Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6425-6429, December 1979 Cell Biology

Tetrodotoxin-sensitive sodium channels in normal human fibroblasts and normal human glia-like cells (sodium ionophore/veratridine/scorpion toxin) ROBERT MUNSON, JR. *, BENGT WESTERMARKt, AND LUIS GLASER* *Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110; and tThe Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden Communicated by William D. Phillips, September 10, 1979

ABSTRACT -sensitive sodium channels are MATERIALS AND METHODS detectable in normal human fibroblasts and in "glia-like" cells at appreciable levels when compared to what is observed in Normal human lung fibroblasts (line IMR 91, Institute for established neuronal cell lines in culture. Two- to 3fold stimu- Medical Research, Camden, NJ) and normal human dermal lations of sodium influx are observed in the presence of 0.2 mM fibroblasts initiated from skin biopsies were obtained from W. veratridine and scorpion venom at 0.1 mg/mi. Tetrodotoxin (2 Sly (Washington University School of Medicine). Cultures were MM) inhibits the observed stimulation of sodium influx. Previous grown in minimal essential medium containing 15% fetal bo- work has indicated that these act on the voltage- vine serum, glutamine (0.1 mg/ml), sodium pyruvate (0.11 sensitive sodium ionophore of excitable cells, and the presence of such channels in cells generally considered nonexcitable mg/ml), and 100 units of penicillin and 100 jig of streptomycin raises questions regarding both the uniqueness of this ionophore per ml. Normal human glia-like cells (CG series) were isolated as a property of excitable cells and the origin of the cells gen- as described by Ponten and MacIntyre (5). Glial cells were erally described as fibroblasts. grown as above without pyruvate and with the substitution of 10% calf serum for the fetal calf serum. 3T3 and simian virus Neuronal cells are characterized by the presence of an action- 40-transformed 3T3 cultures were grown in Dulbecco's mod- potential Na+ ionophore or voltage-dependent Na+ channel. ified Eagle's medium containing 10% calf serum. C6 glioma This channel is by definition the structure responsible for the and N18 neuroblastoma cells were grown in this medium increase in permeability to Na+ during the depolarizing phase containing 10% fetal bovine serum. All cultures were supple- of the nerve action potential. This ionophore can be activated mented with glutamine and antibiotics as described above. by veratridine and synergistically with veratridine by scorpion Sodium uptake assays were performed as described by toxin. This activation is specifically blocked by the addition of Stallcup and Cohn (6) with the addition of bovine serum al- tetrodotoxin. The presence of this ionophore can be determined bumin (1 mg/ml) and scorpion (Lelurus quinquestriatus) electrophysiologically or by measurements of Na+ influx into venom (0.1 mg/ml) (Sigma) (7). Assays were routinely carried cells stimulated by veratridine (with or without the addition out on confluent cultures in 35-mm Falcon tissue culture dishes. of scorpion toxin) and by the inhibition of this effect by tetro- Uptake was terminated after 5 min, unless otherwise indicated, dotoxin. Where it has been examined, there has been an ex- by four washes at room temperature with the sodium-free wash cellent correlation between the presence of the Na+ ionophore medium described by Catterall (8). detected electrophysiologically and the measurements obtained Protein was determined by absorbance at 230 and 260 nm by veratridine stimulation of Na+ uptake (1, 2). as described by Kalb and Bernlohr (9) after solubilization of the We and others have therefore been interested in using such cell layer with 1% sodium dodecyl or by the method of measurements as a criterion for the presence of neuronal cells Lowry et al. (10). or neuronally derived cells in culture. In addition to muscle RESULTS cells, only two examples of non-neuronal cells with tetrodo- toxin-sensitive channels have been reported in the literature. Addition of veratridine and scorpion venom to IMR 91 cells, The Na+ ionophore