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Andrographis paniculata (Nees) selectively blocks voltage-operated calcium channels in rat vas deferens
Journal of Ethnopharmacology 71 (2000) 115 – 121 www.elsevier.com/locate/jethpharm
Andrographis paniculata (Nees) selectively blocks voltage-operated calcium channels in rat vas deferens Rafael A. Burgos *, Marcelo Imilan, Nury S. Sa´nchez, Juan L. Hancke Institute of Pharmacology, Faculty of Veterinary Science, Uni6ersidad Austral de Chile, P.O. Box 567, Valdi6ia, Chile Received 6 July 1999; received in revised form 8 November 1999; accepted 10 November 1999
Abstract The possible blockade of voltage-operated calcium channels (VOCs) by Andrographis paniculata dried extract in vas deferens smooth muscle was investigated in rats. The tissues were incubated in Ca2 + -free Kreb’s solution and stimulated with KCl (40 mM) to produce depolarisation of the membrane. The isometric contractile response to cumulative concentrations of CaCl2 was effectively blockaded by 0.2 and 0.4 mg/ml A. paniculata. In other experiments, the maximum contractile response induced by norepinephrine was not antagonised by 0.2, 0.4 or 0.8 mg/ml A. paniculata. The possible blockade of Ca2 + entry by A. paniculata was evaluated with 45Ca2 + uptake in vas deferens treated with reserpine (5 and 2.5 mg/kg) 48 and 24 h before the experiments. Epididymal segments were incubated with Ca2 + -free Kreb’s solution with KCl, 25 and 50 mM. The influx was completely blockaded with 0.4 mg/ml A. paniculata. These results suggest that A. paniculata selectively blockades VOCs, hence inhibiting the 45Ca2 + influx. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Andrographis paniculata; Vas deferens; Norepinephrine; Calcium channels
1. Introduction Andrographis paniculata (Barm.f.) (Nees) is a medicinal plant from the Far East (Subtropical Asia, Southeast Asia, India and China) that possesses cardiovascular activity (Chang and But, 1987). In normotensive rats (Zhang and Tan, 1997) and in spontaneously hypertensive rats (Zhang and Tan, 1996), a water extract of A. paniculata produced a significant decrease in systolic blood * Corresponding author. E-mail address: [email protected] (R.A. Burgos)
pressure. On the other hand, A. paniculata herb prevented myocardial ischemia induced by reperfusion injury in dogs (Guo et al., 1996), an effect that is associated with a decrease in the Ca2 + overloading (Guo et al., 1994, 1995). The effect on calcium homeostasis also has been observed with 14-deoxyandrographolide, a diterpene lactone isolated from A. paniculata. In rat thoracic aorta, this lactone produced vasorelaxation, and reduced the contractile response of CaCl2 in the presence of high K+ (Zhang and Tan, 1998). This would indicate that A. paniculata exerts its cardiovascular effect by an inhibition of Ca2 + entry. Therefore, the hypotension induced by A. panicu-
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lata could be produced by an L-type calcium channel blocking mechanism, as the entry of calcium through the L-type calcium channels is one of the mechanisms that help to maintain the basal tone of smooth muscle (Guyton and Hall, 1997) in the arteries of normal (Bhugra and Gulati, 1996) and spontaneously hypertensive rats (Asano et al., 1993). Preliminary results demonstrated that A. paniculata can reduce the contractile response mediated by calcium, and block partially the acetylcholine contractile response in rat uterus (Aguila et al., 1998). In the present study, we demonstrate that A. paniculata is able to blockade selectively voltage-operated calcium channels (VOCs) and inhibit the 45Ca influx in rat vas deferens, without affecting the response of norepinephrine.
2. Methodology
2.1. Materials and reagents Plant extracts were provided by Swedish Herbal Institute, Gothenburg, Sweden. The dried plant was purchased from Zhaoqing Native Produce, Guangdong, China. This plant was collected in September–October 1994 and sun dried. A dried voucher (95012) specimen was identified by Dr Georg Wikman and deposited in the herbarium of Swedish Herbal Institute. The dried herb was powdered and extracted in a soxhlet with 70% ethanol at a temperature of 50°C for 12 h. The extract was filtered and maltodextrin was added before spray drying. The yield of the extract (ratio between the dried starting material and the final powdered product) was 5:1 (w/w from the dry plant); the concentration of andrographolide in the extract was 6%. The batch number was 20190 and the colour was dark green, with a fine texture. Thin layer chromatography (Sharma et al., 1992) and high-performance liquid chromatography (Burgos et al., 1997) identifications were performed. Verapamil and norepinephrine were purchased from Sigma®, USA and 45CaCl2 was obtained from NEN Dupont®, USA. LaCl3 and other reagents were purchased from Merck® (Chile).
2.2. Animals Male Sprague–Dawley rats, weighing approximately 200 g, were used. A group of rats were pretreated with reserpine 48 h (5 mg/kg) and 24 h (2.5 mg/kg) before death in order to deplete endogenous norepinephrine (NE), and thus reduce the potential indirect effects of K+. The animals were killed by decapitation, and the epididymal vasa deferentia half were removed and cleaned of vascular and connective tissue while being kept in oxygenated Kreb’s solution (see later).
