Endothelium-independent Pathway Research Articles

Stimulation of the calcium-sensing receptor induces relaxations of rat mesenteric arteries by endothelium-dependent and -independent pathways via BKCa and KATP channels.

Stimulation of the calcium-sensing receptor (CaSR) induces both vasoconstrictions and vasorelaxations but underlying cellular processes remain unclear. This study investigates expression and effect of stimulating the CaSR by increasing external Ca2+ concentration ([Ca2+ ]o ) on contractility of rat mesenteric arteries. Immunofluorescence studies showed expression of the CaSR in perivascular nerves, vascular smooth muscle cells (VSMCs), and vascular endothelium cells. Using wire myography, increasing [Ca2+ ]o from 1 to 10 mM induced vasorelaxations which were inhibited by the calcilytic Calhex-231 and partially dependent on a functional endothelium. [Ca2+ ]o -induced vasorelaxations were reduced by endothelial NO synthase (eNOS, L-NAME) and large conductance Ca2+ -activated K+ channels (BKCa , iberiotoxin), with their inhibitory action requiring a functional endothelium. [Ca2+ ]o -induced vasorelaxations were also markedly inhibited by an ATP-dependent K+ channel (KATP ) blocker (PNU37883), which did not require a functional endothelium to produce its inhibitory action. Inhibitor studies also suggested contributory roles for inward rectifying K+ channels (Kir ), Kv7 channels, and small conductance Ca2+ -activated K+ channels (SKCa ) on [Ca2+ ]o -induced vasorelaxations. These findings indicate that stimulation of the CaSR mediates vasorelaxations involving multiple pathways, including an endothelium-dependent pathway involving NO production and activation of BKCa channels and an endothelium-independent pathway involving stimulation of KATP channels.

New paeonol derivative C302 reduces hypertension in spontaneously hypertensive rats through endothelium-dependent and endothelium-independent vasodilation

Hypertension is a major risk factor for cardiovascular disease and Chinese herb monomers could provide new structural skeletons for anti-hypertension new drug development. Paeonol is a Chinese herbal monomer extracted from Cortex moutan, exhibited some anti-hypertensive activity. The study focused on the structural optimization of paeonol to provide promising lead compounds for anti-hypertension new drug development. Herein, twelve new paeonol derivatives (PD) were designed and synthesized and their vasodilation activity was evaluated by in vitro vasodilation drug screening platform based on Myograph. Its anti-hypertension activity, PD-C302 (2-hydroxy-4-methoxyvalerophenone) as a representative with the optimal vasodilation activity, was determined by its response to blood pressure in spontaneously hypertensive rats (SHR) in vivo. Moreover, its molecular mechanism was probed by the vasodilation activity of rat superior mesenteric artery rings with or without endothelium pre-contracted by potassium chloride (KCl) or phenylephrine hydrochloride (PE). It was indicated that PD-C302 significantly reduced the blood pressure in SHR, which would involve in PD-C302-induced vasodilation. Furthermore, endothelium-dependent pathways and endothelium-independent pathways both contributed importantly to PD-C302-induced vasodilation at low concentration of PD-C302. Endothelium-independent pathways (vascular smooth muscle cell-mediated vasodilation), were mainly responsible for the PD-C302-induced vasodilation at high concentration of PD-C302, which involved in opening multiple K+ channels to restrain Ca2+ channels, and then triggered vasodilation to reduce blood pressure. PD-C302 has a simple structure and favorable anti-hypertensive activity in vivo, which could be a promising lead compound for anti-hypertension new drug development.

A novel endothelium-independent effect of insulin on basal vascular tone in cafeteria diet-induced hypertensive rats

Insulin vasorelaxant effect in metabolic syndrome has been shown on precontracted vessels. However, the insulin effects on basal vascular tone and its interrelationship with nitric oxide (NO) and K-channels are unknown. To test the effect of insulin on the basal vascular tone in isolated aortic rings from the cafeteria diet-induced hypertensive rats and to determine the role of NO and K-channels on this insulin effect. Male Wistar rats were randomized into two groups: one group fed with a cafeteria diet (CafR) and another fed with a standard chow diet (control rats: CR). Then, in isolated aortic rings, the insulin effect on the basal tone and the role of K-channels were evaluated. Also, the endothelial function, NO levels, and resting membrane potential were measured. CafR increased blood pressure (138 ± 6.2 mmHg; n = 9 vs. CR: 109 ± 1.4 mmHg; n = 9; p < 0.001) and vascular basal tone. Insulin 400 mU/ml reduced basal tone in aortic rings (−284 ± 47 mg; n = 9). This effect was unaffected by endothelium removal or NG-nitro-l-arginine methyl ester (L-NAME) treatment. Likewise, CafR showed low NO levels and a hyperpolarized resting membrane potential. Insulin decreased the resting membrane potential and the KCa and Kv channels blockers abolished this effect. In CafR, endothelial dysfunction is accompanied by an increased basal tone. Insulin reduced it by Kv and KCa channels dependent mechanisms, using an endothelium-independent pathway. These results highlight a novel insulin effect on basal tone of aortic rings from animals with metabolic syndrome and endothelial dysfunction, pathophysiological conditions associated with human hypertension.

