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Inhibition by tetrandrine of calcium currents at mouse motor nerve endings
112
Brain Research, 524 (1990) 112-118 Elsevier
BRES 15736
Inhibition by tetrandrine of calcium currents at mouse motor nerve endings H. Wiegand, Susanne Meis and Ursula Gotzsch Medical Institute of Environmental Hygiene at the Heinrich-Heine-University Diisseldorf, Department of Neurotoxicology, Dasseldorf 1 (ER.G.)
(Accepted 6 February 1990) Key words: Tetrandrine; Cobalt; Calcium current; Inhibition; Nerve terminal; Mouse
The inhibition by the bis-benzyl-isoquinoline alkaloid tetrandrine of motor terminal calcium currents has been studied using extracellular, perineuronal electrodes in the M. triangularis preparation of the mouse. The calcium plateau current was irreversibly blocked, whereas the fast calcium current remained unaffected. From these results a calcium antagonism on neuronal calcium channels involved in transmitter release at motor nerve terminals is suggested. INTRODUCTION Tetrandrine, a bis-benzyl-isoquinoline alkaloid isolated from the roots of Stefania tetrandra sp. moore has been used in traditional medicine in China because of its analgesic and antiphlogistic properties for many centuries. Recent findings suggest its therapeutic use in the treatment of angina pectoris and hypertension 8"9. Tetrandrine has negative inotopic effects on isolated heart muscles and inhibits calcium-induced contractions of isolated coronary arteries and isolated smooth muscles of the uterus 1°. It has hypotensive activity in different hypertension in vivo models 28'33. In addition it shortens the action potential of heart cells 34 and inhibits the calcium-induced contractions of potassium-depolarized smooth muscles of the guinea pig caecum 31. It has therefore been suggested, that tetrandrine may be a calcium entry blocker. In recent experiments using the patch clamp technique on G H 3 anterior pituitary cells King et a1.18 described a blockade by tetrandrine of calcium currents through L-type calcium channels, which was half-maximal using a concentration of 6 /~molar tetrandrine. Similar results were obtained using nisoldipine 4. The chemical structure of this alkaloid is quite different from the other known chemical classes of calcium antagonists (for review see Ref. 29), so that its precise mechanism of action on calcium channels of heart muscle and smooth muscle remains enigmatic. The purpose of the present work was
tO explore the effects of tetrandrine on neuronal calcium channels, especially those, which are thought to be involved in transmitter release. There is a bulk of information on the abundance and function of calcium channels in neuronal tissue (for review see Refs. 5, 16, 19, 25, 26). Among the 3 commonly accepted subtypes of neuronal calcium channels it is the N-type, which apparently plays a dominant role in transmitter release 17. This high-threshold-type channel is inhibited by cadmium and unsensitive for dihydropyridine blockade, whereas dihydropyridine agonists activate it. N-channels are supposed to be clustered in the presynaptic nerve terminal membrane near the so called 'active zones' where transmitter is released and they may regulate the fusion of synaptic vesicles releasing the transmitter from the presynaptic terminal. In order to characterize the calcium-antagonizing properties of tetrandrine in neuronal calcium channels involved in transmitter release we choose the triangularis sterni muscle of the mouse 23 for perineuronal recording presynaptic membrane currents of the invading action potential as first described by Mallart 22 and further developed by Penner and Dreyer 27. This technique allows wave forms from the perineurium to be recorded which correspond to the ion fluxes through ion channels within the preterminal and the terminal, respectively. The action of tetrandrine on the different ion fluxes has been characterized. A brief report on the action of tetrandrine on presynaptic motor nerve currents has been given 24'31.
Correspondence: H. Wiegand, Medical Institute of Environmental Hygiene at the Heinrich-Heine-University Dfisseldorf, Department of Neurotoxicology, Auf 'm Hennekamp 50, D-4000 DfJsseldorf 1, ER.G.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
113 MATERIALS AND METHODS The experiments were performed on the M. triangularis sterni nerve muscle preparation of adult mice23. The preparation was visualized at a x400 magnification with a Zeiss microscope equipped with Nomarski inference contrast ~3. The preparations were perfused at a rate of 3 ml/min with a Krebs-solution of the following composition (mmolar): NaCI 118; KCI 4.8; CAC12.2 H20 2.6; MgSO4.7 H20 1.2; NaHCO3 25; KH2PO4 1.2; glucose 6. Krebssolution with reduced calcium ion content had the following composition (mmolar): NaCI 125; KCI 4.8; CAC12.2 H2O 1.0; MgSO4.7 H2O 1.2; NaHCO 3 25; KH2PO4 1.2; glucose 6. The solutions were gassed with 95% 02/5% CO 2 (pH 7.3). In all experiments curare (50/~molar) was used to abolish neuromuscular transmission and procaine (100 Mmolar) was used to prevent repetitive firing of motor terminals which occurs with potassium channel blockers. We saw no influence of tubocurarine or of procaine on the recorded wave forms with the concentrations used here. Nerve stimulation was with a suction-electrode. Extracellular signals were picked up by microelectrodes (0.5 molar NaCI, 3-7 MI2) positioned against a terminal ramification. A large Ag-AgCI wire within the bath served as reference electrode. Although this kind of recording is definitely not recording of membrane currents, the signals picked from within the perineurium have similar time constants to the membrane currents induced by ion fluxes through the different ion channels in the different parts of the terminal22"27. It should be noted, that the signals recorded from the preterminal perineurium have a reversed polarity as compared to the signals recorded from the exposed terminal22. Investigations on the calcium plateau potentials were done during the application of tetraethylammonium (TEA, 1 ~M) and 3,4-diaminopyridine (3,4-DAP, 100 mM). After conventional amplification the signals were fed into an oscilloscope for visual inspection as well as to a LSIll/03 computer for signal analysis with a modified software as described previously32. The area between positive going calcium signals and zero voltage line was calculated by integration; the reduction of the integrated signal area was normalized to untreated control signals and the percent inhibition of the values was plotted versus concentrations for tetrandrine or cobalt, respectively. From these values, concentration-effect relationship curves were fitted and parameters were estimated using Allfit". The following drugs were used: tetrandrine was a gift of the Institute of Health, Peking, People's Republic of China. It was usually dissolved in distilled water, acidified with HCI (final pH 2) to obtain a 10-mmolar stock solution. This was added to the superfusion Krebs-solution to obtain final concentrations of tetrandrine between 100 nmolar to 50/~molar, the maximal pH-deviation being 0.18 units. Stock-solutions with a higher content of tetrandrine than 10 mmolar could not be produced because tetrandrine precipitated. 3,4-DAP, procainehydrochlorid, TEA and d-tubocurarine were from Sigma. All other reagents were of analytical quality.
