Aromatherapy is widely known to be useful for the prevention and treatment of diseases by using the essential oils. A potential effect on arteriosclerosis and hypertension has been suggested from experimental reports on Lavendula angustifolia, a lavender essential oil.1 However, underlying mechanisms of action remain to be clarified.
There are roughly 2 mechanisms relaxing vascular smooth muscle: One is relaxation depending on the vascular endothelial cells, which release several relaxing factors such as nitric oxide (NO),2 prostacyclin (PGI2), and an endothelium-derived hyperpolarizing factor (EDHF).3 The other is a relaxation occurring directly on vascular smooth muscle. Contraction and relaxation of vascular smooth muscle are regulated by myosin light chain (MLC) kinase and MLC phosphatase. MLC kinase mediates MLC phosphorylation and causes contraction in a Ca2+/calmodulin-dependent manner.4 Conversely, MLC phosphatase mediates dephosphorylation of phosphorylated MLC and causes relaxation.5,6
In a preliminary experiment, we found that lavender essential oil relaxed rabbit carotid artery. Lavender essential oil is obtained by the steam distillation of the leaf and tip of the lavender flower and is composed of linalyl acetate, linalol, cis-β-ocimen, lavandulyal acetate, terpinene-4-ol and other minor components. Thus, we designed the present experiment to investigate whether linalyl acetate as a major ingredient of lavender essential oil exerts relaxing activity in the rabbit carotid artery specimens, and if so, to analyze the mechanisms pharmacologically and biochemically.
All experiments were performed in accordance with the Animal Welfare Regulations of Tokyo Medical and Dental University and the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society. Japanese White male rabbits weighing 2 to 3 kg were used in the present study.
Lavendula angustifolia was used as pure lavender essential oil with the following composition: linalyl acetate 38.4%, linalol 32.8%, cis-β-ocimene 4.4%, trans-β-caryophyllene 4.2%, lavandulyl acetate 3.3%, and terpinene-4-ol 2.9%. Because only linalol and linalyl acetate are commercially available (Wako Pure Chemicals, Osaka, Japan) and are major ingredients of lavender essential oil, these 2 substances were used for the analyses. Phenylephrine hydrocholoride was purchased from Kowa Pharmaceutical (Tokyo). Acetylcholine chloride (ACh) was purchased from Daiichi Pharmaceutical (Tokyo). NG-nitro-L-arginine (nitroarginine), indomethacin, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) were from Sigma (St. Louis, MO). Tetrathylamonium came from Nacalai Tesque (Kyoto, Japan). ML-9 came from Hokuriku Seiyaku (Fukui, Japan), and calyculin A came from Calbiochem (La Jolla, CA). Anti-MLC20 rabbit polyclonal immunoglobulin G (IgG) antibody was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), peroxidase-linked donkey anti-rabbit IgG and enhanced chemiluminescence Western blotting detection reagents were from Amersham (Buckinghamshire, England). All other chemicals were reagent grade. Phenylephrine, ACh, nitroarginine, and indomethacin were dissolved in distilled water, whereas lavender essential oil, linalol, linalyl acetate, ODQ, and indomethacin were dissolved in dimethylsulfoxide (DMSO). Lavender essential oil, linalol, and linalyl acetate were prepared immediately before use and handled in light-safe conditions. The concentrations of lavender essential oil, linalol, and linalyl acetate were calculated on the basis of the volume, their specific gravity (0.833, 0.860, and 0.904, respectively), and molecular weight, which for linalol and linalyl acetate were 154.25 and 196.29, respectively.
