Cell Tiss. Res. 166, 413420 (1976)

Cell and Tissue Research 9 by Springer-Verlag 1976

An Ultrastructural Examination of the Action of Vinblastine on Microtubules, Neurofilaments and Muscle Filaments in vitro D.R. Tomlinson and T. Bennett* Department of Physiology and Pharmacology, University Hospital and Medical School, Clifton Boulevard, Nottingham, England

Summary. In vitro incubation of autonomic nerves with vinblastine sulphate (1 • 10-4 M) caused a disappearance of microtubules within 15 min; during the following 15 rain paracrystalline arrays appeared within the axons. An increase in the abundance of microfilaments was also observed, but these did not appear to arise from disaggregated microtubules since the increase in microfilament numbers was noted at an incubation time when crystal formation was extensive. Pretreatment of autonomic nerves with colchicine (2.5 • 10- 4 M) caused a reduction of approximately 80% in the numbers of microtubules, but did not prevent the formation of crystals on subsequent exposure to vinblastine. No ultrastructural changes were observed in myofilaments on incubation with vinblastine.

Key words: Vinblastine - Microtubules - Neurofilaments - Muscle filaments - Microtubular crystals.

Introduction The interaction of high concentrations of vinca alkaloids with the microtubules of the mitotic spindle leads to the formation of regular crystals as seen with the electron microscope (Bensch and Malawista, 1968). Similar crystal formation has also been observed on exposure of central neurones to vinca alkaloids (Kotorii et al., 1971). It has also been suggested that the vinca alkaloids promote the formation of 'neurofibrillary tangles' (Sell and Lampert, 1968) and that this may occur as a result of conversion of disaggregated microtubules to microfilaments (neurofibrils) (Wisniewski etal., 1968). Send offprint requests to: Dr. D.R. Tomlinson, Department of Physiology and Pharmacology,

University Hospital and Medical School, Clifton Boulevard, Nottingham, NG7 2UH, England. * We gratefully acknowledge the use of the electron microscopes in the Departments of Pathology and of Human Morphology, University of Nottingham School and thank Dr. Graham Robinson and Annette Tomlinson for their co-operation.

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Biochemical studies have demonstrated that vinblastine precipitates a number of proteins (Wilson et al., 1970). It is of interest that, in addition to tubulin (the sub-unit of microtubules), actin was found to precipitate. Blitz and Fine (1974) have demonstrated the presence, in neurones, of an actin-like protein which may comprise the neurofilaments. Thus, the disruption of autonomic neuronal function by the vinca alkaloids (see, for example Banks et aI., 1971; Bennett, 1975) could arise from an action on microtubules and/or neurofilaments. In the present investigation an ultrastructural examination was made of the effects of high concentrations of vinblastine on the microtubules and microfilaments in autonomic neurones and on the filamentous structures in skeletal, cardiac and smooth muscle.

Materials and Methods All tissues were incubated in Krebs' solution containing vinblastine sulphate ('Velbe', Lilly) at a concentration of 1 x 10 - 4 M. The solution was gassed with 95% Oz, 5% COz and kept at 37 ~ C. In the first series of experiments, guinea pig hypogastric nerves were incubated with vinblastine for different periods of time from 5 min to 4 hr in order to examine the time course of the action of the drug. A second series was performed in which hypogastric nerves, right atria, vasa deferentia and segments of gastroeneminus muscle from rats and guinea pigs were incubated with vinblastine for 4 hr, since at this time neuronal function is impaired (eg Bennett, 1975). For both series of experiments, control tissues were incubated in normal Krebs' solution for the same period as the experimental tissues were incubated with vinblastine. A third series of experiments was performed in order to determine whether pre-treatment of autonomic nerves with colchicine altered their response to vinblastine. Segments of guinea pig hypogastric nerve were incubated in Krebs' solution containing colchicine (Sigma Chemical Company) at a concentration of 2.5 x 10 4 M for 5 hr and then transferred to Krebs' solution containing vinblastine (1 x 10- ~ M) for a further 11/2 hr. Control tissue was incubated with colchicine for 5 hr and then transferred to Krebs' solution containing no drugs for 11/2 hr; other control nerves were fixed after the 5 hr incubation with colchicine alone. At the end of all incubations the tissues were placed in a pool of fixative and cut into 1 m m cubes. Tissues were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer for 4 hr at r o o m temperature. After washing for 24 hr in several changes of 0.1 M phosphate buffer containing 10% sucrose, the tissues were post-fixed in 1% OsO4 in Millonig's buffer for 2 h r at 4~ Tissue blocks were then dehydrated in graded ethanols, passed through epoxypropane and embedded in epoxy resin ('Araldite': Ciba Ltd., UK). Sections were cut on a Reichert O M U 2 ultramicrotome, stained on the grid with aqueous lead citrate (Reynolds, 1963) and examined and photographed in a Philips EM300 electron microscope, In addition to a thorough subjective examination of the ultrastructure of all specimens, changes in the abundance of microtubules were quantitated. Micrographs of fields selected at r a n d o m were taken and prints at a magnification of 40,000x were made. The microtubule profiles in all non-myelinated axon profiles in these prints were counted. It was readily apparent that the number of microtubules present in an axon increased with its cross-sectional area. Therefore the n u m b e r of microtubules in each axon profile was related to the profile area by cutting out the profiles from the prints and weighing them. The results are expressed as microtubules/~tm z axon profile area. The identification and enumeration of microfilaments was much more difficult. Reliable counts could be made only in those profiles containing microfilaments cut transversely. Since these were

