Tetrodotoxin-sensitive sodium channels in normal human fibroblasts ...

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of such channels in cells generally considered nonexcitable raises questions ... potential Na+ ionophore or voltage-dependent Na+ channel. This channel is by ...
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 Tetrodotoxin-sensitive sodium channels are detectable in normal human fibroblasts and in "glia-like" cells at appreciable levels when compared to what is observed in established neuronal cell lines in culture. Two- to 3fold stimulations of sodium influx are observed in the presence of 0.2 mM veratridine and scorpion venom at 0.1 mg/mi. Tetrodotoxin (2 MM) inhibits the observed stimulation of sodium influx. Previous work has indicated that these neurotoxins act on the voltagesensitive sodium ionophore of excitable cells, and the presence of such channels in cells generally considered nonexcitable raises questions regarding both the uniqueness of this ionophore as a property of excitable cells and the origin of the cells generally described as fibroblasts.

Neuronal cells are characterized by the presence of an actionpotential Na+ ionophore or voltage-dependent Na+ channel. This channel is by definition the structure responsible for the increase in permeability to Na+ during the depolarizing phase of the nerve action potential. This ionophore can be activated by veratridine and synergistically with veratridine by scorpion toxin. This activation is specifically blocked by the addition of tetrodotoxin. The presence of this ionophore can be determined electrophysiologically or by measurements of Na+ influx into cells stimulated by veratridine (with or without the addition of scorpion toxin) and by the inhibition of this effect by tetrodotoxin. Where it has been examined, there has been an excellent correlation between the presence of the Na+ ionophore detected electrophysiologically and the measurements obtained by veratridine stimulation of Na+ uptake (1, 2). We and others have therefore been interested in using such measurements as a criterion for the presence of neuronal cells or neuronally derived cells in culture. In addition to muscle cells, only two examples of non-neuronal cells with tetrodotoxin-sensitive channels have been reported in the literature. The Na+ ionophore has been detected in secretory cells from the pancreas (3), and voltage-independent, veratridine-stimulated Na+ channels were detected in squid Schwann cells (4). As part of our investigation we examined various cultured cells for the presence of the Na+ ionophore. In addition to confirming the results of others that several established non-neuronal lines do not contain the Na+ ionophore, we were surprised to find this ionophore in a number of cell lines described as fibroblasts as well as in normal human "glia-like" cells when assayed with the combination of veratridine and scorpion toxin. These results raise important questions about the uniqueness of this ionophore for excitable cells as well as about the possible origin of these cells. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6425

MATERIALS AND METHODS Normal human lung fibroblasts (line IMR 91, Institute for Medical Research, Camden, NJ) and normal human dermal fibroblasts initiated from skin biopsies were obtained from W. Sly (Washington University School of Medicine). Cultures were grown in minimal essential medium containing 15% fetal bovine serum, glutamine (0.1 mg/ml), sodium pyruvate (0.11 mg/ml), and 100 units of penicillin and 100 jig of streptomycin per ml. Normal human glia-like cells (CG series) were isolated as described by Ponten and MacIntyre (5). Glial cells were grown as above without pyruvate and with the substitution of 10% calf serum for the fetal calf serum. 3T3 and simian virus 40-transformed 3T3 cultures were grown in Dulbecco's modified Eagle's medium containing 10% calf serum. C6 glioma and N18 neuroblastoma cells were grown in this medium containing 10% fetal bovine serum. All cultures were supplemented with glutamine and antibiotics as described above. Sodium uptake assays were performed as described by Stallcup and Cohn (6) with the addition of bovine serum albumin (1 mg/ml) and scorpion (Lelurus quinquestriatus) venom (0.1 mg/ml) (Sigma) (7). Assays were routinely carried out on confluent cultures in 35-mm Falcon tissue culture dishes. Uptake was terminated after 5 min, unless otherwise indicated, by four washes at room temperature with the sodium-free wash medium described by Catterall (8). Protein was determined by absorbance at 230 and 260 nm as described by Kalb and Bernlohr (9) after solubilization of the cell layer with 1% sodium dodecyl sulfate or by the method of Lowry et al. (10). RESULTS Addition of veratridine and scorpion venom to IMR 91 cells, a normal human lung fibroblast cell line, in the presence of ouabain resulted in an increased rate of Na+ entry (Fig. 1). This surprising observation is not a unique property of IMR 91 cells. Veratridine and scorpion toxin also stimulated the entry of Na+ into three other human fibroblast lines (Fig. 2). In all cases, the effect was abolished by tetrodotoxin, a specific inhibitor of the Na+ ionophore. By contrast, Swiss 3T3 cells, simian virus 40transformed 3T3 cells, and C6 cells (rat glioblastoma) did not show this response; N18, an established neuronal cell line, showed a strong response to the addition of veratridine and scorpion toxin (Fig. 3). We have also detected low levels of stimulated Na+ influx in secondary chick embryo fibroblasts (data not shown). Stallcup (11) has classified Na+ channels (Na+ ionophores) in cultured cell lines into three categories based on their response to the combination of scorpion toxin and veratridine and their sensitivity to tetrodotoxin inhibition. Type B and type C channels show poor response to veratridine alone but a marked