Antihypertensive and vasorelaxant effects of Rumex vesicarius (L.) through receptor-operated calcium channels in hypertensive rats.

The aim of the study was to assess the antihypertensive activity of Rumex vesicarius. The genus Rumex (sorrel, Polygonaceae), containing approximately 200 species, is distributed worldwide (African, European, Asian, and American countries). It is widely used in traditional medicine as analgesic, diuretic, antispasmodic, and antihypertensive plants. This study aimed to assess the possible antihypertensive vasorelaxant capacity and effect on angiotensin-converting enzyme 2 (ACE-2) of the aqueous extract of Rumex vesicarius (R. vesicarius). In the present study, the aqueous extract of R. vesicarius (AERV) was prepared, its antihypertensive activity was examined in N(ω)-nitro-L-arginine methyl ester(L-NAME)-induced hypertensive rats, and its vasorelaxant ability along with its effect on stimulating or inhibiting ACE-2 were performed in isolated rat thoracic aorta. The results indicated that AERV decreased the systolic, diastolic, mean, and mean arterial blood pressure in hypertensive rats. The data revealed that AERV exerted its antihypertensive effect through vasodilatory properties via an endothelium-independent pathway. Interestingly, the study demonstrated that the vasorelaxation ability of AERV might be mediated through receptor-operated calcium channels (ROCC). However, AERV extract had no effect on either stimulating or inhibiting ACE-2. The present study demonstrates clearly the antihypertensive and vasorelaxant activities of R. vesicarius in hypertensive rats, supporting its beneficial action as an antihypertensive agent.

Antihypertensive and Vasorelaxant Effects of Hibiscus rosa-sinensis through Angiotensin-Converting Enzyme-2 (ACE-2), and Ca2+ channels Pathways.

The aim of the study was to assess the antihypertensive activity of Hibiscus rosa-sinensis. Hibiscus rosa-sinensis is used traditionally to treat hypertension. The goal of the study was to investigate the effect of the aqueous extract of Hibiscus rosa-sinensis flowers (AEHRS) on resting blood pressure in rats. In the present study, AEHRS was prepared and its antihypertensive activity was evaluated using in vivo and in vitro studies. In the in vivo study, hypertensive and normotensive rats were treated by AEHRS (100 mg/kg) orally for 6 hours in the acute treatment and for 7 days in the subchronic treatment. Systolic, diastolic, and mean arterial blood pressure values and heart rate were then recorded using a tail cuff and a computer-assisted monitoring device. To assess the vasorelaxant activity of AEHRS, isolated thoracic aortic rings were suspended in a tissue bath and changes in tension were recorded using a data acquisition system. Potential pathways involved in the vasorelaxant activity were evaluated using several standard pharmacological agents. The results indicated that repeated oral administration of AEHRS during 7 days lowered systolic, diastolic, and mean arterial blood pressure in hypertensive rats without affecting normotensive rats. Furthermore, the data revealed that AEHRS exerts vasorelaxant properties via an endothelium-independent pathway. More interestingly, the study demonstrates that the vasorelaxant capacity of AEHRS seems to be exerted through the stimulation of angiotensin-converting enzyme-2 (ACE-2) and the inhibition of Ca2+ channels pathway. The present study revealed that aqueous extract of Hibiscus rosa-sinensis has a significant antihypertensive activity and that its vasorelaxant effect may be mediated through stimulation of ACE-2, and inhibition of the Ca2+ channels.

Vasorelaxant Effect of Boesenbergia rotunda and Its Active Ingredients on an Isolated Coronary Artery.

Cardiovascular diseases are a major cause of death in developed countries. The regulation of vascular tone is a major approach to prevent and ameliorate vascular diseases. As part of our ongoing screening for cardioprotective natural compounds, we investigated the vasorelaxant effect of rhizomes from Boesenbergia rotunda (L.) Mansf. [Boesenbergia pandurata (Roxb.) Schltr.] used as a spice and herbal medicine in Asian countries. The methanol extract of B. rotunda rhizomes (BRE) exhibited significant vasorelaxation effects ex vivo at EC50 values of 13.4 ± 6.1 μg/mL and 40.9 ± 7.9 μg/mL, respectively, with and without endothelium in the porcine coronary artery ring. The intrinsic mechanism was evaluated by treating with specific inhibitors or activators that typically affect vascular reactivity. The results suggested that BRE induced relaxation in the coronary artery rings via an endothelium-dependent pathway involving NO-cGMP, and also via an endothelium-independent pathway involving the blockade of Ca2+ channels. Vasorelaxant principles in BRE were identified by subsequent chromatographic methods, which revealed that flavonoids regulate vasorelaxant activity in BRE. One of the flavonoids was a Diels-Alder type adduct, 4-hydroxypanduratin A, which showed the most potent vasorelaxant effect on porcine coronary artery with an EC50 of 17.8 ± 2.5 μM. Our results suggest that rhizomes of B. rotunda might be of interest as herbal medicine against cardiovascular diseases.

