rendered paraplegic following a single subarachnoid injection of either batrachotoxin or 6-aminonicotinamide into the spinal cord, J. Neurobiol., I I (1980) 2833290. 6 Bottenstein, J. E. and Sato, G. H., Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. nat. Acad. Sri. ( Wash.), 76 (1979) 5 14-5 17. 7 Breuer, A. C., Christian, C. N., Henkart, M. and Nelson, P. G., Computer analysis of organelle translocation in primary neuronal cultures and continuous cell lines, J. Cell Biol., 65 (1975) 562-576.
247 8 Calvin, W. H. and Hartline, D. K., Retrograde invasion of lobster stetch receptor somata in control of firing rate and extra spike patterning, J. Neurophysiol., 40 (1977) 106-l 18. 9 Carpenter, D. 0. and Gunn, K., The dependence of pacemaker discharge of Aply& neurons upon Na+ and Ca++, J. Ceil Physiol., 75 (1970) 121-128. 10 Catterall, W. A., Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin, J. biol. Chem., 250 (1975) 4053-4059. 11 Chalazonitis, A. and Greene, L. A., Enhancement in excitability properties of mouse neuroblastoma cells cultured in the presence of dibutyryl cyclic AMP, Bruin Research, 72 (1974) 340-345. 12 Chan, S. Y., Ochs, S. and Worth, R. M., The requirement for Ca2+ and the effect of other ions on axoplasmic transport in mammalian nerve, J. Physiol. (Lond.), 301 (1980) 477-504. 13 Cooper, P. D. and Smith, R. S., The movement of optically detectable organelles in myelinated axons of Xenopus laevis, J. Physiol. (Lo&), 242 (1974) 77-97. 14 Dvorak, J. A. and Stotler, W. F., A controlled-environment culture system for high resolution light microscopy, Exp. CeN Res., 68 (1971) 144148. 15 Edstriim, A., Rapid axonal transport in vitro. Effects of derivatives of cyclic AMP and other agents acting on the cyclic AMP system, J. Neurobiol., 8 (1977) 371-380. 16 Forman, D. S., Padjen, A. L. and Siggins, G. R., Axonal transport of organelles visualized by light microscopy: cinemicrographic and computer analysis, Bruin Research, 136 (1977) 197-213. 17 Geduldig, D. and Junge, D., Sodium and calcium components of action potentials in the Aplysia giant neuron, J. Physiol. (Lond.), 199 (1968) 347-365. 18 Grafstein, B. and Forman, D. S., Intracellular transport in neurons, Physiol. Revs., 60 (1980) 1167-1283. 19 Hammond, G. R. and Smith, R. S., Inhibition of the rapid movement of optically detectable axonal particles by colchicine and vinblastine, Bruin Research, 128 (1977) 227-242. 20 Horn, R., Propagating calcium spikes in an axon of Ap[ysiu, J. Physiol. (Lond.), 281 (1978) 513-534. 21 Junge, D. and Miller, J., Different spike mechanisms in axon and soma of molluscan neurone, Nature (Land.), 252 (1974) 155-156. 22 Kado, R. T., Ap/ysia giant cell: soma-axon voltage clamp current differences, Science, 182 (1973) 843-845. 23 Kirkpatrick, J. B., Bray, J. J. and Palmer, S. M., Visualization of axoplasmic flow in vitro by Nomarski microscopy. Comparison to rapid flow of radioactive proteins, Brain Research, 43 (1972) l-10. 24 Kumara-Siri, M. H., Batrachotoxin inhibits axonal transport without affecting membrane potential in single neurons of Aplysiu Californica, J. Neurobiol., 10 (19798) 509-512. 25 Ochs, S. and Worth, R., Batrachotoxin block of fast axoplasmic transport in mammalian nerve fibers, Science, 187 (1975) 1087-1089. 26 Peacock, J., Minna, J., Nelson, P. and Nirenberg, M., Use of aminopterin in selecting electrically active neuroblastoma cells, Exp. Cell Res., 73 (1972) 367-377. 27 Rebhun, L. I., Polarized intracellular particle transport: saltatory movements and cytoplasmic streaming, ht. Rev. Cytol., 32 (1972) 93-137. 28 Smith, R. S., The short term accumulation of axonally transported organelles in the region of localized lesions of single myelinated axons, J. Neurocytol., 9 (1980) 39-65. 29 West, G. J. and Catterall, W. A. Selection of variant neuroblastoma clones with missing or altered sodium channels, Proc. nut. Acad Sci. (Wash.), 76 (1979) 4136-4140. 30 Willingham, M. C. and Pastan, I., The visualization of fluorescent proteins in living cells by video intensification microscopy (VIM), Cell, 13 (1978) 501-507.