Isometric Tension Measurement
Rabbits were sacrificed by exsanguination from femoral arteries under anesthesia with sodium pentobarbital (25 mg/kg, IV). The carotid arteries were rapidly removed and placed in ice-cold modified Krebs' solution of the following composition (in mmol/L): 118.0 NaCl, 4.7 KCl, 1.2 MgSO47H2O, 1.2 KH2PO4, 25.0 NaHCO3, 2.5 CaCl22H2O, and 10.0 glucose (pH 7.4). The vessel was dissected free of adipose and connective tissues and cut into transverse strips (1-2 mm wide and 3-4 mm long). Both endothelium-intact (E+) and endothelium-denuded strips (E−) were used for the experiments. Vascular endothelium was denuded by rubbing the surface with a moistened cotton swab. The transverse strips were mounted in 10-mL organ chambers filled with modified Krebs' solution, maintained at 37°C, and oxygenated continuously with 95% O2 and 5% CO2. Changes in isometric tension were recorded on a pen-writing oscillograph through force-displacement transducer. After 1 h of equilibration under a resting tension of 1 g, the transverse strips were contracted by adding 10−5 mol/L phenylephrine. The presence or absence of the functioning endothelium was then confirmed by adding 10−6 mol/L ACh. A contraction of ≥750 mg in response to phenylephrine was considered viable, relaxation of ≥60% in response to ACh was considered endothelium-intact, whereas relaxation <20% was considered successfully denuded. Transverse strips were exposed to lavender essential oil, linalol, or linalyl acetate during the phenylephrine-induced contraction to test the potency to relax with these agents. Involvement of the endothelium in the linalyl acetate-induced relaxation was also examined by comparing the magnitude of relaxation between E+ and E− specimens. After washing out and returning to the baseline tension, specimens were pretreated with vehicle or inhibitors including nitroarginine (NO synthase inhibitor), tetraethylammonium (nonselective K channel blocker), indomethacin (cyclooxygenase inhibitor), ODQ (soluble guanylyl cyclase inhibitor), ML-9 (myosin light chain kinase inhibitor), and calyculin A (myosin light chain phosphatase inhibitor) for 20 min before the phenylephrine-induced contraction to characterize the relaxation with linalyl acetate.
Measurement of MLC Phosphorylation
Carotid artery specimens were pretreated with vehicle or linalyl acetate at a concentration of 3 × 10−4 mol/L for 20 min to analyze the inhibitory effect on the phenylephrine-induced contraction. After treatment with each agent, specimens were immersed in acetone containing 10% trichloroacetic acid and 10 mmol/L dithiothreitol (DTT) cooled in dry ice immediately before, 1, 2, and 5 min after the addition of phenylephrine. The frozen specimens were washed twice with acetone containing 10 mmol/L DTT to remove the trichloroacetic acid and dried as described by Seto et al.7,8 The dried specimens were kept at -80°C. Changes in isometric tension were simultaneously recorded in the same way described above. Frozen specimens were placed at room temperature, cut into small pieces, and homogenized with a plastic homogenizer in 50 μL of glycerol-polyacrylamide gel electrophoresis (PAGE) sample buffer containing 20 mmol/L Tris base, 22 mmol/L glycine (pH 8.6), 10 mmol/L DTT, 8 mol/L urea, and 0.1% bromophenol blue.7-9 The samples were then passed through a 0.45-μm membrane filter (Millipore, Billerica, MA) to remove undissolved substances.9 The urea-solubilized samples (2.5 μL) were electrophoresed at 400 V and 4°C for 4 h in polyacrylamide gels (5 × 9 cm) containing 12.5% acrylamide, 0.75% bisacrylamide, 40% glycerol, 20 mmol/L Tris base, and 22 mmol/L glycine, pH 8.6. Before loading the samples, pre-electrophoresis at 400 V for 1 h was performed.10 The reservoir buffer contained 20 mmol/L Tris base, 22 mmol/L glycine, and 1 mmol/L DTT, pH 8.6.7,11 Proteins from glycerol-PAGE gels were transferred to polyvinylidene fluoride (PVDF) membrane in a buffer containing 20 mmol/L Tris base, 20% methanol, pH 7.5,10 with application of 200 mA for 2 h at 4°C. Transferred membranes were incubated with a blocking reagent at 4°C overnight and then with a specific anti-MLC antibody (1:7000 in blocking solution). Membranes were washed with Tris-buffered saline containing 1.0% Tween-20 (TBS-T) and incubated with horseradish peroxidase-conjugated anti-rabbit IgG for 1 h at room temperature (1:5000 in blocking solution). The bands were visualized by the addition of enhanced chemiluminescence detection reagents and exposed to x-ray film. The relative amount of phosphorylated MLC to the total MLC was quantified by scanning laser densitometry (Scion image).
