ing a contraction on the guinea pig Taenia coli by using a previously described technique (17). A fragment of Taenia coli was mounted in a chamber of 1 ml ...
Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7228-7232, November 1984 Neurobiology
The presence in pig brain of an endogenous equivalent of apamin, the bee venom peptide that specifically blocks Ca2+-dependent K+ channels (endogenous ligand/neuropeptide)
MICHEL FOSSET, HEIDY SCHMID-ANTOMARCHI, MICHEL HUGUES, GEORGES ROMEY, AND MICHEL LAZDUNSKI Centre de Biochimie, Centre National de la Recherche Scientifique, Facult6 des Sciences, Parc Valrose, 06034 Nice Cedex, France
Communicated by Pierre Joliot, July 23, 1984
125I-apamin (14 pM) in a sodium citrate buffer (pH 6.25) containing bovine serum albumin (1 mg/ml) in the presence of brain extracts or of the different fractions obtained during the purification procedure (final volume of the incubation, 0.2 ml). After 2 hr of incubation at 40C, the complex formed was precipitated by addition of 0.25 ml of 25% polyethylene glycol 6000 and 0.05 ml of rabbit plasma as carrier. The mixture was vigorously shaken and stored at -200C for 10 min. The incubations were then centrifuged for 10 min at 12,000 x g in a Heraus Haemofuge. The supernatants were discarded, and the different pellets were assayed for radioactivity with an Intertechnique CG4000 gamma counter. Experiments were run in duplicate. Electrophysiological Experiments. Effects of the different fractions purified from pig brain on the Ca2 -dependent K+ conductance of rat muscle cells in culture were measured by techniques as described (15). Contraction of the Isolated Guinea Pig Taenia Coli. Apamin has been shown to convert to a contraction the relaxation induced by myorelaxant agents in intestinal smooth muscle tissue (17, 20-22). The different fractions obtained during the purification have been tested for their potency in inducing a contraction on the guinea pig Taenia coli by using a previously described technique (17). A fragment of Taenia coli was mounted in a chamber of 1 ml thermostated at 37°C. The Taenia coli was first relaxed by addition of epinephrine (3 uM); then an aliquot of the test fraction was added in the chamber, and the contraction induced was estimated as described (17). Extraction of the Apamin-Like Factor from Pig Brain. Pig brains without cerebellum were collected at the local slaughterhouse just after death and immediately were stored in liquid nitrogen until processed. The collection and storage of brains took about 30 min after the death of the animal. Pig brain tissues were homogenized with a Polytron PT20 (setting 6, 1 min) in 3 volumes of 9% HC1/5% formic acid/1% CF3COOH (vol/vol) at 4°C. The homogenate was centrifuged at 12,000 x g for 15 min, the pellet was discarded, and the supernatant was centrifuged in a Beckman rotor type 35 at 80,000 x g for 30 min. The supernatants were pooled and lyophilized. The resulting powder was resuspended in distilled water (30 ml per brain) at 4°C, and the extract was defatted by three extractions with diethyl ether. After the last lipid extraction, traces of ether in the aqueous extract were eliminated by a 15-min evaporation under reduced pressure at 30°C in a Buchi Rota vapor instrument. The extract was then diluted with a total of 50 ml of water per brain and extensively dialyzed for 2 days against distilled water at 4°C with dialysis tubing Spectrapor 6, with a molecular weight cut-off at 1000. The dialysis step decreased the conductivity from 23 to 0.15 mS. The preparation of an extract of low conductivity is essential because monovalent and divalent
ABSTRACT An apamid-like factor has been isolated from pig brain after extraction of the tissue and purification on sulfopropyl-Sephadex C-25 and on reversed-phase high pressure liquid chromatography. The apamin-like factor has the following properties: (i) it prevents l 5I-labeled apamin binding to its specific receptor site present on rat brain synaptosomes, (it) it is active in the radioimmunoassay for apamin (i.e., it prevents 1251-labeled apamin precipitation by anti-apamin antibodies), (iil) it induces contraction of guinea pig intestinal smooth muscle previously relaxed with epinephrine, and (iv) it blocks Ca2+-dependent K+ channels responsible for the long-lasting afterpotential hyperpolarization following the action potential in rat skeletal muscle cells in culture. All these properties are those of apamin itself. The apamin-like factor is a peptide that, like apamin, is destroyed by trypsin and unaffected by chymotrypsin. These results suggest the presence in mammalian brain of a potent Ca2+-dependent K+-channel modulator.