Vasorelaxant effects of benzodiazepines, non-benzodiazepine sedative-hypnotics, and tandospirone on isolated rat arteries

Benzodiazepines (BDZs) and non-BDZ sedative-hypnotics are effective for the management of chronic insomnia; however, they are associated with adverse effects such as headache, dizziness, and palpitations. Furthermore, long-term use of these medications is associated with decreased blood pressure (BP) or depressed baroreflex function. Therefore, here, we assessed whether BDZs and non-BDZs cause vasorelaxation directly. Vasorelaxation in response to 22 BDZs, 2 non-BDZs, and tandospirone was determined by myograph methods using isolated Wistar rat thoracic aortas. All the drugs relaxed phenylephrine-contracted rat aortas in a concentration-dependent manner. Zolpidem and tandospirone caused over 80% relaxation at a concentration of 10 μM; diazepam, estazolam, etizolam, and tofisopam caused 60–70% relaxation; whereas 18 other BDZs (alprazolam, bromazepam, brotizolam, chlordiazepoxide, clobazam, clonazepam, clorazepate, ethyl loflazepate, flunitrazepam, flurazepam, lorazepam, lormetazepam, midazolam, nimetazepam, nitrazepam, oxazepam, temazepam, and triazolam) and zaleplon caused less than 50% relaxation. The relaxation was partially but significantly inhibited to the same extent by a nitric oxide (NO) synthase antagonist and after endothelium removal. Binding assay of gamma-aminobutyric acid type A receptors was performed using [3H]flunitrazepam. No correlation was observed between vasorelaxation at a concentration of 10 μM and the binding affinities for 23 drugs. The study demonstrated that zaleplon, zolpidem, tandospirone, and many BDZs cause vasorelaxation to different extents via endothelial NO-dependent and endothelium-independent pathways. In conclusion, the direct vasodilatory effects of these drugs may be involved in the mechanisms underlying their adverse effects. Additionally, the decreased BP observed in persons who take BDZs or non-BDZs may be partly due to direct vasodilation.

Vasorelaxant effect of the dichloromethane fraction from Lippia thymoides involves voltage-gated potassium channels and the suppression of intracellular calcium in rat aortae

The crude extract and fractions from the leaves and stems of Lippia thymoides Mart. & Schauer, Verbenaceae, were evaluated to determine their vasorelaxant activity on isolated rat aortae. The chromatographic profile from the crude extract and the most potent fraction was determined by HPLC-DAD. The dichloromethane fraction (LtSD) displayed the more potent vasorelaxant effect on phenylephrine-induced tonic contractions in isolated rat aortae with (EC50 = 38.5 (26.3–56.5) μg/ml) or without (EC50 = 37.9 (28.5–50.5) μg/ml) functional endothelium. LtSD-mediated vasorelaxation in endothelium-intact aortic rings was not altered in the presence of atropine, l-NAME, indomethacin, or methylene blue. Preincubation of endothelium-denuded aortic rings with CsCl, glibenclamide, or tetraethylammonium did not alter the effects of LtSD-induced relaxation, but in the presence of 4-aminopyridine, the concentration-response curve of LtSD (EC50 = 37.63 (32.88–43.06) μg/ml in the absence of blocker) shifted to the right and the EC50 value was significantly increased to 156.0 (129.5–187.8) μg/ml. In addition, preincubation with LtSD plus nifedipine inhibited phenylephrine contractions similarly to LtSD alone. These results indicated that the vasorelaxant mechanisms of LtSD were mediated via endothelium-independent pathways, probably involving the activation of KV channels and the reduction of Ca2+ influx through intracellular stores.

Biochanin-A elicits relaxation in coronary artery of goat through different mechanisms

