Mechanism of vasorelaxation induced by Achillea wilhelmsii in rat isolated thoracic aorta.

BACKGROUND: Achillea wilhelmsii (A. wilhelmsii) is used in Iraninan folk medicine for the treatment of hypertension; also, in previous reports, the hypotensive and antihypertensive effects of this plant have been indicated. The aim of the present study is to investigate the vasorelaxant effect of the hydroalcholic extract of A. wilhelmsii and its underlying mechanisms in isolated rat aorta.
MATERIALS AND METHODS: The effect of the hydroalcholic A. wilhelmsii extract was tested on the contractile response of Wistar rat aorta induced by potassium chloride (KCl) and phenylephrine (PE) using a pressure transducer that is connected to the PowerLab.
RESULTS: The cumulative concentrations of A. wilhelmsii (0.5-8 mg/ml) induced a vasorelaxation both in endothelium-intact and endothelium-denuded aortas precontracted by high K(+) (6 × 10(-2) M) or 10(-6) M PE. A. wilhelmsii, at a concentration of 4 mg/ml, reduced Ca(2+)-induced contraction (P < 0.001 vs. control) after PE or KCl had generated a stable contraction in the Ca(2+)-free solution. Furthermore, after incubation with diltiazem, the vasorelaxant effect of A. wilhelmsii reduced in the endothelium-denuded aortas precontracted by PE or KCl (P < 0.001 vs. control). In contrast, A. wilhelmsii-induced relaxation was not affected by glibenclamide, BaCl2, ruthenium red, methylene blue, or heparin.
CONCLUSIONS: The results showed that A. wilhelmsii had a vasorelaxation effect, which was not endothelium-dependent. The relaxation was mediated by inhibition of extracellular Ca(2+) influx through voltage- and receptor-operated Ca(2+) channels (VDDCs and ROCCs) in vascular smooth muscle cells.

INTRODUCTION

High blood pressure is the leading risk factor for mortality around the world, and lowering blood pressure greatly reduces the main risk of developing arterial coronary disease, heart failure, cerebral vascular disease, and renal damage.[1] The interest of the general public in the use of dietary herbs has risen exponentially, due to their presumed low toxicity and good therapeutic performance.

Achillea, is one of the most important genera of the Compositae family and comprises of more than 100 species. The pharmacological effects of the Achillea genus, such as, anti-inflammatory,[23] antibacterial,[45] antitumor,[6] antispasmodic,[78] choleretic,[9] antiulcer,[10] reducing gastric acidity and motility,[1112] and hepatoprotective,[8] have been reported.

In recent times, evidence of the cardiovascular and vasorelaxant effects of Achillea have been accumulated by several in vivo and in vitro studies.[1314151617] Achillea wilhelmsii (A. wilhelmsii) is the major species of the Achillea genus. It is grown in Iran (domestic name: Boomadaran) and widely used in Iranian traditional medicine to treat symptoms associated with gastrointestinal and cardiovascular disorders. Our previous studies have shown the hypotensive and cardiac-depressant effects of A. wilhelmsii.[1819] A. wilhelmsii contains components including carvacrol, luteolin, apigenin, and 1,8-cineole,[202122] which can influence the vascular smooth muscle tone. In many studies the vasorelaxant effects of carvacrol,[23] luteolin,[24] apigenin,[25] and 1,8-cineole[26] have been demonstrated. However, the mechanism/s of the vasorelaxant effect of A. wilhelmsii has not been clarified. Therefore, the present study has been carried out to examine the effects of the hydroalcholic extract of A. wilhelmsii on the vasomotor tone of the aortic rings, and its possible mechanism of action.

MATERIALS AND METHODS

Chemicals and drugs

All chemicals were of analytical grade (Merck). Phenylephrine hydrochloride (PE Hcl), acetylcholine (ACh), methylene blue, ruthenium red (RR), heparin (HP), and diltiazem were obtained from Sigma (Germany).

