Mechanism of vasorelaxation caused by N-Benzylsecoboldine in rat thoracic aorta

The vasorelaxing effect of N-benzylsecoboldine on the rat thoracic aorta was investigated, and we also compare it with nifedipine and cromakalim. In high K⁺ (60 mM) medium, Ca²⁺ (0.03–3 mM)-induced vasoconstriction was inhibited concentration-dependently by N-benzylsecoboldine, whereas this contraction was not altered by cromakalim. Cromakalim relaxed aortic rings precontracted with 15 but not 60 mM of K⁺. N-benzylsecoboldine and nifedipine were more potent and effective in producing relaxation in 60 mM than in 15 mM K⁺-induced contraction. N-benzylsecoboldine was found to be an ₁-adrenoceptor-blocking agent in rat thoracic aorta as revealed by its competitive antagonism of phenylephrine (PE)-induced contraction (pA₂=6.31 ± 0.04, pA₁₀=5.41 ± 0.03). This relaxing effect of N-benzylsecoboldine was not antagonized by indomethacin or methylene blue, and still persisted in endothelium-denuded aorta or in the presence of nifedipine (1 µM). The increase of inositol monophosphate caused by PE in rat aorta was significantly suppressed by N-benzylsecoboldine, but not by nifedipine or cromakalim. High concentration of N-benzylsecoboldine (100 µM) did not affect the contraction induced by B-HT 920, serotonin or PGF₂. Glibenclamide and charybdotoxin did not affect the relaxation of N-benzylsecoboldine in aortic rings precontracted with PE. Neither cGMP nor cAMP levels were changed by N-benzylsecoboldine. We suggest that N-benzyl-secoboldine relaxes rat thoracic aorta by suppressing the Ca²⁺ influx and also has antagonistic effect on ₁-adrenoceptors.     

Endothelium-dependent vasorelaxation in rat thoracic aorta by Mansoa hirsuta D.C.

The vasodilator effect of the ethanolic extract of Mansoa hirsuta leaves (EEF) was assayed in rat aortic rings. EEF produced a concentration-dependent vasodilatation (pIC₅₀=5.1±0.2), which was absent in endothelium-denuded vessels. The vasodilator effect of EEF was similar to a standardized ethanolic extract of Hancornia speciosa Gomes (pIC₅₀=5.1±0.1). The endothelium-dependent vasodilatation induced by EEF was abolished by L-NAME (100 μM), a nitric oxide (NO) synthase inhibitor, but not by indomethacin (10 μM; pIC₅₀=4.9±0.2), a cyclooxygenase inhibitor. The concentration–response curve of EEF was not modified by the addition of superoxide dismutase (SOD; 300 U/ml). In addition, EEF (50 μg/ml) displaced the 3-morpholino-sidnonimine (SIN-1; p<0.05) concentration–effect curve to the left, as well as SOD (300 U/ml). These findings lead us to conclude that EEF induces a NO- and endothelium-dependent vasodilatation in rat aortic preparations, and that this effect is, at least in some extent, due to an increase in the NO bioavailability as consequence of its antioxidant activity. The HPLC-DAD profile recorded for EEF indicates the presence of four major peaks with close retention times, exhibiting similar UV spectra with wavelength maxima compatible with heterogeneous proanthocyanidins.     

Endothelium-dependent vasorelaxation effect of rutin-free tartary buckwheat extract in isolated rat thoracic aorta

The aim of this study was to point out the potential of tartary buckwheat on vascular functions. A nonabsorbed fraction of hot-water extract of tartary buckwheat on a SP70 column (TBSP-T), which was free from rutin, was used for this aim. In a contractile experiment using Sprague-Dawley rat thoracic aorta rings contracted by 1.0 microM phenylephrine (PE) or 50 mM KCl, TBSP-T evoked a significant vasorelaxation [EC50 (mg/ml): PE; 2.2; KCl, 1.9]. By a further fractionation of TBSP-T by liquid-liquid partitioning into basic, neutral and acidic fractions, a marked enhancement of vasorelaxation effect was observed only for acidic fraction (EC50, 0.25 mg/ml). The action of acidic fraction was significantly attenuated in endothelium-denuded aortic rings and in the presence of nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 microM). The fraction also enhanced the cyclic guanosine monophosphate (cGMP) production in aortic rings contracted with PE [cGMP (pmol/mg protein): PE, 7.2+/-2.3; PE+Acidic fraction, 35+/-8]. These results indicate that acidic fraction could mediate NO/cGMP pathways, thereby exerting endothelium-dependent vasorelaxation action. In conclusion, tartary buckwheat was proven to regulate vascular tones and have latent acidic candidates except for rutin.     

Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^2 + channel blocker through both intracellular and extracellular Ca^2 + release.     

Effects of alismol isolated from Alismatis Rhizoma on calcium-induced contraction in the rabbit thoracic aorta

We examined the inhibitory effects of alismol, a sesquiterpenoid isolated from Alismatis Rhizoma, on vascular contractions induced by high concentrations of K+ and Ca2+, and on 45Ca2+ retention in normal and in high K+-containing medium. Alismol affected neither the resting tension nor the 45Ca2+ retention in normal medium, but it inhibited sustained contraction and increased 45Ca2+ retention induced by high K+ concentrations. Alismol did not affect norepinephrine-induced contractions in normal medium, nor phasic contractions in Ca2+-free medium. These results suggested that alismol inhibited mainly Ca2+ influx through a voltage-dependent Ca2+ channel.     

Vasodilatory action mechanisms of apigenin isolated from Apium graveolens in rat thoracic aorta.

The effect of apigenin, isolated from Apium graveolens, on the contraction of rat thoracic aorta was studied. Apigenin inhibited the contraction of aortic rings caused by cumulative concentrations of calcium (0.03-3 mM) in high potassium (60 mM) medium, with an IC50 of about 48 microM. After pretreatment it also inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contraction in a concentration (35-140 microM)-dependent manner with an IC50 of 63 microM. At the plateau of NE-induced tonic contraction, addition of apigenin caused relaxation. This relaxing effect of apigenin was not antagonized by indomethacin (20 microM) or methylene blue (50 microM), and still existed in endothelial denuded rat aorta or in the presence of nifedipine (2-100 microM). Neither cAMP nor cGMP levels were changed by apigenin. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine in the Ca(2+)-free solution were unaffected by apigenin. 45Ca2+ influx caused by either NE or K+ was inhibited by apigenin concentration-dependently. It is concluded that apigenin relaxes rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage- and receptor-operated calcium channels.     

Interleukin-1beta causes different levels of nitric oxide-mediated depression of contractility in different positions of rat thoracic aorta.

Interleukin-1beta (IL-1beta) can be synthesized by macrophages, endothelial cells and vascular smooth muscle cells when stimulated by bacterial lipopolysaccharide (endotoxin) during septic shock. The IL-1beta levels in the blood vessel wall are also elevated in atherosclerosis. IL-1beta can cause induction of inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cells and produce vasorelaxation, hypotension and ultimately tissue damage. We studied the depressions of vascular smooth muscle contractions at 3 hours after exposure to IL-1beta in different positions of rat thoracic aorta. The data show that the aortic rings from the cranial end of rat thoracic aorta had little response to IL-1beta (0.5 and 1.0 ng/ml) while those from the caudal end of thoracic aorta had larger depressant response. S-methylisothiourea sulfate (SMT), an iNOS inhibitor, completely blocked the depression of contraction caused by IL-1beta in intact aortic rings. If the endothelium was removed from the aortic rings before exposure to IL-1beta, all rings from different parts of the thoracic aorta showed an equal amount of vasodepression. Thus, the difference in the depressant response of IL-1beta in different portions of thoracic aorta is endothelium-dependent and involves induction of NOS.     

Bis-N-nitroso-caged nitric oxides: photochemistry and biological performance test by rat aorta vasorelaxation.

Three new caged nitric oxides (NOs)-BNN3, BNN5Na, and BNN5M were tested for biological use. BNNs have a strong ultraviolet (UV) absorption band (lambda(max): 300 nm, epsilon: 13.5 mM(-1) cm (-1)) extended to 420 nm and produce NO upon irradiation with 300-360 nm light in quantum yields about 2. A photoexcited BNN molecule yields two NOs with time constants of less than 10 ns for phase 1 and less than 20 micros for phase 2 at 37 degrees C, suggesting usefulness of BNNs for measuring in vivo and in vitro fast NO reactions. Upon irradiating with UV light, caged nitric oxides-loaded rat aortic strips maintained in a state of active tonic contraction effectively relaxed ( < 3 microM BNN5M loading solution concentration). BNN3 is incorporated in the lipid membrane. BNN5Na, insoluble in organic solvents but water soluble, localizes in the water phase. BNN5M, is muscle-cell-permeable and hydrolysed to BNN5Na to remain in cytosol. BNNs were thermally stable and demonstrated no observable toxicity.     