2.3. Inhibitory contractile response of 6as deferens to A. paniculata induced by CaCl2 The vasa deferentia from rats treated with reserpine were placed in a 50 ml organ bath. The administration of reserpine blocks the release of NE from nerve endings by depolarisation, this can be produced during the CaCl2 cumulative curves, constructed in a Ca2 + -free Kreb’s solution with high K (40 mM KCl) (Khoyi et al., 1988). Briefly, the tissues were first preincubated during 60 min in Ca2 + -free Kreb’s solution containing: NaCl, 120 mM; KCl, 4.69 mM; NaHCO3, 25 mM; MgSO4 xH20, 1.18 mM; KH2PO4, 1.02 mM; and D-glucose, 11.1 mM at 37°C, gassed with 5% CO2 in O2. The initial muscle tension was 1 g, recorded with an isometric transducer coupled to a polygraph 7 Grass. Afterwards, the tissues were incubated in Ca2 + -free Kreb’s solution with high KCl (40 mM) during 10 min at 30°C, gassed with 5% CO2 in O2. After a period of 60 min, cumulative doses of CaCl2 were added alone or in the presence of 0.2 and 0.4 mg/ml A. paniculata. Similar sets of experiments were performed with verapamil.
2.4. Contractile response of 6as deferens to NE alone or with A. paniculata The effect of A. paniculata on contractile response of NE was evaluated in vas deferentia. The vas deferens were incubated in Kreb’s solution containing: NaCl, 120 mM; KCl, 4.69 mM; NaHCO3, 25 mM; MgSO4xH20, 1.18 mM; KH2PO4, 1.02 mM; CaCl2, 2.43 mM; and D-glu-
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cose, 11.1 mM, during 60 min at 30°C, gassed with 5% CO2 in O2. After a 60 min stabilisation period, dose –response curves were constructed for NE alone, or in the presence of A. paniculata or verapamil. These drugs were added to the bath 5 min before the agonist.
2.5.
Ca 2 + influx in 6as deferens
45
The method was that previously described by Khoyi et al., (1988) with some modifications. Briefly, the tissues were equilibrated in Kreb’s solution containing: NaCl, 120 mM; KCl, 4.69 mM; NaHCO3, 25 mM; MgSO4xH20, 1.18 mM; KH2PO4, 1.02 mM; CaCl2, 0.2 mM; and D-glucose, 11.1 mM (37°C) at pH 7.4, gassed with 5% CO2 in O2. The tissues were pre-equilibrated by 90 min, and the solution was changed every 10 min. The tissues were then exposed during 5 min to Kreb’s solution with 25 and 50 mM KCl and 0.4 mg/ml A. paniculata. Afterwards, this solution was labeled with 0.5 mCi/ml 45Ca2 + . For determination of the 45 Ca2 + influx, exposure time was limited to 4 min. After incubation, the tissues were immediately transferred to a 10 ml calcium-free solution of ice-cold Tris–LaCl3/actic acid (pH 6.0)-free Ca2 + that contained: NaCl, 122 mM; KCl, 5.9 mM;
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LaCl3, 50 mM; MgCl2, 1.25 mM; D-glucose, 11 mM; trizma 6 mM, at 4°C, and incubated for 20 min to remove the extracellular 45Ca2 + . The tissue were rinsed twice in ice-cold Tris–LaCl3/actic acid (pH 6.0)-free Ca2 + , laid in filter paper and cut into pieces of 3–4 mm, and weighed. The tissues were put in vials of 5 ml and solubilised in 1 ml of 0.1 N KOH at 90°C for 12 h. Finally, 0.5 ml water and 4 ml scintillation cocktail containing 0.5 g POPOP (1,4-bis[S-phenyl-2-oxazolyl]benzene; 2,2-pphenylene-bis[S-phenyloxazole]), 4 g PPO 2,5diphenyloxadiazole, dissolved in 500 ml toluene and 250 ml triton X-100 was added; 45Ca2 + was counted in a liquid scintillation counter. Tissue counts were converted to tissue calcium content by using the specific activity of 45Ca2 + in the incubation medium. The influx was expressed in micromoles Ca2 + per kg wet weight, and all experiments were carried out in duplicate.
2.6. Statistical analysis Statistical analysis was carried out by a Student’s t-test, and analysis of variance using Dunnet test for multiple comparisons. PB0.05 was considered significant. The dose–response curves were fitted using GraphPad Prism Software 2.0. 3. Results
3.1. Inhibitory contractile response of 6as deferens to A. paniculata induced by CaCl2
Fig. 1. Effects of A. paniculata on contractile response with cumulative concentrations of CaCl2 in KCl depolarised vas deferens (40 mM KCl). CaCl2 contractile response was measured in the absence () or presence of A. paniculata, ( ) 0.2 mg/ ml and ( ) 0.4 mg/ml. Points are the means of nine animals. Bars indicate the S.E.M.
Fig. 1 and Table 1 show the inhibitory effects of A. paniculata on cumulative contractile response of CaCl2. A dose-dependent decrease in the maximum contractile response with 0.2 and 0.4 mg/ml A. paniculata, 78.6492.79 and 47.06 9 2.66% (mean9 S.E.M, n= 9), was observed, respectively. However, the ED50 (− log M CaCl2) was not modified in the control, and A. paniculata groups (Table 1). In Fig. 2 and Table 1, the effect of verapamil on vas deferens CaCl2-induced contractile response is depicted. A dose-dependent decrease in the maximum contractile response, was observed with verapamil. The latter produced a significant decrease (P B0.01) in ED50 (− log M CaCl2) compared with the control (Table 1).
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Table 1 Effect of A. paniculata and verapamil on Emax and ED50 with cumulative CaCl2 concentrations Verapamil
A. paniculata
Control 0.2 mg/ml 0.4 mg/ml
Emax (%)
ED50 (−log M CaCl2)
94.59 92.92 78.64 9 2.79** 47.06 9 2.66**
3.14 9 0.03 3.11 9 0.03 3.149 0.05
Control 5×10−8 M 1×10−7 M
Emax (%)
ED50 (−log M CaCl2)
96.31 92.35 72.92 9 2.39** 57.78 9 6.12**
3.09 90.03 2.84 90.03* 2.67 9 0.09**
* PB0.05 as compared with the control group, n=7. ** PB0.01 as compared with the control group, n= 7.
