Calcium-dependent mechanisms mediate the vasorelaxant effects of Tridax procumbens

Transcription

1 DOI /jbcpp J Basic Clin Physiol Pharmacol 214; 25(2): Hussein M. Salahdeen, Gbolahan O. Idowu, Omoniyi K. Yemitan, Babatunde A. Murtala and Abdul-Rasak A. Alada Calcium-dependent mechanisms mediate the vasorelaxant effects of Tridax procumbens (Lin) aqueous leaf extract in rat aortic ring Abstract Background: Tridax procumbens leaf extract has a folk reputation as an antihypertensive agent in Nigeria. Evidence suggests that it has a relaxant effect on smooth muscles. The present study was designed to investigate the role of calcium in the vasorelaxant effect of this extract. Methods: Concentration-response studies with noradrenaline (NA), KCl and CaCl 2 were carried out in rat aortic rings with and without the extract in physiological salt solution (PSS) (n = 6 each). Also, the role of intracellular calcium mobilization was studied by measuring the phasic response to NA in Ca 2+ -free N,N-ethylene glycol tetraacetic acid (EGTA) PSS (n = 6). Results: The results showed that the contractile responses to either NA or KCl were attenuated (p <.5) in the presence of the extract. Also, the extract attenuated the contractile response to CaCl 2 in the presence of NA or KCl (p <.5) in the Ca 2+ -free EGTA PSS, while the phasic response to NA was significantly (p <.5) diminished. Conclusions: These results suggest that the vasorelaxant effect of T. procumbens leaf extract may be mediated by a non-specific, non-competitive inhibition of Ca 2+ influx as well as by inhibition of Ca 2+ mobilization from intracellular stores. This implies that it may contain vasorelaxant agents that may have calcium antagonistic potential. Keywords: aortic ring; Ca 2+ ; EGTA; hypertension; Tridax procumbens; vasorelaxant. Corresponding author: Hussein M. Salahdeen, Cardiovascular and Smooth Muscles Unit, Department of Physiology, College of Medicine, Lagos State University, P.M.B 21266, Ikeja, Lagos, Nigeria, Phone: , , hmsalahdeen@gmail.com; hussein.salahdeen@lasunigeria.org Gbolahan O. Idowu: Department of Physiology, Olabisi Onabanjo University, Ogun State, Nigeria Omoniyi K. Yemitan: Department of Pharmacology, College of Medicine, Lagos State University, Ikeja, Lagos Babatunde A. Murtala: Department of Physiology, College of Medicine, Lagos State University, Ikeja, Lagos, Nigeria Abdul-Rasak A. Alada: Department of Physiology, University of Ibadan, Ibadan, Nigeria Introduction The extract of the leaves of Tridax procumbens (family: Asteraceae) has been used to treat various ailments for centuries in traditional medicine in many parts of the world. Experimental studies suggest that it has a variety of biological effects, which include anti-inflammatory [1] and immunestimulating effects [2]. In Nigeria, the extract of the leaves of T. procumbens is used in traditional medicine for the treatment of several ailments including hypertension [3]. Previous studies on the cardiovascular effects of T. procumbens showed that an intravenous injection of the crude extract of the leaves of T. procumbens caused a reduction in both blood pressure and heart rate in anesthetized healthy normotensive rats [3, 4]. Indeed, a preparation of T. procumbens leaf extract has been shown to exhibit a non-specific relaxant activity in isolated aortic smooth muscle preparations [5]. We have previously reported that the vasorelaxant effect is endothelium-dependent involving the release of nitric oxide [6]. However, the extract showed a relaxant effect also in endothelium-denuded preparations, suggesting that it might also act directly on the vascular smooth muscle. The contractile mechanism in smooth muscle is known to be activated by a rise in the concentration of free intracellular Ca 2+ concentration ([Ca 2+ ]I [7 9] and calcium (Ca) sensitization [1]. The source of [Ca 2+ ]i is of dual origin: influx from the extracellular fluid or release from internal pools [7 9]. Thus, vascular smooth muscle relaxation agents may produce their effects by inhibiting either or both sources of Ca 2+ [11]. This study was therefore designed to test the hypothesis that T. procumbens leaf extract may exert its smooth muscle-relaxant effect by inhibiting Ca 2+ influx or its release from the cellular stores. Materials and methods Ethical considerations Experimental protocols and procedures used in this study were approved by the Animal Ethics Committee of the Lagos State