Procyanidins in crataegus extract evoke endothelium-dependent vasorelaxation in rat aorta

The extract of Crataegus, a mixture of flavonoids and procyanidins extracted from hawthorn, Crataegus oxyacantha, L. and C. monogyna Jacq., relaxed vascular tone or increased production of cyclic GMP in the rat aorta, but flavonoid components of Crataegus extract, hyperoside, rutin and vitexin, did not affect the vascular tone. The aim of the present study was to characterize the endothelium-dependent relaxation elicited by procyanidins fractionated from Crataegus extract in isolated rat aorta. Procyanidins caused endothelium-dependent relaxation which was associated with the production of cyclic GMP. Both responses to these procyanidins were inhibited by methylene blue or N(G)-nitro-L-arginine, but not by indomethacin. Relaxation in response to procyanidins was not affected by atropine, diphenhydramine, [D-Pro2,D-Trp7,9]substance P, propranolol, nifedipine, verapamil and glibenclamide, but were markedly reduced by tetraethylammonium. These findings showed that procyanidins in Crataegus extract may be responsible for the endothelium-dependent nitric oxide-mediated relaxation in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K+ channels.     

Effects of activated neutrophils on isolated rings of rat thoracic aorta

Local inflammation and respiratory burst of polymorphonuclear leukocytes generate reactive oxygen species (ROS). The aim of our study was to analyze the effects of peritoneal neutrophils on changes of the muscle tension of isolated aorta and compare their effects with those of different ROS. While native neutrophils did not influence muscle tension, the N-formyl-methionyl-leucyl-phenylalanine activated ones evoked a biphasic response on the KCl-precontracted aorta. The effects of activated neutrophils were in both respects similar to those evoked by xanthine/xanthine oxidase (X/XO) and differed from the effects evoked by H(2)O(2) and Fe(2)SO(4)/H(2)O(2). Using H(2)O(2) we demonstrated that the effects of ROS were dependent on the KCl induced initial tension. To exclude the effect of extensive depolarization the action of different ROS was studied also on tissues precontracted by phenylephrine. Under such condition activated neutrophils caused a marked contraction similar to that evoked by X/XO. Their effects differed however, from those of H(2)O(2) and Fe(2)SO(4)/ascorbic acid. These findings and elimination of activated neutrophil-induced contractions as well as the chemiluminiscence by superoxide dismutase suggest that the primarily activated neutrophil-released ROS was superoxide, which can be transformed to peroxynitrite, and other ROS including H(2)O(2). Reduction of all followed-up contractions caused by nordihydroguaiaretic acid, indicate that 5-lipoxygenase metabolites unselectively reduce contractions. In contrast, selective inhibition of activated neutrophil-evoked contraction by indomethacin suggests that cyclooxygenase metabolites are involved mainly in their action on vascular smooth muscle.     

A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery.

In this report, we deal with the isolation of a novel vasorelaxing peptide from a chymotryptic digest of ovalbumin and its vasorelaxing activities. This peptide is composed of Arg-Ala-Asp-His-Pro-Phe (RADHPF) in its sequence, corresponding to residues 359-364 of ovalbumin. This peptide (30-300 microM) exerted a dose-dependent vasodilation in an isolated mesenteric artery from a spontaneously hypertensive rat which was pre-constricted by phenylephrine, besides the relaxation being endothelium-dependent. It is noteworthy that the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester inhibited this relaxation, implying involvement of nitric oxide in its mechanism of action. Following oral administration of RADHPF at a dose of 10 mg/kg, the systolic blood pressure in a spontaneously hypertensive rat was significantly lowered.     