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10 5 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 of neurotoxins (0) or in the presence of 0.2 mM veratridine and scorpion venom (0.1 mg/ml) (0) for the indicated times. After four washes with sodium-free medium (8), the cell layer was dissolved with 1% sodium dodecyl sulfate and assayed in a gamma counter.

response to the combination of veratridine and scorpion venom. The Km-for veratridine in type C channels is independent of scorpion venom concentration; the Km in type B channels can decrease as the concentration of venom is increased. Type B and type C channels are also distinguished by tetrodotoxin sensitivity. Type B channels exhibit an average Ki of approximately 3 X 10-8 M whereas type C channels are approximately 1/30th as sensitive to the drug. Type B channels have been described for a number of cell lines classified as neuronal (11). 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 of scorpion venom per ml) and 29 MiM (at 100Mg of scorpion venom per ml) were found for veratridine (Fig. 5). Due to the

FIG. 3. 22Na influx in neuronal and non-neuronal cell lines. Confluent cultures in 35-mm dishes were assayed as described in the text. Symbols are as in Fig. 2. The cell density was as follows: established neuronal cell line (N18), 1.3 X 106 cells per dish; rat glioblastoma (C6), 2.8 X 106; Swiss 3T3 (3T3), 4.3 X 105; and simian virus 40-transformed 3T3 (SV3T3), 2.7 X 106.

high basal influx rate, we have not attempted to determine an apparent Km for veratridine with scorpion venom at less than 25 Mg/ml. Tetrodotoxin sensitivity of the IMR 91 channel is shown in Fig. 6. Half-maximal inhibition of stimulated uptake occurred at 7 X 10-8 M tetrodotoxin. The results of Catterall (12) suggest that the methods used to measure Na+ influx underestimate the true Na+ permeability, particularly with respect to the maximally stimulated

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FIG. 2. 22Na uptake by several normal human fibroblast lines, measured for 5 min as described in Fig. 1. O, No toxin addition; 0.2 mM veratridine; *, veratridine plus scorpion venom (0.1 mg/ml); 0, both stimulatory toxins plus 2 ,uM tetrodotoxin. The cell number and protein content were: IMR 91, 8.2 X 105 cells per dish, 0.28 mg; S468, 7 X 105 cells per dish, 0.29 mg; S471, 3.1 X 105 cells per dish, 0.18 mg; S475, 5.5 X 105 cells per dish, 0.31 mg. Data are mean + SD of triplicate determinations. m,

FIG. 4. Determination of apparent Km for scorpion venom. IMR 91 cultures were assayed for Na+ influx as described in Fig. 1. Measurements were made at 5-min time points in the presence of 0.2 mM veratridine and the indicated levels of scorpion venom. In the absence of scorpion venom, 67 nmol of Na+ was taken up and this value has been subtracted from the data presented. S/V is scorpion toxin concentration per velocity. The apparent Km was determined by least squares analysis.

Proc. Natl. Acad. Sci. USA 76 (1979)

Cell Biology: Munson et al.

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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 veratridine 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 of scorpion 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 the external Na+ concentration and appears to be directly proportional to the number of Na+ channels in the cell (12). We therefore measured the rate of Na+ influx as a function of external Na+ concentration in IMR 91 cells (Fig. 7). At 5 mM external Na+, Na+ flux appeared to be directly proportional to Na+ concentration, and we observed a net Na+ influx of 11 nmol/min per mg of protein. Under the same conditions with our cultures of N18, the rate of Na+ influx was 20 nmol/min per mg of protein. In addition to fibroblasts, we have also examined normal