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the exception rather than the norm no statistically admissable randomisation procedure could be adopted for axon profile sampling. Assessments of microfilament abundance were therefore made by careful subjective evaluation of counts made from non-randomly selected axon profiles.

Results

Hypogastric Nerves. Normal hypogastric nerves from either rats or guinea pigs contained a mixture of myelinated and non-myelinated fibres. Both types of fibre contained microtubules (Table 1 and Fig. 1 a). Incubation of hypogastric nerves in normal Krebs' solution for periods of time up to 71/2 hr caused no measurable alteration in the appearance or abundance of these organelles. Nerves incubated with vinblastine for 5 min were indistinguishable in appearance, at the ultrastructural level, from untreated or control nerves. After 10 rain incubation with vinblastine, however, the number of microtubules in both myelinated and non-myelinated fibres was significantly reduced (Table 1). This reduction had progressed further by 15 min, after which time most of the a x o n s especially those in the peripheral parts of the nerve trunk-contained no microtubule profiles (see Table 1 and Fig. 1 b). Incubation with vinblastine for 15 rain also promoted the appearance of ill-defined crystals in a few non-myelinated axons (Fig. 1 b). These structures were much more clearly defined and present in many more fibres in nerve trunks incubated for 30 rain. At this time they were also present in myelinated fibres and normal microtubles were absent from both fibre types (Fig. 2 a, b). Crystals were also present in the cytoplasm of many Schwann cells (Fig. 2 a). After incubations with vinblastine of longer than 30 min, the appearance of the crystals was little altered. The crystals displayed a characteristically regular structure. In sections parallel to their long axis, the crystals were seen to consist of regularly spaced parallel laminae with a periodicity of 27.5 to 30 nm. Crystal profiles cut perpendicularly to their long axis displayed a honeycomb structure (see Fig. 2 b). The

Table 1. The effect of incubation with vinblastine sulphate on the numbers of microtubules in profiles of non-myelinated axons from guinea pig hypogastric nerves. The figures in brackets denote the number of axon profiles in each sample Treatment

Microtubules/gm 2 of axon profile ( _+SEM)

Percentage reduction of control number by treatment

Untreated nerves

90.6+ 4.7 (80)

Control nerves - incubated for 15 min in Krebs' solution

95.1 +- 6.3 (60)

Nerves incubated with vinblastine sulphate (1 x 10-4 M) for 10 min

46.7+- 11.9 (80)

50.9

Nerves incubated with vinblastine sulphate (1 • 10-4 M) for 15 min

10.2+- 1.3 (80)

89.3

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Fig. 1 a and b. Both electron micrographs are taken from transverse sections of guinea pig hypogastric nerves. (a) Untreated nerve, showing profiles of non-myelinated axons (A) invaginated into Schwann cells (S). The axons contain many microtubule profiles (m). The magnification is x 36,300. (b) Nerve incubated with vinblastine sulphate (1 • 10 4 M) for 15 min. A few microtubules (m) remain in some fibres whilst others (p) contain none. An early stage in the formation of a crystal is visible in the portion of axoplasm indicated by the large arrow heads, x 34,500. Marker lines on both micrographs denote 0.5 tim