3.2. Contractile response of 6as deferens to NE alone or with A. paniculata Fig. 3 shows the contractile response elicited by NE. The contraction was done in Kreb’s solution as described in Section 2. The ED50 of NE was not modified by A. paniculata in the doses of 0.2, 0.4 and 0.8 mg/ml: 5.3391.42 ×10 − 6, 3.409 1.28× 10 − 6 and 3.06 91.52 ×10 − 6 M, respectively, compared with the control, 5.26 91.27×10 − 6 M (mean9S.E., n = 7). The maximum contractile response of NE did not change with the treatment of 0.2, 0.4 and 0.8 mg/ml A. paniculata: 118.9911.87, 110.396.60 and 97.209 9.91%, respectively. The administration of verapamil (5×10 − 8 and 1 ×10 − 7 M) did not modify the contractile response (data not shown). However, the administration of 1×10 − 6 M verapamil produced a significant decrease in NE, ED50 2.12491.55 ×10 − 6 M, compared with the control group, 5.257 9 1.26 ×10 − 6 M (P B 0.05). Also, 1×10 − 6 M verapamil reduced significantly (P B0.01) the maximum contractile response of NE, 79.86 96.45% compared with the control (mean9 S.E., n =7).
3.3.
ulated vas deferens with 25 and 50 mM KCl, as described in Section 2, in order to assess the selective blocking of A. paniculata on VOCs. Fig. 4 shows that 45Ca2 + uptake in vas deferens stimulated by CaCl2 (0.2 mM) containing 45CaCl2 (about 0.5 mCi/tube) for 4 min in Kreb’s solution at different KCl concentrations (25 and 50 mM KCl). The stimulation with KCl increased significantly the uptake of 45CaCl2, in a dose-dependent manner (PB 0.05), and A. paniculata in a doses of 0.4 mg/ml inhibited the influx of 45Ca2 + .
Ca 2 + influx in 6as deferens
45
As already described, the contractile response to high KCl of cumulative doses of CaCl2 seemed to depend on the influx of Ca2 + ions into the vas deferens smooth muscle cells, from the outside medium. We examined the 45Ca2 + uptake of stim-
Fig. 2. Effects of verapamil on contractile response to cumulative concentrations of CaCl2 in vas deferens depolarised with 40 mM KCl. CaCl2 contractile response was measured in the absence () or presence of verapamil, ( ) 5 ×10 − 8 M and ( ) 1× 10 − 7 M. Points are the means of nine animals. Bars indicate the S.E.M.
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Fig. 3. Contractile response as a function of the concentration of norepinephrine. (A) Contractile response was measured in the absence () or presence of A. paniculata, ( ) 0.2 mg/ml, ( ) 0.4 mg/ml or () 0.8 mg/ml. (B) Contractile response was measured in the absence () or presence of verapamil, ( ) 1 ×10 − 6 M. Points are the means of seven animals. Bars indicate the S.E.M.
4. Discussion and conclusions In the present study, A. paniculata reduced, in a dose-dependent and noncompetitive manner, the contractile response of CaCl2 in vas deferens. On the other hand, verapamil, a phenylalkylamine that blocks the L-type Ca2 + channels (Hockerman et al., 1997), had a similar effect. However, the latter reduced the ED50 of CaCl2, indicating a small competitive antagonism effect on the CaCl2 contractile response. This response has also been observed with verapamil in uterus (Aguila et al., 1998). The depolarisation induced by KCl favors the opening of calcium channel operated by voltage, the entry of Ca2 + and the muscle contractility of the vas deferens (Karaki et al., 1997). Exposure to high K+ increases the influx of 45 Ca2 + in rat (Hay and Wadsworth, 1984; Khoyi et al., 1988; Fig. 4) and guinea pigs vas deferens (Saad and Huddart, 1981). This effect of K+ is direct: it does not depend on the release of NE or cotransmitters from adrenergic nerves. The effect occurs after NE depletion by reserpine (Khoyi et al., 1988; our results) or adrenergic nerve degeneration produced by 6-hydroxydopamine (Khoyi et al., 1988). In the present study, the K+ induced influx of 45Ca2 + was dose dependent, and A. paniculata (0.4 mg/ml) significantly reduced the
uptake of 45Ca2 + (Fig. 4). It is known that the increase of 45Ca2 + uptake by the smooth muscle is due to an increased 45Ca2 + influx from the external medium to the muscle cells through VOCs ( Hay and Wadsworth, 1984; Ichida et al., 1984; Khoyi et al., 1988). This strongly suggests that the inhibition of CaCl2 contractile response induced by A. paniculata is due to a blockade of VOCs. In vas deferens, the receptor-operated calcium channels (ROCs) also participate in the smooth muscle contractions. A. paniculata could be block-
Fig. 4. Inhibition of A. paniculata 45Ca2 + uptake in vas deferens induced by KCl. Values are the mean 9 S.E.M. n =at least 3. * PB 0.05 compared with Kreb’s solution.
