Isolated Rat Aortic Rings Research Articles

Inhibition of Endothelin-1 and Hypoxia-induced Pulmonary Pressor Responses in the Rat by a Novel Selective Endothelin-A Receptor Antagonist, Di-n-butylaminocarbamyl-l-leucyl-d-tryptophanyl-d-4-chloro-Phe

Pulmonary hypertension is a kind of disease associated with a very high rate of mortality, and there are not many effective drugs for the treatment. Today, endothelin (ET)-1 receptor antagonists were proved to be effective in the treatment of pulmonary hypertension. Aiming at developing new endothelin-A receptor (ETA) antagonist for treatment of pulmonary hypertension, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe, named GF063, was synthesized at base of selective ETA receptor antagonist BQ485 and selected for the further pharmacological characterization. The preliminary pharmacodynamics of GF063 was evaluated by radioligand receptor binding assay and test of antivasoconstriction effects in vitro and in vivo. The integrative pharmacodynamics was evaluated in hypoxia-induced rat pulmonary hypertension. In vitro, GF063 bound to ETA receptor with 100,000-fold higher affinity than to ETB receptor. GF063 concentration dependently inhibited contraction of isolated rat aortic ring induced by ET-1 and shifted the cumulative concentration-contraction response curve to right with no change in the maximal response. In vivo, GF063 inhibited the increase of mean systemic arterial pressure induced by ET-1 in anesthetized rat. In hypoxia-induced rat pulmonary hypertension model, pretreatment with GF063 (40 mg/kg, s.c.) significantly decreased pulmonary artery pressure and right ventricular hypertrophy, also significantly inhibited the increase of ET-1 level in lung, improved hemodynamics, and alleviated the wall thickness of pulmonary vessels. This study indicated that GF063, as a selective ETA receptor antagonist, could inhibit vasoconstriction effects in vivo and in vitro, could prevent pulmonary hypertension induced by hypoxia, and may have great potential to be developed as a new drug of antipulmonary hypertension.

Mechanisms of vasorelaxation induced by Ziziphora clinopodioides Lam. (Lamiaceae) extract in rat thoracic aorta

Ziziphora clinopodioides Lam. (ZC) is widely used in Uyghur folk medicine for the treatment of hypertension diseases in Xinjiang, an autonomous region of China. To provide pharmacological basis for this traditional use, we explored the vasodilating effects of ZC and investigated the underlying mechanisms. Activity of hexane (ZCHE), dichloromethane (ZCDE) and aqueous (ZCAE) extracts of ZC were evaluated on isolated rat aortic rings pre-contracted with phenylephrine (PE) or high KCl. The mechanisms were evaluated on ZCDE, the most potent extract. ZCDE-induced relaxation in endothelium-intact aortic rings pre-contracted with phenylephrine (PE, 10(-6) M) or high KCl (6×10(-2) M), with respective EC(50) values of 0.27±0.03 and 0.34±0.04 g/l. Mechanic removal of the endothelium did not significantly modify ZCDE-induced relaxation. In endothelium-denuded aorta pre-contracted with PE (10(-6) M), the vasorelaxant effect of ZCDE was significantly decreased by 4-amino-pyridine (10(-3) M), but not by glibenclamide (10(-4) M), iberiotoxin (3×10(-8) M) and thapsigargin (10(-7) M). In Ca(2+) free solution, ZCDE significantly inhibited extracellular Ca(2+)-induced contraction in high KCl and PE pre-contracted rings. Additionally ZDCE inhibited the intracellular Ca(2+) release sensitive to PE (10(-6) M). The results demonstrate that ZDCE exhibits endothelium-independent vasodilating properties that are mediated by inhibition of extracellular Ca(2+) influx through voltage- and receptor-operated Ca(2+) channels (VDDCs and ROCCs), by inhibition of Ca(2+) release from intracellular stores, and also by the opening of voltage-dependent K(+) channels.