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417

Fig. 2 a and b. Both electron micrograptts are taken from transverse sections of guinea pig hypogastric nerves incubated for 1 hr with vinblastine sulphate (1 x 10-aM), (a) Crystals are present both in axoplasm (ax) and in the cytoplasm of Schwann cells (sx). Large numbers of microfilaments (D are present in all axons, x 39,000. (b) A single non-myelinated axon profile contains a well-defined crystal (x) and many microfilaments 09. No microtubules are present, x 54,250. Marker lines on both micrographs denote 0.5 ~tm

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long axes of the crystals were frequently parallel to the long axes of their axons. In axon profiles, from nerves treated for 10-15 min with vinblastine, the disappearance of microtubules was accompanied by no apparent change in the abundance of microfilaments. However, nerves incubated for longer periods of time (1-4 hr), in which crystal formation was extensive, contained axons showing many more microfilaments than normal (see Fig. 2a, b). No microfilament 'tangles' (as characteristed by the micrographs of Seil and Lampert, 1968) were observed, though the presence of crystals restricted the axoplasm available for occupation by micro filaments causing them to be more densely packed than normal (Fig. 2b). In rat hypogastric nerves incubated for 4 hr with vinblastine, the appearance of crystals and microflaments together with the complete absence of microtubules were exactly similar to those described above for guinea pig nerves. Muscle Tissue. No signifcant ultrastructural changes were observed in skeletal, cardiac or smooth muscle cells. There was no evidence of disorientation of the myofilaments in the striated muscles. The autonomic nerve fibres present in the interstitial spaces in atria and vasa deferentia, however, contained crystals and were devoid of microtubules indicating that the vinblastine had attained a high concentration within the tissues. In these fibres the crystals were confined to the non-varicose portions of the fibres-none were found in the terminals. Pre-Treatment with Colchicine. In the control guinea pig hypogastric nerves incubated with colchicine, but not subsequently with vinblastine (see 'Methods'), the number of microtubules was markedly reduced (16.3 + 4.9 (SEM) per axon; n--60 axons). However, when this colchicine treatment was followed by incubation for 11/2 hr with vinblastine, crystal formation was as extensive as was seen with vinblastine treatment alone (see above). Discussion

The concomitant disappearance of microtubules and appearance of crystals on treatment of nervous tissue with vinblastine indicates that the crystals might arise from disrupted microtubules. Other interpretations are, of course, possible, but recent work indicates that the above conclusions are justified. The reaction of purified tubulin with vinblastine produces ordered paracrystalline structures (Bensch et al., 1969; Marantz and Shelanaski, 1970) and the available evidence indicates that the process is a straightforward chemical reaction between the two substances, involving magnesium ions (Weisenberg and Timasheff, 1970). Berry and Shelanski (1972) have suggested that tubulin is stabilised in the microtubule form by guanosine triphosphate (GTP) and that vinblastine interferes with GTP binding, leading to release of the nucleotide and the coalescence of the labilised tubulin to form crystals. In the present study, the time course of the action of vinblastine on the microtubules was clearly divided into two stages, the first stage being the disappearance of the microtubules and the second the appearance of crystals. It is interesting to note that Banks et al. (1971), using lower concentrations of