If~ I A
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Fig. 1. (A) Typical subendoneuronally recorded nerve signal. Neuromuscular transmission blocked by 50 #M d-tubocurarine. 1: stimulation artefact; 2: sodium-influx within the next proximal node of Ranvier; 3: sodium-influx in the heminode; 4: potassium°efflux at the terminal. In this and all following figures single signals without averaging. (B) M. Triangularis preparation as viewed by Nomarski optics (x400). A typical microelectrode position is given by a symbol. was so n e a r to the e n d p l a t e regions, that the s e c o n d negativity resulting f r o m the p o t a s s i u m influx into the t e r m i n a l s had a larger a m p l i t u d e t h a n the first negativity resulting f r o m the s o d i u m influx w i t h i n the h e m i n o d e 22.
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RESULTS F i g u r e 1 A shows a typical, s u b e n d o n e u r a l l y r e c o r d e d n e r v e signal following s u p r a m a x i m a l s t i m u l a t i o n 2'22"24'27. T h e m i c r o e l e c t r o d e h a d b e e n p o s i t i o n e d into the perin e u r i u m of a n intercostal n e r v e ramification in the vicinity of the m u s c l e e n d p l a t e s of a b u n d l e of 4 - 6 axons, as c a n be s e e n in a typical e x a m p l e in Fig. l B . D e p e n d i n g o n the p o s i t i o n of the m i c r o e l e c t r o d e tip within the p e r i n e u r o n a l s h e a t h a n d o n the n u m b e r of axons, the signal v a r i e d f r o m 1 to 8 mV. U s u a l l y the r e c o r d i n g site
J
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2 ms Fig. 2. Development of positive voltage deflections in the presence of 10 mM TEA and 250/~M 3,4-DAP. a: control; b: depression of potassium signal 6 rain after application of TEAI3,4-DAP; c: positive going short calcium current 9 min after potassium channel blockade; d: long-lasting calcium plateau 12 min after potassium channel blockade.
114
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Fig. 4. Reduction of the slow calcium signal by stimulation frequency increase, a: 0.004 Hz; b: 0.017 Hz; c: 0.03 Hz; d: 0.05 Hz; e: 0.1 Hz; f: 1 Hz. Potassium channels were blocked by 250 MM 3,4-DAP and 10 mM TEA.
Arise of calcium currents upon potassium channel blockade Application of 3,4-DAP (250 Mmolar) and TiEA (10 mmolar)27 to the preparation via superfusion-solution caused the second negative deflection of the subendoneural signal to vanish within 6-9 rain. A relatively short lasting (few ms) positivity then developed which gave
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40ms
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Fig. 3. (A) Effects of different TEA/3,4-DAP concentrations on positive voltage deflections, a: 50 MM 3,4-DAP and 1 mM TEA induce two dearly separated calcium signals, b: increasing potassium channel blockade by 100 # M 3,4-DAP and 5 mM TEA starts merging of fast and slow calcium signal component, c: further increase of potassium blockade by 100 MM 3,4-DAP and 10 mM TEA enhances duration of the slow calcium signal drastically. Stimulation frequency was 0.017 Hz. (B) Effects of extracellular calcium concentration on calcium signals, a: 2.6 mM calcium; b: 1.0 mM calcium; c: 2.6 mM calcium. Potassium channels were blocked by 250 #M 3,4-DAP and 10 mM TEA. Stimulation frequency was 0.006 Hz.
115
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Fig. 5. (A) Effect of tetrandrine upon subendoneuronally recorded nerve signal without potassium channel blockade, a: control signal (a) is not different from treatment with 10/~M tetrandrine (b). b: control signal (c) is not different from treatment with 50 ~M tetrandrine (d). (B) Blockade by 50/~M tetrandrine of the slow calcium plateau signal. way to a long-lasting positive voltage deflection (Fig. 2). These positive going wave forms c o r r e s p o n d to calcium currents entering the m o t o r nerve terminals 27'3°. T h e y were u n m a s k e d with the following physiological and pharmacological treatments. T h e time-course of action of 3 , 4 - D A P and T E A on the calcium currents was concent r a t i o n - d e p e n d e n t . Using 50 ~ m o l a r 3 , 4 - D A P and 1 m m o l a r T E A , the short- and the long-lasting calcium currents s e p a r a t e d in time. T h e short-lasting calcium current usually had a higher a m p l i t u d e than the long-
100
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TABLE I
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Fig. 6. Concentration-effect relationship of tetrandrine (square) and cobalt (triangle). Percent inhibition of the slow calcium plateau signal (ordinate) is plotted versus negative logarithm of tetrandrine or cobalt concentration, respectively. Open square- and filled triangle values are from single experiments. The curves were fitted using AIIfitlL
Parameters derived from non-linear least square fitting to concentration effect relationships using Allfit 11for slow calcium plateau currents
The values are given as • + S.D.
