Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas.

Elsholtzia splendens (ES) is, rich in flavonoids, used to repair copper contaminated soil in China, which has been reported to benefit cardiovascular systems as folk medicine. However, few direct evidences have been found to clarify the vasorelaxation effect of total flavonoids of ES (TFES). The vasoactive effect of TFES and its underlying mechanisms in rat thoracic aortas were investigated using the organ bath system. TFES (5-200mg/L) caused a concentration-dependent vasorelaxation in endothelium-intact rings, which was not abolished but significantly reduced by the removal of endothelium. The nitric oxide synthase (NOS) inhibitor N(ω)-nitro-l-arginine methyl ester (100µM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (30µM) significantly blocked the endothelium-dependent vasorelaxation of TFES. Meanwhile, NOS activity in endothelium-intact aortas was concentration-dependently elevated by TFES. However, indomethacin (10µM) did not affect TFES-induced vasorelaxation. Endothelium-independent vasorelaxation of TFES was significantly attenuated by KATP channel blocker glibenclamide. The accumulative Ca(2+)-induced contraction in endothelium-denuded aortic rings primed with KCl or phenylephrine was markedly weakened by TFES. These results revealed that the NOS/NO/cGMP pathway is likely involved in the endothelium-dependent vasorelaxation induced by TFES, while activating KATP channel, inhibiting intracellular Ca(2+) release, blocking Ca(2+) channels and decreasing Ca(2+) influx into vascular smooth muscle cells might contribute to the endothelium-independent vasorelaxation conferred by TFES.

Total flavonoids of Flos Chrysanthemi protect arterial endothelial cells against oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Total flavonoids of Flos Chrysanthemi (TFFC) are known to modulate vascular functions, but their effect on endothelial cells injured by oxidative stress is unknown. Our objective was to investigate the vasoprotective effect and mechanism of action of TFFC on rat mesenteric artery exposed to superoxide anions produced by pyrogallol. MATERIALS AND METHODS: The vasoprotective effect and mechanism of action of TFFC on primary cultured rat mesenteric arterial endothelial cells and small mesenteric arteries was investigated using small-vessel myography, fluorescent Ca(2+) measurement, fluorescent membrane potential measurement and oxidative fluorescent studies. RESULTS: Experiments using small-vessel myography of third-order rat mesenteric arterial rings showed that pretreatment with pyrogallol (10-1000µM), an auto-oxidizing source of superoxide anions, dose-dependently decreased ACh-induced endothelium-dependent relaxation. TFFC (2.5-320mg/L) evoked a concentration-dependent dilation (pD(2): 29.6±0.276mg/L), which was weakened by ChTX plus apamin. TFFC markedly attenuated the inhibition of vasorelaxation induced by pyrogallol (E(max) elevated from 50.4±7.36% to 86.2±3.61%, and pD(2) increased from 6.74±0.06 to 7.28±0.12). Furthermore, in primary cultured endothelial cells, fluorescent Ca(2+) measurement, fluorescent membrane potential measurement and oxidative fluorescent studies demonstrated that ACh-induced endothelial Ca(2+) influx and hyperpolarization were significantly weakened by the increased basal superoxide level induced by pyrogallol. When the endothelial cells were concurrently exposed to TFFC, the impairment effect of oxidative stress on ACh-induced Ca(2+) influx, hyperpolarization and vasorelaxation were attenuated due to its superoxide-lowering activity. CONCLUSION: This study shows that oxidative stress has a pronounced deleterious effect on EDHF-mediated vasorelaxation to ACh in rat mesenteric artery. TFFC has vasodilating effect and protects EDHF-mediated vasodilator reactivity from oxidative stress. Thus, our experiments suggest that TFFC is potentially useful for the development of therapeutic treatments for cardiovascular diseases associated with oxidative stress.

Antioxidant action of a Chrysanthemum morifolium extract protects rat brain against ischemia and reperfusion injury.

The present study evaluated the potential neuroprotective effect and underlying mechanism of the total flavones extracted from Chrysanthemum morifolium (TFCM) against ischemia/reperfusion (I/R) injury. An animal model of cerebral ischemia was established by occluding the right middle cerebral artery for 90 minutes followed by reperfusion for 22 hours. The neurobehavioral scores, infarct area, and hemispheric edema were evaluated. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and reactive oxygen species (ROS) level in brain were also measured. The results showed that pretreatment with TFCM significantly decreased the neurological deficit scores, percentage of infarction, and brain edema and attenuated the decrease in SOD activity, the elevation of MDA content, and the generation of ROS. In isolated brain mitochondria, Ca(2+)-induced swelling was attenuated by pretreatment with TFCM, and this effect was antagonized by atractyloside. These results showed that pretreatment with TFCM provides significant protection against cerebral I/R injury in rats by, at least in part, its antioxidant action and consequent inhibition of mitochondrial swelling.