‘42 0
Batrachotoxin neuroblastoma
blocks saltatory organelle
211 (1981) 242-247
Elsevier/North-Holland
movement
Biomedical Press
in electrically
excitable
cells
DAVID S. FORMAN
and WILLIAM G. SHAIN, JR.*
Naval Medical Research Institute and Armed Forces Radiobiology Medical Center, Bethesda, Md. 20014 (U.S.A.)
Research
Institute,
National
Naval
(Accepted November 2Oth, 1980) Key
words:
axonal transport - batrachotoxin - neuroblastoma - saltatory organelle movement tetrodotoxin - action potential Na+ channel - video intensification microscopy
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 PM) 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 to; (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 Naf are responsible for the inhibition of fast axonal transport by batrachotoxin.
The fast axonal transport of intra-axonal organelles that can be observed by light microscopy proceeds by a series of saltatory movements*,7,13,16~“3,“*. Saltatory organelle movements (movement in a series of rapid jumps, or saltations, interrupted by pauses and occasional brief reversals of direction) are seen in a variety of other cell typese7. It is not known whether all fast axonal transport, including the transport of organelles that are too small to be directly observed in the light microscope, is also saltatory. If all fast axonal transport and saltatory organelle movements are driven by the same mechanisms, then treatments that alter fast axonal transport should have similar effects on the microscopically visible organelle movements. Several metabolic inhibitors and antimitotic drugs that block fast axonal transport have been shown to also block saltatory movements inside axons 19y23. Another agent that has been reported to inhibit the fast axonal transport of labeled materials is batrachotoxin5,24@. This finding is of special interest because some observations24J5 suggested * Present address: Toxicology Center, Division of Laboratories Department
and Research, New York State of Health, Empire State Plaza, Albany, N.Y. 12201, U.S.A.
243 that this inhibition is independent of the well-established major pharmacological action of batrachotoxin, which is to increase membrane permeability by activating the action potential Naf channels 1~2.We have examined the effect of batrachotoxin on saltatory organelle movements, and found that it does block saltatory organelle movements but that this block is dependent on the presence of action potential Na+ channels. Saltatory organelle movement was studied in neurites and cell bodies of N115 neuroblastoma cells. These cells exhibit a number of neuronal properties including electrical excitabilityll. The cells were grown on glass coverslips in a fully defined, serum-free (N2) mediums; in some instances the cells were stimulated to differentiate morphologically by X-irradiation (1000 rads). The coverslips were transferred to a Dvorak-Stotler observation chamber14 which was continuously perfused with medium at 0.4 ml/h and warmed to 37 “C with an air-stream incubator. Movements of organelles inside the cells were visualized with phase optics, using a Zeiss 63 x/1.4 objective. The images were observed and recorded with a Video Intensification Microscopy system30 consisting of an image-intensifying video camera (RCA TC1030H) and a time-lapse video tape recorder (Panasonic NV-8030). Pharmacological agents (in N2 medium unless otherwise noted) were introduced into the chamber at 0.4 ml/min until 2.0 ml flowed through the 0.2 ml chamber, after which the perfusion rate was returned to 0.4 ml/h. A variety of organelles in the neuroblastoma cells (including large phase-dark organelles, small phase-grey particles, elongated mitochondria, and phase-light vesicles of variable size) all underwent rapid, bidirectional saltatory movements. These movements closely resemble those which have been seen in acutely dissected axons 13J6923, cultured neuron&‘J*, and other types of cell@‘. Batrachotoxin inhibited these movements. In 0.5 PM batrachotoxin movement was noticably reduced by 30 min and had virtually stopped in almost all cells within an hour. At the time when almost all saltatory movement was blocked, membrane ruffling and microspike movements continued, demonstrating that all energy-dependent cell motility was not inhibited. The effects of 60 min in 0.5 ,uM batrachotoxin were reversible in many cells, but with longer incubations the cells eventually died. During the period when saltatory movement was rapidly declining, other changes were seen in some but not all cells, including neurite retraction, swelling of vesicular organelles, Brownian movement, and