has been detected in secretory cells from a normal human lung fibroblast cell line, in the presence of the pancreas (3), and voltage-independent, veratridine-stim- ouabain resulted in an increased rate of Na+ entry (Fig. 1). This ulated Na+ channels were detected in squid Schwann cells (4). surprising observation is not a unique property of IMR 91 cells. As part of our investigation we examined various cultured cells Veratridine and scorpion toxin also stimulated the entry of Na+ for the presence of the Na+ ionophore. In addition to con- into three other human fibroblast lines (Fig. 2). In all cases, the firming the results of others that several established non-neu- effect was abolished by tetrodotoxin, a specific inhibitor of the ronal lines do not contain the Na+ ionophore, we were surprised Na+ ionophore. By contrast, Swiss 3T3 cells, simian virus 40- to find this ionophore in a number of cell lines described as fi- transformed 3T3 cells, and C6 cells (rat glioblastoma) did not broblasts as well as in normal human "glia-like" cells when show this response; N18, an established neuronal cell line, assayed with the combination of veratridine and scorpion toxin. showed a strong response to the addition of veratridine and These results raise important questions about the uniqueness scorpion toxin (Fig. 3). We have also detected low levels of of this ionophore for excitable cells as well as about the possible stimulated Na+ influx in secondary chick embryo fibroblasts of these (data not shown). origin cells. Stallcup (11) has classified Na+ channels (Na+ ionophores) in cultured cell lines into three categories based on their re- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- sponse to the combination of scorpion toxin and veratridine and vertisement" in accordance with 18 U. S. C. §1734 solely to indicate their sensitivity to tetrodotoxin inhibition. Type B and type C this fact. channels show poor response to veratridine alone but a marked 6425 Downloaded by guest on September 28, 2021 6426 Cell Biology: Munson et al. Proc. Natl. Acad. Sci. USA 76 (1979)

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5 10 Time, min FIG. 1. Time course of 22Na uptake by IMR 91 fibroblasts. Confluent cultures (6.8 X 105 cells per dish; 0.3 mg protein) on 35-mm Falcon tissue culture dishes were assayed for Na+ influx in the absence FIG. 3. 22Na influx in neuronal and non-neuronal cell lines. of neurotoxins (0) or in the presence of 0.2 mM veratridine and Confluent cultures in 35-mm dishes were assayed as described in the scorpion venom (0.1 mg/ml) (0) for the indicated times. After four text. Symbols are as in Fig. 2. The cell density was as follows: estab- washes with sodium-free medium (8), the cell layer was dissolved with lished neuronal cell line (N18), 1.3 X 106 cells per dish; rat glioblas- 1% sodium dodecyl sulfate and assayed in a gamma counter. toma (C6), 2.8 X 106; Swiss 3T3 (3T3), 4.3 X 105; and simian virus 40-transformed 3T3 (SV3T3), 2.7 X 106. response to the combination of veratridine and scorpion venom. The Km-for veratridine in type C channels is independent of high basal influx rate, we have not attempted to determine an scorpion venom concentration; the Km in type B channels can apparent Km for veratridine with scorpion venom at less than decrease as the concentration of venom is increased. Type B and 25 Mg/ml. Tetrodotoxin sensitivity of the IMR 91 channel is type C channels are also distinguished by tetrodotoxin sensi- shown in Fig. 6. Half-maximal inhibition of stimulated uptake tivity. Type B channels exhibit an average Ki of approximately occurred at 7 X 10-8 M tetrodotoxin. 3 X 10-8 M whereas type C channels are approximately 1/30th The results of Catterall (12) suggest that the methods used as sensitive to the drug. Type B channels have been described to measure Na+ influx underestimate the true Na+ perme- for a number of cell lines classified as neuronal (11). ability, particularly with respect to the maximally stimulated The Na+ channel in cell line IMR 91 most closely resembles the type B channel as described by Stallcup. The data in Fig. 4 indicate an apparent Km of 22 Atg/ml for scorpion venom (at 200 tiM veratridine). Apparent Km values of 37 ,uM (at 25 ,ug 150 of scorpion venom per ml) and 29 MiM (at 100Mg of scorpion 0.7 venom per ml) were found for veratridine (Fig. 5). Due to the