human glia-like cell lines prepared by the method of Ponten and MacIntyre (5). These are cells that have been described as predominantly astrocyte-like in shape and whose growth control 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 protein. They have a high-affinity y-aminobutyric acid uptake system (unpublished data) but do not contain glial fibrillary acidic protein (unpublished data), a highly specific marker for fibrous astrocytes (18). Neurotoxin-sensitive Na+ channels were detectable in these lines (Fig. 8). Finally, similar sodium channels have been observed in three separate cell lines obtained from human malignant gliomas, two of which (U-251 MG CL 1 and U-343 MGa) have been found to contain glial fibrillary acid protein (unpublished data) (Fig. 9). Characteristically, all of the non-neuronal cells that we have examined have shown no measurable stimulation of Na+ uptake by veratridine alone and significant stimulation with veratridine and scorpion venom (Figs. 2 and 8).

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Na+, mM FIG. 7. Na+ uptake as a function of external Na+ concentration. The Na+ concentration was varied as indicated by substituting choline for Na+ in the medium such that the total of Na+ plus choline equaled 130 mM. IMR 91 cells were washed prior to assay with sodium-free medium, and Na+ uptake was measured as described in Fig. 1. Veratridine was added to the uptake medium in ethanol, resulting in a 1% final concentration. Ethanol was also added to toxin-free uptake medium to yield the same final concentration. 22Na uptake was linear for 5 min at 37°C with 2.5 mM external Na+ as well as with 130 mM external Na+ (Fig. 1). Dishes contained 0.32 mg of protein. 0, Na+ uptake in absence of toxins; 0, Na+ uptake in presence of toxins.

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FIG. 6. Tetrodotoxin sensitivity of the IMR 91 channel. Confluent 35-mm dishes were assayed for Na+ influx in the presence of 0.2 mM veratridine and scorpion venom (100 ug/ml) with the indicated levels

of tetrodotoxin (Sigma). Na+ influx in the absence of stimulatory

toxins was 57 nmol per dish, and this value was subtracted from the data presented.

These observations demonstrate the presence of Na+ channels or Na+ ionophores in cell lines that cannot be clearly identified as being of neuronal or muscular origin. The function of these channels is not required for growth under our culture conditions because we have observed that IMR 91 cells grow normally in the presence of 2 ,M tetrodotoxin. Transformed cells of neuronal origin have frequently been noted to have both neuronal and glial characteristics (19-21), and malignant cells such as those involved in Fig. 9 might be expected to share this "abnormal" behavior. The presence of

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Proc. Natl. Acad. Sci. USA 76 (1979)

Cell Biology: Munson et al.

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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.

Na+ channels in fibroblast cells such as IMR 91 and normal human glia-like cells, which behave normally in that they show growth control and senescence, is more difficult to explain. The 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 often is used to describe any cell that grows from an initial tissue explant. It is possible that all of the cells examined by us are really not derived from connective tissue but may be detived from smooth muscle. (ii) The presence of Na+ channels or Na+ ionophores may be a characteristic of relatively undifferentiated cells, and various cells under culture conditions may express this phenotype. The number of Na+ channels per cell is considerable when compared to cells assumed to be of neuronal origin and assayed under similar conditions. For example, N18, an established mouse neuroblastoma, shows a veratridine- and scorpion venom-stimulated Na+ uptake of 20 nmol/min per mg of protein at 5 mM external Na+, a value one-fourth that reported by Catterall (11) under different assay conditions.t IMR 91 has a toxin-stimulated permeability equal to 55% of that of the neuroblastoma clone when assayed on a protein basis at 5 mM external Nua+. Our results therefore demonstrate that these cell lines have lower, but significant, numbers of tetrodotoxinsensitive Na+ channels. It is important to point out that we do not know the electrophysiological characteristics of the cells that we have examined, which indeed may make them different from neurons. Most cell lines that have been characterized as being of neuronal origin (2, 19-21) have been classified as such after characterization of several markers including measurement of Na+ influx in the presence of veratridine without scorpion The difference also may reflect differences in the N18 cells available in the two laboratories. § Our findings are supported in a qualitative way by unpublished data from W. Stallcup (personal communication) who has examined a number of cell lines, derived from the central nervous system, thought to be glial on the basis of failure to generate an action potential (19) and lack of detectable stimulation of Na+ influx in the presence of veratridine alone (2). Most of these lines respond positively to a combination of veratridine and scorpion venom or to batrachotoxin (7). The magnitude is generally 1/20th to 1/1Oth of the flux found in neuronal cells under comparable conditions.