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ing these ROCs. a-Adrenergic receptors in vas deferens can be activated by NE (Khoyi et al., 1988; Karaki et al., 1997), and the mechanical contractile response to NE can be produced even in Ca2 + -free solution (Ashoori and Tomita, 1983), affecting other mechanisms such as the release of Ca2 + of the intracellular store. A. paniculata did not reduce the contractile response induced by NE, and verapamil only at higher doses produced inhibition of contraction. Verapamil would produce this effect by other mechanisms such as interference at the a-adrenergic receptor (Blackmore et al., 1979), or inhibition of phosphodiesterase (Epstein et al., 1982). This indicates a selective effect of A. paniculata on VOCs and an absence of a-adrenergic antagonist response. The active compound(s) of A. paniculata involved in this effect are unknown. However, 14deoxyandrographolide, a diterpene lactone isolated from A. paniculata, has been described to produce vasorelaxation in rat thoracic aorta, by reducing the contractile response of KCl and NE, an action that is lower than verapamil (Zhang and Tan, 1998). The absence in NE response with A. paniculata extract can be explained by the presence of other active compounds or differences in the tissue response. In support of the latter, the smooth muscle of vas deferens exhibits several unusual features in comparison with the vascular smooth muscle. In vas deferens, NE not only fails to promote 45Ca2 + influx by itself (Khoyi et al., 1987, 1988), it also attenuates the K+-induced 45 Ca2 + influx while promoting release from the sarcoplasmic reticulum (Khoyi et al., 1988). Studies with vascular smooth muscle have shown that NE increases the 45Ca2 + influx and that this effect is additive to the K+-induced 45Ca2 + influx (Meisheri et al., 1981). NE may open VOCs directly or indirectly through GTP-binding proteins in the absence of membrane depolarisation (Benham and Tsien, 1988; Nelson et al., 1988). Moreover, NE and other agonists seem to open the same verapamil-sensitive channels, L-type Ca2 + channels, and may represent the major Ca2 + influx pathway in the aorta during contraction (Karaki et al., 1997). Therefore, a blockade of VOCs in vascular smooth muscle can produce an
indirect and partial inhibition of a-adrenergic contractile response in the aorta. Our experiments and those of others (Aguila et al., 1998) conclude that A. paniculata blocks the entry of extracellular calcium, by blocking VOCs. The effect of A. paniculata on blockade of VOCs of smooth muscle could be a possible mechanism that explains its relaxing action and its use in the treatment of diseases such as diarrhoea (Gupta et al., 1990) and arterial hypertension (Chang and But, 1987).
Acknowledgements We are grateful to Laboratorios Garden House for financial support, Swedish Herbal Institute for the supply of A. paniculata dried extract and Dario Salazar for animal care.
References Aguila, M.J., Imilan, M., Sa´nchez, N., Hancke, J.L., Burgos, R.A., 1998. Effects of Andrographis paniculata on voltage operated calcium channels in uterine smooth muscle. In: XX Reunion Anual de la Sociedad de Farmacolgı´a de Chile, Santiago, Chile, pp. 48. Asano, M., Matsuda, T., Hayakawa, M., Ito, K.M., Ito, K., 1993. Increased resting Ca2 + maintains the miogenic tone and activates K+ channels in arteries from young spontaneously hypertensive rats. European Journal of Pharmacology 247, 295 – 304. Ashoori, F., Tomita, T., 1983. Mechanical response to noradrenaline in calcium-free solution in the rat 6as deferens. Journal of Physiology (London) 338, 165 – 178. Benham, C.D., Tsien, R.W., 1988. Noradrenaline modulation of calcium channels in single smooth muscle cells from rabbit ear artery. Journal of Physiology (London) 404, 767 – 784. Bhugra, P., Gulati, O.D., 1996. Interaction of calcium channel blockers with different agonists in aorta from normal and diseased rats. Indian Journal of Physiology and Pharmacology 40, 109 – 119. Blackmore, P.F., El-Refai, M.F., Exton, J.H., 1979. Adrenergic blockade and inhibition of A23187 mediated Ca2 + uptake by the calcium antagonist verapamil in rat liver cells. Molecular Pharmacology 15, 598 – 606. Burgos, R.A., Caballero, E.E., Sa´nchez, N.S., Schroeder, R.A., Wikman, G.K., Hancke, J.L., 1997. Testicular toxicity assessment of Andrographis paniculata dried extract in rats. Journal of Ethnopharmacology 58, 219 – 224.
R.A. Burgos et al. / Journal of Ethnopharmacology 71 (2000) 115–121 Chang, H.M., But, P.P.H., 1987. Pharmacology and Applications of Chinese Materia Medica, vol. II. World Scientific, Singapore, pp. 918 – 928. Epstein, P.M., Fiss, K., Hachisu, R., Adrenyak, D.M., 1982. Interaction of calcium antagonist with cyclic AMP phosphodiesterases and calmodulin. Biochemical and Biophysical Research Communication 105, 1142–1149. Guo, Z.L., Zhao, H.Y., Zheng, X.H., 1994. The effect of Andrographis paniculata nees (apn) in alleviating the myocardial ischemic reperfusion injury. Journal of Tongji Medical University 14, 49–51. Guo, Z.L., Zhao, H.Y., Zheng, X.H., 1995. An experimental study of the mechanism of Andrographis paniculata nees (apn) in alleviating the Ca2 + -overloading in the process of myocardial ischemic reperfusion. Jounal of Tongji Medical University 15, 205 – 208. Guo, Z., Zhao, H., Fu, L., 1996. Protective effects of API0134 on myocardial ischemia and reperfusion injury. Journal of Tongji Medical University 16, 193–197. Gupta, S., Choudhry, M.A., Yadava, J.N.S., 1990. Antidiarrhoeal activity of diterpenes of Andrographis paniculata (Kal-Megh) againts Eschericia coli enterotoxin in in 6i6o models. International Journal of Crude Drug Research 28, 273 – 283. Guyton, A.C., Hall, J.E., 1997. Contraccio´n y exitacio´n del mu´sculo liso. In: Guyton, A.C., Hall, J.E. (Eds.), Tratado de Fisiologı´a Me´dica. McGraw-Hill Interamericana de Espan˜a, S.A.Me´xico, pp. 103–111. Hay, D.W., Wadsworth, R.M., 1984. Effects of KCl on 45Ca uptake and efflux in the rat 6as deferens. British Journal of Pharmacology 81, 441–447. Hockerman, G.H., Peterson, B.Z., Johnson, B.D., Catterall, W.A., 1997. Molecular determinant of drug binding and action on L-type calcium channels. Annual Review of Pharmacology and Toxicology 47, 361–396. Ichida, S., Moriyama, M., Terao, M., 1984. Characteristics of Ca influxes through voltage-and receptor-operated Ca channels in uterine smooth muscle. Journal of Pharmacology and Experimental Therapeutics 228, 439–445.