were incubated with
Ca2'-dependent K+ channels have been identified in a variety of cellular types (1-13). A class of these channels is responsible for the generation of long-lasting-hyperpolarizations that follow the action potential (1, 4, 8). Ca2+-dependent K+ channels that generate afterpotential hyperpolarizations in neuroblastoma cells and in rat muscle cells in culture (6, 8) are specifically inhibited by apamin (14, 15), an 18amino-acid-long neurotoxic polypeptide from bee venom. It binds to Ca2+-dependent K+ channels with a high affinity (14-19) and has been used in the radiolabeled form to characterize the presence and the biochemical properties of Ca2+dependent K+ channels in a variety of preparations (16-19). This paper describes the existence of an apamin-like peptide in mammalian brain and its purification. MATERIALS AND METHODS Binding Assays. Mono[125I]iodoapamin (125I-apamin) used in binding assays to rat brain synaptosomes was prepared as described (16). Competition experiments between 25I-apamin and the different fractions purified from brain extracts were carried out as described for competition between 125I1 apamin and unlabeled apamin (16) with a 125I-apamin concentration between 30 and 50 pM in an incubation medium buffered by 100 mM Tris HCl at pH 8.4 and at a synaptosome concentration of 0.5 mg of protein per ml. Radioimmunoassay. The different brain extracts also were tested for their ability to compete with 125I-apamin in a RIA. Rabbit anti-apamin immunoglobulins were prepared by first covalently binding apamin (8 mg) to bovine serum albumin (0.44 mg) with disuccinimyl suberate (3 mM) before injecting the resulting product into New Zealand rabbits (34). Rabbit anti-apamin gamma globulins at a final dilution of 1:7600 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.
Abbreviation: SPC-25, SP-Sephadex C-25. 7228
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cations at high enough concentrations are able to prevent 251I-apamin binding to its receptor (14, 16, 23). Once dialyzed, the extract was lyophilized, and the powder was resuspended in distilled water (1 ml per brain). Purification on SP-Sephadex C-25 (SPC-25) Column. The extract obtained as described above was loaded on a SPC-25 column (2.5 x 14 cm for 10 ml of extract). Before starting the purification of the brain extract, the SPC-25 gel was equilibrated in 1 M NH4OAc (pH 8), degased, and reequilibrated with 20 mM NH4OAc (pH 5.0). The brain extract then was loaded on the column (flow rate, 1 ml/min), and the column was eluted stepwise at 40C at a flow rate of 5 ml/min. The column was first washed with 5 column volumes of a 20 mM NH4OAc (pH 5.0). The elution was then continued using successively 200 mM NH4OAc (pH 7.0) and 500, 600, and 700 mM NH4OAc (pH 7.5) (5 column volumes for each step). Fractions resulting from each step of elution were collected and Iyophilized twice to remove NH4OAc. The different lyophilized fractions were solubilized in distilled water at 40C in a volume equal to the volume of the crude extract before the SPC-25 step. Purification on C18 Reversed-Phase HPLC. Fractions from SPC-25 containing an apamin-like activity (detected by the binding assay to the apamin receptor and by the RIA) were submitted to a first step of purification using a semi-preparative C18 reversed-phase column (Merck Hibar Lichrosorb RP18; 2.5 x 25 cm, 7-,Am mesh size). The SPC-25 fraction was loaded at a flow rate of 1 ml/min on the column previously equilibrated with 0.05% CF3COOH in water. The column was then washed with 0.05% CF3COOH/15% acetonitrile in water (1 column volume) at a flow rate of 8 ml/min. Further elution of the column was carried out with a linear 15-50% gradient of acetonitrile in 0.05% CF3COOH for 100 min. At the end of the gradient, the elution was continued for 20 min with 50%/acetonitrile/0.05% CF3COOH. Fractions of 16 ml were collected throughout the elution. All fractions were Iyophilized and then put in solution in distilled water (100 ,l per brain) just before being assayed. The second step of purification used an analytical column (Merck Hibar Lichrosorb RP 18; 4 x 250 mm, 7 ,um mesh size). The active fraction coming from the first HPLC step was purified under isocratic conditions: 15% acetonitrile/0.05% CF3COOH in water at a flow rate of 1 ml/min. Fractions (1 ml) were collected for 1 hr; they were lyophilized and solubilized in distilled