2 162 Salahdeen et al.: Calcium-dependent effect of Tridax procumbens University College of Medicine and conform to the 1985 Guidelines for Laboratory Animal Care of the National Institute of Health (NIH). Plant materials The plant was collected within the Lagos metropolis in Nigeria and authenticated by a traditional medical practitioner and the Department of Botany of Lagos State University, Lagos, Nigeria. A sample specimen (voucher number FHI 18879) was deposited with the Forestry Research Institute of Nigeria (FRIN) herbarium for reference purposes. The leaves were air-dried at room temperature for a week, the dried leaves were pulverized with a laboratory mortar and pestle, and 4 g of the powdered leaves was transferred into a conical flask containing 4 ml of distilled water; the mixture was shaken and allowed to stand for 24 h before filtration using a dry Whatman filter paper into a measuring cylinder. The filtrate was then concentrated by evaporation in a water bath and stored at 4 C until used. Tissue preparation Inbred adult male and female Wistar albino rats, weighing between 15 and 2 g, were obtained from the Laboratory Animal Centre of the Lagos State University College of Medicine, Ikeja, Lagos, Nigeria. The rats were sacrificed by cervical dislocation. The thoracic aorta was quickly removed, freed of connective tissue and placed in a Petri dish containing physiological salt solution (PSS) consisting of 119 mmol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/l KH 2 PO 4, 1.2 mmol/l MgSO 4, 15. mmol/l NaHCO 2, 1.6 mmol/l CaCl 2 and 11.5 mmol/l glucose. The aorta was cut into 2-mm rings and mounted in 5-mL jacketed tissue baths containing PSS. The PSS was continuously bubbled with 95% O 2 and 5% CO 2 gas mixture with the ph maintained at 7.4 using a ph meter model 35L (N. Boyer, UK). The bath temperature was kept at 37 C. The aortic ring was mounted on a fine stainless steel rod which was connected to a force transducer (model 74; Ugo Basile, Varese, Italy) connected to a four-channel data acquisition system (DataCapsule 174, Ugo Basile) for the recording of isometric contractions. The ring was under an initial tension of 2 g and was allowed to equilibrate for a period of 9 min during which the tissue was stimulated three times with a stabilized dose of either 1 7 mol/l noradrenaline (NA) or 6 mmol/l potassium chloride (KCl) to obtain a reproducible response. After a stable response was achieved, the following protocols were carried out. Concentration-response of aortic rings to T. procumbens The effects of T. procumbens on baseline were determined, after which the ring was contracted using 1 7 mol/l NA or 6 mmol/l KCl. After the contraction of the ring had stabilized, T. procumbens leaf extract ( mg/ml) was added cumulatively into the bath solution. The effect of each concentration was allowed to reach a steadystate level before the addition of the next dose. Concentration-response studies with NA and KCl The rings were exposed to cumulative concentrations of NA (1 9 to 1 5 mol/l) after incubating with the vehicle (.2 ml distilled water) for 15 min, and the contractile responses were recorded. The tissues were washed by flushing with PSS at 15-min intervals several times until the pen returned to baseline. After adequate rest, the tissues were incubated with the extract (.45 mg/ml) for 15 min and the concentration-response study with NA was repeated. The same procedure was used for the concentration-response study with KCl (1 1 mmol/l) with and without the extract (.75 mg/ml) in another set of freshly stabilized aortic rings. Concentration-response study with CaCl 2 Aortic rings were stimulated with 1 7 mol/l NA in normal PSS. After the contraction had peaked, the normal PSS was substituted with Cafree PSS containing 5 μmol/l