- Email: [email protected]
The effects of Securidaca longepedunculata root extract on ionic currents and contraction of cultured rat skeletal muscle cells
Journal of Ethnopharmacology 65 (1999) 157 – 164
The effects of Securidaca longepedunculata root extract on ionic currents and contraction of cultured rat skeletal muscle cells A.P. Mouzou a, L. Bulteau b, G. Raymond b,* b
a Faculte´ des Sciences, De´partement de Physiologie Animale, Uni6ersity of Benin, Lome´, Togo L.B.S.C., CNRS UMR 6558, Uni6ersity of Poitiers, Baˆtiment P, 40 a6enue du Recteur Pineau, F-86022 PoitiersCedex, France
Received 23 March 1998; received in revised form 9 November 1998; accepted 20 November 1998
Abstract The effects of the primary extract roots of Securidaca longepedunculata were tested on sodium, calcium and potassium currents in rat skeletal muscle cells developed in culture. In addition, they were tested on depolarisation-induced contraction and resting intracellular calcium levels. S. longepedunculata extract (10 − 6 g/l) increases sodium current at all potentials. No clear effect was observed on calcium current except for a slight increase at negative potentials ( − 30, − 10 mV) revealing a 5 mV shift towards negative potentials of the ICa/V curve, as with potassium current. In contrast, at the same concentration, S. longepedunculata enhanced the contractile response elicited by durable depolarisation. This was not attributable to the slight increase in resting intracellular free calcium concentration which did not change during and following S. longepedunculata application. These results strongly suggest that S. longepedunculata root extract contains one or more components acting on the voltage-sensor of excitation – contraction coupling (dihydropyridine receptors), regardless of its implication as a calcium channel. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Skeletal muscle cells; Whole-cell patch-clamp; Sodium current; Calcium current; Securidaca longepedunculata; Excitation–contraction coupling; Intracellular free calcium
1. Introduction The exploitation of substances of local and natural origin has great promise for the develop* Corresponding author. Fax: +33-5-49-45-40-14. E-mail address: [email protected] (G. Raymond)
ment of the biomedical industry in Africa. It is known that such substances are well accepted by Africans when used in medicine. Among the 2000 species of known snakes, about 400 species are venomous and may be responsible for at least 400 000 human fatalities annually. Many plants are known to be useful against snakebites in countries throughout the world. Among them,
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 2 1 - 9
158
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
Securidaca longepedunculata (Diakite, 1977; Kone et al., 1979; Houghton and Isibogun, 1993) root extract is used by traditional healers. S. longepedunculata is a plant from the Polygalaceae common to most countries of West Africa (Ayensu, 1978; Abbiw, 1990) and the root contains ‘saponines’ (Kerharo, 1972). The root extract has been shown to have many effects on frog muscle such a curare-like effect at neuromuscular junction, an increase in action potential duration which seems to be attributable to a large decrease in potassium permeability and some competition with Naja nigricollis venom (Kone, 1980). However, traditional practitioners use S. longepedunculata root extract on humans and so far nothing is known about the possible electrophysiological effects on mammalian muscle. Thus, in order to eventually isolate the active biological component(s) of S. longepedunculata roots, we have undertaken the present work to test the effects of root extract on ionic currents underlying electrical activity and associated functions (contraction, free calcium homeostasis) in mammalian (rat) cultured skeletal muscle cells.
2. Materials and methods
2.1. Materials The roots from S. longepedunculata were collected from Atakpame (Togo, West Africa) in May 1996 and authenticated by Mr Kokou, Department of Botany, University of Benin, Lome´ (Togo). A voucher species of plant has been deposited in a herbarium of this Department of Botany. The roots were sun-dried and powdered before maceration in deionized water (100 mg/100 ml) with agitation for 4 h at 37°C. The resulting extract was filtered at 0.20 mm and kept at 4°C before addition to test solutions at a final concentration of 10 − 6 g/l.
2.2. Cell culture Primary cultures of mammalian skeletal muscle cells were initiated from satellite cells obtained by trypsinisation of muscle pieces of 1- to 3-day-old
rats hindlimbs (for details see Cognard et al., 1993a). Cells were maintained for 2 days in growth medium, consisting of HamF12 (Gibco, Cergy Pontoise, France), supplemented with 10% heat-inactivated horse serum (Gibco), 10% fetal calf serum (Boehringer Mannheim, Meylan, France), and 1% antibiotics (penicillin-G, 100 U/ ml, Sigma, and streptomycin, 50 mg/ml, Sigma). The growth medium was then exchanged for a fusion promoting medium constituted by Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 5% heat-inactivated horse serum and 1% antibiotics. This medium exchange operation was used as time zero to age cells in culture. Colchicine (30 nM; Sigma) was added when elongated myotubes appeared (around day 2) to favour the formation of rounded cells (‘myoballs’) suitable for patch-clamp experiments. Experiments were carried out on 5- to 7-day-old myoballs which present characteristics of adult muscle cells (Cognard et al., 1993a).
2.3. Current recording Voltage-clamp experiments were performed at room temperature (209 2°C) in the whole-cell configuration of the patch-clamp technique. Pipettes (2–5 mV) were connected to the head stage of a patch-clamp amplifier (RK300, Biologic, Grenoble, France), driven by a PC compatible microcomputer equipped with a Labmaster A/D conversion board (Scientific Solutions, Solon, USA). Membrane voltage clamping, data acquisition, and analysis were performed by means of a software package (pClamp, Axon Instruments, Foster City).