water (10 ,ul per brain) just before being tested. Degradation of the Apamin-Like Activity by Protease Digestion. The active fraction with an apamin-like activity was incubated in the presence of trypsin or chymotrypsin (0.20 mg/ml) at 37°C in 100 mM Tris HCl (pH 7.5). After 30 min of incubation, proteases were -inhibited by addition of soybean trypsin inhibitor (0.25 mg/ml), and the remaining activity of the apamin-like factor was measured by both the RIA and the apamin-receptor binding assay as described above. Control experiments were made to check if the presence of the complex formed between the protease and the soybean trypsin inhibitor in the incubation medium did not modify the radioreceptor assay and the RIA; other control was performed to see the effect of the incubation at 37°C without proteolytic enzymes on the behavior of both the apamin receptor and of the apamin itself in the binding assay and the RIA. Native apamin also was submitted to degradation by trypsin and chymotrypsin under the same conditions as those used for the apamin-like factor.
RESULTS Evidence for the Existence of an Apamin-Like Activity in
Pig Brain. Apamin-like activity was demonstrated by competition with 125I-apamin for binding to the apamin receptor, by RIA, and by the physiological activity on smooth muscle.
Proc. Natl. Acad. Sci. USA 81 (1984)
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Increasing amounts of the dialyzed brain extract (150-200 mg of protein per ml) of low conductivity gradually prevented the binding of 125I-apamin to rat brain synaptosomes (Fig. 1). The amount of crude dialyzed extract able to inhibit 50% of 125I-apamin binding to the apamin receptor in the experiment was about 0.45 p1. If we consider that 1 unit of apaminlike factor is the amount of extract able to displace 50% of 125I-apamin specifically bound to its receptor, then the extract from 10 brains contains 25,000 units of apamin-like factor (12.5 units/mg of protein). The properties of displacement of 125I-apamin by competition with unlabeled apamin (14, 16) are presented in Fig. 1 Inset for comparison. Fig. 2 shows the elution profile of the SPC-25 column loaded with a dialyzed extract obtained for 10 pig brains. Very little of the apamin-like activity measured by the 1251. apamin receptor assay was eluted from the gel at the first step of elution with 20 mM NH4OAc (pH 5.0) (fraction 1) or at the second step of elution with 200 mM NH4OAc (pH 7.0) (fraction 2). Most of the activity was detectable in the step corresponding to the elution in 500 mM NH4OAc (fraction 3), and very little activity was eluted in 600 and 700 mM NH4OAc (fractions 4 and 5). Fig. 1 shows the dose-response curve of the inhibition of I251-apamin binding to the apamin receptor when using fraction 3 (20 mg of protein per ml) of the eluate from the SPC-25 column. The half-maximal inhibition occurred with 1.6 ,ul of fraction 3, which corresponds to 6200 units of apamin-like activity (30-35 units/mg of protein). Fjg. 2B shows the presence of an apamin-like structure detected by the RIA in both the dialyzed extract and in the eluate from the SPC-25 column. It also shows that most of the apamin-like activity detected by the RIA was eluted in fraction 3. Fraction 3 from the chromatographic step on SPC-25 was further purified by two successive HPLC steps. The first step consisted of an elution on a semipreparative column by a gradient from 15% to 50% acetonitrile in 0.05% CF3COOH in water. Fig. 3A shows that under these conditions, six peaks of apamin-like activity were detected by the binding assay to the apamin receptor. Eight peaks were able to inhibit significantly (>20%) the recognition of apamin by the antiapamin antibody in the RIA (Fig. 3B). All of the peaks able to interact with the apamin receptor (Fig. 3A) were submitted to a screening assay using the contraction of the isolated
(100
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FIG. 1. Binding of 1251I-apamnin (30-50 pM) to synaptosomal membranes. Competition experiments were with: the dialyzed brain extract (150-200 mg of protein per ml) (e), fraction 3 from the SPC25 purification step (20 mg of protein per ml) (A), and fraction 3-16 from the HPLC purification step on C18 reversed-phase column (65 -g of protein per ml) (o) (extract and fractions from 10 pig brains). (Inset) Competition between 125I-apamin and unlabeled apamin on rat brain synaptosomes.