Flavonoids have shown beneficial effects in various disease conditions as reported by various previous studies. Biochanin-A is a flavonoid present in various plants in nature. Present investigation was done to assess the vasorelaxant potential of biochanin-A on isolated coronary artery of goat and its possible mechanism of action. Vascular reactivity experiments were done on circumflex coronary artery of goats using the tension experiments. Goat coronary arterial rings were relaxed with biochanin-A in concentration (0.1–100 μM)-dependent manner. Endothelium had no effect on biochanin-A-induced relaxation. Maximum relaxation induced by biochanin-A was 116.54 ± 12.21% in endothelium-intact artery and it was not significantly different with maximal relaxation (108.22 ± 1.88%) of endothelium-denuded vessel. L-NAME (100 μM) did not show any effect on biochanin-A-induced relaxation. TEA (BKCa channel blocker), and BaCl2 (KIR blocker) had no effect on biochanin-A-induced relaxation. However, biochanin-A-induced maximal relaxation (71.72 ± 4.50%) was reduced significantly (P < .001) in the presence of 4-aminopyridine (KV channel blocker, 3 mM) in comparison with control (114.07 ± 4.33%). Glibenclaminde (KATP channel blocker), H89 (PKA inhibitor), ICI182780 (estrogen receptor antagonist) showed partial attenuation in the biochanin-A-induced relaxation. ODQ (sGC blocker) and HC067047 (TRPV4 channel blocker) had no effect on biochanin-A-induced relaxation. In K+-depolarized endothelium-denuded arterial rings, biochanin-A (30 μM) significantly (P < .05; P < .001) decreased CaCl2-induced contractions (0.02 ± 0.01 g vs. control 0.73 ± 0.30 g). Biochanin-A did not influence the fasudil (rho kinase inhibitor) and SNP (NO-donor)-induced relaxation in this vessel. Biochanin-A showed relaxation in goat coronary artery in endothelium-independent pathways and showed the partial involvement of KATP, protein kinase A and estrogen receptors and full involvement of Cav1.2 channels.

Curcumane C and (±)-curcumane D, an unusual seco-cadinane sesquiterpenoid and a pair of unusual nor-bisabolane enantiomers with significant vasorelaxant activity from Curcuma longa

A new seco-cadinane sesquiterpenoid (curcumane C, 1) and a pair of new nor-bisabolene enantiomers [(+)- and (−)-curcumane D, 2a and 2b] were isolated from C. longa. Compound 1 possesses an unusual 4,5-seco-cadinane skeleton with a tetrahydrophthalide moiety, while 2a and 2b contain an unusual 15-nor-bisabolene skeleton with a chromone core. All compounds exhibited significant vasorelaxant effects against KCl-induced contraction of rat aortic rings. Compound 1 also exhibited a vasorelaxant effect against phenylephrine-induced contraction of rat aortic rings. Meanwhile, compound 1 showed a stronger vasorelaxant effect in endothelium-intact rat aortic rings compared with endothelium-denuded rat aortic rings, indicating that vasodilation by 1 involved both endothelium-dependent and endothelium-independent pathways. Furthermore, compound 1 increased the NO content in human umbilical vein endothelial cells and its vasorelaxant effect could be attenuated by treatment with L-NAME, an endothelium NO synthase inhibitor. Thus, the underlying vasodilatory mechanisms of 1 may be mediated via abrogation of extracellular Ca2+ influx and regulation of NO release in vascular endothelial cells.

Antihypertensive activity of Petroselinum crispum through inhibition of vascular calcium channels in rats

Ethnopharmacological relevanceParsley (Petroselinum crispum; P. crispum) is among the popular aromatic vegetables and a part of the daily diet in the Mediterranean area. This plant is widely used in alternative medicine as a remedy against hypertension. The aim of the studyThe aim of the study was to evaluate the antihypertensive activity of the aqueous extract of this plant. Material and methodsIn the current study, the aqueous extract of the aerial parts of parsley (AEPC) was prepared and its antihypertensive activity was evaluated using in vivo and in vitro studies. In the in vivo investigation, anesthetized L-NAME-hypertensive and normotensive rats received orally AEPC (160 mg/kg) during 6 h for the acute experiment and during seven days for the sub-chronic treatment. Thereafter, systolic, diastolic, mean arterial blood pressure and heart rate were recorded using a tail cuff and a computer-assisted monitoring device. Concerning the in vitro investigation, isolated thoracic aortic rings were suspended in a tissue bath and the tension changes were recorded to a data acquisition system. ResultsThe results indicated that AEPC extract decreased the systolic, diastolic, mean arterial blood pressure in normotensive and hypertensive rats. The data revealed that parsley extract exerts its hypotensive effects through vasodilatory properties via an endothelium-independent pathway. More interestingly, the study demonstrated here that the vasorelaxing ability of AEPC is exerted through both Voltage Operated and Receptor Operated Calcium Channels (VOCC and ROCC). ConclusionThe study illustrates the beneficial action of P. crispum as an antihypertensive agent.