2.4. Mechanical recording Contractile responses were recorded simultaneously with membrane currents (Rivet et al., 1989) by means of a photomultiplier tube (IP28, Hamamatsu, Japan) mounted on an auxiliary light-path exit of an inverted microscope. This device measured the changes in light transmission resulting from cell deformation during contraction. Relative contractions were not calibrated, but expressed in arbitrary units which corre-
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
sponded to the electrical potential at the exit of the current/voltage converter following the IP28 tube.
2.5. Resting free calcium concentration estimation Intracellular free Ca2 + concentration was measured at regulated room temperature (22°C) by means of a ratiometric fluorescence method using a laser scanning cytofluorimeter (ACAS 570, Meridian Instruments) and Indo-1 as calcium probe. Details of the technique have already been reported (Cognard et al., 1993b). Muscle cells were loaded by exposure to the membrane-permeant form of Indo-1 (Indo-1/AM) for 60 min in the dark at room temperature, then washed with control solution and incubated for 10 min at 37°C to complete the de-esterification of Indo-1. Indo-1, excited in UV range (351 – 364 nm wavelengths) with a laser beam (5 W pulsed Argon laser), maximally fluoresces at 485 and 405 nm in the absence and presence of calcium respectively. Calcium activity in cells was then estimated as the ratio of the 405 and 485 nm fluorescence emissions collected by means of a dichroic filter and two photomultiplier tubes. Calcium measurements were obtained from laser scanning images. The ratio was calculated in the entire cell as the average of pixel ratio values measured in a hand-delimited area. The cytosolic free calcium activities were calculated from the Grynkiewicz equation (Grynkiewicz et al., 1985): [Ca2 + ]i = Kd ß[(R − Rmin)/(Rmax −R)] where R is the ratio of the fluorescence signals. Kd was assumed to be fixed at the in vitro calculated value of 250 nM. The ß constant, Rmin and Rmax values were determined in vivo as already described (Cognard et al., 1993b).
2.6. Solutions and experimental conditions The culture medium was exchanged before each experiment for a saline bath solution allowing for sodium (solution 1), calcium (solution 2) or potassium (solution 3) current recording. The extract was added to the saline bath solution at a final concentration of 10 − 6 g/l. The determination (by
159
spectrometric emission technique in the Chemistry Laboratory, University of Poitiers) of the concentrations of sodium and potassium in saline bath solution (control solution) and after addition of extract (test solution) showed no difference between the concentration of these ions in the solutions (27 and 26 mM Na + ) in control and test solution respectively, 0 mM K + in both. Pipettes were filled with solution 4 for INa and ICa, and solution 5 for IK. Intracellular calcium measurements were performed in solution 3. Solutions contained (in mM): 1. NaCl; 100 TEACl; 2.5 CaCl2; 10 HEPES; 5.6 glucose; 2 CoCl2; pH 7.4; 2. TEACl; 2.5 CaCl2; 10 HEPES; 5.6 glucose; 0.8 MgCl2; pH 7.4; 3. NaCl; 2.5 CaCl2; 10 HEPES; 5.6 glucose; 0.8 MgCl2; 5.4 KCl; 2 CoClo2 ; 2 TTX; pH 7.4; 4. CaCl2; 10 HEPES; 5.6 glucose; 1 MgCl2; 145 CsCl; 1 EGTA; pH 7.2; 5. CaCl2; 10 HEPES; 5.6 glucose; 1 MgCl2; 140 KCl; 1 CsCl; pH 7.2. The S. longepedunculata root extracts were perfused for 5 min before stimulation. Currents and contraction were elicited by a series of increasing depolarising steps from a holding potential (HP) of − 90 mV. Data were corrected for leakage currents from a linear extrapolation of membrane current magnitudes (assumed to be ohmic) for small depolarising, which did not induce any dynamic currents. The current amplitude was measured as the difference between peak amplitude of inward current and the background current level before the test pulse.
3. Results
3.1. Effects on sodium current (INa) Fig. 1A shows examples of inward sodium current elicited by depolarising pulses from − 90 to −30 mV (left) and 0 mV (right), in the absence (filled circles) or presence (open circles) of S. longepedunculata. For all the potentials tested, the S. longepedunculata root extract induced a clear increase in the amplitude of INa. The mean I/V curves of Fig. 1B show that INa is significantly
160
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
increased at all potentials between −60 and 40 mV, the increase ranging from 40 to 300%. Additionally it can be seen from the recordings of A that in all cases the inactivation phase of INa is accelerated by the drug.
3.2. Effects on calcium current (ICa) The effect of 10 − 6 g/l S. longepedunculata was tested on the calcium current of skeletal muscle cells. Fig. 2A shows examples of inward calcium current elicited by depolarising pulses from −90 to −40 mV (T-type current, left) and 0 mV (L-type current, right), in the absence (filled circles) or presence (open circles) of S. longepedunculata. No significant effect on calcium current peak amplitude was observed except a slight increase
Fig. 2. Effects of 10 − 6 g/l S. longepedunculata on calcium current of rat skeletal muscle myoballs. (A) Calcium current induced by two different depolarisations from −90 to − 40 mV and 0 mV under control conditions (filled circles) and in the presence of extract (open circles). (B) Mean I/V curves for calcium current in control conditions and in the presence of extract (n =8).
between − 40 and 0 mV and a decrease above this, as illustrated by the I/V curve in B. Obviously this results from a 5 mV-shift in the I/V curve towards negative potentials rather than some effect on the channel conductance. This seems to be related to the L-type current and not to the T-type since the shift is not present for potentials below the threshold for L-type.