Neurobiology: Fosset et aLPProc. Nad Acad Sd USA 81
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FIG. 2. Ion-exchange chromatography on SPC-25 column of the dialyzed extract from 10 brains. The column was eluted by increasing the ionic strength stepwise by using successively the NH4OAc buffers of different composition indicated on the top of the figure. (A) Percentage inhibition of 1251I-apamin binding to its receptors. Elution of the apamin-like activity was followed by the binding assay to the apamin receptor. Each eluted fraction was lyophilized, solubilized in 10 ml of distilled water, and assayed in competition with 125I-apamin [10 Ml of each fraction of the incubation medium (final volume, 1 ml) for the binding assay]. (B) Percentage inhibition of 1251 -apamin precipitation in the RIA. Elution of apamin-like activity was followed by RIA. The different fractions were lyophilized, solubilized in 10 ml of distilled water, and tested in the RIA [20 1.d of each fraction of the incubation medium (final volume, 200 gld) for the
RIA].
guinea pig Taenia coli. This screening showed that only one among the six fractions able to prevent 1251-apamin binding to its receptor was able to produce a contraction as apamin itself does (Fig. 4). The effect of both apamin and of the apammn-like factor were reversed by washing. This active fraction, called "fraction 3-16," was eluted at 23% acetonitrile (see Fig. 3) with a retention time of 32 min. This fraction was the most active on both the radioreceptor assay and in the RIA. The dose-response curve describing the inhibitory action of fraction 3-16 on the binding of 1211-apamin to its receptor is presented in Fig. 1. At this stage of purification, it was impossible to measure the protein content of the active fraction. If one assumed that the protein concentration below the detection threshold of the techniques used, for instance, about 5 g.g of protein per ml, the activity of fraction 3-16 is then at least 15,000-20,000 units/mg of protein. The second step of purification using the HPLC technique consisted of chromatographing fraction 3-16 on an analytical column under isocratic elution conditions corresponding to 15% acetonitrile/0.05% CF3COOH in water. A major peak of apamin-like activity (fraction 3-16-17) could be detected by the binding assay to the apamin receptor (Fig. 5). There were four other small peaks of activity. All of the peaks were as-
FIG. 3. C18 reversed-phase chromatography of fraction 3 from the SPC-25 column step. (A) Percentage inhibition of 1251I-apamin binding to its receptor. Each fraction was lyophilized, solubilized in 2.5 ml of distilled water, and assayed for binding activity to the apamin receptor in competition with 125I-apamin (50 A.l of each fraction in 1 ml of the binding assay incubation medium. ---, Linear acetonitrile gradient (15-50%). (B) Percentage inhibition of 125I-apamin precipitation in the RIA. The same fractions were also tested for their capacity to prevent 125I-apamin precipitation by the rabbit anti-apamin immunoglobulins (60 ,ul of each fraction in 0.2 ml of RIA incubation medium).