clumping of organelles in the cell body. These non-specific toxic changes suggest that the block of movement by batrachotoxin is a sequel of a more general derangement of the internal environment of the cell, rather than a specific action on the axonal transport mechanism. The effects of batrachotoxin were dependent on time and concentration. Inhibition of movement began more rapidly in 1.O,uM batrachotoxin than in 0.5 PM, and all movement stopped in many cells within 30 min. In 0.1-0.2 PM batrachotoxin, on the other hand, movement appeared normal in some cells after 60 min although complete inhibition was seen by 3 h. Movement was not inhibited after 3 h in 0.01 ,uM batrachotoxin. This dose-response relationship is similar to that reported for batrachotoxin activation of action potential Na + Phannels in N18 neuroblastoma cellslo. In
244 N18 cells, batrachotoxin
has a half-maximal
effect at a concentration
0.5 ,uM. This suggests that the inhibition consequence of the increase in Na+ permeability
of approximately
of saltatory movement caused by batrachotoxin,
may be a perhaps due
to changes of internal ion concentrations which follow. Although depolarization of the cell membrane would follow an increase in Nat permeability, we think that the inhibition
of saltatory
organelle
movement
is not directly related to membrane
depola-
rization but rather is the result of increased ion permeability. Two observations are consistent with this interpretation. First, fast axonal transport is not inhibited by high external K+ concentrations which depolarize the axonsr2,2*. Second, when growth phase N115 cells are studied, batrachotoxin is still effective. These cells should have a lower resting membrane potential than differentiated cellslr. Several other observations support the hypothesis that the inhibition of organelle movements by batrachotoxin is a result of activation of the action potential Na+ channels : (1) when tetrodotoxin is present (1 .O ,uM), movement continues normally in 0.5 ,uM batrachotoxin for at least 5 h. Tetrodotoxin prevents the effects of batrachotoxin by inhibiting the movements of Nat ions through the batrachotoxin-activated action potential Nat channel@; (2) batrachotoxin (1 .O ,uM) has no effect on saltatory organelle movement in cells that do not exhibit action potential Nam+channels, such as N103as and LB42g neuroblastoma cells, and 3T3 mouse fibroblasts; and (3) if the batrachotoxin-induced increase in permeability is the first step leading to inhibition of saltatory movement, then batrachotoxin should be ineffective in low Nat or Na’free media. When N 115 cells are exposed to batrachotoxin (0.5 PM) in Na+-free, choline-substituted Hanks’ saline, saltatory organelle movements are not inhibited. Batrachotoxin did appear to have some effect on ceils in the choline-substitute medium, since the addition of batrachotoxin was followed by the retraction of neurites in a few cells. Nevertheless, saltatory movements continued for at least 3 h in the cell bodies of these cells, as well as in all of the neurities which did not retract. Although we are unable to explain this apparent effect of batrachotoxin leading to neurite retraction in some cells in the Na+-free, choline-substituted saline, our main finding is that this medium does protect the cells from blockage of saltatory organelle movement. The effects of batrachotoxin were also tested in Hanks’ salines in which Na+ was replaced by sucrose or mannitol. Salines in which all of the NaCl was replaced by isoosmotic concentrations of these sugars (216 mM) could not be used, because saltatory movement ceased in them after 2 h. However, low-Na + salines (13 mM Na+, 194 m M sucrose or mannitol) would support saltatory organelle movement for at least 6 h. Batrachotoxin(0.5pM)did not block saltatoryorganelle movements in these low-Nai salines, and did not induce neurite withdrawal. (In the low-Nat salines there were changes in the shape of the cells, and the cells filled with large pinocytotic vacuoles which, like other vesicular organelles, displayed saltatory movements. However, these morphological changes were not affected by the presence of batrachotoxin.) Our evidence (summarized in Table I) supports the conclusion that batrachotoxin blocks saltatory organelle movement in electrically excitable neuroblastoma cells (and by inference, fast axonal transport in neurons) as a consequence of its activation of the action potential Na+ channels and the subsequent influx of Nat into the cells. A
245 TABLE I Eflect of batrachotoxin Abbreviations:
on saltatory
organelle movement
BTX, batrachotoxin;
TTX, tetrodotoxin.
