Galangin (3,5,7-trihydroxyflavone, Fig. 1) is a bioflavonoid derived primarily from propolis, a natural compound produced by honeybee and rhizome of
The vascular contractility is is regulated via both Ca2+-dependent and Ca2+ sensitization mechanisms (Kuriyama
However, the specific protein kinases and associated cellular pathways primarily responsible for increased calcium desensitization in response to galangin remain unknown. Therefore, the purpose of this study was to investigate the specific protein kinase and associated cellular signaling pathways responsible for myosin phosphatase reactivation and calcium desensitization induced by galangin.
Male Sprague-Dawley rats (210-240 g) were anesthetized with etomidate (0.3 mg/kg i.v.) and euthanized by thoracotomy and exsanguination according to the guidance approved by the Institutional Committee at Chung-Ang University and Daegu Catholic University (IACUC-2016-040). After euthanasia performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals, the thoracic aorta was carefully and rapidly isolated and placed in oxygenated physiological saline solution consisting (mM) of 115.0 NaCl, 4.7 KCl, 25.0 NaHCO3, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, and 10.0 glucose. The aorta was separated from the surrounding connective tissue and the endothelia were cleaned by gentle abrasion using a pipette tip and NG-monomethyl-L-arginine (L-NMMA) if necessary.
To examine functional changes of the muscle in response of a vasoconstrictor, each muscle was incubated with the vasoconstrictor
The relaxation effect of galangin was determined by its application after KCl- (50 mM), phenylephrine- (1 μM), thromboxane A2- (0.1 μM), phorbol ester- (1 μM) or fluoride- (6 mM) evoked contractions had plateaued in normal Krebs’ solution.
Protein expression was quantiﬁed using immunoblotting, as reported previously (Jeon
Sodium chloride, potassium chloride, sodium fluoride, acetylcholine, galangin, U46619 and phorbol 12,13-dibutyrate were obtained from Sigma (St. Louis, MO, USA). DTT, TCA and acetone were purchased from Fisher Scientific (Hampton, NH, USA). Enhanced chemiluminescence (ECL) kits were purchased from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, phospho-phosphorylation-dependent inhibitory protein of myosin phosphatase (phospho-CPI-17) at Thr38 (1:1,000), CPI-17, adducin or phospho-adducin at Ser662, ERK or phosphoERK at Thr202/Tyr204 (Upstate Biotechnology, Lake Placid, NY, USA or Cell Signaling Technology, Danvers, MA, USA) were used to determine levels of RhoA/ROCK activity (Kitazawa
The data are presented as mean ± standard error of the mean (SEM). Statistical evaluations between two groups were performed using student’s unpaired t-test or ANOVA. These statistical analyses were made using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Differences were considered significant when
Removal of endothelium, the regulator of vascular homeostasis, was achieved by gently rubbing with a pipette tip and NG-mono-methyl-L-arginine (L-NMMA) to identify the relaxation effect of galangin on vascular smooth muscle. The absence of endothelium was identified by a lack of relaxation after treating contracted muscle segments with acetylcholine (1 μM). Galangin had no observable effect on basal tension (data not shown), but it significantly inhibited the contraction evoked by a ROCK activator fluoride, regardless of the absence of endothelial nitric oxide synthesis in denuded (Fig. 2A) or intact (Fig. 2B) muscles. This suggests that the relaxation mechanism of galangin might include the repression of ROCK activity and myosin phosphatase reactivation besides endothelial nitric oxide synthesis and the activation of guanylyl cyclase.
Galangin inhibited thromboxane A2 mimetic U46619-induced contraction in denuded muscles (Fig. 3), suggesting that the mechanism includes repression of ROCK activity and myosin phosphatase reactivation and a dual activator (thromboxane mimetic) acts similar to a full activator targeting ROCK.
Phorbol esters are primarily MEK activators and partial ROCK activators (Goyal
To identify the role of galangin on thick filament regulation of vascular contractibility, we measured levels of myosin phosphatase targeting subunit 1 (MYPT1) and phospho-MYPT1 in aortas quick-frozen after a 60-min exposure to galangin for equilibration. Each relaxing muscle was contracted with 6 mM fluoride. This work was conducted using quick frozen galangin (0.1 mM)-treated muscles in the absence of endothelium, and levels were compared to those of vehicle-treated muscles (Fig. 5). A significant decrease in fluoride-induced MYPT1 phosphorylation at Thr855 in response to galangin treatment was observed (Fig. 5). Furthermore, a decrease in fluoride-induced LC20 phosphorylation was found in response to galangin treatment (Fig. 6). Therefore, thick filament regulation, including myosin phosphatase reactivation via RhoA/ROCK inactivation might be involved in the decreased contractility of galangin-treated rat aortas.