sayed for their physiological activity on Taenia coli. Only the major peak was active (Fig. 5 Inset). The active peak was eluted after a retention time of 18 min, which corresponds to 5.5 column volumes. By comparison with the contraction induced by 2 nM apamin on the guinea pig Taenia coli (Fig. 4), it has been possible to calculate that, at this step of purification, the amount of the apamin-like factor recovered corresponded to an equivalent quantity of apamin of 1 + 0.5 pmol per pig brain. Electrophysiological Properties of the Apamin-Like Factor for Pig Brain. In rat muscle cells in culture, the action potential is followed by a long-lasting hyperpolarization (8). This afterpotential hyperpolarization has been demonstrated to be mediated by the activation of an apamin-sensitive Ca2t_ dependent K' channel (15). Fig. 6 shows a comparison of the effects of apamin and of the apamin-like factor on the electrical activity of rat myotubes in culture. The apamin-like factor contained in fraction 3-16-17 from the last isocratic HPLC step blocked the afterpotential hyperpolarization as apamin itself does (six experiments). All other fractions active in the 125I-apamin binding assay to its receptor were inactive in the electrophysiological assay (not shown). The Sensitivity of the Apamin-Like Factor from Pig Brain to Proteases. The apamin-like factor was degraded by proteases. Trypsin under the conditions described in Materials
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FIG. 4. Effect of fraction 3-16 on the contraction of a preparation of guinea pig Taenia coli relaxed with epinephrine (3 A.M). Traces: 1, control; 2, contraction induced by 5 ,ul of fraction 3-16; 3, contraction induced by 10 Al of fraction 3-16; 4, contraction induced by 5 Al. of fraction 3-16; 5, contraction induced by apamin at 2 nM. All of these successive experiments were made with the same Taenia coli preparation. A washing step (W) has been carried out between each experiment in the series. Volume of the organ bath, 1 ml; tension scale, 0.5 g; time scale, 5 min. The preparation was allowed to rest for 3-8 min between each test.
and Methods completely destroyed (100%) the binding activity of the apamin-like factor to the apamin receptor. Whereas chymotrypsin was without any effect. These properties are identical to those found with apamin itself. Apamin is not degraded by chymotrypsin because of a lack of aromatic amino acids in its sequence (24, 25).
DISCUSSION Apamin is a polypeptidic neurotoxin isolated from bee venom. This peptide consists of 18 amino acids with two disul-
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FIG. 6. Action potentials and afterpotential hyperpolarization (arrow) from rat myotubes evoked by anodal break stimulation. (A Left) Control. (A Right) Ten minutes after the application of 10 nM apamin. (B Left) Control. (B Right) Fifteen minutes after the addition of 50 jAl of fraction 3-16-17 to the external medium (2 ml). Horizontal solid lines indicate the zero-voltage line.
fide bridges (24, 25). The active site of the toxin involves primarily the two contiguous amino acids arginine-13 and -14 (26). The lethal dose (LD50) of the toxin by i.p. injection in Swiss mouse is near 4 mg/kg of body weight (27, 28). The toxicity of apamin is tremendously increased by intracisternal injection (LD50 near 2 ,ug/kg). The difference in efficien-
100
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Fraction FIG. 5. C18 reversed-phase chromatography under isocratic conditions (15% acetonitrile) of fraction 3-16 from the HPLC purification shown in Fig. 3. Fractions of 1 ml were collected, lyophilized, and solubilized in distilled water (250 gl). The elution of the apamin-like activity was followed by submitting each fraction to the 125I-apamin binding assay. A major peak of activity was eluted around fraction 17. This very active fraction was named "fraction 3-16-17." (Inset) Contraction was induced by fraction 3-16-17 on a preparation of guinea pig Taenia coli relaxed with 3 ,uM epinephrine. Traces: 1, control; 2, 50 t.d of fraction 3-16-17; 3, control. W, washing; tension scale, 0.5 g; time scale, 5 min.