N,N-ethylene glycol tetraacetic acid (EGTA) for 3 min. Midway into this period, the tissue was stimulated with 1 7 mol/l NA to further deplete its Ca stores. At the end of 3 min, the Ca-free EGTA PSS was replaced with a Ca-free PSS without EGTA, after which 1 7 mol/l NA was added to open the ligandgated Ca 2+ channels, followed almost immediately by the addition of.2 ml of the vehicle or extract (.45 mg/ml). The tissues were incubated with the vehicle or extract for 15 min, and then CaCl 2 (1 5 to 1 1 mol/l) was added cumulatively [12, 13]. Concentration-response study with CaCl 2 was carried out in a different set of freshly stabilized aortic rings with or without the extract (.75 mg/ml) using the above protocol. However, 6 mmol/l KCl was used to open the voltagegated Ca 2+ channels before the cumulative addition of CaCl 2. Phasic contraction to noradrenaline Phasic contraction in 1 7 mol/l NA was carried out in a Ca-free EGTA PSS [12, 13]. The tissues were stimulated with 1 7 mol/l NA in normal PSS. At the peak of the contraction, the normal PSS was replaced with Ca-free PSS containing 5 μmol/l EGTA for 15 min in order to deplete the cellular stores of Ca. This was followed by incubation of the rings in PSS containing 2 mmol/l CaCl 2 for another 1 min (Ca loading), after which this was substituted with Ca-free EGTA PSS and incubated with the vehicle or extract (.45 mg/ml) for 15 min before they were stimulated with 1 7 mol/l NA and the contractile response recorded. Such phasic contraction is a useful index of Ca 2+ mobilization from intracellular stores [11, 12]. Drugs Drugs used in this study were NA and EGTA. Both were from Sigma (Sigma-Aldrich Corporation, St. Louis, MO, USA). The drugs were dissolved in distilled water. They were freshly prepared on the day of the experiment. Statistical analysis Data are expressed as means ± SE, where n equals the number of animals from which blood vessels were isolated. The data were analyzed using one-way analysis of variance. The Student-Newman-Keuls post hoc test was used to identify the differences between individual means. The confidence interval was set at 95%, so that, in all cases,

3 Salahdeen et al.: Calcium-dependent effect of Tridax procumbens 163 results with a value of p <.5 were considered to indicate statistical significance. Results Relaxation response to T. procumbens Figure 1 shows typical tracings of relaxation responses to T. procumbens leaf extract recorded in aortic rings precontracted with NA or KCl Concentration-response studies with NA and KCl The concentration-response of aortic rings to NA with and without T. procumbens leaf extract is shown in Figure 2. The curve, obtained in the presence of T. procumbens leaf extract, was significantly (p <.5) attenuated compared to the control curve. The concentration-response curve for KCl in the presence T. procumbens leaf extract was also Noradrenaline concentration, Log M Figure 2 Concentration-response curves for NA in aortic rings incubated with and without T. procumbens (.45 mg/ml). Each point represents the mean ± SEM (n = 6). p <.5 at all concentrations. significantly (p <.5) attenuated compared to the control curve (Figure 3). A 2 min 2 g Concentration-response to CaCl 2 The concentration-response curves for CaCl 2 in the presence and absence of T. procumbens leaf extract following stimulation by 1 7 mol/l NA and 6 mmol/l KCl, NE (1-7 M) Washed 1 8 B 2 g 1 2 min KCl (6 mm) Washed Figure 1 Typical tracings showing the vasorelaxant effects of graded concentrations of T. procumbens aqueous leaf extract on (A) noradrenaline (NA)-induced and (B) KCl-induced contractions in isolated aortic ring preparations obtained from normotensive rat. Arrows 1 6 represent cumulatively administered T. procumbens leaf extracts (.3,.45,.6,.75,.9 and 1.5 mg/ml, respectively). NA, KCl and T. procumbens were washed out (open upward arrow) KCl concentration, Log M Figure 3 Concentration-response curves for KCl in aortic rings in the absence of (control) and following exposure to T. procumbens (.75 mg/ml). Each point represents the mean ± SEM (n = 6). p <.5 at all concentrations.