The temporal effect of a wild blueberry (Vaccinium angustifolium)-enriched diet on vasomotor tone in the Sprague-Dawley rat

Background and aimsWe have previously reported that wild blueberry (Vaccinium angustifolium)–enriched diets (WB) attenuate aortic adrenergic response through endothelial-mediated pathways. The duration of dietary intervention necessary to induce the positive changes on vasomotor tone has not been studied to date. Thus, our objective was to investigate the temporal effect of WB consumption on vascular function and reactivity in Sprague-Dawley (SD) rat aorta after 4 and 7 weeks of dietary treatment. Methods and resultsForty male SD rats were randomly assigned to a control (AIN-93) (C) or a WB diet for 4 or 7 weeks. Vascular ring studies were conducted in 3-mm isolated rat aortic rings to investigate vasoconstriction induced by six doses of the α1-adrenergic agonist, l-phenylephrine (Phe, 10−8–3×10−6 M) alone or in the presence of the NOS inhibitor, l-NG-monomethyl-arginine (l-NMMA, 10−4M). The maximum force of contraction (Fmax) and vessel sensitivity (pD2) were determined. Analysis of variance revealed no significant differences on Fmax after 4 weeks of the WB diet but only a significant increase in pD2 in the absence of l-NMMA. Seven week WB consumption significantly attenuated contraction in response to l-Phe and resulted in lower pD2. Inhibition of NOS induced a significant increase in the constrictor response in both diet groups at both time periods, with the WB group fed for 7 weeks having the greater response. ConclusionThus wild blueberries incorporated into the diet at 8% w/w positively affect vascular smooth muscle contractility and sensitivity but these effects are evident only after 7 weeks of WB consumption.

Vascular dilation by paeonol — A mechanism study

The goal of this study was to investigate the mechanism underlaying the vasodilatory effect of paeonol, a major active element from the root bark of Chinese herbs Paeonia suffruticosa Andr. and Cynanchum paniculatum (Bunge) Kitagawa. Paeonol relaxed isolated rat aorta rings by 95.6% while the 10−6M forskolin-induced vasodilatation used as 100%. The EC50 of vasodilatation by paeonol is 2.9×10−4M. Although paeonol exerted endothelium-independent relaxation, L-NAME treatment inhibited paeonol-induced vasodilation of endothelium intact rings, while indomethacin did not. Both L-NAME and ODQ did not affect paeonol relaxation in the rings without endothelium. In addition, paeonol markedly elevated NO generation in cultured endothelial cells. Pre-treatment of propranolol, glibenclamide, TEA and BaCl2 did not affect paeonol relaxation of endothelium removed rings. On the other hand, pre-treated of rings (without endothelium) with paeonol markedly blocked vasoconstriction induced by AngII, PGF2α, 5-HT, dopamine, vasopressin, endothelin-1 and PE. The paeonol incubation also significantly attenuated KCl-induced contraction which mainly depended on Ca2+ influx. In Ca2+-free medium (containing 10−4M of EGTA and 60mM of KCl), paeonol suppressed the contraction curve of CaCl2. In addition, paeonol also inhibited contraction by PE in Ca2+ free solution (containing 10−4M of EGTA) which mainly relied on intracellular Ca2+ release. Whole-cell patch-clamp experiment showed that paeonol shifted the I–V curve and the peak value of calcium currents was significantly inhibited. In conclusion, our study suggested that voltage-dependent and receptor-operated Ca2+ channel, as well as intracellular Ca2+ release were all inhibited by paeonol. An intracellular Ca2+ regulatory mechanism may be responsible to potent vasodilatory effect of paeonol.

The vasodilatory effect of a synthetic polymer‐based root canal material on thoracic aorta