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measured for 5 min as described in Fig. 1. O, No toxin addition; m, 0.2 91 cultures were assayed for Na+ influx as described in Fig. 1. Mea- mM veratridine; *, veratridine plus scorpion venom (0.1 mg/ml); 0, surements were made at 5-min time points in the presence of 0.2 mM both stimulatory toxins plus 2 ,uM tetrodotoxin. The cell number and veratridine and the indicated levels of scorpion venom. In the absence protein content were: IMR 91, 8.2 X 105 cells per dish, 0.28 mg; S468, of scorpion venom, 67 nmol of Na+ was taken up and this value has 7 X 105 cells per dish, 0.29 mg; S471, 3.1 X 105 cells per dish, 0.18 mg; been subtracted from the data presented. S/V is scorpion toxin con- S475, 5.5 X 105 cells per dish, 0.31 mg. Data are mean + SD of tripli- centration per velocity. The apparent Km was determined by least cate determinations. squares analysis. Downloaded by guest on September 28, 2021 Cell Biology: Munson et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6427

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50 1 00 150 2-00 5.0 100 150 20C Veratridine, Aim FIG. 5. Determination of apparent Km for veratridine. Confluent cultures of IMR 91 were assayed for Na+ influx as described in Fig. 1. (Left) Velocity as a function of scorpion venom concentration. (Right) Veratridine concentration per velocity as a function of vera- tridine concentration. Scorpion venom was present at 25 (0) or 100 jig/ml (0). Apparent Km values were determined from least squares analysis of the data. Na+ influx in the absence ofscorpion venom was 57 nmol per dish, and this value was subtracted from the data shown. rates, because the observed rate of Na+ influx may be limited by the rate at which other ions can move across the membrane. At low Na+ concentrations the rate of entry is proportional to Na+, mM the external Na+ concentration and appears to be directly FIG. 7. Na+ uptake as a function of external Na+ concentration. proportional to the number of Na+ channels in the cell (12). We The Na+ concentration was varied as indicated by substituting choline therefore measured the rate of Na+ influx as a function of ex- for Na+ in the medium such that the total of Na+ plus choline equaled ternal Na+ concentration in IMR 91 cells (Fig. 7). At 5 mM 130 mM. IMR 91 cells were washed prior to assay with sodium-free Na+ flux to be directly proportional medium, and Na+ uptake was measured as described in Fig. 1. Ver- external Na+, appeared atridine was added to the uptake medium in , resulting in a to Na+ concentration, and we observed a net Na+ influx of 11 1% final concentration. Ethanol was also added to toxin-free uptake nmol/min per mg of protein. Under the same conditions with medium to yield the same final concentration. 22Na uptake was linear our cultures of N18, the rate of Na+ influx was 20 nmol/min for 5 min at 37°C with 2.5 mM external Na+ as well as with 130 mM per mg of protein. external Na+ (Fig. 1). Dishes contained 0.32 mg of protein. 0, Na+ In addition to fibroblasts, we have also examined normal uptake in absence of toxins; 0, Na+ uptake in presence of toxins. human glia-like cell lines prepared by the method of Ponten and MacIntyre (5). These are cells that have been described as 200 predominantly astrocyte-like in shape and whose growth con- trol and senescence have been studied extensively (13-15). They produce fibronectin (16), glycosaminoglycans (17), and low levels of S-100 (unpublished observations), a glia-specific pro- tein. They have a high-affinity y-aminobutyric acid uptake 150 system (unpublished data) but do not contain glial fibrillary acidic protein (unpublished data), a highly specific marker for .C fibrous astrocytes (18). -sensitive Na+ channels were LO\E detectable in these lines (Fig. 8). Finally, similar sodium channels have been observed in three separate cell lines ob- E tained from human