Results are given as the mean ± SEM. When percentage of relaxation is mentioned it refers to the percentage of the phenylephrine-induced contraction. The concentration to produce half of the maximum relaxation (EC50) and maximum relaxation (Emax) were obtained from concentration-relaxation curves. The extent of relaxation during the phenylephrine contraction was expressed as percentage decrease to the steady-state contraction induced by phenylephrine. Results were compared using paired and unpaired t test. P < 0.05 was regarded as significantly different.
Relaxation with Lavender Essential Oil during the Phenylephrine-induced Contraction
The potencies of lavender essential oil to relax rabbit carotid artery specimens were tested. After resting tension of each specimen was stabilized, sustained and stable contraction of 750 ± 108 mg (n = 5) was maintained by adding 10−5 mol/L phenylephrine. When the contraction had stabilized (10 min after the addition of phenylephrine), lavender essential oil was added in concentrations from 2.6 × 10−8 to 2.6 × 10−7 g/mL (Fig. 1), resulting in a sustained and progressive relaxation within 90 min. Lavender essential oil produced a concentration-dependent relaxation with the maximum value of 105.7% ± 2.7% (n = 5) at a concentration of 2.6 × 10−7 g/mL. The EC50 value for lavender essential oil was determined to be 8.9 × 10−2 g/mL. At this concentration of lavender essential oil solution, concentrations of linalol and linalyl acetate were estimated to be 1.85 × 10−4 and 1.78 × 10−4 mol/L, respectively. The relaxation caused by lavender essential oil was significantly greater (P < 0.005; n = 3) than the spontaneous relaxation that occurred after addition of vehicle (DMSO) without test compound.
Relaxation with Linalyl Acetate during the Phenylephrine-induced Contraction
Linalyl acetate produced a sustained and progressive relaxation during the contraction caused by phenylephrine (Fig. 2), and the relaxation was significantly greater than the relaxation in the control (0.3% DMSO). The contraction caused by 10−5 mol/L phenylephrine was reversible after washing out of linalyl acetate (data not shown). The Emax value for linalyl acetate was determined to be 88.8% ± 0.6% in E+ (n = 7) and 90.4% ± 1.2% in E− specimens (n = 6) at a concentration of 10-3 mol/L each. The EC50 values was determined to be 3.6 × 10−4 and 4.3 × 10−4 mol/L, for E+ and E− specimens, respectively. Linalyl acetate at a concentration of 3 × 10−4 mol/L produced a definite relaxation of 41.1% ± 5.8% (n = 7) in E+ and 23.1% ± 2.8% (n = 6) for E− specimens; thus this concentration was chosen to pharmacologically analyze the relaxing effect of linalyl acetate. Linalol also produced relaxation during the phenylephrine-induced contraction, whereas the agent was less effective than linalyl acetate in producing relaxation. The extent of relaxation with 10-3 mol/L linalol was determined to be 55.5% ± 4.7% (n = 7) in E+ and 61.3% ± 6.3% (n = 6) in E− specimens.
Pharmacological Characterization of the Endothelium-dependent Relaxation with Linalyl Acetate
Endothelium-dependent relaxation with linalyl acetate at a concentration of 3 × 10−4 mol/L was pharmacologically characterized in E+ specimens. As shown in Figure 3, 10−4 mol/L nitroarginine significantly attenuated the relaxation with linalyl acetate by 68.6% ± 5.7% of the control (P < 0.05, n = 10) without affecting the phenylephrine-induced contraction. In the presence of 3 × 10−5 mol/L ODQ, linalyl acetate-induced relaxation was 66.2% ± 7.2% of the control (n = 10), the value of which was significantly (P < 0.05) lower than the control (n = 14). Tetraethylammonium 10−4 mol/L as a nonselective blocker of potassium channels and 10−5 mol/L indomethacin as a cyclooxygenase inhibitor did not affect relaxation significantly (70.0% ± 8.2% of the control, n = 4, and 70.0% ± 10.9% of the control, n = 4, respectively).
Pretreatment of linalyl acetate at a concentration of 3 × 10−4 mol/L significantly inhibited the phenylephrine-induced contraction at 2 and 5 min after the application of the agonist, with the contraction ratio being inhibited to 81.1% ± 3.9% (P < 0.05, n = 24) and 79.7% ± 4.8% (P < 0.05, n = 24) from 100% with phenylephrine alone (n = 4). These results are shown in Figure 4A, B.