IC50 ~M) Slope factor
Tetrandrine
Cobalt
6.4 + 0.8 -0.6 + 0.07
20 + 2.5 -0.7 + 0.07
116
lmV lOms
Fig. 7. Effect of tetrandrine upon fast and slow calcium signals. Moderate potassium channel blockade (50/~M 3,4-DAP and 1 mM TEA) induced the two clearly separated calcium signals, a: control; b: reduction of signal duration 10 min after application of 10/~M tetrandrine; c: same 20 rain after 10 pM tetrandrine-application. Stimulation frequency was 0.017 Hz.
lasting calcium current. With higher concentrations (100 ~molar 3,4-DAP and 5 mmolar TEA) both calcium currents merged with the increase in signal duration (Fig. 3A). The long-lasting calcium signal will be called the calcium plateau signal.
Influence of extracellular calcium concentration on presynaptic calcium currents When the calcium content of the superfusion medium was reduced from the 2.6 mmolar control to 1.0 mmolar, the amplitude as well as the duration of the calcium plateau was drastically reduced (0.006 Hz, Fig. 3B). The calcium plateau was again lengthened within 12-15 min with superfusion of 2.6 mmolar calcium medium.
Influence of tetrandrine on presynaptic currents Sodium and potassium signal: Tetrandrine applied to nerve muscle preparations at concentrations up to 50 /~molar was without effect onto the recorded wave forms (Fig. 5A). Calcium plateau signal: The duration of the calcium plateau was reduced within 30 min with 50 /~molar tetrandrine (Fig. 5B). This action resembled that of verapamil on the calcium plateau (see Fig. 6c of Ref. 27). The reduction in the slow calcium plateau was irreversible for 6 h of washing. The dose-response curve over 3 orders of magnitude was sigmoid (Fig. 6). The IC50 for tetrandrine was 4 times less than for cobalt (Table I). Short calcium current: The analysis of the tetrandrine effect on the perineuronal signals showed a fundamental difference in the behavior of the two calcium signal components. 3,4-DAP and T E A (50 ~molar 3,4-DAP and 1 mmolar TEA) induced a moderate calcium plateau, on which the fast calcium component is visible (see Fig. 7, curve a). Ten/~molar tetrandrine progressively shortened the signal (Fig. 7) with little or no change in the amplitude and rising phase of the fast signal (Fig. 7). One Hz stimulation with tetrandrine at 10 ~molar was without any effect even after 23 min (Fig. 8, cf. diltiazem 10/~molar; see Fig. 6e of Ref. 27). Influence of cobalt on presynaptic currents The fast calcium current remained unaffected by cobalt application. The IC5o and slope factor for cobalt are 20 ktmolar and -0.7, respectively (Fig. 6, Table I). DISCUSSION
Influence of stimulation frequency on presynaptic calcium currents When the stimulation frequency was increased from 0.006 Hz to 1 Hz, the long-lasting calcium plateau progressively diminished (Fig. 4), and this effect was completely reversible.
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Fig. 8. Effect of tetrandrine on the fast calcium signal. Stimulation frequency was 1 Hz. a: control; b: 23 min after application of 10/~M tetrandrine. Potassium channels were blocked by 50/~M 3,4-DAP and 1 mM TEA.
The present results show the influence of tetrandrine on presynaptic membrane currents as compared to the influence of cobalt. Though the method by which these currents were recorded do not allow their unequivocal attribution directly to special ion channels, to our knowledge it is the best available method for recording presynaptic events from mammalian motor nerve terminals. The mode of action of neurotoxicants on these currents depends on pharmacological identification because voltage clamping of the presynaptic terminal membrane is not feasible. The application of tetrandrine to muscle preparations shows that neither the first negative peak attributed to sodium influx within the heminode nor the second negative peak known as a potassium current in the nerve terminals were influenced. This means tetrandrine has no sodium channel blocking activity and does not block potassium channels in the presynaptic membrane (for review see Ref. 12). There was no effect of tetrandrine
117 on the fast presynaptic calcium current (Penner and Dreyer27). Rather there was a blocking activity on the slow calcium current. Tetrandrine has a moderate affinity and specificity as compared to other organic and the inorganic calcium channel blockers. The calcium plateau signal has been shown to be carried by calcium and barium, and is blocked by verapamil, diltiazem, cadmium, manganese 27 as well as by cobalt (this paper). If one compares the activities of the different inorganic calcium channel blockers (cadmium, manganese, cobalt) on the one site and the low affinity binding sites of the organic calcium blockers verapamil and diltiazem on the other site one can establish the following sequence of blocking activity: cadmium > tetrandrine > verapamil > cobalt > diltiazem > manganese (see Table I of Ref. 27; this paper Table I). The slow calcium plateau signal was not sensitive to 1,4-dihydropyridine27 nor to omegaconotoxin ~. There is pharmacological evidence27 that the slow calcium plateau signal is carried by a distinct class of slow calcium channels. Mallart 22 has suggested that the fast and the slow calcium components can be separated by the combination of moderate potassium channel blockers (3,4-DAP and TEA) and that they are carried by one and the same calcium current becoming regenerative. Therefore it is not possible to decide, which calcium channel is affected by tetrandrine and cobalt. Different types of calcium currents have been demonstrated in neuronal as well as in non-neuronal cells. In mammalian CNS neurons low threshold channels were differentiated from high threshold channels 7. The high threshold calcium channels could be separated into two kinds, the so called N-type and L-type, whereas there seemed to be only one low-threshold channel, the so called T-type channel. Distinction between these 3 channel-types was based on biophysical as well as pharmacological means and usually was done in patch and whole cell clamp recordings. L-type channels in D R G neurons carry large barium currents which were long-lasting and activated by strong depolarizations from relatively low holding potentials. In contrast T-type calcium channels carried small barium currents; they are evoked only from negative holding potentials and require small depolarizations for activation. They inactivate more slowly than N- or L-type channels. Whereas N-type or T-type calcium channels are not affected by 1,4-dihydropyridines these drugs select strongly for L-type channels, but whether they completely block L-currents is unclear especially if these are REFERENCES 1 Anderson, A.J. and Harvey, A.L., Omega-conotoxin does not block the verapamil-sensitive calcium channel at mouse motor nerve terminals, Neurosci. Lett., 82 (1987) 177-180.