Plant material and preparation of the extract

The aerial part of A. wilhelmsii was collected in spring from the Khorasan Province, Neyshabour, Iran, and identified by the Ferdowsi University Herbarium (voucher No. 164-2218-2) and then dried at room temperature. Four hundred grams of the aerial parts of the plant were soaked in ethanol (50%) for 48 hours and filter paper was used to filter the solute after mixing. The solution was then dried using a 40°C oven for 72 hours. The dried extract was dissolved in distilled water to make 0.5, 1, 2, 4, and 8 mg/ml concentrations.

Animals

The experiment was conducted using 91 male Wistar rats (weighing 200-250 g). The animals were kept in a 22 ± 2°C temperature with a 12 hour light/dark cycle and fed with a standard diet and drinking tap water. All experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and care and, with institutional guidelines.

Preparation of rat aortas

After anesthesia with Ketamin (50 mg/kg), the animals were decapitated by guillotine. The chest was opened and the descending thoracic aorta was rapidly dissected out and immersed in chilled Krebs solution (composition (mM): NaCl 118.5, KCl 4.74, MgSO 4 1.18, NaHCO3 24.9, CaCl 2 2.5, and glucose 10. pH = 7.4) and gassed with carbogen (95% O2, 5% CO2). After the perivascular tissue was carefully removed, aortic rings approximately 5 mm in length were cut. The aortic rings were suspended in organ chambers containing 10 ml Krebs’ solution at 37° C, pH = 7.4, and aerated with 95% O2 + 5% CO2. After a resting tension of 2 g, the vessel segments were allowed to equilibrate for one hour. Changes in tension were recorded by isometric transducers connected to a data acquisition system (AD instrument, Australia). When required, the endothelium was removed by gently rubbing the intimal space with a thin metal rod. The absence of a functional endothelium was verified by the inability of ACh (10−5 M) to induce the relaxation of rings precontracted with PE (10−6 M).

Experimental procedure

Aortic contraction induced by Phenylephrine and Potassium Chloride

In this series of experiments, 10−6 M PE or 6 × 10−2 M KCl were used to induce a steady contraction in rings with the endothelium intact or denuded, and A. wilhelmsii was added cumulatively (0.5, 1, 2, 4, and 8 mg/ml). The A. wilhelmsii extract induced relaxation in the aortic rings, which was calculated as a percentage of the relaxation in response to PE and KCl.

A. wilhelmsii extract induced relaxation, the roles of influx of Ca2+ and Ca2+ channels

In the first set of these experiments, an attempt was made to verify that the relaxation induced by A. wilhelmsii involved Ca2+ influx. The endothelium-denuded aortic rings were washed four to five times with Ca2+-free Krebs’ solution (containing 5 × 10−5 M EGTA) before PE (10−6 M) or KCl (6 × 10−2 M) was applied, to produce a steady contraction, and then Ca2+ was added cumulatively to obtain a concentration-response curve (10−5 to 10−2 M). In the second set of experiments, the aim was to evaluate the roles of voltage-dependent calcium channels in extract-induced relaxation. Endothelium-denuded aortic rings were exposed to diltiazem (10−5 M), an L-type Ca2+ channel inhibitor, for 30 minutes, before the application of PE (10−6 M) or KCl (6 × 10−2 M), to induce a steady contraction; subsequently the A. wilhelmsii extract (4 mg/ml) was added to evoke a relaxation.

A. wilhelmsii extract induced relaxation and intracellular sources of Ca2+

In this set of experiments, the aim was to clarify whether the relaxation induced by A. wilhelmsii was related to the inhibition of intracellular Ca2+ release. Endothelium-denuded aortic rings were exposed to diltiazem (10−5 M) and Ruthenium red (10−5 M), a ryanodine receptor inhibitor (RR)[27] or heparin (50 mg/l). An IP3 receptor inhibitor (HP)[28] was added 30 minutes before the application of PE (10−6 M) to induce a steady contraction; subsequently the A. wilhelmsii extract (4 mg/ml) was added to evoke a relaxation in a separate experimental group.