To test the hypothesis that, Epiphany, either in its mixed form or as separate components, can alter the vascular reactivity of isolated rat thoracic aorta. The possible mechanism of its vascular action was also investigated. The relaxant effect of the base, the catalyst and mixed Epiphany on isolated rat aortic rings pre-contracted with phenylephrine (PE) was tested. The aortic rings were then incubated with either nitric oxide synthase (NOS) inhibitor, cyclooxygenase (COX) inhibitor or K(+) channel inhibitors; after pre-contraction with PE, relaxations to the various compounds of Epiphany were examined. In another set of experiments, to investigate the Ca(2+)channel antagonistic effect of the Epiphany, the effect of these compounds in Ca(2+)-free solution on extracellular Ca(2+)(CaCl(2))-induced contraction in high-K(+) pre-challenged rings (in K(+)-depolarized rings) was examined to determine whether the direct inhibition of [Ca(2+)] influx increase accounted for the vasodilatory effects of these compounds. For comparison, L-type Ca(2+)channel blocker nifedipine (1 micromol L(-1)), instead of Epiphany compounds, was assayed in adjacent rat aortic rings in parallel. The catalyst and the mixture of Epiphany induced concentration-dependent relaxations. However, the base of Epiphany did not cause relaxation in rat aorta. The relaxation responses were not significantly altered by incubation of aorta with NOS, COX and potassium channel inhibitors. Whilst nifedipine, the catalyst and the mixture of Epiphany inhibited CaCl(2)-induced contractions (P < 0.05), the base of Epiphany did not inhibit CaCl(2)-induced contractions significantly (P > 0.05). Epiphany induced relaxation of rat aorta via a calcium antagonistic effect. Provided that the vasodilatory effect elicited by Epiphany can be reversed by the circulation, its haemorrhagic potential by virtue of permanent vascular dilatation can be ignored.

Synergistic vasorelaxant and antihypertensive effects of Ligusticum wallichii and Angelica gigas

Aim of the study The synergistic vasorelaxant and antihypertensive effects of Ligusticum wallichii and Angelica gigas were examined in isolated rat aorta rings and spontaneously hypertensive rats (SHRs). Materials and methods The ethanol extract of Ligusticum wallichii (LwEx) or Angelica gigas (AgEx) or their combinations at ratios Ligusticum wallichii: Angelica gigas = 1:1 (MxEx11), 1:3 (MxEx13), and 3:1 (and MxEx31), and their successive water soluble (LwDw, AgDw, MxDw11, MxDw13 and MxDw31) or n-butanol soluble fractions (LwBt, AgBt, MxBt11, MxBt13, and MxBt31) were examined for their vasorelaxant effects. In an antihypertensive study, LwEx, AgEx, or MxEx11 (100 mg/kg) was orally administered to SHRs, and the systolic, diastolic, and mean blood pressure were measured using the tail-cuff method before and 1, 3, 5, 7, and 24 h after oral administration. Results Each of the ethanol extracts caused long-term relaxation in endothelium-intact or endothelium-denuded rat aorta preconstricted with norepinephrine (NE, 300 nM). All of the water phases of the ethanol extracts elicited an endothelium-dependent acute relaxation, and the water phase of MxDw11 (EC 50 values: 1.08 mg/mL, P < 0.05) had the highest activity. MxDw11-induced acute relaxation was abolished by pretreatment with N G -nitro- l-arginine (10 μM), methylene blue (1.0 μM), or atropine (0.1 μM), indicating that the response to MxDw involves the enhancement of the nitric oxide-cGMP system. On the other hand, all of the butanol phases showed an endothelium-independent long-term relaxation, and MxBt11 (85 ± 7% relaxation of NE-preconstricted active tone at 20 min after the addition, P < 0.05) displayed the highest activity. MxBt11-induced gradual relaxation was significantly attenuated by an inward rectifier potassium-channel inhibitor, but not by an ATP-sensitive or a large conductance Ca 2+-activated potassium-channel blocker. Calcium concentration-dependent contraction curves in high-potassium, depolarizing medium were shifted significantly to the right and downward after incubation with MxBt11 (0.03, 0.1, and 0.3 mg/mL), implying that MxBt11 is also involved in the inhibition of extracellular calcium influx to vascular smooth muscle. MxEx11 (100 mg/kg) significantly reduced systolic blood pressure of SHRs at 3, 5, and 7 h after oral administration, but this effect was not induced by Ligusticum wallichii or Angelica gigas alone. Conclusions The combination of Ligusticum wallichii and Angelica gigas elicits a synergistic effect on vasorelaxation in isolated rat aortas and antihypertension in SHRs. The ratio of Ligusticum wallichii: Angelica gigas = 1:1 was the most effective of all combinations tested.