evoked from negative holding potentials. All 3 types of channels are found in central neurons, for example hippocampal pyramidal cells. Some other cells appeared to express only one of the 3 types, for example L-type in adrenal chromaffine cells or T-type in neoplastic Blymphocytes 14A5. In peripheral neurons, N-type channels were suggested to play a dominant if not exclusive role in controlling transmitter release. Norepinephrine release from sympathetic neurons is resistant to dihydropyridines, but sensitive to cadmium and omega-conotoxin 17. N-type channels might also explain the dihydropyridine resistance of transmitter release from neuromuscular junctions 25'26. There is evidence, that N- and L-type calcium channels coexist in motor nerve terminals 3. Sometimes calcium channels seem to be clustered in hot spots, which contain one type of channel exclusively21. These hot spots were suggested to play an important role in triggering transmitter release in synaptic terminals. It has been proposed 17 that calcium current through calcium channels of the N-type may be involved in triggering exocytosis of synaptic vesicles, whereas L-type channels regulate exocytosis of large dense core vesicles. Tetrandrine acted on calcium channels within the presynaptic terminal which: (1) could be blocked by the anorganic channel blockers cadmium, manganese, cobalt; (2) could be blocked by the organic channel blockers verapamil and diltiazem; (3) could not be blocked by 1,4-dihydropyridines; (4) could not be blocked by omega-conotoxin; (5) inactivated only within hundreds of ms. Obviously such kind of pharmacological profile did not fit precisely into any scheme of neuronal calcium channel pharmacology known as yet (see above). Moreover, it was definitely different from that of presynaptic L-type calcium channels as described recently in lizard nerve endings 2°. In view of these findings on the role of calcium channels in transmitter release our results at the mouse neuromuscular junction in combination with others L 6,13,27 do not define the calcium channel tetrandrine might influence. Acknowledgements. Prof. F. Dreyer, Giessen, ER.G., introduced us to Nomarski optic techniques, R. Penner, G6ttingen, ER.G., to the triangularis preparation. Dr. D. Rodbard, Bethesda, MD, U.S.A., kindly provided the programme Allfit. Karolina Sveinsson prepared the figures.
2 Anderson, A.J. and Harvey, A.L., Effects of the facilitatory compounds catechole, guanidine, noradrenaline and phencyclidine on presynaptic currents of mouse motor nerve terminals, Naunyn-Schmiedeberg's Arch. Pharmacol., 338 (1988) 133-137. 3 Atchison, W.D. and O'Leary, S.M., Bay K 8644 increases
118 release of acetyl-choline at the murine neuromuscular junction, Brain Research, 419 (1987) 315-319. 4 Barros, E, Katz, G.M., Kaczorowski, G.J., Vandelen, R.L. and Reuben, J.P., Calcium currents in GH 3 cultured pituitary cells under whole cell voltage clamp: inhibition by voltage dependent potassium currents, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 1108-1112. 5 Bean, B.P., Classes of calcium channels in vertebrate cells, Annu. Rev. PhysioL, 51 (1989) 367-384. 6 Bourret, C. and Mallart, A., Depression of calcium current at mouse motor nerve endings by polycationic antibiotics, Brain Research, 478 (1989) 403-406. 7 Carbone, E. and Lux, H.D., A low voltage activated fully inactivating Ca ++ channel in vertebrate sensory neurons, Nature, 310 (1984) 501-502. 8 Chang Tan-Mu, Chao Kuo-Chue and Lue-Fu Hua, The cardiovascular effects of tetrandrine and d-methyl-tetrandrine, Acta Pharm. Sin., 6 (1956) 147-153. 9 Chan Tan-Mu, Chao Kuo-Chue and Lue Fu-Hua, The mechanism of the hypotensive effect of tetrandrine and d-methyltetrandrine, Acta Pharm. Sin., 8 (1958) 163-168. 10 Da-Chao Fang and Ming-Zing Jiang, A new calcium antagonist of chinese medicinal origin: tetrandrine, J. Hypertens., 4 (1986) S1500S152. 11 De Lean, A., Munson, P.J., Guardabasso, V. and Rodbard, D., Allfit. Simultaneous Fitting of Families of Sigmoidal DoseResponse Curves using the Four-Parameter Logistic Equation, Program version 2.7, Bethesda, MD, U.S.A., 1988. 12 Dreyer, F. and Penner, R., The action of presynaptic snake toxins on membrane currents of mouse motor nerve terminals, J. Physiol. (London), 386 (1987) 455-463. 13 Dreyer, F., Miiller, K.D., Pepper, K. and Sterz, R., The m.omohyoideus of the mouse as a convenient mammalian muscle preparation. A study of junctional and extrajunctional acetylcholine receptors by noise analysis and cooperativity, Pfliig. Arch. Eur. J. Physiol., 367 (1979) 115-122. 14 Fenwick, E.M., Marty, A. and Neher, E., Sodium and calcium channels in bovine chromaffin cells, J. Physiol. (London), 311 (1982) 599-635. 15 Fukoshima, Y. and Hagiwara, S., Currents carried by monovalent actions through calcium channels in mouse neoplastic B-lymphocytes, J. Physiol. (London), 358 (1985) 255-284. 16 Hagiwara, S. and Byerly, L., Calcium channel, Annu. Rev. Neurosci., 4 (1981) 69-125. 17 Hirning, L.D., Fox, A.P., McCleskey, E.W., Olivera, B.M., Thayer, S.A., Miller, R.J. and Tsien, R.W., Dominant role of N-type channels in evoked release of norepinephrine from sympathetic neurons, Science, 239 (1988) 57-61. 18 King, V.F., Garcia, M.L., Himmel, D., Reuben, J.P., Lam, Y.T., Pan, J., Han, G. and Kaczorowski, G.J., Interaction of tetrandrine with slowly inactivating calcium channels. Characterization of calcium channel modulation by an alkaloid of chinese medicinal herb origin, J. Biol. Chem., 263 (1988)