A. wilhelmsii extract induced relaxation and guanylate cyclase

To examine the role of guanylate cyclase in the extract-induced relaxation, the aortic rings were rinsed and exposed to 10-5 M methylene blue, an inhibitor of the cGMP-mediated pathway, for 30 minutes before the application of 10−6 M PE to induce a steady contraction, and finally the effects of the cumulative concentrations of the extract (0.5, 1, 2, 4 and 8 mg/ml) were evaluated for 25 minutes.

A. wilhelmsii extract induced relaxation and K+ channels

To examine the role of K+ channels in the extract induced relaxation, the aortic rings were rinsed and exposed to glibenclamide (10-5 M), an inhibitor of the ATP-dependent K+ channels (KATP) and barium chloride (4 × 10−3 M), an inhibitor of the inward rectifier K+ channels (KIR), for 30 minutes before the application of 10−6 M PE, to induce a steady contraction, and finally the effects of the cumulative concentrations of the extract (0.5, 1, 2, 4 and 8 mg/ml) were evaluated for 25 minutes.

Statistical analysis

All data are expressed as mean ± S.E.M. Student's t-test was used to compare the data. Curves were compared using one-way ANOVA followed by the Tukey's test. P-values less than 0.05 were considered to be statistically significant.

RESULTS

Effect of A. wilhelmsii on phenylephrine and potassium chloride contracted aorta

Figure 1 shows the effect of the cumulative concentrations of the A. wilhelmsii extract (0.5, 1, 2, 4 and 8 mg/ml) on aortic smooth muscle contracted by KCl (6 × 10−2 M) and PE (10-6 M) in the intact and denuded endothelium.

Figure 1

Effect of cumulative concentrations of A. wilhelmsii extract (0.5, 1, 2, 4, and 8 mg/ml) on phenylephrine (PE) (10−6 M) (a) and KCl (6 × 10−2 M) (b) precontracted rat aortic rings with (E+) or without (E-) endothelium. Data are expressed as mean ± S.E.M., using the unpaired t-test (n = 7); *P < 0.05, **P < 0.01, ***P < 0.001, compared to the baseline

Extract-induced vasorelaxation incidents at 0.5, 1, 2, 4, and 8 mg/ml in intact and denuded rings contracted with KCl were 13 ± 1.2%, 17.8 ± 3.4%, 36 ± 5.9%, 50 ± 4.2%, and 54 ± 4.3%, and 10 ± 2.4%, 12.4 ± 3.1%, 33 ± 3%, 49 ± 5%, and 52 ± 3.4%, respectively [Figure 1a] compared to PE, where they were, 9.8 ± 1.4%, 11.5 ± 2.3%, 29.7 ± 3.1%, 51.3 ± 2.6, and 50 ± 2.4% and 13.7 ± 2.01, 16.2 ± 2.7, 32.3 ± 3.2%, 52.8 ± 2.6%, and 51.2 ± 3.7%, respectively [Figure 1b].

Effect of A. wilhelmsii on extracellular Ca2+-induced contraction and Ca2+ channels

The cumulative addition of Ca2+ in a Ca2+-free medium containing PE or KCl induced a concentration-dependent contraction of aortic rings. Pre-incubation of the rings with 4 mg/ml of A. wilhelmsii significantly inhibited the Ca2+-induced contraction in both KCl [Figure 2a] and PE [Figure 2b] constricted rings. In the endothelium-denuded rings pretreated for 30 minutes with diltiazem (10−5 M) and subsequently contracted by PE or KCl, the relaxant effect of A. wilhelmsii (4 mg/ml) was significantly reduced (P < 0.001) [Figure 3].