Vasorelaxant and antihypertensive effects of Tianshu Capsule on rats: An in vitro and in vivo approach

BackgroundBoth Chuanxiong (Ligusticum chuanxiong Hort) and Tianma (Gastrodia elata Blume) have the effects of vasorelaxation and antihypertension. However, the effects of Tianshu Capsule (TSC, composed of Chuanxiong and Tianma in the mass ratio of 4:1) on antihypertensive activity have not been explored. This study aimed to investigate the eff ;ects of TSC on vascular tension and blood pressure in rats and to explore the underlying mechanisms. MethodsThe vasorelaxant effect of TSC was explored on thoracic aortic rings (both intact endothelium and denuded) preincubated with phenylephrine (Phe) or potassium chloride (KCL). The mechanism was investigated in the presence of antagonists or blockers on aorta isolated from normotensive rats. The in vivo antihypertensive effect was assessed using a tail-cuff method on spontaneously hypertensive rats (SHRs). ResultsTSC (0.125–4 mg/mL) produced a concentration-dependent vasorelaxation on aortic rings preincubated with Phe (1 μM) or KCL (60 mM). Removal of aorta endothelium markedly attenuated the TSC activity. Pretreatment of aortic rings with β-adrenoceptor blocker propranolol (1 μM), muscarinic receptor antagonist atropine (1 μM), cyclooxygenase inhibitor indomethacin (IDO, 1 μM), adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 μM), K+ channel blockers 4-aminopyridine(4-AP, 1 mM) or barium chloride(BaCl2, 1 mM) followed by addition of Phe (1 μM) prior to TSC did not influence the TSC-induced relaxation. In contrast, the vasorelaxant effects of TSC were markedly inhibited by the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 10 μM), guanylyl cyclase inhibitor 1H- [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 μM), K+ channel blockers, glibenclamide (100 μM) and clotrimazole (5 mM). Moreover, TSC (2 mg/mL, 4 mg/mL) inhibited CaCl2-induced contractions and caused a concentration-dependent rightward shift of the response curves. Additionally, TSC (2 mg/mL, 4 mg/mL) depressed the constriction caused by Phe (1 μM) in the absence of extracellular Ca2+. Furthermore, TSC (2.15 g/kg) lowered the systolic blood pressure (SBP), with no alteration in heart rate (HR) in SHRs. ConclusionsThese findings demonstrated that TSC induced vasorelaxant effects via both endothelium-dependent and endothelium-independent pathways. The NO/sGC/cGMP pathway, ATP-sensitive K+ channels, Ca2+-activated K+ channels, inhibition of extracellular Ca2+ influx and intracellular Ca2+ release were probably involved in this relaxation. The vasorelaxant effects of TSC may make the greatest contribution to the reduction in high blood pressure.

Tetrahydroxystilbene glucoside-induced relaxation of the superior mesenteric artery via both endothelium-dependent and endothelium-independent mechanisms

Tetrahydroxystilbene glucoside (TSG) is the main water-soluble component in Polygonum multiflorum Thunb, and it has many cardioprotective effects. Although TSG is able to relax blood vessels, its relaxation of rat superior mesenteric arteries and the underlying mechanism of this process are not clearly understood. The aim of the present study was to use in vivo and in vitro models to investigate the arterial relaxation effect of TSG on rat superior mesenteric arteries and the mechanisms involved. We found that TSG concentration-dependently relaxed the superior mesenteric artery with or without endothelium. The vasorelaxation induced by TSG is not related to the vasodilator derived factor NO but is rather by the inhibition of COX-2 activity and decreased TXA2. We also found that the vasorelaxation induced by TSG was attenuated by 4‑AP. Moreover, TSG also inhibited the contraction induced by an increase in external calcium concentration in Ca2+-free medium plus KCl (60 mM). These results suggest that TSG induces relaxation in mesenteric arterial rings through an endothelium-dependent pathway that involves the inhibition of COX-2 activity and decreased in TXA2 and through an endothelium-independent pathway via opening of a voltage-dependent K+ channel, blockade of Ca2+ influx and release of intracellular Ca2+.

Kaempferol-induces vasorelaxation via endothelium-independent pathways in rat isolated pulmonary artery.

Kaempferol, a flavonoid, is the essential part of human diet. Flavonoids have different pharmacological activities like cardioprotective, anti-inflammatory and anti-oxidant. The aim of current study was to investigate vasorelaxant potential of kaempferol on rat isolated pulmonary artery and to assess the underling mechanisms. Tension experiments were conducted on both the branches of main pulmonary artery of rats. Experiments were done using isolated organ bath system by recording tension with the help of data acquisition system, Power Lab. Kaempferol (10-8-10-4.5M) caused concentration-dependent relaxation (Emax 124.33±4.37%; pD2 5.03±0.084) of endothelium-intact pulmonary artery. In endothelium-denuded arterial rings, relaxation produced by kaempferol was not different from intact artery. L-NAME, indomethacin, combination of L-NAME and indomethacin did not show any effect on kaempferol-induced relaxation. Kaempferol-induced relaxation was reduced (Emax 55.53±7.72%) in 60mMK+ pre-contracted pulmonary arterial rings. Iberiotoxin significantly decreased (Emax 71.68±11.84%) the relaxation response. However, glibenclamide, BaCl2, 4-AP (1mM) and ICI182780 did not reduce the kaempferol-induced relaxation. TEA (10mM) and 4-AP (5mM) significantly reduced relaxation. Kaempferol-induced relaxation was significantly attenuated (Emax 94.92±19.60%) in presence of ODQ. H89 significantly decreased (Emax, 98.38±8.55%) the kaempferol-induced relaxation in rat pulmonary arterial rings. HC067047 and apamin did not show any effect on kaempferol-induced relaxation. In endothelium-denuded K+ (80mM)-depolarized arterial rings, kaempferol (10μM) markedly reduced CaCl2-induced contractions (Emax 35.14±6.53% vs. control 69.04±15.19%). Kaempferol relaxes rat pulmonary artery in endothelium-independent manner through involvement of BKCa channel, sGC, PKA pathways and inhibition of Ca2+-influx through L-type calcium channels.