3.3. Effects on potassium current (IK) Fig. 1. Effects of 10 − 6 g/l S. longepedunculata on sodium current of rat skeletal muscle myoballs. (A) Sodium current induced by two different depolarisations from − 90 to − 30 mV and 0 mV under control conditions (filled circles) and in the presence of extract (open circles). (B) Mean I/V curves for sodium current in control conditions and in the presence of extract (n =8).
The extract was tested at the same concentration on the potassium currents elicited by long duration pulses (1500 ms) from −90 to + 60 mV without any effect whatever the potential tested (data not shown).
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
161
3.4. Effects on contractile acti6ity
3.5. Resting intracellular calcium acti6ity
The contraction was simultaneously recorded with current as shown in Fig. 3A in the absence (filled circles) and presence (open circles) of 10 − 6 g/l S. longepedunculata for depolarising pulses from − 90 to −20 and 0 mV. A drastic increase in peak tension was observed at all potentials. From the comparison of the normalized T/V curves of Fig. 3B, it is obvious that S. longepedunculata extract induced a 20 mV-shift towards negative potentials and an increase in tension ranging between 50 and 100%.
Since the increase in tension could be due to some increase in resting cytoplasmic calcium level, 10 − 6 g/l S. longepedunculata extract was applied in 30 myotubes from different cultures in conditions allowing measurement of cytosolic calcium. The extract did not induce any change in the calcium level, which was estimated at 959 4 nM before S. longepedunculata application and 989 6 nM after. These resting values fully agree with those obtained by Cognard et al. (1993a,b) in similar experimental conditions.
3.6. The re6ersible and dose-dependent effects on sodium current Fig. 4A shows examples of the reversibility of the effects of extract on inward sodium current elicited by depolarising pulses from − 90 to − 30 mV (left) and 0 mV (right). After control conditions (filled circles) the extract was perfused for 5 min and then the preparation was stimulated (open circles). Reversibility was tested upon washing at 3 (open squares) and at 5 min (open triangles) and was obvious after 5 min of washing. From the values of Fig. 4B it is demonstrated that the effect of extract is dose-dependent with an optimum at 5× 10 − 7 g/l. Higher concentrations appeared to be toxic for cells.
4. Discussion
Fig. 3. Effects of 10 − 6 g/l S. longepedunculata on the contractile response induced by long depolarisation. (A) Recordings of contraction induced by depolarising from − 90 to −20 mV and 0 mV in control conditions (filled circles) and in the presence of extract (open circles). (B) Mean T/V curves for the contractile response in control conditions and in the presence of extract (n =8).
The first evidence from the present results concerns the sodium current which is drastically increased by the root extract from S. longepedunculata. Compared to the results of Kone (1980) obtained with S. longepedunculata in voltage-clamped skeletal fibres of frog which showed a weak increase in the sodium current, it is clear that S. longepedunculata is considerably more efficient in mammalian muscle cells than in batracian ones (30% over 10%). Such an increase of the sodium current provides some interesting potentialities of the S. longepedunculata extract concerning the acceleration of the action potential transmission. However a comparative study with
162
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
Fig. 4. (A) Reversibility of the effects of S. longepedunculata on sodium current of rat skeletal muscle myoballs. Sodium current induced by two different depolarisations from − 90 to − 30 mV and 0 mV under control conditions (filled circles) and in the presence of extract (open circles). Reversibility of the effects of extract appeared upon washing at 3 min (open squares) and at 5 min (open triangles) (n = 6). (B) The table of the effects: increase ( + ) and decrease ( − ) of extract at different concentrations (n = 5).