Figure 2

Effect of A. wilhelmsii extract at 4 mg/ml on the Ca2+-induced (10−5 to 10−3 M) contraction of rat aortic rings without endothelium, pretreated with phenylephrine (PE) (10−6 M) (a) and KCl (6 × 10−2 M) (b). Data are expressed as mean ± S.E.M., using unpaired t-test (n = 7); **P < 0.01, ***P < 0.001 compared to the control

Figure 3

Effects of A. wilhelmsii extract, at 4 mg/ml, on the endothelium-denuded rat aortic rings contracted with phenylephrine (PE) (10−6 M) or KCl (6 × 10−2 M), after diltiazem (10−5 M) pretreatment. Data are expressed as mean ± S.E.M., using one way ANOVA (n = 7); ***P < 0.001 compared to PE, +++P < 0.001 compared to KCl

Effect of A. wilhelmsii on intracellular sources of Ca2+

The results of 30 minutes of pre-incubation of the endothelium-denuded aortic rings with diltiazem and heparin or RR, with subsequent contraction by PE showed that the relaxant effect of A. wilhelmsii (4 mg/ml) was the same as diltiazem [Figure 4].

Figure 4

Effects of A. wilhelmsii extract at 4 mg/ml on endothelium-denuded rat aortic rings contracted with phenylephrine (PE) (10−6 M), after diltiazem (10−5 M), heparin (50 mg/l), and Ruthenium red (10−5 M) pretreatment. Data are expressed as mean ± S.E.M., using one way ANOVA (n = 7). +Dil: Denuded endothelium pretreated with diltiazem, +Dil + HP: denuded endothelium pretreated with diltiazem and heparin, +Dil+RR: denuded endothelium pretreated with diltiazem and Ruthenium red. ***P < 0.001 compared to PE

Effect of A. wilhelmsii on guanylate cyclase

The relaxant effect of the cumulative concentrations of A. wilhelmsii showed no difference when compared with A. wilhelmsii alone after 30 minutes of pre-incubation of the aortic rings with methylene blue, with subsequent contraction by PE [Figure 5a].

Figure 5

Effect of cumulative concentrations of A. wilhelmsii extract (0.5, 1, 2, 4 and 8 mg/ml) on rat aortic rings contracted with phenylephrine (PE) (10−6 M) after being pretreated with a guanylate cyclase inhibitor methylene blue (10-5 M)(a) and K+ channel inhibitor, glibenclamide (10−5 M) and barium chloride (4 × 10−3 M)(b). Data are expressed as means ± S.E.M., using unpaired t-test (a) and one way ANOVA (b) (n = 7). +En: With endothelium, MB: Methylene blue, Gly: glibenclamide, BaCl2: Barium chloride

Effect of A. wilhelmsii on K+ channels

After 30 minutes of pre-incubation of the aortic rings with glibenclamide or barium chloride and with a subsequent contraction by PE, the relaxant effect of the cumulative concentrations of A. wilhelmsii showed no difference when compared with A. wilhelmsii alone [Figure 5b].

DISCUSSION

In these experiments we found that A. wilhelmsii evoked relaxation in aortic rings precontracted by KCl and PE, regardless of the presence or absence of the endothelium. This indicated that the action of A. wilhelmsii to induce relaxation was directly on the vascular smooth muscle cells (VSMCs) and not on the endothelium-derived vasodilator factors, such as, NO and prostacyclin.

Ca2+ is a critical factor in the excitation-contraction coupling in smooth muscle cells.[2930] Influx of extracellular Ca2+ through receptor-operated Ca2+ channels (ROCCs), voltage-dependent Ca2+ channels (VDCCs), and release of Ca2+ from the sarcoplasmic reticulum by activation of 1,4,5 triphosphate inositol (IP3) and ryanodine receptors (RYR),[313233] result in increased intracellular Ca2+, which causes contraction.