malignant gliomas, two of which (U-251 C 100\ MG CL 1 and U-343 MGa) have been found to contain glial fibrillary acid protein (unpublished data) (Fig. 9). Character- istically, all of the non-neuronal cells that we have examined + \ have shown no measurable stimulation of Na+ uptake by ver- z atridine alone and significant stimulation with veratridine and 50 scorpion venom (Figs. 2 and 8). DISCUSSION These observations demonstrate the presence of Na+ channels or Na+ ionophores in cell lines that cannot be clearly identified .'I ' as being of neuronal or muscular origin. The function of these 10 1i0- 107 106 channels is not required for growth under our culture conditions Tetrodotoxin, M because we have observed that IMR 91 cells grow normally in FIG. 6. Tetrodotoxin sensitivity ofthe IMR 91 channel. Confluent the presence of 2 ,M tetrodotoxin. 35-mm dishes were assayed for Na+ influx in the presence of 0.2 mM Transformed cells of neuronal origin have frequently been veratridine and scorpion venom (100 ug/ml) with the indicated levels of tetrodotoxin (Sigma). Na+ influx in the absence of stimulatory noted to have both neuronal and glial characteristics (19-21), toxins was 57 nmol per dish, and this value was subtracted from the and malignant cells such as those involved in Fig. 9 might be data presented. expected to share this "abnormal" behavior. The presence of Downloaded by guest on September 28, 2021 6428 Cell Biology: Munson et al. Proc. Natl. Acad. Sci. USA 76 (1979)

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U787CG U622CG U854CG FIG. 8. Na+ influx in normal human glia-like cells, measured and presented as in Fig. 2. Cell number: 1.8 X 105 cells per dish for U787 CG; 2.8 X 105 for U622 CG; and 1.0 X 105 for U854 CG. Results are the mean of duplicate determinations. as IMR 91 and normal FIG. 9. Na+ influx in human malignant gliomas, measured and Na+ channels in fibroblast cells such presented as in Fig. 2. Cell number: 6.3 X 105 cells per dish for U-251 human glia-like cells, which behave normally in that they show MG CL 1; 1.2 X 106 for U-178 MG; and 1.9 X 106 for U-343 MGa. growth control and senescence, is more difficult to explain. The Results are the mean of duplicate determinations. following possibilities can be considered, although we have no easy method to distinguish among them at this time. (i) The definition of a fibroblast is at best vague and the term venom. The type of channel described in this communication often is used to describe any cell that grows from an initial tissue would not have been detected by these workers.§ However, explant. It is possible that all of the cells examined by us are existence of these tetrodotoxin-sensitive Na+ channels in cells really not derived from connective tissue but may be detived that by most criteria appear not to be of neuronal or muscular from smooth muscle. origin suggests that a great deal of caution needs to be exercised (ii) The presence of Na+ channels or Na+ ionophores may in assigning cellular origins on the basis of the presence of Na+ be a characteristic of relatively undifferentiated cells, and channels. In particular we have noted in preliminary experi- this ments that BHK(C13) cells have Na+ channels that, under our various cells under culture conditions may express pheno- assay conditions, can be stimulated by veratridine in the absence type. of scorpion venom. Characterization of cells as being of neu- The number of Na+ channels per cell is considerable when ronal origin requires as minimal criteria the demonstration of compared to cells assumed to be of neuronal origin and assayed the ability of these cells to synthesize neurotransmitters or to under similar conditions. For example, N18, an established soluble such as the mouse neuroblastoma, shows a veratridine- and scorpion synthesize neuronal specific antigens, either venom-stimulated Na+ uptake of 20 nmol/min per mg of protein designated as 14-3-2 (22) or surface (23-25). protein at 5 mM external Na+, a value one-fourth that reported We are grateful to Dr. William Sly for the generous gift of fibroblast by Catterall (11) under different assay conditions.t