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Karaki, H., Ozaki, H., Hori, M., Mitsui-Saito, M., Amano, K.I., Harada, K.I., Miyamoto, S., Nakazawa, H., Won, K.J., Sato, K., 1997. Calcium movements, distribution, and functions in smooth muscle. Pharmacology Review 49, 157 – 239. Khoyi, M.A., Westfall, D.P., Gerthoffer, W.T., 1987. Effects of potassium and norepinephrine on calcium influx in guinea-pig 6as deferens. European Journal of Pharmacology 140, 55 – 62. Khoyi, M.A., Westfall, D.P., Buxton, I.L.O., Akhtar-khavari, F., Rezaei, E., Salaices, M., Sanchez-Garcia, P., 1988. Norepinephrine and potassium induced calcium translocation in rat Vas deferens. Journal of Pharmacology and Experimental Therapeutics 246, 917 – 923. Meisheri, K.D., Hwang, O.K., Van Breemen, C., 1981. Evidence for two separate Ca + 2 pathways in smooth muscle plasmalemma. Journal of Membrane Biology 59, 19 – 25. Nelson, M.T., Standen, N.B., Brayden, J.E., Worley, J.F., 1988. Noradrenaline contracts arteries by activating voltage-dependent calcium channels. Nature 336, 382 – 385. Saad, K.H.M., Huddart, H., 1981. Influence of noradrenaline and KCl on calcium translocation in rat vas deferens smooth muscle and its subcellular fractions. General Pharmacology 12, 373 – 380. Sharma, A., Lal, K., Handa, S.S., 1992. Standardization of the indian crude drug Kalmegh by high pressure liquid chromatographic determination of andrographolide. Phytochemical Analysis 3, 129 – 131. Zhang, C.Y., Tan, B.K., 1996. Hypotensive activity of aqueous extract of Andrographis paniculata in rats. Clinical and Experimental Pharmacology and Physiology 23, 675 – 678. Zhang, C.Y., Tan, B.K., 1997. Mechanism of cardiovascular activity of Andrographis paniculata in the anaesthetized rat. Journal of Ethnopharmacology 56, 97 – 101. Zhang, C.Y., Tan, B.K., 1998. Vasorelaxation of rat thoracic aorta caused by 14-deoxyandrographolide. Clinical and Experimental Pharmacology and Physiology 25, 424 – 429.
Andrographis paniculata (Nees) selectively blocks voltage-operated calcium channels in rat vas deferens Rafael A. Burgos *, Marcelo Imilan, Nury S. Sa´nchez, Juan L. Hancke Institute of Pharmacology, Faculty of Veterinary Science, Uni6ersidad Austral de Chile, P.O. Box 567, Valdi6ia, Chile Received 6 July 1999; received in revised form 8 November 1999; accepted 10 November 1999
Abstract The possible blockade of voltage-operated calcium channels (VOCs) by Andrographis paniculata dried extract in vas deferens smooth muscle was investigated in rats. The tissues were incubated in Ca2 + -free Kreb’s solution and stimulated with KCl (40 mM) to produce depolarisation of the membrane. The isometric contractile response to cumulative concentrations of CaCl2 was effectively blockaded by 0.2 and 0.4 mg/ml A. paniculata. In other experiments, the maximum contractile response induced by norepinephrine was not antagonised by 0.2, 0.4 or 0.8 mg/ml A. paniculata. The possible blockade of Ca2 + entry by A. paniculata was evaluated with 45Ca2 + uptake in vas deferens treated with reserpine (5 and 2.5 mg/kg) 48 and 24 h before the experiments. Epididymal segments were incubated with Ca2 + -free Kreb’s solution with KCl, 25 and 50 mM. The influx was completely blockaded with 0.4 mg/ml A. paniculata. These results suggest that A. paniculata selectively blockades VOCs, hence inhibiting the 45Ca2 + influx. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Andrographis paniculata; Vas deferens; Norepinephrine; Calcium channels
1. Introduction Andrographis paniculata (Barm.f.) (Nees) is a medicinal plant from the Far East (Subtropical Asia, Southeast Asia, India and China) that possesses cardiovascular activity (Chang and But, 1987). In normotensive rats (Zhang and Tan, 1997) and in spontaneously hypertensive rats (Zhang and Tan, 1996), a water extract of A. paniculata produced a significant decrease in systolic blood * Corresponding author. E-mail address: [email protected] (R.A. Burgos)
pressure. On the other hand, A. paniculata herb prevented myocardial ischemia induced by reperfusion injury in dogs (Guo et al., 1996), an effect that is associated with a decrease in the Ca2 + overloading (Guo et al., 1994, 1995). The effect on calcium homeostasis also has been observed with 14-deoxyandrographolide, a diterpene lactone isolated from A. paniculata. In rat thoracic aorta, this lactone produced vasorelaxation, and reduced the contractile response of CaCl2 in the presence of high K+ (Zhang and Tan, 1998). This would indicate that A. paniculata exerts its cardiovascular effect by an inhibition of Ca2 + entry. Therefore, the hypotension induced by A. panicu-
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lata could be produced by an L-type calcium channel blocking mechanism, as the entry of calcium through the L-type calcium channels is one of the mechanisms that help to maintain the basal tone of smooth muscle (Guyton and Hall, 1997) in the arteries of normal (Bhugra and Gulati, 1996) and spontaneously hypertensive rats (Asano et al., 1993). Preliminary results demonstrated that A. paniculata can reduce the contractile response mediated by calcium, and block partially the acetylcholine contractile response in rat uterus (Aguila et al., 1998). In the present study, we demonstrate that A. paniculata is able to blockade selectively voltage-operated calcium channels (VOCs) and inhibit the 45Ca influx in rat vas deferens, without affecting the response of norepinephrine.