2238-2244. 19 Kostyuk, P.G., Diversity of calcium ion channels in cellular membranes, Neuroscience, 28 (1989) 263-271. 20 Lindgren, C.A. and Moore, J.W., Identification of ionic currents at presynaptic nerve endings of the lizard, J. Physiol. (London), 414 (1989) 201-222. 21 Lipscombe, D., Madison, D.V., Poenie, M., Reuter, H., Tsien, R.Y. and Tsien, R.W., Spatial distribution of calcium channels and cytosolic calcium transients in growth cones and cell bodies of sympathetic neurones, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 2398-2402. 22 Mallart, A., Electric current flow inside perineurial sheaths of mouse motor nerves, J. Physiol. (London), 368 (1985) 565-575. 23 McArdle, J.J., Angaud-Petit, D., Mallart, A., Bournaud, R., Faille, L. and Brigant, J.L., Advantages of the triangularis sterni muscle of the mouse for investigations of synaptic phenomena, J. Neurosci. Methods, 4 (1981) 109-115. 24 Meis, S., Uhlig, S. and Wiegand, H., Presynaptic recordings from motor nerve endings in the M. triangularis preparation as a tool in neurotoxicoiogy. In N. Eisner and EG. Barth (Eds.), Sense Organs. Interfaces between Environment and Behavior, Thieme, Stuttgart, 1988, p. 107. 25 Miller, R.J., Calcium channels in neurons. In J.C. Venter and D. Triggle (Eds.), Structure and Physiology of the Slow Inward Calcium Channel, A.R. Liss, New York, NY, 1987, pp. 161-246. 26 Miller, R.J., Multiple calcium channels and neuronal function, Science, 235 (1987) 46-52. 27 Penner, R. and Dreyer, E, Two different presynaptic calcium currents in mouse motor nerve terminals, Pflag. Arch. Eur. J. Physiol., 406 (1986) 190o197. 28 Oian, J.Q., Thoolen, M.J.M.C., Van Meel, J.C.A., Timmermans, P.B.M.W.M. and Van Zwieten, P.A., Hypotensive activity of tetrandrine in rats. Investigation into its mode of action, Pharmacology, 26 (1983) 187-197. 29 Spedding, M., Calcium antagonist subgroups, Trends Pharmacol. Sci., 6 (1985) 109-114. 30 Tabti, N., Bourret, C. and Mallart, A., Three potassium currents in mouse motor nerve terminals, Pflag. Arch. Eur. J. Physiol., 413 (189) 395-400. 31 Wiegand, H., Calcium antagonism of tetrandrine in intestinal smooth muscle preparations in mouse motor nerve terminals, Naunyn-Schmiedeberg's Arch. Pharmacol., $338 (1988) R 37. 32 Wiegand, H., Lohmann, H. and Chandra, S.V., The action of thallium acetate on phasic transmitter release in the mouse neuromuscular junction, Arch. Toxicol., 58 (1986) 265-270. 33 Zeng, D., Shaw, Jr., D.H. and Ogilvie, R.I., Kinetic disposition and hemodynamic effects of tetrandrine in unaesthetized dogs, J. Cardiovasc. Pharmacol., 7 (1985) 1034-1039. 34 Zong, X.G., Jin, N.W., Xia, G.J., Fang, D.C. and Jiang, M.X., Effects of tetrandrine on action potential and contraction of isolated guinea pig papillary muscles, Acta Pharmacol. Sin., 4 (1983) 258-261.
Brain Research, 524 (1990) 112-118 Elsevier
BRES 15736
Inhibition by tetrandrine of calcium currents at mouse motor nerve endings H. Wiegand, Susanne Meis and Ursula Gotzsch Medical Institute of Environmental Hygiene at the Heinrich-Heine-University Diisseldorf, Department of Neurotoxicology, Dasseldorf 1 (ER.G.)
(Accepted 6 February 1990) Key words: Tetrandrine; Cobalt; Calcium current; Inhibition; Nerve terminal; Mouse
The inhibition by the bis-benzyl-isoquinoline alkaloid tetrandrine of motor terminal calcium currents has been studied using extracellular, perineuronal electrodes in the M. triangularis preparation of the mouse. The calcium plateau current was irreversibly blocked, whereas the fast calcium current remained unaffected. From these results a calcium antagonism on neuronal calcium channels involved in transmitter release at motor nerve terminals is suggested. INTRODUCTION Tetrandrine, a bis-benzyl-isoquinoline alkaloid isolated from the roots of Stefania tetrandra sp. moore has been used in traditional medicine in China because of its analgesic and antiphlogistic properties for many centuries. Recent findings suggest its therapeutic use in the treatment of angina pectoris and hypertension 8"9. Tetrandrine has negative inotopic effects on isolated heart muscles and inhibits calcium-induced contractions of isolated coronary arteries and isolated smooth muscles of the uterus 1°. It has hypotensive activity in different hypertension in vivo models 28'33. In addition it shortens the action potential of heart cells 34 and inhibits the calcium-induced contractions of potassium-depolarized smooth muscles of the guinea pig caecum 31. It has therefore been suggested, that tetrandrine may be a calcium entry blocker. In recent experiments using the patch clamp technique on G H 3 anterior pituitary cells King et a1.18 described a blockade by tetrandrine of calcium currents through L-type calcium channels, which was half-maximal using a concentration of 6 /~molar tetrandrine. Similar results were obtained using nisoldipine 4. The chemical structure of this alkaloid is quite different from the other known chemical classes of calcium antagonists (for review see Ref. 29), so that its precise mechanism of action on calcium channels of heart muscle and smooth muscle remains enigmatic. The purpose of the present work was
tO explore the effects of tetrandrine on neuronal calcium channels, especially those, which are thought to be involved in transmitter release. There is a bulk of information on the abundance and function of calcium channels in neuronal tissue (for review see Refs. 5, 16, 19, 25, 26). Among the 3 commonly accepted subtypes of neuronal calcium channels it is the N-type, which apparently plays a dominant role in transmitter release 17. This high-threshold-type channel is inhibited by cadmium and unsensitive for dihydropyridine blockade, whereas dihydropyridine agonists activate it. N-channels are supposed to be clustered in the presynaptic nerve terminal membrane near the so called 'active zones' where transmitter is released and they may regulate the fusion of synaptic vesicles releasing the transmitter from the presynaptic terminal. In order to characterize the calcium-antagonizing properties of tetrandrine in neuronal calcium channels involved in transmitter release we choose the triangularis sterni muscle of the mouse 23 for perineuronal recording presynaptic membrane currents of the invading action potential as first described by Mallart 22 and further developed by Penner and Dreyer 27. This technique allows wave forms from the perineurium to be recorded which correspond to the ion fluxes through ion channels within the preterminal and the terminal, respectively. The action of tetrandrine on the different ion fluxes has been characterized. A brief report on the action of tetrandrine on presynaptic motor nerve currents has been given 24'31.