Patchouli alcohol isolated from Pogostemon cablin mediates endothelium-independent vasorelaxation by blockade of Ca2+ channels in rat isolated thoracic aorta

Ethnopharmacological relevanceThe aerial parts of Pogostemon cablin (Blanco) Benth. for the treatment of cardiodynia have been documented in Mingyi Bielu of late Han Dynasty, in addition to that the Ca2+ antagonized activities of P. cablin and its critically pharmacological ingredient patchouli alcohol (PA) were reported previously. Aim of the studyTo investigate the relaxant effects of PA on rat isolated thoracic aortas and further explore its potential mechanisms of actions. Materials and methodsThe aortas with endothelium and without endothelium were prepared and suspended in the organ bath for isometric tension recordings. The responses to accumulative concentrations of PA in endothelium-intact (E + ) aortas with basal tension and in different treated aortas pre-contracted with potassium chloride (KCl) or phenylephrine (PHE) were observed; the effects of L-NAME and indomethacin in aortas with intact endothelium, and of L-NAME, propranolol, tetraethtylamine (TEA), 4-aminopyridine (4-AP), barium chloride (BaCl2), glyburide in aortas with endothelial denudation on PA-produced vasorelaxation were assessed; the influences of PA on extracellular Ca2+ influx and intracellular Ca2+ release were measured in Ca2+-free medium. Finally, the abilities of PA to inhibit KCl- and PHE-induced contractions were compared to that of verapamil in E- aortas. ResultsPA produced vasorelaxant effects in KCl- and PHE-precontracted E + aortas in a concentration-dependent manner, which had no statistically different from that in KCl- and PHE-precontracted E- aortas. PA (10 μM) significantly reduced KCl-induced contractions while PHE-induced contractions were significantly reduced by 100 μM of PA instead of 10 μM and 30 μM in aortas with endothelium. Pre-incubation of E + aortas with L-NAME as well as indomethacin and of E- aortas with L-NAME, propranolol, TEA, 4-AP, BaCl2 and glyburide had no obvious effects on vasorelaxation of PA. In endothelium-removed aortas around Ca2+-free solution, PA remarkably lowered the contractions stimulated with Ca2+ and PHE, and application of ruthenium red and heparin further enhanced the abilities of PA to reduce PHE-caused contractions. In aortas without endothelium, 100 μM of PA markedly attenuated KCl-induced contractions but not affect PHE-induced contractions. Verapamil at the equal dose markedly attenuated KCl- and PHE-induced contractions, and the inhibitory effects on KCl-induced contractions were more forceful than that on PHE-induced contractions. In combined usage, the inhibitory effects on the contractions elicited by KCl were evidently weaker than that of verapamil alone and indifferently stronger than that of PA alone, and the inhibitory effects on the contractions elicited by PHE were evidently weaker than that of single verapamil but stronger than that of single PA. ConclusionPA may act as a Ca2+ antagonist to exert an intensively vasorelaxant effects through endothelium-independent pathway, and its mechanisms underlying the vasoactivities seem to be associated with the blockade of extracellular Ca2+ influx through VDCCs and ROCCs in vascular smooth muscle cells (VSMCs) membrane and intracellular Ca2+ releases through IP3R- and RYR-mediated Ca2+ channels in sarcolemma.