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FIG. 9. Na+ influx in human malignant gliomas, measured and presented as in Fig. 2. Cell number: 6.3 X 105 cells per dish for U-251 MG CL 1; 1.2 X 106 for U-178 MG; and 1.9 X 106 for U-343 MGa. Results are the mean of duplicate determinations.

venom. The type of channel described in this communication would not have been detected by these workers.§ However, existence of these tetrodotoxin-sensitive Na+ channels in cells that by most criteria appear not to be of neuronal or muscular origin suggests that a great deal of caution needs to be exercised in assigning cellular origins on the basis of the presence of Na+ channels. In particular we have noted in preliminary experiments that BHK(C13) cells have Na+ channels that, under our assay conditions, can be stimulated by veratridine in the absence of scorpion venom. Characterization of cells as being of neuronal origin requires as minimal criteria the demonstration of the ability of these cells to synthesize neurotransmitters or to synthesize neuronal specific antigens, either soluble such as the protein designated as 14-3-2 (22) or surface (23-25). We are grateful to Dr. William Sly for the generous gift of fibroblast cultures. This work was supported by grants from the National Science Foundation (BM 77-15972) and the National Institutes of Health (GM 18405). R.M. was supported by a grant from the National Institutes of Health (5T32NS(7071). Some tissue culture media were obtained from the Washington University Basic Cancer Center supported by Grant CH 16217A from the National Institutes of Health. 1. Catterall, W. A. & Nirenberg, M. (1973) Proc. Natl. Acad. Sci. USA 70,3759-3763. 2. Stallcup, W. B. & Cohn, M. (1976) Exp. Cell Res. 98,285-297. 3. Lowe, D. A., Richardson, B. P., Taylor, P. & Daratsch, P. (1976) Nature (London) 260,337-338. 4. Villegas, J., Sevcik, C., Barnola, F. V. & Villegas, R. (1976) J. Gen. Physiol. 67, 369-380. 5. Ponten, J. & MacIntyre, E. H. (1968) Acta Path. Microbiol. Scand. 74, 465-486. 6. Stallcup, W. B. & Cohn, M. (1976) Exp. Cell Res. 98,277-284. 7. Catterall, W. A. (1975) Proc. Natl. Acad. Sd. USA 72, 17821786. 8. Catterall, W. A. (1976) J. Biol. Chem. 251, 5528-5536. 9. Kalb, V. F., Jr. & Bernlohr, R. W. (1977) Anal. Biochem. 82, 362-371. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 11. Stallcup, W. B. (1977) Brain Res. 135,37-53. 12. Catterall, W. A. (1977) J. Biol. Chem. 252, 8669-8676.

Cell Biology: Munson et al. 13. Westermark, B. (1971) Exp. Cell Res. 69,259-264. 14. Lindgren, A., Westermark, B. & Pont6n, J. (1975) Exp. Cell Res.

95,311-319. 15. Westermark, B. (1977) Proc. Natl. Acad. Sci. USA 74, 16191621. 16. Vaheri, A., Ruoslahti, E., Westermark, B. & Ponten, J. (1976) J. Exp. Med. 143, 64-72. 17. Glimelius, B., Norling, B., Westermark, B. & Wasteson, A. (1978) Biochem. J. 172,443-456. 18. Bignami, A., Eng, L. F., Dahl, D. & Uyeda, C. T. (1972) Brain Res. 43, 429-435. 19. Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., Kimes, B., Patrick, J., Steinbach, J. H., Culp, W. & Brandt, B. L. (1974) Nature (London) 249, 224-227.

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20. West, G. J., Uki, J., Stahn, R. & Herschman, H. R. (1977) Brain Res. 130, 387-392. 21. Tomozawa, Y. & Sueoka, N. (1978) Proc. Natl. Acad. Sci. USA 75, 6305-6309. 22. Moore, B. W. (1973) in Proteins of the Nervous System, eds. Schneider, D. J., Angeletti, R. H., Bradshaw, R. A., Grasso, A. & Moore, B. W. (Raven, New York), pp. 1-12. 23. Fields, K. L., Brockes, J. P., Mirsky, R. & Wendon, L. M. B. (1978) Cell 14, 43-51. 24. Schachner, M., Wortham, K. A., Carter, L. D. & Chaffee, J. K. (1975) Dev. Biol. 44, 313-325. 25. Raff, M. C., Mirsky, R., Fields, K. L., Lisak, R. P., Dorfman, S. H., Silberberg, D. H., Gregson, N. A., Leibowitz, S. & Kennedy, M. C. (1978) Nature (London) 274, 813-816.