Tryptic amaranth glutelin digests induce endothelial nitric oxide production through inhibition of ACE: Antihypertensive role of amaranth peptides

Amaranth seed proteins have a better balance of essential amino acids than cereals and legumes. In addition, the tryptic hydrolysis of amaranth proteins generates, among other peptides, angiotensin converting enzyme (ACE) inhibitory (ACEi) peptides. ACE converts angiotensin I (Ang I) into Ang II, but is also responsible for the degradation of bradykinin (BK). In contrast to Ang II, BK stimulates vasodilation modulated through endothelial nitric oxide (NO) production. The aim of the present study was to characterize the ACEi activity of amaranth trypsin-digested glutelins (TDGs) and their ability to induce endothelial NO production. An IC 50 value of 200 μg ml −1 was measured for TDG inhibition of ACE. TDGs stimulated endothelial NO production in coronary endothelial cells (CEC) by 52% compared to control. The effects of TDGs were comparable to those of BK and Captopril, both used as positive controls of NO production. Consistent with these effects, TDGs induced, in a dose-dependent manner, endothelial NO-dependent vasodilation in isolated rat aortic rings. These results suggest that TDGs induce endothelial NO production and consequent vasodilation through their ACEi activity. Amaranth TDGs have a high potential as a nutraceutical food in prevention of cardiovascular diseases. Further molecular, cellular and physiological studies are currently under way and the results may contribute to a better understanding and control of cardiovascular disorders.

(±)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis

Praeruptorin A is a coumarin compound naturally occurring in the roots of Peucedanum praeruptorum Dunn., a commonly used traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. Although previous studies indicated the relaxant effects of (±)-praeruptorin A on tracheal and arterial preparations, little is known about the functional characteristics of the enantiomers. In the present study, the two enantiomers were successfully isolated and identified by using a preparative Daicel Chiralpak AD-H column, and their relaxant effects on aorta rings were observed and compared. (+)-Praeruptorin A showed more potent relaxation than (−)-praeruptorin A against KCl- and phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. Removal of the endothelium remarkably reduced the relaxant effect of (+)-praeruptorin A but not that of (−)-praeruptorin A. Pretreatment of aortic rings with N ω-nitro- l-arginine methyl ester ( l-NAME, an inhibitor of nitric oxide synthase) or methylene blue (MB, a soluble guanylyl cyclase inhibitor) resulted in similar changes of the relaxant effects of the two enantiomers to endothelium removal. Molecular docking studies also demonstrated that (+)-praeruptorin A was in more agreement to nitric oxide synthase pharmacophores than (−)-praeruptorin A. On the other hand, the two enantiomers of praeruptorin A could slightly attenuate the contraction of rat aortic rings induced by internal Ca 2+ release from sarcoplasmic reticulum (SR). These findings indicated that (+)-praeruptorin A and (−)-praeruptorin A exerted distinct relaxant effects on isolated rat aorta rings, which might be mainly attributed to nitric oxide synthesis catalyzed by endothelial nitric oxide synthase.

Vascular relaxation by ethanol extract of Xanthoceras sorbifolia via Akt‐ and SOCE‐eNOS‐cGMP signaling

ObjectiveXanthoceras sorbifolia Bunge has long been used as a folk medicine in China. The purpose of the present study was to define the effects of ethanol extract of leaves of X. sorbifolia (EXS) on the vascular tone and the mechanisms by which EXS relaxes vascular smooth muscle in isolated rat aortic rings.Methods and ResultsExperiments were performed in isolated rat aortic rings. Aortic ring was suspended in organ bath and isometric tension was recorded in response to drugs. EXS (0.1–100 ug/ml) produced concentration‐dependent relaxation of aortic rings precontracted with phenylephrine. Endothelium‐denudation abolished the EXS‐induced vasorelaxation. L‐NAME and ODQ blocked the EXS‐induced vasorelaxation. Diltiazem had no effect on the EXS‐induced vasorelaxation. However, Ca2+ ‐free buffer attenuated the EXS‐induced vasorelaxation. Thapsigargin, 2‐APB, and Gd3+, modulators of store‐operated Ca2+ entry (SOCE), and Wortmannin, an inhibitor of Akt, significantly attenuated the EXS‐induced vasorelaxation. However, indomethacin, atropine, and propranolol had no effect on the EXS‐induced vasorelaxation. EXS increased cGMP levels of the aortic rings in a concentration‐dependent manner.ConclusionThese data demonstrate that EXS relaxes vascular smooth muscle via endothelium‐dependent Akt‐ and SOCE‐eNOS‐cGMP signaling pathways. Supported by NSFC (No30570669; 30971080).