The Curcumin-Induced Vasorelaxation in Rat Superior Mesenteric Arteries

Curcumin (Cur) is a natural lipophilic polyphenol compound extracted from the rhizome of turmeric. Recently, protective effect of Cur on cardiovascular system is paid close attention. Some researches demonstrated that Cur could induce vascular relaxation in many arterial beds. However, relaxant effect of Cur on rat superior mesenteric artery is not clear. This present study will investigate the vasorelaxant effect of Cur on rat superior mesenteric arteries and the mechanisms involved. The isometric tension of rat superior mesenteric arterial rings was recorded by a sensitive myograph system invitro. The vasodilation of Cur at various concentrations (range: 10-8-10-4 M) on potassium chloride (KCl; 60mmol/L)-precontracted or phenylephrine hydrochloride (PE; 10μmol/L)-precontracted arterial rings were observed. We also observed vasorelaxant effect of Cur on KCl (60mM)-preconstricted rat superior mesenteric arterial rings after incubating the inhibitors of endothelial mechanism, including the endothelial nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester, the guanylate cyclase inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, and the cyclooxygenase inhibitor indomethacin, and inhibitors of potassium ion channel, including 4-aminopyridine (Voltage-sensitive K+ channel blockers), and tetraethylammonium chloride (Ca2+ activated K+ channel blockers), and BaCl2 (Inward rectifying K+ channel blockers), and glibenclamide (ATP -sensitive K+ channel blockers), respectively. The effects of Cur are expressed as percentage of relaxation from the precontraction induced by KCl (60mmol/L) or PE (10μmol/L). The Emax value refers to the maximum relaxation. The pD2 value refers to the negative logarithmic value of the drug concentration that produces 50% Emax. Cur concentration dependently relaxed the superior mesenteric artery rings with endothelium precontracted by PE (Emax=84.33±1.11 and pD2=5.03±0.02) or KCl (Emax=80.96±2.12% and pD2=4.32±0.01). The vasorelaxant effect of Cur on the superiormesenteric artery rings relied on endothelium partially. Indomethacin (5μM) significantlyinhibited the effect. However, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (10μM) and Nω-nitro-L-arginine methyl ester (100μM) had no effect on the action. In artery rings without endothelium, vasorelaxation induced by Cur was attenuated by 4-aminopyridine (100μM). However, barium chloride dehydrate (10μM), glibenclamide (10μM), and traethylammonium chloride (1mM) did not affect vasorelaxation induced by Cur. Moreover, Cur also significantly inhibited contraction induced by increasing external calcium in Ca2+-free medium plus K+ (60mM) and releasing intracellular Ca2+ in the Ca2+-free solution. Our results suggested that Cur induces relaxation in superior mesenteric arterial rings through an endothelium-dependent pathway, involving prostanoid, and also through an endothelium-independent pathway, opening K+ channel, and blockade of Ca2+ influx and intracellular Ca2+ release.

P13 Pinocembrin isolated from Propolis induces relaxation of rat basilar artery rings through both endothelium-dependent and -independent pathways

Objective: Pinocembrin (5,7-dihydroxyflavanone) is one of the primary flavonoids in propolis with various biological activities. We found that pinocembrin increased cerebral blood flow and improved the outcome after focal or global cerebral ischemia/reperfusion injury in rats. In the present study, the vasorelaxant effect of pinocembrin on the rat basilar artery and its potential mechanisms were studied. Methods: Endothelium-intact and -denuded basilar artery rings were prepared from SD rats, and then stimulated with KCl, 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1). The tension of the basilar artery rings was measured through an isometric force transducer. Results: Pinocembrin induced vasorelaxation in a dose-dependent manner in endothelium-intact rings contracted by KCl, 5-HT, and ET-1. However, partial loss of the vasorelaxation induced by pinocembrin was observed in endothelium-denuded rings contracted by KCl. The vasorelaxant effect of pinocembrin was significantly reduced by pre-incubation with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME), the guanylate cyclase inhibitor ODQ and the cyclooxygenase inhibitor indomethacin. In addition, the pinocembrin-induced relaxations were remarkably attenuated by the voltage-dependent K+ channel blocker 4-aminopyridine, the ATP-sensitive K+ channel blocker glibenclamide and the Ca2+-activated K+ channel blocker tetraethylammonium. Moreover, pre-incubation with pinocembrin dramatically suppressed the high K+-induced extracellular Ca2+ influx and ET-1-induced intracellular Ca2+ release in endothelium-denuded rings. Conclusion: These results demonstrated that pinocembrin has a vasorelaxant effect in isolated rat basilar artery rings. It may exert its effects by an endothelium-dependent pathway involving NO-cGMP, and also through an endothelium-independent pathway, opening K+ channels and blockade of Ca2+ channels.

Mechanisms of simvastatin-induced vasodilatation of rat superior mesenteric arteries.

Independent of its lipid-lowering properties, simvastatin (Sim) induces vasorelaxation; however, the underlying mechanisms have remained elusive. The aim of the present study was to investigate the vasorelaxant effects of Sim on rat superior mesenteric arteries and the mechanisms involved. The isometric tension of rat superior mesenteric arterial rings was recorded in vitro on a myograph. The results showed that Sim concentration-dependently relaxed the superior mesenteric artery rings with endothelium pre-contracted by phenylephrine hydrochloride [maximum relaxation (Emax)=51.05±4.09%; negative logarithm of the concentration that caused 50% of the maximum response (pD2)=4.17±0.18] or KCl (Emax=41.65±1.32%; pD2=3.55±0.1). Nω-nitro-L-arginine methyl ester (100 µM) significantly inhibited this effect, while it was not affected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 µM) and indomethacin (5 µM). In artery rings without endothelium, vasorelaxation induced by Sim was attenuated by 4-aminopyridine (100 µM), but was not affected by barium chloride dehydrate (10 µM), glibenclamide (10 µM) and traethylammonium chloride (1 mM). Moreover, Sim also inhibited the contraction induced by increasing external calcium in Ca2+-free medium with added KCl (60 mM). These results suggested that Sim induces relaxation of superior mesenteric arterial rings through an endothelium-dependent pathway, involving nitric oxide release and also through an endothelium-independent pathway, involving the opening of voltage-dependent K+ channels and blockade of extracellular Ca2+ influx.