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
Naja nigricollis venom would be of interest to determine to what extent this effect could counteract the venom one, since the results of Kone (1980) show the increase of sodium current by this venom. The reversibility of the effects of extract on sodium current agrees with the results of Kone (1980) on the action potential in voltage-clamped skeletal muscle fibres of frog. The other discrepancy with the results obtained on voltage-clamped frog muscle fibres concerns the absence of the effect of S. longepedunculata extract on the potassium current and also the effect on contractile tension which was not altered in frog muscle whereas it is drastically increased in mammalian muscle cells. Since the amplitude of calcium currents and that of cytosolic free calcium are not altered by S. longepedunculata, such an increase in tension might be attributable to the increase in sodium current. Indeed, Caille´ et al. (1978, 1985) had shown that a component of the contraction could be related to the T-tubular sodium current of frog muscle and Potreau and Raymond (1982) have reported the existence of a mechanism of sodium-induced calcium release at the triadic level of such frog muscle fibres. However this possibility can be discarded because an increase in tension due to an increase in T-tubular sodium-current would have slowed down the inactivation phase of INa for potentials above the mechanical threshold rather than accelerating it as observed. Thus it is highly probable that one component of S. longepedunculatal root extract acts on the L-type calcium channel (dihydropyridine receptor), on voltage-sensor of excitation – contraction coupling function but not on the T-type calcium channel function. The effects of extract on the L-type calcium channel could be due to the acceleration of cinetic activation complement of channel. This is strongly reinforced by the observation of a shift of the L-type ICa/V curve towards negative potential (facilitation of contraction) upon S. longepedunculata application whereas neither ICaT nor ICaL amplitudes are modified. Thus the root extract differentiates between the two functions of dihydropyridine receptor which is known (Rios and Pizarro, 1991) to act as a voltage-sensor of EC coupling and as a voltage-
163
dependent L-type calcium channel. A similar discriminative effect between L-type activity and excitation–contraction coupling has already been observed with SR33557 (Bois et al., 1991), an indolizinsulphone which, used at low concentrations, blocked contraction but had little effect on current. A more precise knowledge of the component of S. longepedunculata root presently acting and the comparison of its structure with that of SR33557, could be of great interest for studying the dual role of dihydropyridine receptor in muscle physiology. In conclusion, the present results clearly demonstrate the existence of an agonistic effect of S. longepedunculata extract on the sodium current of skeletal muscle myoballs. In addition the present work provides some interesting results concerning the possible action on the dihydropyridine receptor acting as voltage-sensor (Agnew, 1988; Campbell et al., 1988). Therefore, the extraction and isolation of the different components of S. longepedunculata root extract could be interesting in providing tools for further insights into the excitation–contraction coupling mechanisms. Moreover, the possibility of isolation of specific activating agents for sodium current or voltagesensor would be highly relevant for the development of the biomedical industry in Africa. Acknowledgements This work was supported by CNRS/Universite´ de Poitiers UMR 6558, Professor Legube of the Chemistry Laboratory/Universite´ de Poitiers and the French Agency for Cooperation (Togo). References Abbiw, W., 1990. Useful Plants of Ghana. Intermediate Technology, London, pp. 218 – 220. Agnew, W.S., 1988. Proteins that bridge the gap. Nature 334, 299 – 300. Ayensu, E.S., 1978. Medical Plants of West Africa. Reference Publications, Algonae, M I, 231. Bois, P., Romey, G., Lazdunski, M., 1991. Indolizinsulphones. A class of blockers with dual but discriminative effects on L-type Ca2 + channel activity and excitation – contraction coupling in skeletal muscle. Pflu¨gers Archiv 419, 651 – 656.
164
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
Caille´, J., Ildefonse, M., Rougier, O., 1978. Existence of a sodium current in the tubular membrane of frog skeletal muscle fibre; its possible role in the activation of contraction. Pflu¨gers Archiv 374, 167–177. Caille´, J., Ildefonse, M., Rougier, O., 1985. Excitation–contraction coupling in skeletal muscle. Progress in Biophysics and Molecular Biology 46, 185–239. Campbell, K.P., Leung, A.T., Sharp, A.H., 1988. The biochemistry and molecular biology of the dihydropyridinesensitive calcium channel. Trends in Neurological Sciences 11, 425 – 430. Cognard, C., Constantin, B., Rivet-Bastide, M., Imbert, N., Besse, C., Raymond, G., 1993. Appearance and evolution of calcium currents and contraction during the early postfusional stages of rat skeletal muscle cells developing in primary culture. Development 117, 1153–1161. Cognard, C., Constantin, B., Rivet-Bastide, M., Raymond, G., 1993. Intracellular calcium transients induced by different kinds of stimulus during myogenesis of rat skeletal muscle cells studied by laser cytofluorimetry with Indo-1. Cell Calcium 14, 333 – 348. Diakite, D., 1977. Premier inventaire de la faune ophidienne du Mali. Etude e´pide´miologique, clinique et the´rapeutique des accidents d’envenimation. The`se de Me´decine, Bamako. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2 + indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260,
.
3440 – 3450. Houghton, P.J., Isibogun, M., 1993. Flowering plants used against snakebite. Journal of Ethnopharmacology 39, 1 – 29. Kerharo, 1972. La pharmacope´e et la me´decine traditionnelle se´ne´galaises. Kone, P., 1980. Etude toxicologique, e´lectrophysiologique et pharmacologique du venin de Naja nigricollis (Elapide´ de Coˆte d’Ivoire) et d’une substance antivenimeuse de la pharmacope´e traditionnelle africaine (extrait de Securidaca longepedunculata, Polygalace´e). The`se d’e´tat Abidjan. Kone, P.P., Tricoche, R., Kone, Y., 1979. Mode d’action de quelques venins de serpents de Coˆte d’Ivoire (Dendroaspis 6inidis, Naja nigrocollis, Bitis gabonica) et d’un antivenimeux de la pharmacope´e traditionnelle africaine, au niveau des proprie´te´s e´lectriques et me´caniques de la pre´paration musculaire et neuromusculaire. Troisie`me symposium interafricain OUA/CSTR sur la pharmacope´e traditionnelle et les plantes africaines. Abidjan AMP 79, 109. Potreau, D., Raymond, G., 1982. Existence of a sodium-induced calcium release mechanism on frog skeletal muscle fibres. Journal of Physiology London 333, 463 – 480. Rios, E., Pizarro, G., 1991. Voltage sensor of excitation – contraction coupling in skeletal muscle. Physiological Review 71, 849 – 907. Rivet, M., Cognard, C., Raymond, G., 1989. The slow inward calcium current is responsible for a part of contraction of patch-clamped rat myoballs. Pflu¨gers Archiv 413, 316 – 318.