For further analyses of the inhibitory effect of linalyl acetate on the contraction caused by phenylephrine in E− specimens, we measured MLC phosphorylation. The MLC phosphorylation ratio at 2 and 5 min after the application of phenylephrine was significantly decreased from 56.0% ± 3.2% and 49.8 ± 5.4% (n = 6) in phenylephrine alone to 44.7% ± 4.0% and 41.3% ± 3.4% (P < 0.05, n = 6) by the pretreatment with linalyl acetate, respectively, as shown in Figure 4C, D.
Pharmacological Analyses of the Endothelium-independent Relaxation with Linalyl Acetate
To investigate the endothelium-independent relaxation with linalyl acetate, ML-9 (3 × 10−5 mol/L), calyculin A (3.6 × 10−8 mol/L) or vehicle was applied before the phenylephrine-induced contraction, and its influence to the relaxation caused by 3 × 10−4 mol/L linalyl acetate during the contraction was analyzed in the E− specimens. Pretreatment with 3 × 10−5 mol/L ML-9 attenuated the phenylephrine-induced contraction, whereas it did not significantly affect the relaxation with linalyl acetate (118.8% ± 10.7% of the control, n = 4; Fig. 5). In the presence of 3.6 × 10−8 mol/L calyculin A, the linalyl acetate-induced relaxation was 56.0% ± 3.7% of the control (n = 4), which was significantly (P < 0.05) lower than the control (n = 8). Slight and sustained contraction (490 ± 102 mg, n = 4) was produced by adding 3.6 × 10−8 mol/L calyculin A.
Restoration with Calyculin A of the Linalyl Acetate-mediated Inhibition of Phenylephrine-induced Contraction and MLC Phosphorylation Ratio
For further analysis of the mechanism of the endothelium-independent relaxation with linalyl acetate, effects of calyculin A (3.6 × 10−8 mol/L) as an inhibitor of MLC phosphatase on the phenylephrine-induced contraction and the MLC phosphorylation ratio were determined in E− specimens. Pretreatment with calyculin A attenuated the inhibitory effect of linalyl acetate on the phenylephrine-induced contraction at 1, 2, and 5 min after the application of phenylephrine, with the contraction ratio being increased significantly to 127.9% ± 16.3% (P < 0.005, n = 4), 110.5% ± 6.2% (P < 0.05, n = 4), and 131.0% ± 13.1% (P < 0.005, n = 4) versus the corresponding inhibited values of 88.5% ± 3.1%, 81.1% ± 3.9%, and 79.7% ± 4.8% (n = 24) with linalyl acetate, respectively. As shown in Figure 6B and C, the MLC phosphorylation ratio at 1, 2, and 5 min after the application of phenylephrine was significantly increased from the respective values of 46.5% ± 1.3%, 46.2% ± 2.4%, and 39.9% ± 2.4% (n = 10) in the linalyl acetate group to the corresponding values of 58.9% ± 1.3% (P < 0.005, n = 4), 57.9% ± 2.5% (P < 0.05, n = 4), and 50.3% ± 2.8% (P < 0.005, n = 4) by the pretreatment with calyculin A. Calyculin A alone slightly increased the MLC phosphorylation ratio, which was accompanied by the increased tension as described above. Both the phenylephrine-induced contraction and the MLC phosphorylation ratio were recovered to the control level attained with phenylephrine alone by the pretreatment with calyculin A.
The aim of the present study was to investigate whether linalyl acetate as the main ingredient of lavender essential oil exerts a relaxing activity or an inhibitory effect on the contraction caused by phenylephrine and to analyze their mechanism. The main findings were as follows. First, linalyl acetate exerted a sustained and progressive relaxation during the contraction caused by phenylephrine and an inhibitory effect on the phenylephrine-induced contraction. Second, the relaxation by linalyl acetate was partially endothelium dependent, possibly associated with activation of NO-cyclic guanosine monophosphate pathway, and partially endothelium independent. Third, the inhibitory effect of linalyl acetate on the phenylephrine-induced contraction was associated with alterations in the MLC phosphorylation ratio, and the relaxation and inhibition of the phenylephrine-induced contraction by linalyl acetate were associated with the dephosphorylation of phosphorylated MLC, possibly through activation of MLC phosphatase.