Our results showed that the relaxing activity of A. wilhelmsii is evident after both PE- and KCl-induced contraction. PE is an alpha adrenergic agonist that induces VSMC contractions by a Ca2+ influx through the ROCCs and by the release of intracellular Ca2+ from the sarcoplasmic reticulum after activation of IP3 receptors (IP3R).[313334] By contrast, the contraction elicited by KCl mainly results from the influx of extracellular Ca2+ induced by depolarization of the cell membrane and subsequent opening of the VDCCs.[32]

To determine whether A. wilhelmsii modified the extracellular Ca2+ influx, experiments were conducted on rings contracted with PE or KCl in a Ca2+-free Krebs solution, in which Ca2+ was added subsequently. Our data reporting that A. wilhelmsii decreased Ca2+-induced contractions after both PE- and KCl-induced contractions argue that the blockade of both ROCCs and VDCCs are a part of the vasodilating effects of A. wilhelmsii. These results were verified by PE- and KCl-induced contractions in the presence of diltiazem, in which the vasorelaxant effect of A. wilhelmsii decreased significantly.

The results of this study are consistent with the previous findings in the gastrointestinal or vascular smooth muscles. Previous studies on the effect of various species of Achillea genus on gastrointestinal smooth muscle have revealed its antispasmodic effects.[735] Yaeesh and colleagues showed that A. millefolium inhibits calcium channels and its antispasmodic effect contributes to the antagonistic roles on VDCCs.[8] The vasorelaxant effect of A. millefolium, which is mainly mediated by calcium channel inhibition was also shown.[16] Furthermore, we previously demonstrated the cardiac depressant and hypotensive effects of A. wilhelmsii[19] and the negative inotropic and chronotropic effect of A. millefolium in isolated heart.[17]

To investigate whether A. wilhelmsii could exert its vasorelaxant effects by interfering with the calcium release from the intracellular source the experiments were conducted on rings precontracted with PE. It is well known that the PE-induced release of intracellular calcium is attributable to the receptor-mediated formation of IP3.[36] Heparin and RR did not affect the vasorelaxant effect of A. wilhelmsii. These results indicate the IP3 signaling pathway and ryanodine receptors have any role in the vasorelaxant effect of A. wilhelmsii.

It is well established that cGMP provides the signal that elicits vascular relaxation via cGMP-dependent protein kinase signaling. Activation of the soluble guanylate cyclase (sGC) in the VSMCs results in an increase in intracellular cGMP levels, which elicits cGMP-dependent protein kinase (PKG) signaling. It has been reported that PKG inhibits Ca2+ influx, augments Ca2+ sequestration, and decreases the sensitivity of contractile elements to Ca2+.[37] Our results showed that Methylene blue did not reduce the relaxation induced by A. wilhelmsii, therefore, it suggests that the vascular relaxation evoked by A. wilhelmsii was not mediated by cGMP signaling. It is in favor of the endothelium-independent vasorelaxant effect of A. wilhelmsii.

Besides Ca2+ channels, K+ channels contribute to the regulation of the membrane potential in electrically excitable cells, including VSMCs.[38] Membrane hyperpolarization due to an efflux of K+ results from the opening of the K+ channels in the VSMCs. This effect is followed by the closure of voltage-dependent Ca2+ channels, leading to the reduction in Ca2+ entry and vasodilation.[32] VSMCs express both KATP and KIR.[3940] In the present study, the blockade of the KATP or KIR channel with glibenclamide or BaCl2, respectively, did not affect the relaxing properties of A. wilhelmsii, which indicated that these K+ channels may not be involved in A. wilhelmsii-induced vasorelaxation.

Moreover, A. wilhelmsii contains important ingredients, such as, carvacrol, luteolin, apigenin, and 1,8 cineole, which can influence the vascular smooth muscle tone. In many studies the antispasmodic and vasorelaxant effects of carvacrol,[234142] luteolin,[2443] apigenin,[25] and 1, 8 cineole[2644] have been demonstrated. Luteolin has a vasorelaxant effect by the inhibition of sarcolemmal Ca2+ channels, release from the intracellular Ca2+ stores, and activation of K+ channels.[23]

Taken together, this study demonstrates that A. wilhelmsii exhibits vasodilating activity. The relaxant effect does not depend on the presence of the endothelium. The extract acts directly on VSMCs and relaxation is mainly related to the inhibition of extracellular Ca2+ influx through ROCCs and VDCCs.