.
The effects of Securidaca longepedunculata root extract on ionic currents and contraction of cultured rat skeletal muscle cells A.P. Mouzou a, L. Bulteau b, G. Raymond b,* b
a Faculte´ des Sciences, De´partement de Physiologie Animale, Uni6ersity of Benin, Lome´, Togo L.B.S.C., CNRS UMR 6558, Uni6ersity of Poitiers, Baˆtiment P, 40 a6enue du Recteur Pineau, F-86022 PoitiersCedex, France
Received 23 March 1998; received in revised form 9 November 1998; accepted 20 November 1998
Abstract The effects of the primary extract roots of Securidaca longepedunculata were tested on sodium, calcium and potassium currents in rat skeletal muscle cells developed in culture. In addition, they were tested on depolarisation-induced contraction and resting intracellular calcium levels. S. longepedunculata extract (10 − 6 g/l) increases sodium current at all potentials. No clear effect was observed on calcium current except for a slight increase at negative potentials ( − 30, − 10 mV) revealing a 5 mV shift towards negative potentials of the ICa/V curve, as with potassium current. In contrast, at the same concentration, S. longepedunculata enhanced the contractile response elicited by durable depolarisation. This was not attributable to the slight increase in resting intracellular free calcium concentration which did not change during and following S. longepedunculata application. These results strongly suggest that S. longepedunculata root extract contains one or more components acting on the voltage-sensor of excitation – contraction coupling (dihydropyridine receptors), regardless of its implication as a calcium channel. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Skeletal muscle cells; Whole-cell patch-clamp; Sodium current; Calcium current; Securidaca longepedunculata; Excitation–contraction coupling; Intracellular free calcium
1. Introduction The exploitation of substances of local and natural origin has great promise for the develop* Corresponding author. Fax: +33-5-49-45-40-14. E-mail address: [email protected] (G. Raymond)
ment of the biomedical industry in Africa. It is known that such substances are well accepted by Africans when used in medicine. Among the 2000 species of known snakes, about 400 species are venomous and may be responsible for at least 400 000 human fatalities annually. Many plants are known to be useful against snakebites in countries throughout the world. Among them,
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 2 1 - 9
158
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
Securidaca longepedunculata (Diakite, 1977; Kone et al., 1979; Houghton and Isibogun, 1993) root extract is used by traditional healers. S. longepedunculata is a plant from the Polygalaceae common to most countries of West Africa (Ayensu, 1978; Abbiw, 1990) and the root contains ‘saponines’ (Kerharo, 1972). The root extract has been shown to have many effects on frog muscle such a curare-like effect at neuromuscular junction, an increase in action potential duration which seems to be attributable to a large decrease in potassium permeability and some competition with Naja nigricollis venom (Kone, 1980). However, traditional practitioners use S. longepedunculata root extract on humans and so far nothing is known about the possible electrophysiological effects on mammalian muscle. Thus, in order to eventually isolate the active biological component(s) of S. longepedunculata roots, we have undertaken the present work to test the effects of root extract on ionic currents underlying electrical activity and associated functions (contraction, free calcium homeostasis) in mammalian (rat) cultured skeletal muscle cells.
2. Materials and methods
2.1. Materials The roots from S. longepedunculata were collected from Atakpame (Togo, West Africa) in May 1996 and authenticated by Mr Kokou, Department of Botany, University of Benin, Lome´ (Togo). A voucher species of plant has been deposited in a herbarium of this Department of Botany. The roots were sun-dried and powdered before maceration in deionized water (100 mg/100 ml) with agitation for 4 h at 37°C. The resulting extract was filtered at 0.20 mm and kept at 4°C before addition to test solutions at a final concentration of 10 − 6 g/l.
2.2. Cell culture Primary cultures of mammalian skeletal muscle cells were initiated from satellite cells obtained by trypsinisation of muscle pieces of 1- to 3-day-old
rats hindlimbs (for details see Cognard et al., 1993a). Cells were maintained for 2 days in growth medium, consisting of HamF12 (Gibco, Cergy Pontoise, France), supplemented with 10% heat-inactivated horse serum (Gibco), 10% fetal calf serum (Boehringer Mannheim, Meylan, France), and 1% antibiotics (penicillin-G, 100 U/ ml, Sigma, and streptomycin, 50 mg/ml, Sigma). The growth medium was then exchanged for a fusion promoting medium constituted by Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 5% heat-inactivated horse serum and 1% antibiotics. This medium exchange operation was used as time zero to age cells in culture. Colchicine (30 nM; Sigma) was added when elongated myotubes appeared (around day 2) to favour the formation of rounded cells (‘myoballs’) suitable for patch-clamp experiments. Experiments were carried out on 5- to 7-day-old myoballs which present characteristics of adult muscle cells (Cognard et al., 1993a).
2.3. Current recording Voltage-clamp experiments were performed at room temperature (209 2°C) in the whole-cell configuration of the patch-clamp technique. Pipettes (2–5 mV) were connected to the head stage of a patch-clamp amplifier (RK300, Biologic, Grenoble, France), driven by a PC compatible microcomputer equipped with a Labmaster A/D conversion board (Scientific Solutions, Solon, USA). Membrane voltage clamping, data acquisition, and analysis were performed by means of a software package (pClamp, Axon Instruments, Foster City).