Vasorelaxant effect of the flavonoid galangin on isolated rat thoracic aorta

Here we investigated the effect of the flavonoid galangin in isolated rat thoracic aortic rings. Galangin (0.1-100 microM) induced relaxation in rings pre-contracted with phenylephrine (PE 1 microM) or with KCl (100 mM) or pre-treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM), the cyclooxygenase inhibitor indomethacin (10 microM) and the adenylate cyclase inhibitor, SQ 22,536 (100 microM). In another set of experiments, rat aortic rings were incubated with galangin (1-100 microM) and the contractile responses to PE (0.001-3 microM) or to KCl (60 mM) were evaluated. We also evaluated the effect of galangin (100 microM) on PE (10 microM)-induced contraction in a Ca2+-free medium. Galangin relaxed aortic rings with or without endothelium. Galangin effect was significantly inhibited by L-NAME. Galangin inhibited the contractile response to PE, either in presence or in absence of external calcium, and to KCl. In the end, we also found that galangin caused nitric oxide (NO) release from aortic rings and abolished the increase in [Ca2+]i triggered by PE or KCl in aortic smooth muscle cells, either in presence and in absence of external Ca2+. Our results suggest that galangin reduces the contractility of rat aortic rings through an endothelium-dependent mechanism, involving NO, and also through an endothelium-independent mechanism, inhibiting calcium movements through cell membranes.     

不同pH条件下离体大鼠胸主动脉对多巴胺反应性的变化

目的：观察不同pH值条件下大鼠胸主动脉对多巴胺反应性的变化。方法：采用离体血管灌流方法,观察①胸主动脉对不同浓度多巴胺的反应。②检测不同pH值备件下血管对多巴胺反应性的变化。结果：①不同浓度多巴胺（10^-6mol·L-1,10^-5mol·L^-1,10^-4mol·L^-1）均能增加正常大鼠胸主动脉收缩力。②pH值依次降低（7．4,7．3,7．2,7．1,7．0,6．9,618,6．7）,大鼠离体胸主动脉对多巴胺10^-5mol／L反应性下降,血管在pH6．6时完全失去对多巴胺的反应性。结论：大鼠胸主动脉对多巴胺刺激的反应性随环境pH值的变化而变化,表现为随着环境pH值的下降,离体动脉对多巴胺刺激的反应性也随之下降。     

Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta

The potency, structure-activity relationship, and mechanism of vasorelaxation of a series of flavonoids, representing different subclasses (flavonols: fisetin, rutin, quercetin; flavones: chrysin, flavone, baicalein; flavanones: naringenin, naringin; isoflavones: diadzein and flavanes: epigallo catechin gallate), were examined in the isolated rat aorta. Most of the flavonoids tested showed concentration dependent relaxant effects against K+ (80 mM) and phenylephrine (PE, 0.1 microM)-induced contractions with a greater inhibition of the responses to the alpha1-adrenoceptor agonist. The relaxant effects of most of the flavonoids involve in part the release of nitric oxide and prostaglandins from the endothelium as pretreatment with L-NAME and indomethacin attenuated the responses. In addition, the relaxant action of the flavonoids includes inhibition of Ca+2 influx and release of Ca+2 from intracellular stores. A structure-activity relationship amongst the flavonoids was suggested.     

Evidence that lithium induces a glutamatergic: Nitric oxide-mediated response in rat brain

Studies have indicated the involvement of a glutamatergic mechanism in lithium (Li⁺) action. Glutamatergic agonists, such as kainic acid, are known to promote the synthesis of nitric oxide (NO) and to increase cGMP, while Li⁺ has displayed a similar, yet unexplained, ability to increase cGMP. NO synthesis is regarded as the principal prodromal event leading to the activation of the guanyl cyclase-cGMP transduction mechanism. In the present study, the involvement of the NO:cGMP pathway in the action of Li⁺ was examined, while the possibility of a glutamatergic mechanism in this response was also investigated. Parameters examined included cortical accumulation of cGMP and the stable oxidative metabolites of NO, viz. NO ₂ ^– and NO ₃ ^–, collectively expressed as NO ₂ ^–. A significant positive correlation was observed in the in vivo cGMP and NO ₂ ^– data throughout all the groups. Chronic treatment of rats with LiCl (0.3% m/m) engendered a significant increase in cGMP levels which was inhibited by the NO-synthase (NOS) inhibitor, N-nitro-l-arginine methyl ester (L-NAME). Acute administration of kainic acid resulted in an increased accumulation of NO ₂ ^–, also prevented by concomitant L-NAME administration. In addition, a synergistic stimulatory response on cortical NO ₂ ^– was observed in the combination of LiCl and kainic acid. Collectively, these data implicate an involvement of a glutamatergic-mediated NO:cGMP transduction mechanism in the action of Li⁺.     