Cell line
Expresses action potential Na+ channels
Treatment
Saltatory movement
N115 N115 N115 N115 N115 N103 LB4 3T3
+
None BTX BTX + TTX BTX + Na+-free* BTX + 13 mM Na+** BTX BTX BTX
+
+ + + + -
* Choline substituted for Na+.
+ + + + + +
** Sucrose or mannitol substituted for Naf.
similar mechanism probably accounts for the block of axonal transport by veratridiners which affects neurons by a mechanism similar to that of batrachotoxinrs. Two previous publications suggested that batrachotoxin might inhibit fast axonal transport by a mechanism independent of the activation of Na+ action potential channels by the toxin; those results can now also be reinterpreted in terms of an action on Na+ channels : (I) although most of their evidence was compatible with a primary action on the sodium channels, Ochs and Worth25 found that fast transport in sensory axons of cat sciatic nerve in vitro was blocked by batrachotoxin even in a sodium-free medium. However, this result was apparently due to retention of sodium by the nerve sheath, since in a desheathed nerve, batrachotoxin does not block fast transport in a sodium-free mediumis (R. M. Worth and S. Ochs, manuscript in preparation); and (2) Kumara-Siri reported that intracellular injections of batrachotoxin into the soma of giant cerebral neurons of Aplysia californica would block the fast axonal transport of serotonin but did not significantly alter either antidromically elicited action potentials, input resistance, or membrane potential; however, there was a transient decrease in action potential amplitude and input resistance. These observed effects of batrachotoxin may be due to the particular properties of many Aplysia neurons. In those neurons, there are two action potential mechanisms: one is tetrodotoxinsensitive and Na+ is the current-carrying ion; the other is cobalt-sensitive and Casf is the current-carrying ion 9317. Furthermore, it has been observed that these two mechanisms are unequally distributed on a given neuron, with the Ca2+ mechanism being predominantly located in the cell body 213s. There is, however, sufficient density of Ca2+ action potential current in axons to permit antidromic activation of tetrodotoxin-insensitive action potentialsso. Thus, one possible interpretation of Kumara-Siri’s observations that would be compatible with our results is as follows: the injected batrachotoxin may transiently affect the Naf action potential channels, resulting in the observed decrease in the action potential amplitude and input
246 resistance
of the cell soma. There may be no effect on the resting membrane
because of the relative numbers (pump) sites in these neurons. channels,
of action potential
Na+ channels
and Nat-K+
However, the axon, with a greater predominance
would be more sensitive to batrachotoxin.
The duration
potential ATPase of Na+
and the extent of the
effects of batrachotoxin in the axon may not be evident in recordings made in the neuronal soma because of the relatively small size of the axon compared to the large somaao. Thus, the reduction in fast axonal transport of serotonin might still result from batrachotoxin activation of axonal action potential Na-* channels and the resulting
alterations
in intra-axonal
ion distributions,
Our finding that batrachotoxin blocks saltatory organelle movement in electrically excitable cells is further evidence for our hypothesis that the mechanisms which drive fast axonal transport
and saltatory
organelle
movement
are the same. Although
our results show that an increase in permeability to Nat- is a necessary initial event in the inhibition of saltatory organelle movement by batrachotoxin, they do not identify which changes in internal ionic concentration are responsible for the block. Increase in Caa+ are all possible internal Na+, loss of internal K +, or an increase in internal candidates. Naval Medical Research and Development Command, Research Task ZF58.524.013.1023. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. We wish to thank Brenda Bolden for expert technical assistance with the cell culture, and David K. Wood, Donna Mitchell and David Weisman for assistance with the time-lapse video microscopy. Batrachotoxin was a generous gift from Dr. John W. Daly. We wish to thank Dr. William A. Catterall for the LB 4 cells and Dr. Katherine Holmes for the 3T3 cells. We also wish to thank Drs. Daly, Catterall, and Sidney Ochs for helpful discussion.
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