Correspondence: H. Wiegand, Medical Institute of Environmental Hygiene at the Heinrich-Heine-University Dfisseldorf, Department of Neurotoxicology, Auf 'm Hennekamp 50, D-4000 DfJsseldorf 1, ER.G.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
113 MATERIALS AND METHODS The experiments were performed on the M. triangularis sterni nerve muscle preparation of adult mice23. The preparation was visualized at a x400 magnification with a Zeiss microscope equipped with Nomarski inference contrast ~3. The preparations were perfused at a rate of 3 ml/min with a Krebs-solution of the following composition (mmolar): NaCI 118; KCI 4.8; CAC12.2 H20 2.6; MgSO4.7 H20 1.2; NaHCO3 25; KH2PO4 1.2; glucose 6. Krebssolution with reduced calcium ion content had the following composition (mmolar): NaCI 125; KCI 4.8; CAC12.2 H2O 1.0; MgSO4.7 H2O 1.2; NaHCO 3 25; KH2PO4 1.2; glucose 6. The solutions were gassed with 95% 02/5% CO 2 (pH 7.3). In all experiments curare (50/~molar) was used to abolish neuromuscular transmission and procaine (100 Mmolar) was used to prevent repetitive firing of motor terminals which occurs with potassium channel blockers. We saw no influence of tubocurarine or of procaine on the recorded wave forms with the concentrations used here. Nerve stimulation was with a suction-electrode. Extracellular signals were picked up by microelectrodes (0.5 molar NaCI, 3-7 MI2) positioned against a terminal ramification. A large Ag-AgCI wire within the bath served as reference electrode. Although this kind of recording is definitely not recording of membrane currents, the signals picked from within the perineurium have similar time constants to the membrane currents induced by ion fluxes through the different ion channels in the different parts of the terminal22"27. It should be noted, that the signals recorded from the preterminal perineurium have a reversed polarity as compared to the signals recorded from the exposed terminal22. Investigations on the calcium plateau potentials were done during the application of tetraethylammonium (TEA, 1 ~M) and 3,4-diaminopyridine (3,4-DAP, 100 mM). After conventional amplification the signals were fed into an oscilloscope for visual inspection as well as to a LSIll/03 computer for signal analysis with a modified software as described previously32. The area between positive going calcium signals and zero voltage line was calculated by integration; the reduction of the integrated signal area was normalized to untreated control signals and the percent inhibition of the values was plotted versus concentrations for tetrandrine or cobalt, respectively. From these values, concentration-effect relationship curves were fitted and parameters were estimated using Allfit". The following drugs were used: tetrandrine was a gift of the Institute of Health, Peking, People's Republic of China. It was usually dissolved in distilled water, acidified with HCI (final pH 2) to obtain a 10-mmolar stock solution. This was added to the superfusion Krebs-solution to obtain final concentrations of tetrandrine between 100 nmolar to 50/~molar, the maximal pH-deviation being 0.18 units. Stock-solutions with a higher content of tetrandrine than 10 mmolar could not be produced because tetrandrine precipitated. 3,4-DAP, procainehydrochlorid, TEA and d-tubocurarine were from Sigma. All other reagents were of analytical quality.
If~ I A
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Fig. 1. (A) Typical subendoneuronally recorded nerve signal. Neuromuscular transmission blocked by 50 #M d-tubocurarine. 1: stimulation artefact; 2: sodium-influx within the next proximal node of Ranvier; 3: sodium-influx in the heminode; 4: potassium°efflux at the terminal. In this and all following figures single signals without averaging. (B) M. Triangularis preparation as viewed by Nomarski optics (x400). A typical microelectrode position is given by a symbol. was so n e a r to the e n d p l a t e regions, that the s e c o n d negativity resulting f r o m the p o t a s s i u m influx into the t e r m i n a l s had a larger a m p l i t u d e t h a n the first negativity resulting f r o m the s o d i u m influx w i t h i n the h e m i n o d e 22.
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RESULTS F i g u r e 1 A shows a typical, s u b e n d o n e u r a l l y r e c o r d e d n e r v e signal following s u p r a m a x i m a l s t i m u l a t i o n 2'22"24'27. T h e m i c r o e l e c t r o d e h a d b e e n p o s i t i o n e d into the perin e u r i u m of a n intercostal n e r v e ramification in the vicinity of the m u s c l e e n d p l a t e s of a b u n d l e of 4 - 6 axons, as c a n be s e e n in a typical e x a m p l e in Fig. l B . D e p e n d i n g o n the p o s i t i o n of the m i c r o e l e c t r o d e tip within the p e r i n e u r o n a l s h e a t h a n d o n the n u m b e r of axons, the signal v a r i e d f r o m 1 to 8 mV. U s u a l l y the r e c o r d i n g site
J
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2 ms Fig. 2. Development of positive voltage deflections in the presence of 10 mM TEA and 250/~M 3,4-DAP. a: control; b: depression of potassium signal 6 rain after application of TEAI3,4-DAP; c: positive going short calcium current 9 min after potassium channel blockade; d: long-lasting calcium plateau 12 min after potassium channel blockade.
