Cinnamomi ramulus Ethanol Extract Exerts Vasorelaxation through Inhibition of Ca2+ Influx and Ca2+ Release in Rat Aorta

Contraction of vascular smooth muscle cells depends on the induction of cytosolic calcium ion (Ca2+) due to either Ca2+ influx through voltage-gated Ca2+ channels or to receptor-mediated Ca2+ release from the sarcoplasmic reticulum. The present study investigated the vasorelaxation effect of Cinnamomi ramulus ethanol extract (CRE) and the possible mechanisms in rat aorta. CRE (0.1 mg/mL) relaxed vasoconstriction induced by phenylephrine (PE; 1 μM) and angiotensin II (5 μM). Preincubation with CRE significantly reduced the rat aortic contraction by addition of CaCl2 in Ca2+-free Krebs solution and FPL64176 (10 μM). Pretreatment with nifedipine (100 μM) or verapamil (1 μM) significantly reduced the CRE-mediated vasorelaxation of PE-induced vascular contraction. In addition, CRE also relaxed the vascular contraction caused by m-3M3FBS (5 μg/mL), but U73122 (10 μM) significantly inhibited the vasorelaxation of PE precontracted aortic rings. Furthermore, CRE significantly reduced the magnitude of PE- and caffeine (30 mM)-induced transient contraction. In vascular strips, CRE downregulated the expression levels of phosphorylated PLC and phosphoinositide 3-kinase elevated by PE or m-3M3FBS. These results suggest that CRE relaxes vascular smooth muscle through the inhibition of both Ca2+ influx via L-type Ca2+ channel and inositol triphosphate-induced Ca2+ release from the sarcoplasmic reticulum.


Introduction
Increasing cytosolic calcium ion (Ca 2+ ) concentration is essential for the contraction of smooth muscle cells. The increase results from the influx of Ca 2+ through the plasma membrane and release of Ca 2+ from intracellular stores, mainly the sarcoplasmic reticulum (SR) [1][2][3][4].
The herb Cinnamomi ramulus (CR) has traditionally been used in Asia and Europe to treat maladies involving blood circulation and inflammation. In one study, an aqueous extract of CR ameliorated sucrose-induced blood pressure elevation in spontaneously hypertensive rats [18]. Recently, we reported that CR ethanol extract (CRE) reduces vascular contraction through the inhibition of voltage-dependent Ca 2+ channels [19]. However, the possible mechanisms of CRE were not elucidated. The present study explored the suggestion that the vasodilatory effect of CRE is related to Ca 2+ -dependent mechanisms in rat aorta.

Preparation of CRE.
Dried CR (100 g) was extracted with 500 mL of 70% ethanol by heating at 75 • C for 3 h. The extract was filtered through Whatman filter paper (Whatman International, Maidstone, UK) to remove the insoluble materials. After filtration, the extracts were concentrated by rotary evaporation using a model VV2000 apparatus (Heidolph, Walpersdorfer, Germany) at a temperature of 75 • C and then dried using a model FD8508S freeze dryer (Ilshin, Busan, Korea). The yield of dry matter from the extracts was approximately 2.1%. The material was stored at 4 • C until use. The EC 50 value of 0.1 mg/mL CRE was used in all experiments. In a previous research [20], cinnamaldehyde and coumarin were analyzed as main compounds of CRE by gas chromatography-mass spectrometry. Also, cinnamaldehyde was known as major active compound of CR for vasodilation, antitumor, and antifungal activity.

Preparation of Thoracic Aortic
Rings. All procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of Dongguk University. A previously described procedure [21] was employed with some modification. Briefly, rats were sacrificed and their thoracic aortas were immediately excised and immersed in ice-cold Krebs solution (115.0 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgCl 2 , 25.0 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , and 10.0 mM dextrose). The aortas were cleaned of all adherent connective tissue and cut into 3 mm long ring segments. Endothelium was removed from the internal surface of each segment by gentle rubbing with forceps.