4 164 Salahdeen et al.: Calcium-dependent effect of Tridax procumbens CaCl 2 concentration, Log M respectively, are shown in Figures 4 and 5. Again, the curves were significantly (p <.5) attenuated in the presence of T. procumbens leaf extract. Phasic contraction to noradrenaline Figure 4 Concentration-response curves for CaCl 2 in aortic rings incubated with and without T. procumbens (.45 mg/ml) following the opening of ligand-gated channels with 1 7 M NA. Each point represents the mean ± SEM (n = 6). p <.5. The phasic contraction to NA in the presence of T. procumbens leaf extract was significantly (p <.5) lower than that in its absence (Figure 6) CaCl 2 concentration, Log M Figure 5 Concentration-response curves for CaCl 2 in aortic rings incubated with and without T. procumbens (.75 mg/ml) following the opening of voltage-gated channels with 6 mmol/l KCl. Each point represents the mean ± SEM (n = 6). p <.5. Maximum contraction Figure 6 Bar graphs showing the effect of T. procumbens (.45 mg/ml) on phasic contraction to NA in Ca-free PSS (n = 6). p <.5. Discussion In this study, we examined and observed that T. procumbens leaf extract elicited relaxation of precontracted rings and also attenuated intra- and extracellular Ca-dependent contractions. The concentration of T. procumbens leaf extracts produced vasorelaxation of NA (1 7 mol/l) and is comparable to our earlier reports [5]. Two modes of stimulation (NA and high K + ) were used in this study to characterize the relaxant effect of T. procumbens leaf extract. Noradrenaline causes contraction by mobilizing intracellular Ca pools, as well as stimulating Ca 2+ influx through receptor-operated channels, while high K + -induced contractions are non-specific and involve Ca 2+ influx through voltage-dependent channels [9, 11]. As T. procumbens leaf extract inhibited the contraction of aortic smooth muscle produced by these two mechanisms, it thus indicates that the blockade of voltage-gated Ca channels may not be an important factor in the relaxation. In the absence of NA, T. procumbens leaf extract did not produce any changes in the tension of smooth muscle, suggesting that the extract itself does not have direct effects on contractile proteins. Since T. procumbens leaf extract alone was not sufficient to inhibit the NA contraction of aortic smooth muscle, the possibility of a requirement for the co-existence of an unidentified factor being involved in the T. procumbens leaf extract-induced inhibition must not be ruled out [5]. Similarly, our observation in the concentrationresponse curve for CaCl 2 in the presence of T. procumbens leaf extract following the opening of Ca 2+ channels by NA or KCl suggests the non-competitive antagonism effects of the extract [14]. The contractile mechanism in smooth muscle is activated by a rise in the concentration of free intracellular Ca 2+ concentration [Ca 2+ ]I [7, 9]. This

5 Salahdeen et al.: Calcium-dependent effect of Tridax procumbens 165 may occur via Ca 2+ influx through ligand-gated or voltagegated Ca 2+ channels [7, 9], which was opened by the use of NA and KCl, respectively, in this study. Consequently, the present findings suggest that the reduction in contractile response to these agonists by T. procumbens may be through a non-specific, non-competitive antagonistic action on Ca 2+ influx. However, since the increase in [Ca 2+ ]i may also occur through the release from cellular stores, the effect of the extract on this was studied by recording the phasic response to NA in the absence of extracellular Ca. Such phasic contraction is a useful index of Ca 2+ mobilization from intracellular stores [11, 13, 15]. The results from the present study show that the phasic response to NA in Ca 2+ -free EGTA PSS was attenuated by the extract. This suggests that mobilization of intracellular Ca 2+ for contraction by