The myosin phosphatase inhibitor CPI-17 is phosphorylated by ROCK or PKC. CPI-17 phosphorylation is usually increased during contraction as it is one mechanism that increases myoﬁlament Ca2+ sensitivity. Fluoride or phorbol 12,13-dibutyrate was used as a control for CPI-17 phosphorylation as it directly activates ROCK or PKC producing a signiﬁcant increase in CPI-17 phosphorylation. To confirm the role of galangin in thick or thin filament disinhibition of smooth muscle contractility, we measured levels of CPI-17 and phospho-CPI-17 in aortas quick-frozen after a 60-min exposure to galangin for equilibration. Each relaxing muscle was precontracted with 6 mM fluoride or 1 μM phorbol ester. This work was conducted using quick frozen flavonol (0.1 mM)-treated muscles in the absence of endothelium, and levels were compared to those of vehicle-treated muscles (Fig. 7). Interestingly, a significant decrease in fluoride-induced CPI-17 phosphorylation at Thr-38 in response to galangin treatment was observed (Fig. 7A). The decrease in CPI-17 phosphorylation with galangin during fluoride stimulation suggests that ROCK is inactivated in the galangin-induced decrease in contraction and MLC phosphorylation, and myosin phosphatase reactivation.
To identify the role of galangin on thin filament disinhibition of vascular contractibility, we measured levels of adducin and phospho-adducin and ERK1/2 and phospho-ERK1/2 in aortas quick frozen after 60 minutes of exposure to galangin for equilibration. Each relaxing muscle was precontracted with 1 μM phorbol 12,13-dibutyrate. As compared with vehicle-treated muscles, a decrease in adducin and ERK 1/2 phosphorylation at Ser662 and Thr202/Tyr204 was observed in galangin (0.1 mM)-treated muscles in the absence of endothelium (Fig. 8); significant relaxation (Fig. 4) and thin filament regulation were observed. These findings represent that thin filament regulation, including adducin and ERK1/2 phosphorylation via PKC and MEK activation, plays a role in galangin-induced relaxation.
This is the study to suggest that galangin attenuates tonic tension and suppresses Ca2+ sensitization through the blockade of not PKC-mediated CPI-17 phosphorylation but ROCK-mediated CPI-17 phosphorylation. Pharmacological activators of ROCK (fluoride), MEK (phorbol 12,13-dibutyrate) or both (thromboxane mimetic) were used to determine their involvement in suppressed contraction. The CPI-17-mediated and calcium-sensitized contraction, elicited by various agonists, was enhanced consistently. Galangin attenuates tonic tension and suppresses calcium sensitization through the blockade of ROCK-mediated myosin phosphatase inhibition. Importantly, galangin did not affect PDBu-stimulated phosphorylation of CPI-17, but selectively inhibited fluoride-stimulated phosphorylation of CPI-17 and MYPT1, so preventing myosin phosphatase activities, which resulted in a decreased level of LC phosphorylation. With this distinct mode of action, galangin inhibited fluoride, phorbol 12,13-dibutyrate and thromboxane mimetic-induced vasoconstriction; thus revealing a novel therapeutic target for the development of novel antihypertensive agents.
Activation of ROCK or PKC, phosphorylation of CPI-17 or MYPT1, and subsequent inhibition of myosin phosphatase are part of the Ca2+ sensitization pathway that promotes increased MLC phosphorylation without requiring an increase in Ca2+ inﬂux or release. ROCK phosphorylates myosin phosphatase, which inhibits phosphatase activity and leads to an accumulation of phosphorylated MLCs (Johnson
The present study demonstrates that galangin ameliorates contractions induced by vasoconstrictors (phorbol ester or fluoride) in an endothelium-independent manner (Fig. 2-4), and that the mechanism included the PKC/MEK/ERK and RhoA/ROCK pathways. Galangin attenuated the fluoride-evoked phosphorylation of CPI-17 at Thr38, suggesting that CPI-17 included in fluoride-induced contraction is a downstream effector activated by ROCK. Furthermore, galangin significantly decreased the contraction and the phosphorylation of MYPT1 at Thr855 and CPI-17 at Thr-38 evoked by fluoride (Fig. 5, 7A) with the full relaxation (Fig. 2) and α-adducin and ERK 1/2 phosphorylation at Ser662 and Thr202/Tyr204 induced by a phorbol ester (Fig. 8), suggesting that inhibition of PKC/MEK or ROCK activity is a major mechanism underlying the effects of galangin on smooth muscle contractility. Activation of ROCK by fluoride decreases the activity of myosin phosphatase through phosphorylation of MYPT1 and CPI-17, resulting in an increase in MLC20 phosphorylation and contractions (Sakurada
In summary, galangin without newly reported adverse effects (Aloud