Organ Bath Study.
Tension was measured by a modification of a previously described procedure [22]. Briefly, two stainless-steel triangles were inserted through each vessel ring. One triangle was anchored to a stationary support and the other was connected to a FT03 isometric force transducer (Grass, Quincy, Mass, USA). Each vessel ring was incubated in a water-jacketed organ bath (10 mL) that was maintained at 37 • C and aerated with a mixture of 95% O 2 and 5% CO 2 . Each ring was stretched passively by imposing the optimal resting tension of approximately 2.0 g, which was maintained throughout the experiment. Each endothelium-free aortic ring was allowed to equilibrate in the organ bath for at least 50 min before the experiment involving the contractile response to 5 μM Ang II, 1 μM PE, 10 μM FPL64176, 5 μg/mL m-3M3FBS, or 30 mM caffeine. Endothelium-free rings were used because preliminary experiments (data not shown) established that CRE relaxes vascular constriction in an endothelium-independent manner. The denudation of endothelium was assessed by treating the rings with 1 μM acetylcholine. Isometric tension was recorded using a PowerLab/8SP computerized data acquisition system (ADInstruments, Castle Hill, NSW, Australia). The influence of CRE on extracellular Ca 2+ influx was studied in Ca 2+ -free Krebs solution. After equilibration of the ring in Ca 2+free Krebs solution containing 60 mM KCl, cumulative doses of CaCl 2 were added (0.3, 0.6, 1, 1.5, 2.5, 5, and 10 mM, in order) with preincubation of CRE in organ bath. The CaCl 2 dose-dependent maximum constriction of the aortic ring with 60 mM KCl in Ca 2+ -free Krebs solution was expressed as 100%. To determine the influence of CRE on Ca 2+ influx through the L-type Ca 2+ channel, aortic rings were pretreated with nifedipine or verapamil before PE contraction, and were preincubated with CRE before contraction by FPL64176. To investigate the inhibitory effect of CRE on intracellular Ca 2+ release by PE in Ca 2+ -free conditions, and by caffeine in normal Krebs solution, the transient contraction of CRE preincubated aortic rings was measured. To further investigate the relationship with the PLC pathway, aortic rings were constricted with m-3M3FBS, and were preincubated with U73122 prior to contraction by PE. When the constriction reached a plateau, CRE was added to the organ bath.

Preparation of Aorta Protein Extracts and Western Blot
Analysis. A previously described protocol [22] was used for preparation of protein extract with some modifications. Briefly, endothelium-free aortic rings were contracted with 1 μM PE or 5 μg/mL m-3M3FBS, and then treated with CRE for 30 min. The aortic rings were quick frozen by immersion in acetone containing 10% trichloroacetic acid (TCA) and 10 mM dithiothreitol (DTT) precooled to −80 • C. When used, recovered samples were homogenized in buffer containing 320 mM sucrose, 50 mM Tris, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, and the following protease inhibitors: leupeptin (10 μg/mL), trypsin (10 μg/mL), aprotinin (2 μg/mL), or phenylmethylsulphonyl fluoride (100 μg/mL). The protein samples were electrophoresed and the resolved proteins were transferred to a nitrocellulose membrane. The membrane was incubated with primary antibodies and then treated with horseradish peroxidaseconjugated anti-rabbit IgG as a secondary antibody. All bands were detected using an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK).

Statistical Analyses.
Each set of experiments was done at least three times and results are presented as the mean ± SD. The statistical significance of differences between mean values was assessed with Student's t-test or ANOVA. Test values that resulted in P < 0.05 were considered as significant.

Vasorelaxation Effect of CRE on PE-or Ang II-induced
Constricted Aorta. Ang II increases the intracellular Ca 2+ concentration in vascular smooth muscle cells through a sequence of events following activation of Ang II type 1 receptor and L-type calcium channels [6,7]. PE-or Ang II-induced contraction was significantly dilated by 53.6 ± 7.8% and 66.0 ± 5.4%, respectively, as compared to maximal tension (Figure 1), indicating that that CRE-mediated vasodilation may be related to decreased intracellular Ca 2+ concentration.

Effect of CRE on Ca 2+
Influx through L-type Ca 2+ -Channels. To discern the effect of CRE on the L-type calcium channel, the influence of the L-type calcium channel blocker nifedipine (100 μM) or verapamil (1 μM), and the L-type calcium channel activator FPL64176 (10 μM) on vasorelaxation of CRE against PE-induced contraction of aortic rings was measured. Pretreatment of aortic rings with nifedipine or verapamil significantly inhibited the relaxant effect of CRE (Figure 3(a)). Previous studies have shown that FPL64176 increases extracellular Ca 2+ entry, thereby enhancing the cytosolic Ca 2+ concentration [23,24]. Presently, FPL64176 induced contraction, which plateaued at 3.75 ± 0.22 g in 30 min, was inhibited by 2.4 ± 0.1 g with preincubation of CRE (Figure 3(b)).