114
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Fig. 4. Reduction of the slow calcium signal by stimulation frequency increase, a: 0.004 Hz; b: 0.017 Hz; c: 0.03 Hz; d: 0.05 Hz; e: 0.1 Hz; f: 1 Hz. Potassium channels were blocked by 250 MM 3,4-DAP and 10 mM TEA.
Arise of calcium currents upon potassium channel blockade Application of 3,4-DAP (250 Mmolar) and TiEA (10 mmolar)27 to the preparation via superfusion-solution caused the second negative deflection of the subendoneural signal to vanish within 6-9 rain. A relatively short lasting (few ms) positivity then developed which gave
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Fig. 3. (A) Effects of different TEA/3,4-DAP concentrations on positive voltage deflections, a: 50 MM 3,4-DAP and 1 mM TEA induce two dearly separated calcium signals, b: increasing potassium channel blockade by 100 # M 3,4-DAP and 5 mM TEA starts merging of fast and slow calcium signal component, c: further increase of potassium blockade by 100 MM 3,4-DAP and 10 mM TEA enhances duration of the slow calcium signal drastically. Stimulation frequency was 0.017 Hz. (B) Effects of extracellular calcium concentration on calcium signals, a: 2.6 mM calcium; b: 1.0 mM calcium; c: 2.6 mM calcium. Potassium channels were blocked by 250 #M 3,4-DAP and 10 mM TEA. Stimulation frequency was 0.006 Hz.
115
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Fig. 5. (A) Effect of tetrandrine upon subendoneuronally recorded nerve signal without potassium channel blockade, a: control signal (a) is not different from treatment with 10/~M tetrandrine (b). b: control signal (c) is not different from treatment with 50 ~M tetrandrine (d). (B) Blockade by 50/~M tetrandrine of the slow calcium plateau signal. way to a long-lasting positive voltage deflection (Fig. 2). These positive going wave forms c o r r e s p o n d to calcium currents entering the m o t o r nerve terminals 27'3°. T h e y were u n m a s k e d with the following physiological and pharmacological treatments. T h e time-course of action of 3 , 4 - D A P and T E A on the calcium currents was concent r a t i o n - d e p e n d e n t . Using 50 ~ m o l a r 3 , 4 - D A P and 1 m m o l a r T E A , the short- and the long-lasting calcium currents s e p a r a t e d in time. T h e short-lasting calcium current usually had a higher a m p l i t u d e than the long-
100
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TABLE I
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Fig. 6. Concentration-effect relationship of tetrandrine (square) and cobalt (triangle). Percent inhibition of the slow calcium plateau signal (ordinate) is plotted versus negative logarithm of tetrandrine or cobalt concentration, respectively. Open square- and filled triangle values are from single experiments. The curves were fitted using AIIfitlL
Parameters derived from non-linear least square fitting to concentration effect relationships using Allfit 11for slow calcium plateau currents
The values are given as • + S.D.
IC50 ~M) Slope factor
Tetrandrine
Cobalt
6.4 + 0.8 -0.6 + 0.07
20 + 2.5 -0.7 + 0.07
116
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Fig. 7. Effect of tetrandrine upon fast and slow calcium signals. Moderate potassium channel blockade (50/~M 3,4-DAP and 1 mM TEA) induced the two clearly separated calcium signals, a: control; b: reduction of signal duration 10 min after application of 10/~M tetrandrine; c: same 20 rain after 10 pM tetrandrine-application. Stimulation frequency was 0.017 Hz.
lasting calcium current. With higher concentrations (100 ~molar 3,4-DAP and 5 mmolar TEA) both calcium currents merged with the increase in signal duration (Fig. 3A). The long-lasting calcium signal will be called the calcium plateau signal.
Influence of extracellular calcium concentration on presynaptic calcium currents When the calcium content of the superfusion medium was reduced from the 2.6 mmolar control to 1.0 mmolar, the amplitude as well as the duration of the calcium plateau was drastically reduced (0.006 Hz, Fig. 3B). The calcium plateau was again lengthened within 12-15 min with superfusion of 2.6 mmolar calcium medium.
Influence of tetrandrine on presynaptic currents Sodium and potassium signal: Tetrandrine applied to nerve muscle preparations at concentrations up to 50 /~molar was without effect onto the recorded wave forms (Fig. 5A). Calcium plateau signal: The duration of the calcium plateau was reduced within 30 min with 50 /~molar tetrandrine (Fig. 5B). This action resembled that of verapamil on the calcium plateau (see Fig. 6c of Ref. 27). The reduction in the slow calcium plateau was irreversible for 6 h of washing. The dose-response curve over 3 orders of magnitude was sigmoid (Fig. 6). The IC50 for tetrandrine was 4 times less than for cobalt (Table I). Short calcium current: The analysis of the tetrandrine effect on the perineuronal signals showed a fundamental difference in the behavior of the two calcium signal components. 3,4-DAP and T E A (50 ~molar 3,4-DAP and 1 mmolar TEA) induced a moderate calcium plateau, on which the fast calcium component is visible (see Fig. 7, curve a). Ten/~molar tetrandrine progressively shortened the signal (Fig. 7) with little or no change in the amplitude and rising phase of the fast signal (Fig. 7). One Hz stimulation with tetrandrine at 10 ~molar was without any effect even after 23 min (Fig. 8, cf. diltiazem 10/~molar; see Fig. 6e of Ref. 27). Influence of cobalt on presynaptic currents The fast calcium current remained unaffected by cobalt application. The IC5o and slope factor for cobalt are 20 ktmolar and -0.7, respectively (Fig. 6, Table I). DISCUSSION
Influence of stimulation frequency on presynaptic calcium currents When the stimulation frequency was increased from 0.006 Hz to 1 Hz, the long-lasting calcium plateau progressively diminished (Fig. 4), and this effect was completely reversible.