2.4. Mechanical recording Contractile responses were recorded simultaneously with membrane currents (Rivet et al., 1989) by means of a photomultiplier tube (IP28, Hamamatsu, Japan) mounted on an auxiliary light-path exit of an inverted microscope. This device measured the changes in light transmission resulting from cell deformation during contraction. Relative contractions were not calibrated, but expressed in arbitrary units which corre-
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
sponded to the electrical potential at the exit of the current/voltage converter following the IP28 tube.
2.5. Resting free calcium concentration estimation Intracellular free Ca2 + concentration was measured at regulated room temperature (22°C) by means of a ratiometric fluorescence method using a laser scanning cytofluorimeter (ACAS 570, Meridian Instruments) and Indo-1 as calcium probe. Details of the technique have already been reported (Cognard et al., 1993b). Muscle cells were loaded by exposure to the membrane-permeant form of Indo-1 (Indo-1/AM) for 60 min in the dark at room temperature, then washed with control solution and incubated for 10 min at 37°C to complete the de-esterification of Indo-1. Indo-1, excited in UV range (351 – 364 nm wavelengths) with a laser beam (5 W pulsed Argon laser), maximally fluoresces at 485 and 405 nm in the absence and presence of calcium respectively. Calcium activity in cells was then estimated as the ratio of the 405 and 485 nm fluorescence emissions collected by means of a dichroic filter and two photomultiplier tubes. Calcium measurements were obtained from laser scanning images. The ratio was calculated in the entire cell as the average of pixel ratio values measured in a hand-delimited area. The cytosolic free calcium activities were calculated from the Grynkiewicz equation (Grynkiewicz et al., 1985): [Ca2 + ]i = Kd ß[(R − Rmin)/(Rmax −R)] where R is the ratio of the fluorescence signals. Kd was assumed to be fixed at the in vitro calculated value of 250 nM. The ß constant, Rmin and Rmax values were determined in vivo as already described (Cognard et al., 1993b).
2.6. Solutions and experimental conditions The culture medium was exchanged before each experiment for a saline bath solution allowing for sodium (solution 1), calcium (solution 2) or potassium (solution 3) current recording. The extract was added to the saline bath solution at a final concentration of 10 − 6 g/l. The determination (by
159
spectrometric emission technique in the Chemistry Laboratory, University of Poitiers) of the concentrations of sodium and potassium in saline bath solution (control solution) and after addition of extract (test solution) showed no difference between the concentration of these ions in the solutions (27 and 26 mM Na + ) in control and test solution respectively, 0 mM K + in both. Pipettes were filled with solution 4 for INa and ICa, and solution 5 for IK. Intracellular calcium measurements were performed in solution 3. Solutions contained (in mM): 1. NaCl; 100 TEACl; 2.5 CaCl2; 10 HEPES; 5.6 glucose; 2 CoCl2; pH 7.4; 2. TEACl; 2.5 CaCl2; 10 HEPES; 5.6 glucose; 0.8 MgCl2; pH 7.4; 3. NaCl; 2.5 CaCl2; 10 HEPES; 5.6 glucose; 0.8 MgCl2; 5.4 KCl; 2 CoClo2 ; 2 TTX; pH 7.4; 4. CaCl2; 10 HEPES; 5.6 glucose; 1 MgCl2; 145 CsCl; 1 EGTA; pH 7.2; 5. CaCl2; 10 HEPES; 5.6 glucose; 1 MgCl2; 140 KCl; 1 CsCl; pH 7.2. The S. longepedunculata root extracts were perfused for 5 min before stimulation. Currents and contraction were elicited by a series of increasing depolarising steps from a holding potential (HP) of − 90 mV. Data were corrected for leakage currents from a linear extrapolation of membrane current magnitudes (assumed to be ohmic) for small depolarising, which did not induce any dynamic currents. The current amplitude was measured as the difference between peak amplitude of inward current and the background current level before the test pulse.
3. Results
3.1. Effects on sodium current (INa) Fig. 1A shows examples of inward sodium current elicited by depolarising pulses from − 90 to −30 mV (left) and 0 mV (right), in the absence (filled circles) or presence (open circles) of S. longepedunculata. For all the potentials tested, the S. longepedunculata root extract induced a clear increase in the amplitude of INa. The mean I/V curves of Fig. 1B show that INa is significantly
160
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
increased at all potentials between −60 and 40 mV, the increase ranging from 40 to 300%. Additionally it can be seen from the recordings of A that in all cases the inactivation phase of INa is accelerated by the drug.
3.2. Effects on calcium current (ICa) The effect of 10 − 6 g/l S. longepedunculata was tested on the calcium current of skeletal muscle cells. Fig. 2A shows examples of inward calcium current elicited by depolarising pulses from −90 to −40 mV (T-type current, left) and 0 mV (L-type current, right), in the absence (filled circles) or presence (open circles) of S. longepedunculata. No significant effect on calcium current peak amplitude was observed except a slight increase
Fig. 2. Effects of 10 − 6 g/l S. longepedunculata on calcium current of rat skeletal muscle myoballs. (A) Calcium current induced by two different depolarisations from −90 to − 40 mV and 0 mV under control conditions (filled circles) and in the presence of extract (open circles). (B) Mean I/V curves for calcium current in control conditions and in the presence of extract (n =8).
between − 40 and 0 mV and a decrease above this, as illustrated by the I/V curve in B. Obviously this results from a 5 mV-shift in the I/V curve towards negative potentials rather than some effect on the channel conductance. This seems to be related to the L-type current and not to the T-type since the shift is not present for potentials below the threshold for L-type.