Alteration of guanylyl cyclase‐activated vasorelaxation in thoracic aorta from hyperthyroid rats

ObjectiveNitric oxide (NO) and C‐type natriuretic peptide (CNP) activate soluble (sGC) and particulate guanylyl cyclas e (pGC), respectively, and play important roles in the mainte nance of cardiovascular homeostasis. The purpose of the present study was to define the alteration of pGC‐ and sGC‐ac tivated vasorelaxation in the thoracic aorta from hyperthyroid rats.Methods and ResultsHyperthyroidism was induced by administering L‐thyroxine (T4, 0.05 mg/100 g, sc) daily for 16 days. Experiments were performed in isolated rat aortic rings. CNP (0.1–1000 nM) and SNAP (10‐10000 nM) induced vascular relaxation in a concentration‐dependent manner in aortic rings from eu‐and hyperthyroid rats. The responses were significantly attenuated in aortic rings from hyperthyroid rats. ODQ (10 uM), an inhibitor of sGC, blocked the SNAP ‐induced vasorelaxation of aortic rings from both eu‐ and hyperthyroid d rat. To define the changes in response to cGMP, 8‐Br‐Cgmp (1–100 uM), a cell‐permeable cGMP analog, was used. 8‐Br‐cGMP‐induced vasorelaxation was attenuated in aortic rings from hyperthyroid rats compared to those from euthyroid rats.ConclusionsThese data demonstrate that pGC‐ and sGC‐activated vaso relaxation is attenuated in thoracic aorta from hyperthyroid rats which may be a part of the pathophysiology of the disease. Supported by NSFC (No30570669; 30971080).

Evaluation of the NO-releasing properties of NO-donor linkers

This work describes the synthesis of some benzoic (1-4) and alcoholic (5-7) nitrooxy derivatives, which are nitric oxide (NO) donors in themselves, and can also be seen as useful linkers that can be used in multi-target drugs capable of releasing NO. The NO-mediated vasorelaxing effects of the compounds were tested on endothelium-denuded isolated rat aortic rings pre-contracted with KCl. The pharmacological study of these compounds demonstrated that slight structural modification, such as the insertion of (a) methyl group(s) into the nitrooxymethyl chain or into the aromatic ring, and a change in the position of this nitrooxymethyl chain, could exert a marked (and potentially useful) influence on the NO releasing properties.

Effects of Leaf Extracts of Piliostigma thonningii Schum on Aortic Ring Contractility and Bleeding Time in Rats

The leaves of Piliostigma thonningii Schum have been used in herbal medicine to treat inflammations, bacterial infections, and worm infestations and to arrest bleeding. In an investigation of the possible scientific basis for the use of P. thonningii as a hemostatic agent, the contractility of isolated rat aortic rings and wound bleeding times were measured after exposure of the tissues to aqueous and ethanol extracts of P. thonningii leaves. The extracts produced responses on the isolated aortic rings. The maximum contractile response produced by 32 mg mL−1 of the aqueous extract was 30.8% ± 1.6% of the contractile response produced by 1 μM phenylephrine. The ethanol extract was much less potent with 64 mg mL−1 producing only 12.3% ± 0.9% of the contractile response produced with the phenylephrine. In addition, the aqueous extract, but not the ethanol extract, significantly reduced bleeding time as compared with saline solution (p < .05) but was significantly less than the reduction produced with 0.1% epinephrine solution. The constriction of blood vessels may be the basis for P. thonningii's use as a hemostatic agent in traditional herbal medicine.

Cardiovascular activity of the n-butanol fraction of the methanol extract of Loranthus ferrugineus Roxb.