Endothelium-independent vasorelaxant effect of 20(S)-protopanaxadiol on isolated rat thoracic aorta.

Ginsenosides are considered to be the major pharmacologically active ginseng constituents, whereas 20(S)-protopanaxadiol [20(S)-PPD] is the active metabolite of ginsenosides in gut. In this study we investigated the effect of 20(S)-PPD on isolated rat thoracic aortas as well as its vasorelaxant mechanisms. Aortic rings with or without endothelium were prepared from Wistar rats and suspended in organ-chambers. The changes in tension of the preparations were recorded through isometric transducers connected to a data acquisition system. The aortic rings were precontracted with phenylephrine (PE, 1 μmol/L) or high-K+ (80 mmol/L). Application of 20(S)-PPD (21.5-108.5 μmol/L) caused concentration-dependent vasodilation of endothelium-intact aortic rings precontracted with PE or high-K+, which resulted in the EC50 values of 90.4 or 46.5 μmol/L, respectively. The removal of endothelium had no effect on 20(S)-PPD-induced relaxation. The vasorelaxant effect of 20(S)-PPD was also not influenced by the preincubation with β-adrenergic receptor antagonist propranolol, or with ATP-sensitive K+ channel blocker glibenclamide, voltage-dependent K+ channel blocker 4-AP and inward rectifier K+ channel blocker BaCl2, whereas it was significantly attenuated by the preincubation with Ca2+-activated K+ (BKCa) channel blocker TEA (1 mmol/L). Furthermore, the inhibition of NO synthesis, cGMP and prostacyclin pathways did not affect the vasorelaxant effect of 20(S)-PPD. In Ca2+-free solution, 20(S)-PPD (108.5 μmol/L) markedly decreased the extracellular Ca2+-induced contraction in aortic rings precontracted with PE or high-K+ and reduced PE-induced transient contraction. Voltage-dependent Ca2+ channel antagonist nifedipine inhibited PE-induced contraction; further inhibition was observed after the application of receptor-operated Ca2+ channel inhibitor SK&F 96365 or 20(S)-PPD. 20(S)-PPD induces vasorelaxation via an endothelium-independent pathway. The inhibition of voltage-dependent Ca2+ channels and receptor-operated Ca2+ channels and the activation of Ca2+-activated K+ channels are probably involved in the relaxation.

Endothelium-dependent and-independent relaxation induced by resveratrol in rat superior mesenteric arteries.

Resveratrol (Res) is a specific agonist of sirtuin 1, and has many cardioprotective effects. Although Res is able to relax various vascular beds, its pharmacological properties in rat superior mesenteric arteries and the underlying mechanism are not well clarified. The aim of present study was to investigate the vasorelaxant effects of Res on rat superior mesenteric arteries and the mechanisms involved. The isometric tension of rat superior mesenteric arterial rings was recorded in vitro using myography. It was found that Res concentration-dependently relaxed endothelium-intact superior mesenteric artery rings pre-contracted by phenylephrine hydrochloride (Emax, 97.66±0.79%; pD2, 4.30±0.14) or KCl (Emax, 101.3±0.6%; pD2, 4.12±0.03). The vasorelaxant effect of Res on the superior mesenteric artery rings was partially endothelium-dependent. NG-nitro-L-arginine methyl ester (100 µM) significantly inhibited the Res-induced vasorelaxant effect. However, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (10 µM) and indomethacin (5 µM) each had no effect on the Res-induced vasorelaxation. In artery rings without endothelium, the vasorelaxation induced by Res was attenuated by 4-aminopyridine (100 µM) and glibenclamide (10 µM). However, barium chloride dehydrate (10 µM) and tetraethylammonium chloride (1 mM) did not affect the vasorelaxation induced by Res. Moreover, Res also inhibited the contraction induced by an increase in external calcium concentration in Ca2+-free medium plus KCl (60 mM). These results suggest that Res induces relaxation in superior mesenteric arterial rings through an endothelium-dependent pathway, involving nitric oxide release, and also through an endothelium-independent pathway, with opening of voltage-dependent K+ channels and ATP-sensitive K+ channels and blockade of extracellular Ca2+ influx.