3.3. Effects on potassium current (IK) Fig. 1. Effects of 10 − 6 g/l S. longepedunculata on sodium current of rat skeletal muscle myoballs. (A) Sodium current induced by two different depolarisations from − 90 to − 30 mV and 0 mV under control conditions (filled circles) and in the presence of extract (open circles). (B) Mean I/V curves for sodium current in control conditions and in the presence of extract (n =8).
The extract was tested at the same concentration on the potassium currents elicited by long duration pulses (1500 ms) from −90 to + 60 mV without any effect whatever the potential tested (data not shown).
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
161
3.4. Effects on contractile acti6ity
3.5. Resting intracellular calcium acti6ity
The contraction was simultaneously recorded with current as shown in Fig. 3A in the absence (filled circles) and presence (open circles) of 10 − 6 g/l S. longepedunculata for depolarising pulses from − 90 to −20 and 0 mV. A drastic increase in peak tension was observed at all potentials. From the comparison of the normalized T/V curves of Fig. 3B, it is obvious that S. longepedunculata extract induced a 20 mV-shift towards negative potentials and an increase in tension ranging between 50 and 100%.
Since the increase in tension could be due to some increase in resting cytoplasmic calcium level, 10 − 6 g/l S. longepedunculata extract was applied in 30 myotubes from different cultures in conditions allowing measurement of cytosolic calcium. The extract did not induce any change in the calcium level, which was estimated at 959 4 nM before S. longepedunculata application and 989 6 nM after. These resting values fully agree with those obtained by Cognard et al. (1993a,b) in similar experimental conditions.
3.6. The re6ersible and dose-dependent effects on sodium current Fig. 4A shows examples of the reversibility of the effects of extract on inward sodium current elicited by depolarising pulses from − 90 to − 30 mV (left) and 0 mV (right). After control conditions (filled circles) the extract was perfused for 5 min and then the preparation was stimulated (open circles). Reversibility was tested upon washing at 3 (open squares) and at 5 min (open triangles) and was obvious after 5 min of washing. From the values of Fig. 4B it is demonstrated that the effect of extract is dose-dependent with an optimum at 5× 10 − 7 g/l. Higher concentrations appeared to be toxic for cells.
4. Discussion
Fig. 3. Effects of 10 − 6 g/l S. longepedunculata on the contractile response induced by long depolarisation. (A) Recordings of contraction induced by depolarising from − 90 to −20 mV and 0 mV in control conditions (filled circles) and in the presence of extract (open circles). (B) Mean T/V curves for the contractile response in control conditions and in the presence of extract (n =8).
The first evidence from the present results concerns the sodium current which is drastically increased by the root extract from S. longepedunculata. Compared to the results of Kone (1980) obtained with S. longepedunculata in voltage-clamped skeletal fibres of frog which showed a weak increase in the sodium current, it is clear that S. longepedunculata is considerably more efficient in mammalian muscle cells than in batracian ones (30% over 10%). Such an increase of the sodium current provides some interesting potentialities of the S. longepedunculata extract concerning the acceleration of the action potential transmission. However a comparative study with
162
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
Fig. 4. (A) Reversibility of the effects of S. longepedunculata on sodium current of rat skeletal muscle myoballs. Sodium current induced by two different depolarisations from − 90 to − 30 mV and 0 mV under control conditions (filled circles) and in the presence of extract (open circles). Reversibility of the effects of extract appeared upon washing at 3 min (open squares) and at 5 min (open triangles) (n = 6). (B) The table of the effects: increase ( + ) and decrease ( − ) of extract at different concentrations (n = 5).