We investigated the vascular responses and the blood pressure reducing effects of different fractions obtained from the methanol extract of Loranthus ferrugineus Roxb. (F. Loranthaceae). By means of solvent-solvent extraction, L. ferrugineus methanol extract (LFME) was successively fractionated with chloroform, ethyl acetate and n-butanol. The ability of these LFME fractions to relax vascular smooth muscle against phenylephrine (PE)- and KCl-induced contractions in isolated rat aortic rings was determined. In another set of experiments, LFME fractions were tested for blood pressure lowering activity in anesthetized adult male Sprague-Dawley rats (250-300 g, 14-18 weeks). The n-butanol fraction of LFME (NBF-LFME) produced a significant concentration-dependent inhibition of PE- and KCl-induced aortic ring contractions compared to other fractions. Moreover, NBF-LFME had a significantly higher relaxant effect against PE- than against high K+-induced contractions. In anesthetized Sprague-Dawley rats, NBF-LFME significantly lowered blood pressure in a dose-dependent manner and with a relatively longer duration of action compared to the other fractions. HPLC, UV and IR spectra suggested the presence of terpenoid constituents in both LFME and NBF-LFME. Accordingly, we conclude that NBF-LFME is the most potent fraction producing a concentration-dependent relaxation in vascular smooth muscle in vitro and a dose-dependent blood pressure lowering activity in vivo. The cardiovascular effects of NBF-LFME are most likely attributable to its terpenoid content.

Endothelium-Independent Vasorelaxation by Ligusticum wallichii in Isolated Rat Aorta: Comparison of a Butanolic Fraction and Tetramethylpyrazine, the Main Active Component of Ligusticum wallichii

Ligusticum wallichii is an herb widely used to treat vascular disorders in Asian countries, and tetramethylpyrazine (TMP) has been identified as one of its vasorelaxant active components. This study was performed to examine the endothelium-independent relaxation produced by the butanol-soluble fraction of L. wallichii extract (LwBt) and its possible mechanisms of action in isolated rat aortic rings. The effects were compared with those of TMP. LwBt produced vasorelaxation that increased gradually after 2-3 min of LwBt administration and reached a maximum within 30 min. LwBt-induced relaxation was significantly attenuated by pretreatment with 4-aminopyridine and apamin. Additionally, LwBt attenuated CaCl(2)-induced vasoconstriction in high-potassium depolarized medium. Thus, LwBt-induced vasorelaxation apparently involved inhibition of calcium influx, mediated by the opening of voltage-dependent and/or Ca(2+)-activated potassium channels. On the other hand, the effect of TMP was significantly attenuated by pretreatment with glibenclamide, and 4-aminopyridine had no effect. In conclusion, LwBt-induced endothelium-independent vasorelaxation was mediated by the opening of voltage-dependent potassium channels, while TMP-induced relaxation was mediated by the opening of ATP-dependent potassium channels. These effects of LwBt may be due to a substance other than TMP.

The Direct Effect of Levobupivacaine in Isolated Rat Aorta Involves Lipoxygenase Pathway Activation and Endothelial Nitric Oxide Release