A concentration-dependent relaxation response was observed when linalyl acetate was added cumulatively to rabbit carotid artery specimens during the contraction caused by phenylephrine. The 50% effective concentration (EC50) was determined as 3.6 × 10−4 mol/L. The relaxation in response to 3 × 10−4 mol/L linalyl acetate was partially but significantly attenuated by denudation of endothelial cells, nitroarginine as an inhibitor of NOS, or by ODQ as an inhibitor of guanylyl cyclase. However, indomethacin as an inhibitor of cyclooxygenase and tetraethylammonium as a nonselective blocker of K channels failed to modify the relaxation with linalyl acetate. These results suggest that the relaxation in response to linalyl acetate was partially endothelium dependent and possibly mediated by activating the NO/cyclic guanosine monophosphate (Fig. 7) and that neither vasodilator prostanoid(s) nor EDHF was involved in producing the relaxation with linalyl acetate.
Even after using nitroarginine or ODQ, there was residual relaxation (Fig. 3), which could be a direct effect of linalyl acetate on vascular smooth muscle cell. Thus, we tested whether linalyl acetate alters MLC phosphorylation levels in E− artery specimens. It is generally accepted that contraction and relaxation of vascular smooth muscle are regulated by MLC kinase and MLC phosphatase. MLC kinase mediates MLC phosphorylation and causes contraction in a Ca2+/calmodulin-dependent manner.4 Conversely, MLC phosphatase mediates dephosphorylation of phosphorylated MLC and causes relaxation.5,6 In the E− specimens, phenylephrine-induced contraction and MLC phosphorylation were significantly attenuated by pretreatment with linalyl acetate, leading us to assume that linalyl acetate possibly mediates the relaxation and the inhibition of phenylephrine contraction by reducing the MLC phosphorylation ratio.
For further characterization of the endothelium-independent relaxation with linalyl acetate, ML-9 and calyculin A were used. The relaxation in response to linalyl acetate in the E− specimens was clearly inhibited by calyclulin A as an inhibitor of MLC phosphatase, whereas ML-9 as an inhibitor of MLC kinase failed to modify the linalyl acetate-induced relaxation. Calyculin A alone slightly but significantly increased the basal tone, which was accompanied by an increased MLC phosphorylation ratio at the used concentration of 3.6 × 10−8 mol/L. In addition, suppression of the phenylephrine-induced contraction and MLC phosphorylation with linalyl acetate was canceled by adding calyculin A. These results suggest that the relaxation and the inhibition of phenylephrine-induced contraction by linalyl acetate may be mediated by the dephosphorylation of phosphorylated MLC through activation of MLC phosphatase (Fig. 7).
Smooth muscle contraction and relaxation are regulated not only by changes in cytoplasmic calcium concentration ([Ca2+]i) but also by other important signaling mechanisms, that is, independent of the changes in [Ca2+]i, referred to as Ca2+ sensitization.12-14 Although the increase in [Ca2+]i initiates smooth muscle contraction via activating MLC kinase, Ca2+ sensitization mediates smooth muscle contraction by inhibiting MLC phosphatase.15,16 Furthermore, it is widely recognized that the Rho A-Rho kinase pathway plays a crucial role in agonist-induced Ca2+ sensitization during smooth muscle contraction.15,16 As already described, the relaxation effect of linalyl acetate was associated with dephosphorylation of phosphorylated MLC through the activation of MLC phosphatase (Fig. 7). Thus, it is possible to assume that linalyl acetate relaxes rabbit vascular smooth muscle by inhibiting Ca2+ sensitization through the Rho A- Rho kinase pathway. It is also possible that linalyl acetate-mediated relaxation is associated with protein kinase C or CPI-17 that inhibits MLC phosphatase. However, additional experiments should be performed to understand the detailed mechanisms that produce the relaxation with linalyl acetate. Attention has recently been focused on vasodilators involving the activator of MLC phosphatase, which differs from classical vasodilating drugs that decrease [Ca2+]i through hyperpolarization, blocking calcium entry or increasing cyclic guanosine monophosphate. Therefore, it seems interesting that linalyl acetate as the major ingredient of lavender essential oil has a vasodilator effect by activating MLC phosphatase. There may be potential value in the treatment and prevention of cardiovascular diseases such as atherosclerosis and hypertension, if the chemical structure of linalyl acetate is optimized on the basis of the vasodilating mechanism of the chemical.
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