Luteolin reduces high glucose-mediated impairment of endothelium-dependent relaxation in rat aorta by reducing oxidative stress.

While luteolin, a flavone rich in many plants, has some cardiovascular activity, it is not clear whether luteolin has beneficial effects on the vascular endothelial impairment in hyperglycemia/high glucose. Here, we reveal the protective effect of luteolin on endothelium-dependent relaxation in isolated rat aortic rings exposed to high glucose. The thoracic aorta of male Sprague-Dawley rats was rapidly dissected out and the effect of luteolin on the tension of aortic rings pretreated with high glucose (44mM) for 4h was measured in an organ bath system. The levels of nitric oxide (NO), hydroxy radical (OH(-)) and reactive oxygen species (ROS), and the activity of superoxide dismutase (SOD) and nitric oxide synthase (NOS) were measured in aortas. The vasorelaxation after treatment with luteolin for 8 weeks in aortic rings from diabetic rats was also determined. We found that exposure to high glucose decreased acetylcholine-induced endothelium-dependent relaxation. However, high mannitol had no effect on vasorelaxation. Luteolin evoked a concentration-dependent relaxation in aortic rings previously contracted by phenylephrine, and the pD(2) value was 5.24+/-0.04. The EC(50) of luteolin markedly attenuated the inhibition of relaxation induced by high glucose, which was significantly weakened by pretreatment with l-NAME (0.1mM), but not by indomethacin (0.01mM). Luteolin significantly inhibited the increase of ROS level and OH(-) formation, and the decrease of NO level, NOS and SOD activity caused by high glucose. The improving effect of luteolin on endothelium-dependent vasorelaxation in diabetic rat aortic rings was reversed by pretreatment with l-NAME or methylene blue. The results indicate that the decrease of endothelium-dependent relaxation in rat aortic rings exposed to high glucose is markedly attenuated by luteolin, which may be mediated by reducing oxidative stress and enhancing activity in the NOS-NO pathway.

Endothelium-independent relaxation and contraction of rat aorta induced by ethyl acetate extract from leaves of Morus alba (L.).

AIM OF THE STUDY: Based on screening for vasoactive traditional Chinese medicinal herbs, the present study was performed to investigate the vasoactive effects of an ethyl acetate extract from leaves of Morus alba (L.) (ELM) on rat thoracic aorta and the mechanisms underlying these effects. MATERIALS AND METHODS: Isolated rat thoracic rings were mounted in an organ bath system and the effects of ELM on their responses were evaluated. RESULTS: ELM (0.125-32.000g/l) induced a concentration-dependent relaxation (P<0.01 vs. control) both in endothelium-intact and -denuded aortas precontracted by high K(+) (6 x 10(-2)M) or 10(-6)M phenylephrine (PE). In endothelium-denuded aortas, ELM at the EC(50) concentration reduced Ca(2+)-induced contraction (P<0.01 vs. control) after PE or KCl had generated a stable contraction in Ca(2+)-free solution. And after incubation with verapamil, ELM induced contraction in endothelium-denuded aortas precontracted by PE (P<0.01 vs. control); this was abolished by ruthenium red (P<0.01 vs. ELM-treated endothelium-denuded group; P>0.05 vs. control), but not by heparin (P>0.01 vs. ELM-treated endothelium-denuded group; P<0.01 vs. control). CONCLUSIONS: The results showed that ELM had dual vasoactive effects, and the relaxation was greater than the contraction. The relaxation was mediated by inhibition of voltage- and receptor-dependent Ca(2+) channels in vascular smooth muscle cells, while the contraction occurred via activation of ryanodine receptors in the sarcoplasmic reticulum.

Mechanisms underlying biochanin A-induced relaxation of the aorta differ between normotensive and hypertensive rats.