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cy between the two routes of injection probably reflects the fact that apamin does not easily cross the blood-brain barrier (27, 28). Animals succumb after toxin injection to uncoordinated, uninterrupted movement, culminating in generalized convulsions. The apamin receptor has been identified by using 125I-apamin in excitable membranes from the nervous, skeletal, and smooth muscle systems (14-18). The dissociation constant of the apamin-receptor complex has been found to be 20-60 x 10- M in all these tissues. The apamin receptor is also present in hepatocytes (19). Electrophysiological studies including voltage clamp have clearly shown that the target of apamin is the Ca2'-dependent K+ channel responsible for long-lasting hyperpolarizations following action potentials (14, 15, 29). This work clearly demonstrates that an apamin-like factor exists in pig brain. This factor has been extensively purified, and it has the following properties: (i) it antagonizes 1251_ apamin binding to its receptor on the Ca2'-dependent K+ channel of synaptosomes, (ii) it antagonizes 125I-apamin binding to anti-apamin antibodies, (iii) it contracts the intestinal smooth muscle that had been relaxed by epinephrine, and (iv) it blocks the hyperpolarization that follows the action potential in rat skeletal myotubes in culture. All of these properties are those of apamin itself, and the interesting observation is that, as apamin does, the apamin-like factor acts on a variety of excitable membranes (brain synaptosomes, smooth muscle, rat skeletal muscle in culture). For all these reasons there is little doubt concerning the presence of an apamin-like factor in pig brain. This factor also exists in rat brain (unpublished work); therefore, these are all reasons to believe that the apamin-like factor is generally present in mammalian brain. The important question that remains to be solved is to determine the extent to which the apamin-like factor and apamin itself are similar. The final answer will be given when the sequence of the apamin-like factor is known. Large-scale purifications must be done to solve this structural problem. For the moment, the structural indications that we have are the following: (i) the apamin-like factor is a peptide; (ii) it has a Mr > 1000, since it does not pass through dialysis tubing with a molecular weight cut off at 1000; (iii) it is sufficiently similar in structure to apamin to be recognized by anti-apamin antibodies in the RIA; and (iv) the apamin-like factor, as apamin itself, is destroyed by trypsin and resistant to chymotrypsin. It may be that apamin and the endogenous apamin-like factor have very similar structures. The present purification procedure of the apamin-like factor gives an activity that is the equivalent of 1.5 + 0.5 ng of apamin per pig brain. The comparison of Fig. 4 A and B shows that there are peaks that are positive in the RIA and inactive in the receptor assay with 125I-apamin or in the physiological assay involving intestinal smooth muscle contraction. A possible explanation of this situation is that inactive precursors of the apamin-like factor are also purified. Because of their specificity and their high-affinity for structures that are essential for the generation of electrical excitability and for the coupling between excitation and contraction or between excitation and secretion, natural neurotoxins have become essential tools for studying the nervous, cardiac, and skeletal muscle systems (30-33). The discovery of the existence of an apamin-like factor in mammalian brain enhances the possibility that there are also endogeneous equivalents of other natural neurotoxins, in particular those that, like apamin, are specific for ionic channels (30, 32).