2. Methodology
2.1. Materials and reagents Plant extracts were provided by Swedish Herbal Institute, Gothenburg, Sweden. The dried plant was purchased from Zhaoqing Native Produce, Guangdong, China. This plant was collected in September–October 1994 and sun dried. A dried voucher (95012) specimen was identified by Dr Georg Wikman and deposited in the herbarium of Swedish Herbal Institute. The dried herb was powdered and extracted in a soxhlet with 70% ethanol at a temperature of 50°C for 12 h. The extract was filtered and maltodextrin was added before spray drying. The yield of the extract (ratio between the dried starting material and the final powdered product) was 5:1 (w/w from the dry plant); the concentration of andrographolide in the extract was 6%. The batch number was 20190 and the colour was dark green, with a fine texture. Thin layer chromatography (Sharma et al., 1992) and high-performance liquid chromatography (Burgos et al., 1997) identifications were performed. Verapamil and norepinephrine were purchased from Sigma®, USA and 45CaCl2 was obtained from NEN Dupont®, USA. LaCl3 and other reagents were purchased from Merck® (Chile).
2.2. Animals Male Sprague–Dawley rats, weighing approximately 200 g, were used. A group of rats were pretreated with reserpine 48 h (5 mg/kg) and 24 h (2.5 mg/kg) before death in order to deplete endogenous norepinephrine (NE), and thus reduce the potential indirect effects of K+. The animals were killed by decapitation, and the epididymal vasa deferentia half were removed and cleaned of vascular and connective tissue while being kept in oxygenated Kreb’s solution (see later).
2.3. Inhibitory contractile response of 6as deferens to A. paniculata induced by CaCl2 The vasa deferentia from rats treated with reserpine were placed in a 50 ml organ bath. The administration of reserpine blocks the release of NE from nerve endings by depolarisation, this can be produced during the CaCl2 cumulative curves, constructed in a Ca2 + -free Kreb’s solution with high K (40 mM KCl) (Khoyi et al., 1988). Briefly, the tissues were first preincubated during 60 min in Ca2 + -free Kreb’s solution containing: NaCl, 120 mM; KCl, 4.69 mM; NaHCO3, 25 mM; MgSO4 xH20, 1.18 mM; KH2PO4, 1.02 mM; and D-glucose, 11.1 mM at 37°C, gassed with 5% CO2 in O2. The initial muscle tension was 1 g, recorded with an isometric transducer coupled to a polygraph 7 Grass. Afterwards, the tissues were incubated in Ca2 + -free Kreb’s solution with high KCl (40 mM) during 10 min at 30°C, gassed with 5% CO2 in O2. After a period of 60 min, cumulative doses of CaCl2 were added alone or in the presence of 0.2 and 0.4 mg/ml A. paniculata. Similar sets of experiments were performed with verapamil.
2.4. Contractile response of 6as deferens to NE alone or with A. paniculata The effect of A. paniculata on contractile response of NE was evaluated in vas deferentia. The vas deferens were incubated in Kreb’s solution containing: NaCl, 120 mM; KCl, 4.69 mM; NaHCO3, 25 mM; MgSO4xH20, 1.18 mM; KH2PO4, 1.02 mM; CaCl2, 2.43 mM; and D-glu-
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cose, 11.1 mM, during 60 min at 30°C, gassed with 5% CO2 in O2. After a 60 min stabilisation period, dose –response curves were constructed for NE alone, or in the presence of A. paniculata or verapamil. These drugs were added to the bath 5 min before the agonist.
2.5.