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
Naja nigricollis venom would be of interest to determine to what extent this effect could counteract the venom one, since the results of Kone (1980) show the increase of sodium current by this venom. The reversibility of the effects of extract on sodium current agrees with the results of Kone (1980) on the action potential in voltage-clamped skeletal muscle fibres of frog. The other discrepancy with the results obtained on voltage-clamped frog muscle fibres concerns the absence of the effect of S. longepedunculata extract on the potassium current and also the effect on contractile tension which was not altered in frog muscle whereas it is drastically increased in mammalian muscle cells. Since the amplitude of calcium currents and that of cytosolic free calcium are not altered by S. longepedunculata, such an increase in tension might be attributable to the increase in sodium current. Indeed, Caille´ et al. (1978, 1985) had shown that a component of the contraction could be related to the T-tubular sodium current of frog muscle and Potreau and Raymond (1982) have reported the existence of a mechanism of sodium-induced calcium release at the triadic level of such frog muscle fibres. However this possibility can be discarded because an increase in tension due to an increase in T-tubular sodium-current would have slowed down the inactivation phase of INa for potentials above the mechanical threshold rather than accelerating it as observed. Thus it is highly probable that one component of S. longepedunculatal root extract acts on the L-type calcium channel (dihydropyridine receptor), on voltage-sensor of excitation – contraction coupling function but not on the T-type calcium channel function. The effects of extract on the L-type calcium channel could be due to the acceleration of cinetic activation complement of channel. This is strongly reinforced by the observation of a shift of the L-type ICa/V curve towards negative potential (facilitation of contraction) upon S. longepedunculata application whereas neither ICaT nor ICaL amplitudes are modified. Thus the root extract differentiates between the two functions of dihydropyridine receptor which is known (Rios and Pizarro, 1991) to act as a voltage-sensor of EC coupling and as a voltage-
163
dependent L-type calcium channel. A similar discriminative effect between L-type activity and excitation–contraction coupling has already been observed with SR33557 (Bois et al., 1991), an indolizinsulphone which, used at low concentrations, blocked contraction but had little effect on current. A more precise knowledge of the component of S. longepedunculata root presently acting and the comparison of its structure with that of SR33557, could be of great interest for studying the dual role of dihydropyridine receptor in muscle physiology. In conclusion, the present results clearly demonstrate the existence of an agonistic effect of S. longepedunculata extract on the sodium current of skeletal muscle myoballs. In addition the present work provides some interesting results concerning the possible action on the dihydropyridine receptor acting as voltage-sensor (Agnew, 1988; Campbell et al., 1988). Therefore, the extraction and isolation of the different components of S. longepedunculata root extract could be interesting in providing tools for further insights into the excitation–contraction coupling mechanisms. Moreover, the possibility of isolation of specific activating agents for sodium current or voltagesensor would be highly relevant for the development of the biomedical industry in Africa. Acknowledgements This work was supported by CNRS/Universite´ de Poitiers UMR 6558, Professor Legube of the Chemistry Laboratory/Universite´ de Poitiers and the French Agency for Cooperation (Togo). References Abbiw, W., 1990. Useful Plants of Ghana. Intermediate Technology, London, pp. 218 – 220. Agnew, W.S., 1988. Proteins that bridge the gap. Nature 334, 299 – 300. Ayensu, E.S., 1978. Medical Plants of West Africa. Reference Publications, Algonae, M I, 231. Bois, P., Romey, G., Lazdunski, M., 1991. Indolizinsulphones. A class of blockers with dual but discriminative effects on L-type Ca2 + channel activity and excitation – contraction coupling in skeletal muscle. Pflu¨gers Archiv 419, 651 – 656.
164
A.P. Mouzou et al. / Journal of Ethnopharmacology 65 (1999) 157–164
Caille´, J., Ildefonse, M., Rougier, O., 1978. Existence of a sodium current in the tubular membrane of frog skeletal muscle fibre; its possible role in the activation of contraction. Pflu¨gers Archiv 374, 167–177. Caille´, J., Ildefonse, M., Rougier, O., 1985. Excitation–contraction coupling in skeletal muscle. Progress in Biophysics and Molecular Biology 46, 185–239. Campbell, K.P., Leung, A.T., Sharp, A.H., 1988. The biochemistry and molecular biology of the dihydropyridinesensitive calcium channel. Trends in Neurological Sciences 11, 425 – 430. Cognard, C., Constantin, B., Rivet-Bastide, M., Imbert, N., Besse, C., Raymond, G., 1993. Appearance and evolution of calcium currents and contraction during the early postfusional stages of rat skeletal muscle cells developing in primary culture. Development 117, 1153–1161. Cognard, C., Constantin, B., Rivet-Bastide, M., Raymond, G., 1993. Intracellular calcium transients induced by different kinds of stimulus during myogenesis of rat skeletal muscle cells studied by laser cytofluorimetry with Indo-1. Cell Calcium 14, 333 – 348. Diakite, D., 1977. Premier inventaire de la faune ophidienne du Mali. Etude e´pide´miologique, clinique et the´rapeutique des accidents d’envenimation. The`se de Me´decine, Bamako. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2 + indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260,
.
3440 – 3450. Houghton, P.J., Isibogun, M., 1993. Flowering plants used against snakebite. Journal of Ethnopharmacology 39, 1 – 29. Kerharo, 1972. La pharmacope´e et la me´decine traditionnelle se´ne´galaises. Kone, P., 1980. Etude toxicologique, e´lectrophysiologique et pharmacologique du venin de Naja nigricollis (Elapide´ de Coˆte d’Ivoire) et d’une substance antivenimeuse de la pharmacope´e traditionnelle africaine (extrait de Securidaca longepedunculata, Polygalace´e). The`se d’e´tat Abidjan. Kone, P.P., Tricoche, R., Kone, Y., 1979. Mode d’action de quelques venins de serpents de Coˆte d’Ivoire (Dendroaspis 6inidis, Naja nigrocollis, Bitis gabonica) et d’un antivenimeux de la pharmacope´e traditionnelle africaine, au niveau des proprie´te´s e´lectriques et me´caniques de la pre´paration musculaire et neuromusculaire. Troisie`me symposium interafricain OUA/CSTR sur la pharmacope´e traditionnelle et les plantes africaines. Abidjan AMP 79, 109. Potreau, D., Raymond, G., 1982. Existence of a sodium-induced calcium release mechanism on frog skeletal muscle fibres. Journal of Physiology London 333, 463 – 480. Rios, E., Pizarro, G., 1991. Voltage sensor of excitation – contraction coupling in skeletal muscle. Physiological Review 71, 849 – 907. Rivet, M., Cognard, C., Raymond, G., 1989. The slow inward calcium current is responsible for a part of contraction of patch-clamped rat myoballs. Pflu¨gers Archiv 413, 316 – 318.
.