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Fig. 8. Effect of tetrandrine on the fast calcium signal. Stimulation frequency was 1 Hz. a: control; b: 23 min after application of 10/~M tetrandrine. Potassium channels were blocked by 50/~M 3,4-DAP and 1 mM TEA.
The present results show the influence of tetrandrine on presynaptic membrane currents as compared to the influence of cobalt. Though the method by which these currents were recorded do not allow their unequivocal attribution directly to special ion channels, to our knowledge it is the best available method for recording presynaptic events from mammalian motor nerve terminals. The mode of action of neurotoxicants on these currents depends on pharmacological identification because voltage clamping of the presynaptic terminal membrane is not feasible. The application of tetrandrine to muscle preparations shows that neither the first negative peak attributed to sodium influx within the heminode nor the second negative peak known as a potassium current in the nerve terminals were influenced. This means tetrandrine has no sodium channel blocking activity and does not block potassium channels in the presynaptic membrane (for review see Ref. 12). There was no effect of tetrandrine
117 on the fast presynaptic calcium current (Penner and Dreyer27). Rather there was a blocking activity on the slow calcium current. Tetrandrine has a moderate affinity and specificity as compared to other organic and the inorganic calcium channel blockers. The calcium plateau signal has been shown to be carried by calcium and barium, and is blocked by verapamil, diltiazem, cadmium, manganese 27 as well as by cobalt (this paper). If one compares the activities of the different inorganic calcium channel blockers (cadmium, manganese, cobalt) on the one site and the low affinity binding sites of the organic calcium blockers verapamil and diltiazem on the other site one can establish the following sequence of blocking activity: cadmium > tetrandrine > verapamil > cobalt > diltiazem > manganese (see Table I of Ref. 27; this paper Table I). The slow calcium plateau signal was not sensitive to 1,4-dihydropyridine27 nor to omegaconotoxin ~. There is pharmacological evidence27 that the slow calcium plateau signal is carried by a distinct class of slow calcium channels. Mallart 22 has suggested that the fast and the slow calcium components can be separated by the combination of moderate potassium channel blockers (3,4-DAP and TEA) and that they are carried by one and the same calcium current becoming regenerative. Therefore it is not possible to decide, which calcium channel is affected by tetrandrine and cobalt. Different types of calcium currents have been demonstrated in neuronal as well as in non-neuronal cells. In mammalian CNS neurons low threshold channels were differentiated from high threshold channels 7. The high threshold calcium channels could be separated into two kinds, the so called N-type and L-type, whereas there seemed to be only one low-threshold channel, the so called T-type channel. Distinction between these 3 channel-types was based on biophysical as well as pharmacological means and usually was done in patch and whole cell clamp recordings. L-type channels in D R G neurons carry large barium currents which were long-lasting and activated by strong depolarizations from relatively low holding potentials. In contrast T-type calcium channels carried small barium currents; they are evoked only from negative holding potentials and require small depolarizations for activation. They inactivate more slowly than N- or L-type channels. Whereas N-type or T-type calcium channels are not affected by 1,4-dihydropyridines these drugs select strongly for L-type channels, but whether they completely block L-currents is unclear especially if these are REFERENCES 1 Anderson, A.J. and Harvey, A.L., Omega-conotoxin does not block the verapamil-sensitive calcium channel at mouse motor nerve terminals, Neurosci. Lett., 82 (1987) 177-180.
evoked from negative holding potentials. All 3 types of channels are found in central neurons, for example hippocampal pyramidal cells. Some other cells appeared to express only one of the 3 types, for example L-type in adrenal chromaffine cells or T-type in neoplastic Blymphocytes 14A5. In peripheral neurons, N-type channels were suggested to play a dominant if not exclusive role in controlling transmitter release. Norepinephrine release from sympathetic neurons is resistant to dihydropyridines, but sensitive to cadmium and omega-conotoxin 17. N-type channels might also explain the dihydropyridine resistance of transmitter release from neuromuscular junctions 25'26. There is evidence, that N- and L-type calcium channels coexist in motor nerve terminals 3. Sometimes calcium channels seem to be clustered in hot spots, which contain one type of channel exclusively21. These hot spots were suggested to play an important role in triggering transmitter release in synaptic terminals. It has been proposed 17 that calcium current through calcium channels of the N-type may be involved in triggering exocytosis of synaptic vesicles, whereas L-type channels regulate exocytosis of large dense core vesicles. Tetrandrine acted on calcium channels within the presynaptic terminal which: (1) could be blocked by the anorganic channel blockers cadmium, manganese, cobalt; (2) could be blocked by the organic channel blockers verapamil and diltiazem; (3) could not be blocked by 1,4-dihydropyridines; (4) could not be blocked by omega-conotoxin; (5) inactivated only within hundreds of ms. Obviously such kind of pharmacological profile did not fit precisely into any scheme of neuronal calcium channel pharmacology known as yet (see above). Moreover, it was definitely different from that of presynaptic L-type calcium channels as described recently in lizard nerve endings 2°. In view of these findings on the role of calcium channels in transmitter release our results at the mouse neuromuscular junction in combination with others L 6,13,27 do not define the calcium channel tetrandrine might influence. Acknowledgements. Prof. F. Dreyer, Giessen, ER.G., introduced us to Nomarski optic techniques, R. Penner, G6ttingen, ER.G., to the triangularis preparation. Dr. D. Rodbard, Bethesda, MD, U.S.A., kindly provided the programme Allfit. Karolina Sveinsson prepared the figures.
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