Ca 2 + influx in 6as deferens
45
The method was that previously described by Khoyi et al., (1988) with some modifications. Briefly, the tissues were equilibrated in Kreb’s solution containing: NaCl, 120 mM; KCl, 4.69 mM; NaHCO3, 25 mM; MgSO4xH20, 1.18 mM; KH2PO4, 1.02 mM; CaCl2, 0.2 mM; and D-glucose, 11.1 mM (37°C) at pH 7.4, gassed with 5% CO2 in O2. The tissues were pre-equilibrated by 90 min, and the solution was changed every 10 min. The tissues were then exposed during 5 min to Kreb’s solution with 25 and 50 mM KCl and 0.4 mg/ml A. paniculata. Afterwards, this solution was labeled with 0.5 mCi/ml 45Ca2 + . For determination of the 45 Ca2 + influx, exposure time was limited to 4 min. After incubation, the tissues were immediately transferred to a 10 ml calcium-free solution of ice-cold Tris–LaCl3/actic acid (pH 6.0)-free Ca2 + that contained: NaCl, 122 mM; KCl, 5.9 mM;
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LaCl3, 50 mM; MgCl2, 1.25 mM; D-glucose, 11 mM; trizma 6 mM, at 4°C, and incubated for 20 min to remove the extracellular 45Ca2 + . The tissue were rinsed twice in ice-cold Tris–LaCl3/actic acid (pH 6.0)-free Ca2 + , laid in filter paper and cut into pieces of 3–4 mm, and weighed. The tissues were put in vials of 5 ml and solubilised in 1 ml of 0.1 N KOH at 90°C for 12 h. Finally, 0.5 ml water and 4 ml scintillation cocktail containing 0.5 g POPOP (1,4-bis[S-phenyl-2-oxazolyl]benzene; 2,2-pphenylene-bis[S-phenyloxazole]), 4 g PPO 2,5diphenyloxadiazole, dissolved in 500 ml toluene and 250 ml triton X-100 was added; 45Ca2 + was counted in a liquid scintillation counter. Tissue counts were converted to tissue calcium content by using the specific activity of 45Ca2 + in the incubation medium. The influx was expressed in micromoles Ca2 + per kg wet weight, and all experiments were carried out in duplicate.
2.6. Statistical analysis Statistical analysis was carried out by a Student’s t-test, and analysis of variance using Dunnet test for multiple comparisons. PB0.05 was considered significant. The dose–response curves were fitted using GraphPad Prism Software 2.0. 3. Results
3.1. Inhibitory contractile response of 6as deferens to A. paniculata induced by CaCl2
Fig. 1. Effects of A. paniculata on contractile response with cumulative concentrations of CaCl2 in KCl depolarised vas deferens (40 mM KCl). CaCl2 contractile response was measured in the absence () or presence of A. paniculata, ( ) 0.2 mg/ ml and ( ) 0.4 mg/ml. Points are the means of nine animals. Bars indicate the S.E.M.
Fig. 1 and Table 1 show the inhibitory effects of A. paniculata on cumulative contractile response of CaCl2. A dose-dependent decrease in the maximum contractile response with 0.2 and 0.4 mg/ml A. paniculata, 78.6492.79 and 47.06 9 2.66% (mean9 S.E.M, n= 9), was observed, respectively. However, the ED50 (− log M CaCl2) was not modified in the control, and A. paniculata groups (Table 1). In Fig. 2 and Table 1, the effect of verapamil on vas deferens CaCl2-induced contractile response is depicted. A dose-dependent decrease in the maximum contractile response, was observed with verapamil. The latter produced a significant decrease (P B0.01) in ED50 (− log M CaCl2) compared with the control (Table 1).
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Table 1 Effect of A. paniculata and verapamil on Emax and ED50 with cumulative CaCl2 concentrations Verapamil
A. paniculata
Control 0.2 mg/ml 0.4 mg/ml
Emax (%)
ED50 (−log M CaCl2)
94.59 92.92 78.64 9 2.79** 47.06 9 2.66**
3.14 9 0.03 3.11 9 0.03 3.149 0.05
Control 5×10−8 M 1×10−7 M
Emax (%)
ED50 (−log M CaCl2)
96.31 92.35 72.92 9 2.39** 57.78 9 6.12**
3.09 90.03 2.84 90.03* 2.67 9 0.09**
* PB0.05 as compared with the control group, n=7. ** PB0.01 as compared with the control group, n= 7.
3.2. Contractile response of 6as deferens to NE alone or with A. paniculata Fig. 3 shows the contractile response elicited by NE. The contraction was done in Kreb’s solution as described in Section 2. The ED50 of NE was not modified by A. paniculata in the doses of 0.2, 0.4 and 0.8 mg/ml: 5.3391.42 ×10 − 6, 3.409 1.28× 10 − 6 and 3.06 91.52 ×10 − 6 M, respectively, compared with the control, 5.26 91.27×10 − 6 M (mean9S.E., n = 7). The maximum contractile response of NE did not change with the treatment of 0.2, 0.4 and 0.8 mg/ml A. paniculata: 118.9911.87, 110.396.60 and 97.209 9.91%, respectively. The administration of verapamil (5×10 − 8 and 1 ×10 − 7 M) did not modify the contractile response (data not shown). However, the administration of 1×10 − 6 M verapamil produced a significant decrease in NE, ED50 2.12491.55 ×10 − 6 M, compared with the control group, 5.257 9 1.26 ×10 − 6 M (P B 0.05). Also, 1×10 − 6 M verapamil reduced significantly (P B0.01) the maximum contractile response of NE, 79.86 96.45% compared with the control (mean9 S.E., n =7).
3.3.
ulated vas deferens with 25 and 50 mM KCl, as described in Section 2, in order to assess the selective blocking of A. paniculata on VOCs. Fig. 4 shows that 45Ca2 + uptake in vas deferens stimulated by CaCl2 (0.2 mM) containing 45CaCl2 (about 0.5 mCi/tube) for 4 min in Kreb’s solution at different KCl concentrations (25 and 50 mM KCl). The stimulation with KCl increased significantly the uptake of 45CaCl2, in a dose-dependent manner (PB 0.05), and A. paniculata in a doses of 0.4 mg/ml inhibited the influx of 45Ca2 + .