1. The aim of the present study was to investigate the mechanism underlying biochanin A-induced relaxation of the aorta in spontaneously hypertensive rats (SHR). 2. The tension in isolated ring preparations of thoracic aortas from normotensive (Wistar-Kyoto (WKY) rats) and SHR at 5 and 10 weeks of age was measured isometrically. 3. Biochanin A (10(-7) to 10(-4) mol/L) induced a concentration-dependent relaxation in aortic rings from both strains at the age of 5 and 10 weeks and the relaxation was greater in rings from 10-week-old SHR compared with age-matched WKY rats. The vasorelaxation induced by biochanin A was significantly reduced by denudation of the endothelium in aortic rings from SHR, but not WKY rats. Treatment with either indomethacin, a cyclo-oxygenase inhibitor, or N(omega)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, had little effect on the relaxation induced by biochanin A in aortic rings from either strain. Glibenclamide, a selective inhibitor of ATP-sensitive potassium channels, significantly attenuated the relaxation induced by biochanin A in aortic rings from both strains, although the extent of reduction was greater in WKY rats than SHR. Conversely, treatment with 4-aminopyridine, a selective inhibitor of voltage-dependent potassium channels, or tetraethylammonium, an inhibitor of calcium-activated potassium channels, significantly reduced the vasorelaxation induced by biochanin A in rings from SHR but not WKY rats. 4. The greater vasorelaxation produced by biochanin A in aortic rings from 10-week-old SHR is endothelium dependent. Different mechanisms underlie the relaxant effects of biochanin A in aorta from SHR and WKY rats. The mechanisms of biochanin A-induced vasorelaxation in thoracic aortas from both normotensive and hypertensive rats involve ATP-sensitive potassium channels and, in addition, in rings from the hypertensive strain at 10 weeks of age, an endothelium-derived activation of smooth muscle cell potassium channels contributes to the vasorelaxation observed.

Delayed uncoupling is related to cardioprotection induced by kappa-agonist U-50,488H in rat heart.

OBJECTIVES: To determine whether the kappa-opioid receptor agonist U50,488H affects electrical uncoupling during prolonged ischemia and, if so, whether the changes are associated with its cardioprotective action. DESIGN: The isolated rat heart was perfused in a Langendorff apparatus. Formazan content, lactate dehydrogenase (LDH) and hemodynamic parameters were measured to confirm the cardioprotective effect of U50,488H. The effects of U50,488H on electrical coupling during prolonged ischemia were also measured. RESULTS: U50,488H concentration-dependently increased formazan content and reduced LDH release, and the ameliorating effect of 10(-5) mol/L U50,488H was abolished by 5 x 10(-6) mol/L nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist, or 10(-4) mol/L 5-hydroxydecanoate (5-HD), a selective mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker. The onset of electrical uncoupling during prolonged ischemia was delayed by U50,488H, and the delay was not only abolished, but also advanced by nor-BNI or 5-HD relative to the control group. CONCLUSIONS: These results demonstrate that delayed uncoupling during prolonged ischemia is associated with the cardioprotection of U50,488H, and these effects of U50,488H are mediated by mitochondrial K(ATP) channels.

Pinacidil improves contractile function and intracellular calcium handling in isolated cardiac myocytes exposed to simulated cardioplegic arrest.

BACKGROUND: We examined the effects of pinacidil on contractile function and intracellular calcium in isolated rat cardiomyocytes exposed to cardioplegic solution. METHODS: Rat myocytes were incubated at 24 degrees C for 2 hours in cardioplegic solution with or without pinacidil (50 micromol/L), then they were perfused with Krebs-Henseleit solution with a gas phase of 95% O2/5% CO2 at the same temperature. Contraction and intracellular calcium transients were then measured by video tracking and spectrofluorometry. RESULTS: During 20 minutes of perfusion after 2 hours in cardioplegic solution with pinacidil, (1) the recovery of contractile function was significantly increased in terms of both amplitude of contraction (98.30% +/- 9.90% versus 81.00% +/- 11.25%; p < 0.05) and peak velocity of cell shortening (100.90% +/- 13.79% versus 76.89% +/- 18.14%; p < 0.01) when compared with myocytes in cardioplegic solution without pinacidil; (2) the amplitudes of the intracellular calcium transients evoked by electrical stimulation and caffeine (10 mmol/L) increased by 23.31% to approximately 40.72% and 61.73%, respectively, compared with those in cardioplegic solution without pinacidil; and (3) the decay time of the caffeine-induced intracellular calcium transient decreased by 36.64% +/- 15.10% relative to that measured in cardioplegic solution without pinacidil. The effects induced by supplementing the cardioplegic solution with pinacidil were diminished in the presence of glibenclamide (10 micromol/L). CONCLUSIONS: Addition of the adenosine triphosphate-sensitive potassium-channel opener, pinacidil, to a high potassium cardioplegic solution improves recovery of contractile properties and cytosolic calcium in isolated rat cardiac myocytes.