Levobupivacaine is a long-acting local anesthetic with a clinical profile similar to that of racemic bupivacaine but with a greater margin of safety. Levobupivacaine produces dose-dependent vasoconstriction in vivo. Our goal in this in vitro study was to investigate the role of pathways involved in arachidonic acid metabolism in the levobupivacaine-induced contraction of isolated rat aorta and to determine which endothelium-derived vasodilators are involved in the modulation of levobupivacaine-induced contraction. Rat thoracic aortic rings were isolated and suspended for isometric tension recording. Cumulative levobupivacaine dose-response curves over a range of 10(-6) to 3 x 10(-4) M were constructed in 1) aortic rings with no drug pretreatment; 2) endothelium-denuded rings pretreated with quinacrine dihydrochloride (nonspecific phospholipase A(2) inhibitor: 2 x 10(-5), 4 x 10(-5) M), nordihydroguaiaretic acid (NDGA) (lipoxygenase inhibitor: 10(-5), 3 x 10(-5) M), indomethacin (nonspecific cyclooxygenase inhibitor: 10(-5) M), AA-861 (5-lipoxygenase inhibitor: 10(-5), 5 x 10(-5) M), fluconazole (cytochrome P450 epoxygenase inhibitor: 10(-5) M), verapamil (10(-5) M), or calcium-free solution; and 3) endothelium-intact rings pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor: 5 x 10(-5) M), indomethacin, or fluconazole. Levobupivacaine-induced contractile response at each concentration (10(-4), 3 x 10(-4) M) was assessed in endothelium-denuded rings. Dose-response curves for potassium chloride in endothelium-denuded rings were generated in the presence or absence of NDGA and AA-861. Intracellular Ca(2+) levels were monitored by Ca(2+) image analysis using Fluo-4 fluorescence in vascular smooth muscle cells treated with levobupivacaine alone or AA-861 plus levobupivacaine. Levobupivacaine produced a tonic contraction in isolated rat aorta rings; this response was maximal at 10(-4) M levobupivacaine and gradually attenuated at 3 x 10(-4) M levobupivacaine. Levobupivacaine-induced contractions of endothelium-denuded rings were larger than those of endothelium-intact rings. Levobupivacaine-induced contraction of endothelium-denuded rings was attenuated by quinacrine dihydrochloride, NDGA, AA-861, verapamil, and calcium-free solution and, to a lesser extent, by indomethacin. L-NAME enhanced levobupivacaine-induced contraction of endothelium-intact rings and indomethacin slightly attenuated this contraction. NDGA and AA-861 attenuated the potassium chloride-induced contraction. AA-861 attenuated the levobupivacaine-induced intracellular calcium increase in vascular smooth muscle cells. Our data indicate that levobupivacaine-induced contraction of rat aortic smooth muscle is mediated mainly by activation of the lipoxygenase pathway and in part by activation of the cyclooxygenase pathway. In addition, activation of the lipoxygenase pathway seems to facilitate calcium influx via L-type calcium channels. Endothelial nitric oxide attenuates levobupivacaine-induced contraction.

Vasorelaxant effects of extracts of the stem bark of Terminalia superba Engler & Diels (Combretaceae)

Aim of the study The stem bark of Terminalia superba (Combretaceae) (TS) is used in traditional Cameroonian medicine as antihypertensive remedy. In the present study, we investigated the vasorelaxant properties of different extracts of TS and their underlying mechanisms. Materials and methods Activities of aqueous (AQU), methanolic (MET), methylene chloride (MC), and methylene chloride–methanol (MCM) extracts of TS were evaluated on isolated rat aortic rings precontracted with phenylephrine (PE) or high KCl. Results All extracts induced a vasodilating effect both on KCl- and PE-induced contractions. The effects of MC and MCM extracts were greater than those of AQU or MET extracts ( P < 0.05). MC had an endothelium-independent effect and reduced Ca ++-induced contraction following PE or KCl challenge ( P < 0.05). After incubation with verapamil, MC induced a relaxation in rings precontracted by PE ( P < 0.001). By contrast, the effect of MCM was endothelium-dependent and decreased by the nitric oxide synthase inhibitor N W-nitro- l-arginine methyl ester ( P < 0.05). Conclusions These data demonstrate that the MC extract exhibits vasorelaxant effects that are partly due to inhibition of extracellular Ca ++ influx and/or inhibition of intracellular Ca ++ release in vascular smooth muscle cells. By contrast, the effect of the MCM extract was found to be endothelium- and nitric oxide dependent.

Abstract 5218: Corrin-mediated Activation of Nitric Oxide Receptor and Its Cardiovascular Consequences