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vinblastine (1 x 10- 5M) than those used here, observed a disappearance of microtubules without concomitant crystal formation. This was seen in spite of prolonged exposure (48 hr) of the nervous tissue to vinblastine. It would appear therefore that higher concentrations of vinblastine are required to promote crystal formation than those needed to dissolve the microtubules. Thus, in our experiments, it is probable that crystals formed only when a critical amount of the drug had permeated the tissue. Perhaps the hypotheses of Berry and Shelanski (1972), referred to above, may be modified to suggest that low concentrations of vinblastine interfere with GTP binding, leading to disaggregation of the tubule subunits, whilst higher concentrations cause the sub-units to coalesce in the form of crystals. In the present investigation the disappearance of microtubules was not associated with an increase in the numbers of microfilaments. Indeed such an increase in microfilaments occurred only when crystal formation was extensive. It is therefore difficult to see how vinblastine could have promoted conversion of microtubules to microfilaments, an action suggested by Wisniewski et al. (1968). Indeed, if the tubulin formed crystals, such a conversion is unlikely to occur. Furthermore, we observed no aggregation of microfilaments into tightly packed bundles-the so-called 'neurofibrillary tangles' reported by Seil and Lampert (1968). This report of aggregated bundles of microfilaments as well as that made by Wisniewski e t a l . (1968) arise from experiments in which neurons were exposed to vinblastine over periods of time ranging from 31 hr to 14 days. Since no evidence of this phenomenon was found in the present short-term experiments we suggest that microfilament aggregation may be a more complex cellular response to vinblastine treatment rather than a direct effect of the drug on microtubules. It is perhaps significant that bundles of aggregated microfilaments are seen in neurones as a chronic effect of axotomy (Zelena et al., 1968). The formation of microtubule crystals was also noted in several other cell types which were present in the tissues used in this study. These ceils comprised fibroblasts, endothelial cells, polymorph granulocytes, extramedullary chromaffin cells and the perikarya of neurones in the diffuse hypogastric plexus. This ubiquitous action of vinblastine on microtubules may serve as a useful method for specific, though retrospective, identification of these organelles in situations where their occurrence is contentious (see Pellegrino de lraldi and de Robertis, 1968). The absence of any effect of vinblastine on the ultrastructure of muscle filaments is in contrast to the report (Wilson et al., 1970) that vinblastine precipitates actin. These workers, however, used an even higher concentration of drug (1 x 10-3 M) than ourselves and sub-cellular preparations were employed. It remains to be demonstrated what effects vinca alkaloids may exert on the function of muscle proteins. References Banks, P., Mayor, D., Mitchell, M., Tomlinson, D. : Studies on the translocation of noradrenalinecontaining vesicles in post-ganglionic sympathetic neurones in vitro. Inhibition of movement by

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colchicine and vinblastine and evidence for the involvement of axonal microtubules. J. Physiol. (Lond.) 216, 625439 (1971) Bennett, T. : The in vitro effects of vinblastine and vincristine on the responses of the guinea-pig vas deferens to nerve stimulation. Naunyn-Schmiedeberg's Arch. Pharmacol. 287, 413-422 (1975) Bensch, K.G., Malawista, S.E.: Microtubule crystals: a new biophysical phenomenon induced by vinca alkaloids. Nature (Lond.) 218, 1176 1177 (1968) Bensch, K.G., Marantz, R., Wisniewski, H., Shelanski, M.: Induction in vitro of microtubular crystals by vinca alkaloids. Science 165, 495-496 (1969) Berry, R.W., Shelanski, M.L. : Interactions of tubulin with vinblastine and guanosine triphosphate. J. molec. Biol. 71, 71 80 (1972) Blitz, A.L., Fine, R.E.: Muscle-like contractile proteins and tubulin in synaptosomes. Proc. nat. Acad. Sci. (Wash.) 71, 4472-4476 (1974) Kotorii, K., Mori, H., Yoshida, M.: A peculiar crystalline structure in neurons of rabbits treated with vincristine. Kurume med. J. 18, 5743 (1971) Marantz, R., Shelanski, M.L.: Structure of microtubular crystals induced by vinblastine in vitro. J. Cell Biol. 44, 234-238 (1970) Pellegrino de Iraldi, A., de Robertis, E.: Action of reserpine on the submicroscopic morphology of the pineal gland. Experientia (Basel) 17, 122 124 (1961) Reynolds, E.S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208~12 (1963) Seil, F.J., Lampert, P.W.: Neurofibrillary tangles induced by vincristine and vinblastine sulfate in central and peripheral neurons in vitro. Exp. Neurol. 21, 219~30 (1968) Weisenberg, R.C., Timasheff, S.N. : Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine and vinblastine. Biochemistry (Wash.) 9, 4110-4116 (1970) Wilson, L., Bryan, J., Ruby, A., Mazia, D.: Precipitation of proteins by vinblastine and calcium ions. Proc. nat. Acad. Sci. (Wash.) 66, 807 814 (1970) Wisniewski, H., Shelanski, M.L., Terry, R.D. : Effects of mitotic spindle inhibitors on neurotubules and neurofilaments in anterior horn cells. J. Cell Biol. 38, 224-229 (1968) Zelena, J., Lubinska, L., Gutmann, E.: Accumulation of organelles at the ends of interrupted axons. Z. Zellforsch. 91,200-219 (1968)

Received November 10, 1975

An ultrastructural examination of the action of vinblastine on microtubules, neurofilaments and muscle filaments in vitro.

In vitro incubation of autonomic nerves with vinblastine sulphate (1 times 10(-4) M) caused a disappearance of microtubules within 15 min; during the ...
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