Effect of CRE on Ca 2+
Release from SR. To assess whether CRE is involved in Ca 2+ release-mediated vasoconstriction from intracellular stores, the transient contraction by PE or caffeine was examined in CRE preincubated aortic rings. Preincubation reduced the magnitude of contraction by PE from 0.32 ± 0.6 g to 0.1 ± 0.6 g (Figure 4(a)). The transient contraction induced by 30 mM caffeine was also reduced by CRE pretreatment (Figure 4(b)).

Effect of CRE on PLC Pathway.
To evaluate whether the relaxant effect of CRE was involved in the PLC pathway, the PLC pathway inhibitor U73122 and activator m-3M3FBS were used. U73122 pretreatment significantly inhibited the relaxant effect of CRE on PE-induced contraction from 56.9 ± 2.7% to 8.5 ± 1.3% (Figure 5(a)). m-3M3FBS (5 μg/mL)-induced contraction was relaxed significantly with CRE treatment (Figure 5(b)).

Discussion and Conclusions
Traditionally, Cinnamomum cassia has been used as a medicinal herb. Its bark and twig are known as Cinnamomi cortex (CC) and Cinnamomi ramulus (CR), respectively. CC inhibits Helicobacter pylori [25] and ameliorates sucroseinduced blood pressure elevation in spontaneously hypertensive rats [18]. Furthermore, CRE exerts an endotheliumindependent vasodilatory response through inhibition of voltage-dependent Ca 2+ channels [19]. However, the mechanism by which CR exerts vasodilation remains to be elucidated. The present study investigated the vasodilatory effect of CRE resulting from the inhibition of both Ca 2+ influx and release in rat aorta.
PE or Ang II stimulate PLC isoforms to generate IP 3 through the activation of G proteins, causing release of activator Ca 2+ from SR [9,10,[14][15][16]. Presently, CRE markedly and similarly relaxed aortic rings that were precontracted with PE or AngII. These results suggest that CRE-mediated vasodilation may be involved in the regulation of Ca 2+ mobilization. To assess this, the regulation of Ca 2+ influx and release was investigated. Firstly, whether CRE actually inhibits extracellular Ca 2+ influx or not, we measured the * * * * * * *  The SR is the major source of Ca 2+ release into the cytosol [1,16,17]. This Ca 2+ release is induced by the IP 3 second messenger, which is generated by PLC activation [17]. Ca 2+ release from the SR is considered to be the initial mechanism in agonists such as PE-and Ang II-induced vasoconstriction [6,17]. PE-induced transient constriction is dependent on Ca 2+ release from the SR through the IP 3 signal pathway in Ca 2+ -free Krebs solution [26]; however, caffeine is dependent on Ca 2+ -induced Ca 2+ release from the SR [27,28]. To demonstrate the effects of CRE on Ca 2+ release from the SR, transient contractions induced by PE in Ca 2+ -free Krebs solution and induced by caffeine in normal Krebs solution were investigated. CRE significantly reduced the magnitudes of transient contraction by PE and caffeine, suggesting CRE inhibits Ca 2+ release from the SR by blocking the IP 3 -induced Ca 2+ release and Ca 2+ -induced Ca 2+ release mechanisms.
Pretreatment with the PLC inhibitor U73122 significantly reduced the vasorelaxation of CRE on PE-induced vasoconstriction, and CRE relaxed m-3M3FBS-induced vasoconstriction. Additionally, we analyzed the expression levels of the intracellular signaling regulator proteins PI3K and PLC. PI3K generates various 3-phosphorylated phosphoinositides through activation by G-proteins, especially PI(3,4,5)P 3 stimulates the L-type Ca 2+ channel that plays an important role in the regulation of vascular tone [8,11,12]. On the other hand, PLC formats the two potent second messengers IP 3 and DAG. Especially, IP 3 induces the activation of IP 3 receptor on the SR membrane, opening a calcium channel, resulting in the release of Ca 2+ into the cytosol [14,17]. Presently, PE-or m-3M3FBS-induced phosphorylation of PLC and upregulation of PI3K/p85 protein expression were inhibited by CRE ( Figure 6). The collective data supports the idea that CRE dilates vascular contraction through the inhibition of both Ca 2+ influx via the L-type Ca 2+ channel and IP 3 -induced Ca 2+ release from the SR.
In conclusion, the data supports the vasorelaxation of CRE through the inhibition of Ca 2+ influx and Ca 2+ release. Therefore, CRE may be useful as a drug for the treatment and prevention of high blood pressure associated with Ca 2+dependent contraction of smooth muscle.