Ca 2 + influx in 6as deferens
45
As already described, the contractile response to high KCl of cumulative doses of CaCl2 seemed to depend on the influx of Ca2 + ions into the vas deferens smooth muscle cells, from the outside medium. We examined the 45Ca2 + uptake of stim-
Fig. 2. Effects of verapamil on contractile response to cumulative concentrations of CaCl2 in vas deferens depolarised with 40 mM KCl. CaCl2 contractile response was measured in the absence () or presence of verapamil, ( ) 5 ×10 − 8 M and ( ) 1× 10 − 7 M. Points are the means of nine animals. Bars indicate the S.E.M.
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Fig. 3. Contractile response as a function of the concentration of norepinephrine. (A) Contractile response was measured in the absence () or presence of A. paniculata, ( ) 0.2 mg/ml, ( ) 0.4 mg/ml or () 0.8 mg/ml. (B) Contractile response was measured in the absence () or presence of verapamil, ( ) 1 ×10 − 6 M. Points are the means of seven animals. Bars indicate the S.E.M.
4. Discussion and conclusions In the present study, A. paniculata reduced, in a dose-dependent and noncompetitive manner, the contractile response of CaCl2 in vas deferens. On the other hand, verapamil, a phenylalkylamine that blocks the L-type Ca2 + channels (Hockerman et al., 1997), had a similar effect. However, the latter reduced the ED50 of CaCl2, indicating a small competitive antagonism effect on the CaCl2 contractile response. This response has also been observed with verapamil in uterus (Aguila et al., 1998). The depolarisation induced by KCl favors the opening of calcium channel operated by voltage, the entry of Ca2 + and the muscle contractility of the vas deferens (Karaki et al., 1997). Exposure to high K+ increases the influx of 45 Ca2 + in rat (Hay and Wadsworth, 1984; Khoyi et al., 1988; Fig. 4) and guinea pigs vas deferens (Saad and Huddart, 1981). This effect of K+ is direct: it does not depend on the release of NE or cotransmitters from adrenergic nerves. The effect occurs after NE depletion by reserpine (Khoyi et al., 1988; our results) or adrenergic nerve degeneration produced by 6-hydroxydopamine (Khoyi et al., 1988). In the present study, the K+ induced influx of 45Ca2 + was dose dependent, and A. paniculata (0.4 mg/ml) significantly reduced the
uptake of 45Ca2 + (Fig. 4). It is known that the increase of 45Ca2 + uptake by the smooth muscle is due to an increased 45Ca2 + influx from the external medium to the muscle cells through VOCs ( Hay and Wadsworth, 1984; Ichida et al., 1984; Khoyi et al., 1988). This strongly suggests that the inhibition of CaCl2 contractile response induced by A. paniculata is due to a blockade of VOCs. In vas deferens, the receptor-operated calcium channels (ROCs) also participate in the smooth muscle contractions. A. paniculata could be block-
Fig. 4. Inhibition of A. paniculata 45Ca2 + uptake in vas deferens induced by KCl. Values are the mean 9 S.E.M. n =at least 3. * PB 0.05 compared with Kreb’s solution.
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ing these ROCs. a-Adrenergic receptors in vas deferens can be activated by NE (Khoyi et al., 1988; Karaki et al., 1997), and the mechanical contractile response to NE can be produced even in Ca2 + -free solution (Ashoori and Tomita, 1983), affecting other mechanisms such as the release of Ca2 + of the intracellular store. A. paniculata did not reduce the contractile response induced by NE, and verapamil only at higher doses produced inhibition of contraction. Verapamil would produce this effect by other mechanisms such as interference at the a-adrenergic receptor (Blackmore et al., 1979), or inhibition of phosphodiesterase (Epstein et al., 1982). This indicates a selective effect of A. paniculata on VOCs and an absence of a-adrenergic antagonist response. The active compound(s) of A. paniculata involved in this effect are unknown. However, 14deoxyandrographolide, a diterpene lactone isolated from A. paniculata, has been described to produce vasorelaxation in rat thoracic aorta, by reducing the contractile response of KCl and NE, an action that is lower than verapamil (Zhang and Tan, 1998). The absence in NE response with A. paniculata extract can be explained by the presence of other active compounds or differences in the tissue response. In support of the latter, the smooth muscle of vas deferens exhibits several unusual features in comparison with the vascular smooth muscle. In vas deferens, NE not only fails to promote 45Ca2 + influx by itself (Khoyi et al., 1987, 1988), it also attenuates the K+-induced 45 Ca2 + influx while promoting release from the sarcoplasmic reticulum (Khoyi et al., 1988). Studies with vascular smooth muscle have shown that NE increases the 45Ca2 + influx and that this effect is additive to the K+-induced 45Ca2 + influx (Meisheri et al., 1981). NE may open VOCs directly or indirectly through GTP-binding proteins in the absence of membrane depolarisation (Benham and Tsien, 1988; Nelson et al., 1988). Moreover, NE and other agonists seem to open the same verapamil-sensitive channels, L-type Ca2 + channels, and may represent the major Ca2 + influx pathway in the aorta during contraction (Karaki et al., 1997). Therefore, a blockade of VOCs in vascular smooth muscle can produce an
indirect and partial inhibition of a-adrenergic contractile response in the aorta. Our experiments and those of others (Aguila et al., 1998) conclude that A. paniculata blocks the entry of extracellular calcium, by blocking VOCs. The effect of A. paniculata on blockade of VOCs of smooth muscle could be a possible mechanism that explains its relaxing action and its use in the treatment of diseases such as diarrhoea (Gupta et al., 1990) and arterial hypertension (Chang and But, 1987).
Acknowledgements We are grateful to Laboratorios Garden House for financial support, Swedish Herbal Institute for the supply of A. paniculata dried extract and Dario Salazar for animal care.
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