IMR 91 has cultures. This work was supported by grants from the National Science a toxin-stimulated permeability equal to 55% of that of the Foundation (BM 77-15972) and the National Institutes of Health (GM neuroblastoma clone when assayed on a protein basis at 5 mM 18405). R.M. was supported by a grant from the National Institutes of external Nua+. Our results therefore demonstrate that these cell Health (5T32NS(7071). Some tissue culture media were obtained from lines have lower, but significant, numbers of tetrodotoxin- the Washington University Basic Cancer Center supported by Grant sensitive Na+ channels. It is important to point out that we do CH 16217A from the National Institutes of Health. not know the electrophysiological characteristics of the cells that 1. Catterall, W. A. & Nirenberg, M. (1973) Proc. Natl. Acad. Sci. we have examined, which indeed may make them different USA 70,3759-3763. from neurons. 2. Stallcup, W. B. & Cohn, M. (1976) Exp. Cell Res. 98,285-297. Most cell lines that have been characterized as being of 3. Lowe, D. A., Richardson, B. P., Taylor, P. & Daratsch, P. (1976) neuronal origin (2, 19-21) have been classified as such after Nature (London) 260,337-338. characterization of several markers including measurement of 4. Villegas, J., Sevcik, C., Barnola, F. V. & Villegas, R. (1976) J. Gen. Na+ influx in the presence of veratridine without scorpion Physiol. 67, 369-380. 5. Ponten, J. & MacIntyre, E. H. (1968) Acta Path. Microbiol. Scand. 74, 465-486. t The difference also may reflect differences in the N18 cells available 6. Stallcup, W. B. & Cohn, M. (1976) Exp. Cell Res. 98,277-284. in the two laboratories. 7. Catterall, W. A. (1975) Proc. Natl. Acad. Sd. USA 72, 1782- § Our findings are supported in a qualitative way by unpublished data 1786. who has examined a from W. Stallcup (personal communication) 8. W. A. J. Biol. Chem. 251, 5528-5536. derived from the central nervous system, Catterall, (1976) number of cell lines, 9. V. F., Jr. & Bernlohr, R. W. (1977) Anal. Biochem. 82, thought to be glial on the basis of failure to generate an action po- Kalb, and lack of detectable stimulation of Na+ influx in the 362-371. tential (19) N. A. L. & R. presence of veratridine alone (2). Most of these lines respond posi- 10. Lowry, 0. H., Rosebrough, J., Farr, Randall, J. tively to a combination of veratridine and scorpion venom or to ba- (1951) J. Biol. Chem. 193,265-275. trachotoxin (7). The magnitude is generally 1/20th to 1/1Oth of the 11. Stallcup, W. B. (1977) Brain Res. 135,37-53. flux found in neuronal cells under comparable conditions. 12. Catterall, W. A. (1977) J. Biol. Chem. 252, 8669-8676. Downloaded by guest on September 28, 2021 Cell Biology: Munson et al. Proc. Nati. Acad. Sci. USA 76 (1979) 6429 13. Westermark, B. (1971) Exp. Cell Res. 69,259-264. 20. West, G. J., Uki, J., Stahn, R. & Herschman, H. R. (1977) Brain 14. Lindgren, A., Westermark, B. & Pont6n, J. (1975) Exp. Cell Res. Res. 130, 387-392. 95,311-319. 21. Tomozawa, Y. & Sueoka, N. (1978) Proc. Natl. Acad. Sci. USA 15. Westermark, B. (1977) Proc. Natl. Acad. Sci. USA 74, 1619- 75, 6305-6309. 1621. 22. Moore, B. W. (1973) in Proteins of the Nervous System, eds. 16. Vaheri, A., Ruoslahti, E., Westermark, B. & Ponten, J. (1976) J. Schneider, D. J., Angeletti, R. H., Bradshaw, R. A., Grasso, A. & Exp. Med. 143, 64-72. Moore, B. W. (Raven, New York), pp. 1-12. 17. Glimelius, B., Norling, B., Westermark, B. & Wasteson, A. (1978) 23. Fields, K. L., Brockes, J. P., Mirsky, R. & Wendon, L. M. B. (1978) Biochem. J. 172,443-456. Cell 14, 43-51. 18. Bignami, A., Eng, L. F., Dahl, D. & Uyeda, C. T. (1972) Brain 24. Schachner, M., Wortham, K. A., Carter, L. D. & Chaffee, J. K. Res. 43, 429-435. (1975) Dev. Biol. 44, 313-325. 19. Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., Kimes, 25. Raff, M. C., Mirsky, R., Fields, K. L., Lisak, R. P., Dorfman, S. B., Patrick, J., Steinbach, J. H., Culp, W. & Brandt, B. L. (1974) H., Silberberg, D. H., Gregson, N. A., Leibowitz, S. & Kennedy, Nature (London) 249, 224-227. M. C. (1978) Nature (London) 274, 813-816. Downloaded by guest on September 28, 2021