Vascular effects of caffeic acid phenethyl ester (CAPE) on isolated rat thoracic aorta

This study was aimed to investigate the vascular activity of caffeic acid phenethylester (CAPE), one of the major components of honeybee propolis. Experiments were performed on rat thoracic aortic rings, mounted in an isolated organ bath and connected to an isometric force transducer. The effect of CAPE (0.1-300 microM) was evaluated on tissue pre-contracted with phenylephrine (PE, 1 microM) or with KCl (100 mM). In another set of experiments, tissue was incubated with CAPE (1-100 microM) and responses to PE (0.01-3 microM) or KCl (60 mM) were evaluated. The effect of CAPE on cytosolic Ca(2+) concentration in aortic smooth muscle cells stimulated with PE or KCl was also evaluated. CAPE (0.1-300 microM) caused a concentration-dependent relaxation (pEC(50) 4.99 +/- 0.19; Emax 100.75 +/- 1.65%; n = 4) of tissue pre-contracted with PE that was reduced by endothelium removal or by incubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM). CAPE also relaxed KCl-precontracted tissue (pEC(50) 4.40 +/- 0.08; n = 4). CAPE inhibited contractile responses to PE or to KCl, and also inhibited the contractile response to PE obtained in a Ca(2+)-free medium. In addition, CAPE inhibited the increase in cytosolic Ca(2+) concentration triggered by stimulation of aortic smooth muscle cells with PE or KCl. Our results demonstrate a vascular activity for CAPE, that is only partially dependent on nitric oxide. Indeed, at high concentrations, CAPE vasorelaxant effect occurs also in absence of endothelium and it is likely due to an inhibitory effect on calcium movements through cell membranes.     

Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.     

Vibrio vulnificus hemolysin dilates rat thoracic aorta by activating guanylate cyclase

Hemolysin produced by Vibrio vulniflcus caused hypotension and tachycardia in rats and dilated rat thoracic aorta. Hemolysin-induced vasodilatation of the aorta was not affected by Nω-nitro-L-arginine methyl ester and aminoguanidine, NO synthase inhibitors, whereas the vasodilatation was inhibited by LY 83,583, a guanylate cyclase inhibitor. Hemolysin elevated cGMP levels, and the elevation was abolished by LY 83,583. These results suggest that V. vulnificus hemolysin activates guanylate cyclase independently of NO synthase, and the subsequent increase in cGMP levels results in vasodilatation.     

Nitric oxide-mediated inhibition of DNA synthesis was attenuated in hypertrophied neonatal rat ventricular myocytes

The antiproliferative action of nitric oxide (NO) has been well established and increased production was reported in the infarcted rat heart. Concomitantly, increased DNA synthesis and hyperplasia of cardiac myocytes were documented in the hypertrophied myocardium. Despite these observations, the effect of NO on DNA synthesis in hypertrophied cardiac myocytes remains unexamined. Hypertrophy of the non-infarcted left ventricle (NILV) in 1-week post-MI rats was characterized by the increased prepro-ANP and reduction of alpha-myosin heavy chain protein expression. Inducible NO synthase was expressed in the NILV and associated with a concomitant attenuation of MnSuperoxide dismutase protein content. The latter data suggest that an antiproliferative action of NO in the hypertrophied NILV may proceed via either a cyclic GMP-dependent pathway and/or facilitated by a peroxynitrite-dependent mechanism. In neonatal rat ventricular myocytes (NNVM), the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) promoted a dose-dependent attenuation of DNA synthesis via a cyclic GMP-independent pathway. The permeable superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abrogated SNAP-dependent attenuation of DNA synthesis in NNVM. MnTBAP failed to inhibit SNAP-mediated recruitment of extracellular signal regulated kinase 1/2 (ERK1/2) but partially attenuated p38 phosphorylation. In hypertrophied NNVM induced by norepinephrine, SNAP-mediated peroxynitrite-dependent inhibition of DNA synthesis, ERK1/2 and p38 phosphorylation were significantly attenuated. Collectively, these data suggest that despite a favourable environment for NO and subsequent peroxynitrite generation in the NILV, hypertrophied cardiac myocytes may be partially refractory to their biological actions.