Proc. Natl. Acad Sci. USA 81 (1984) We thank Dr. A. Lombet, C. Widmann, and M. Tomkowiak for their help in the preparation of brain extracts. We are grateful to Dr. J. W. Taylor for fruitful discussions at the beginning of this work and for his advice in the elaboration of the HPLC purification steps, to Dr. Starzack for a careful reading of the manuscript, and to M. Valetti for expert secretarial assistance. This work was supported by the Centre National de la Recherche Scientifique (ATP 381). 1. Meech, R. W. (1978) Annu. Rev. Biophys. Bioeng. 7, 1-18. 2. Lew, V. L. & Ferreira, H. G. (1978) Curr. Top. Membr. Transp. 10, 217-277. 3. Methfessel, C. & Boheim, G. (1982) Biophys. Struct. Mech. 9, 35-60. 4. Lew, V. L., ed. (1983) Cell Calcium Vol. 4, (Churchill-Livingstone, Edinburgh, Scotland). 5. Krnjevic, K., Puil, E. & Werman, R. (1978) J. Physiol. 275, 199-223. 6. Moolenaar, W. R. & Spector, 1. (1979) J. Physiol. 292, 307323. 7. Marty, A. (1981) Nature (London) 291, 497-500. 8. Barrett, J. N., Barrett, E. F. & Dribin, L. B. (1981) Dev. Biol. 82, 258-266. 9. Burgess, G. M., Claret, M. & Jenkinson, D. H. (1981) J. Physiol. 317, 67-90. 10. Caroni, P. & Carafoli, E. (1982) Proc. Natl. Acad. Sci. USA 79, 5763-5767. 11. De Peyer, J. E., Cachelin, A. B., Levitan, I. B. & Reuter, H. (1982) Proc. NatI. Acad. Sci. USA 79, 4207-4211. 12. Lebrun, P., Atwater, I., Claret, M., Malaisse, W. J. & Herchuelz, A. (1983) FEBS Lett. 161, 41-44. 13. Marty, A. (1983) Trends Neurosci. 6, 262-265. 14. Hugues, M., Romey, G., Duval, D., Vincent, J. P. & Lazdunski, M. (1982) Proc. Natl. Acad. Sci. USA 79, 1308-1312. 15. Hugues, M., Schmid, H., Romey, G., Duval, D., Frelin, C. & Lazdunski, M. (1982) EMBO J. 9, 1039-1042. 16. Hugues, M., Duval, D., Kitabgi, P., Lazdunski, M. & Vincent, J. P. (1982) J. Biol. Chem. 257, 2762-2769. 17. Hugues, M., Duval, D., Schmid, H., Kitabgi, P., Lazdunski, M. & Vincent, J. P. (1982) Life Sci. 31, 437-443. 18. Haberman, E. & Fisher, K. (1979) Eur. J. Biochem. 94, 355364. 19. Cook, N. S., Haylett, D. N. & Strong, P. (1983) FEBS Lett. 152, 265-269. 20. Maas, A. D. J. J. & Den Hertog, A. (1979) Eur. J. Pharmacol. 58, 151-156. 21. Maas, A. D. J. J., Den Hertog, A., Ras, R. & Van Den Akker, J. (1980) Eur. J. Pharmacol. 67, 265-274. 22. Banks, B. E. C., Brown, C., Burgess, G. M., Claret, M., Cocks, T. M. & Jenkinson, D. H. (1979) Nature (London) 282, 415-417. 23. Habermann, E. & Fisher, K. (1979) Adv. Cytopharmacol. 3, 387-394. 24. Shipolini, R., Bradbury, A. F., Callewaert, G. L. & Vernon, C. A. (1967) Chem. Commun. 14, 679-680. 25. Von Haux, P., Sowerthal, H. & Habermann, E. (1967) HoppeSeyler's Z. Physiol. Chem. 348, 737-743. 26. Vincent, J. P., Schweitz, H. & Lazdunski, M. (1975) Biochemistry 14, 2521-2525. 27. Habermann, E. (1977) Naunyn-Schmiedebergs Arch. Pharmakol. 300, 189-191. 28. Schweitz, H. (1984) Toxicon 22, 308-311. 29. Romey, G. & Lazdunski, M. (1984) Biochem. Biophys. Res. Commun. 118, 669-674. 30. Narahashi, T. (1974) Physiol. Rev. 54, 813-889. 31. Howard, B. D. & Gunderson, C. B., Jr. (1980) Annu. Rev. Pharmacol. 20, 307-336. 32. Sperelakis, N. (1981) in Cardiac Toxicology, ed. Balazs, T. (CRC, Boca Raton, FL) Vol. 1, pp. 39-108. 33. Lazdunski, M. & Renaud, J. F. (1982) Annu. Rev. Physiol. 44, 463-473. 34. Schweitz, H. & Lazdunski, M. (1984) Toxicon 22, 985-988.