NA was inhibited. The result is consistent with an earlier study suggesting that T. procumbens preparation exhibited a non-specific relaxant effect in aortic smooth muscle preparations [5]. However, it is possible that the extract may produce its vasorelaxant action through other mechanisms as well. Earlier reports suggest that its vasorelaxant effect was endothelium-dependent occurring through the nitric oxide/soluble guanylate cyclase pathway [6]. Indeed, since endothelial-intact preparations were used in this study, some of the actions on Ca 2+ influx or mobilization from cellular stores observed for T. procumbens may be partly mediated by nitric oxide (NO). This is because NO inhibits the Ca 2+ influx through ligand-gated Ca 2+ channels as well as through release from cellular stores [16]. Thus, it is conceivable that the extract may achieve vasorelaxation through stimulation of the nitric oxide/soluble guanylate cyclase pathway as well as through Ca 2+ -dependent mechanisms. These dual mechanisms characterized antihypertensive action, thereby suggesting that the extract may be useful in the treatment of hypertension. Hypertension is characterized by an increase in peripheral resistance, which may be due to elevated [Ca 2+ ]i and/or endothelial dysfunction, and the extract may be useful in its treatment conceivably by ameliorating these defects. Therefore, more experiments related to the effects of T. procumbens leaf extract on contractile system such as the alteration of Ca sensitivity or regulatory proteins will be needed to confirm this observation. In spite of this, the present findings provide some evidence suggesting that the use of this extract in Nigerian folk medicine for the treatment of hypertension may be justified. We conclude that, in isolated rat aortic rings, T. procumbens leaf extract-induced relaxation is non-specific and is dependent on the decrease in Ca 2+ supply from the extracellular fluid. Conflict of interest statement Authors conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received March 12, 213; accepted September 6, 213; previously published online October 14, 213 References 1. Diwan PV, Karwande I, Margaret I, Sattur PB. Pharmacology and biochemical evaluation of Tridax procumbens on inflammation. Ind Pharmacol 1989;21: Udupa SL, Udupa AL, Lalkarni DR. Influence of Tridax procumbens on lysl-oxidase activity and wound healing. Planta Med 1991;57: Salahdeen HM, Yemitan OK, Alada AR. Effect of aqueous leaf extract of Tridax procumbens on blood pressure and heart rate in rats. Afri J Biomed Res 24;7: Ikewuchi JC, Onyeike EN, Uwakwe AA, Ikewuchi CC. Effect of aqueous extract of the leaves of Tridax procumbens Linn on blood pressure components and pulse rates of subchronic salt-loaded rats. Pacific J Sci Technol 211;12: Salahdeen HM, Murtala BA. Vasorelaxant effects of aqueous leaf extract of Tridax procumbens on aortic smooth muscle isolated from the rat. J Smooth Muscle Res 212;48: Salahdeen HM, Idowu GO, Murtala BA. Endothelium-dependent and independent vasorelaxant effects of aqueous extract of Tridax procumbens Lin leaf in rat aortic rings. Ind J Exp Biol 212;5: Bohr DF. Vascular smooth muscle: dual effect of calcium. Science 1963;139: Somlyo AP, Somlyo AV. Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J Pharmacol Exp Ther 1968;159: Bohr DF. Vascular smooth muscle updated. Circ Res 1973;32: Hari M, Sato K, Miyamoto S, Ozaki H, Karaka H. Different pathways of calcium sensitization activated by receptor agonists and phorbol esters in vascular smooth muscle. Br J Pharmacol 1993;11: Ebeigbe AB, Aloamaka CP. Mechanism of hydralazineinduced relaxation of arterial smooth muscle. Cardiovasc Res 1985;19: Sofola OA, Obiefuna PC, Adegunloye BJ. Contractile response of normotensive rat aorta to serum from salt-loaded

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