Soluble guanylyl cyclase (sGC) is the recognized receptor for nitric oxide (NO) messenger molecule. In addition to NO, several allosteric regulators, which do not produce NO, can activate sGC heme-dependently or independently. Earlier studies recognized that tetrapyrrole protoporphyrin IX can actively replace sGC heme moiety or bind to the heme-deficient sGC, resulting in a robust activation of sGC ability to produce cGMP from GTP. We investigated the effects of various tetrapyrroles on the ability of sGC to generate cGMP in vitro. Our data demonstrated that not only various protoporphyrins but some corrins can activate sGC. Pro-vitamin B12 dicyanocobinamide (CN2-Cbi) was the most efficient among tested compounds. CN2-Cbi-dependent activation of sGC was synergistically enhanced by BAY41–2272, a heme-dependent allosteric regulator of sGC. CN2-Cbi and several other corrins, including vitamin B12, induces a dose-dependent relaxation on isolated rat aorta rings. Tested corrins were equally effective in endothelium competent or endothelium denuded rings. Effective concentrations of CN2-Cbi were significantly improved with BAY41–2272, and relaxation was achieved even with micromolar CN2-Cbi. Therefore, data are consistent with direct activation of sGC by CN2-Cbi. Following ex-vivo relaxation by CN2-Cbi, we monitored blood pressure (BP) in anesthetized rats in response to CN2-Cbi. CN2-Cbi (5 mg/kg bolus) induced a modest and transient decrease in BP while 15 mg/kg showed a more marked and prolonged response. Decreased BP was associated with increased cardiac output, suggesting vascular relaxation. CN2-Cbi combined with 100 μ g/kg BAY41–2272 induced a more profound and prolonged decrease in BP. Following U-46619, a thromboxane analogue to induce systemic and pulmonary hypertension, combined CN2-Cbi/BAY41–2272 was also effective in restoring systemic and pulmonary pressures to baseline. These data suggest that CN2-Cbi (or corrin)-mediated activation of sGC may be an alternative approach to regulate sGC activity and vascular function. Data indicate that derivatives of vitamin B12 may be used for regulation of BP in normal and hypertensive conditions as part or treatment regimens or dietary supplements.

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 (44 mM) for 4 h 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 (0.1 mM), but not by indomethacin (0.01 mM). 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.

Effects of 45-nm silver nanoparticles on coronary endothelial cells and isolated rat aortic rings

This study was undertaken to determine whether silver nanoparticles (Ag-45nm NPs) induce selective and specific biological effects, such as induction of proliferation and nitric oxide (NO) production, and cytotoxicity in coronary endothelial cells (CECs), and regulation of vascular tone in isolated rat aortic rings. Physical characterization of Ag-45nm NPs by transmission electron microscopy (TEM) demonstrated that nanoparticles ranging in size from 10 to 90nm had biological effects on CECs. Increasing concentrations of Ag-45nm NPs exerted a dual effect on cell proliferation whereby proliferation was inhibited at low concentrations of NPs and stimulated at high concentrations. The effects of high, but not low, concentrations of Ag-45nm NPs were dependent on NO because the effects were partially blocked by NG-nitro-l-arginine methyl ester (L-NAME). We have also shown that high, but not low, concentrations of Ag-45nm NPs induce NO-dependent proliferation through activation of endothelial nitric oxide synthase (eNOS) by phosphorylation of Serine 1177. Moreover, the antiproliferative and proliferative effects of Ag-45nm NPs were concentration-dependent and inversely correlated with cellular toxicity. In isolated rat aortic rings, a low concentration of NPs induced vasoconstriction and a high concentration stimulated vasodilation. The physiologic effects induced by a low concentration of Ag-45nm NPs inhibited acetylcholine- (ACh-) induced NO-mediated relaxation. Vasodilation induced by a high concentration of NPs was partially abolished by L-NAME pretreatment. When the endothelium was removed from the rings, all physiologic responses were blocked. These results clearly demonstrate that the NPs have selective and specific effects on the vascular endothelium in a concentration-dependent manner and suggest that opposite effects could be associated with NPs of different sizes.

Apigenin protects endothelium-dependent relaxation of rat aorta against oxidative stress

Apigenin is shown to have cardiovascular effects, but the effects of apigenin on aortas injured by exogenous oxidants are unknown. The objective of this study was to investigate the effect of apigenin on endothelium-dependent vasorelaxation in isolated rat aortic rings exposed to superoxide anion produced by pyrogallol, and its mechanism. The male Sprague–Dawley rat thoracic aorta was rapidly dissected out and the effect of apigenin on tension of aortic rings pretreated with 500 μM pyrogallol, inducing oxidative stress injury, was measured. The activity of nitric oxide synthase (NOS), the level of nitric oxide (NO) and the inhibition of superoxide anion in aortic tissues were measured. We found that pretreatment with pyrogallol concentration-dependently decreased acetylcholine-induced endothelium-dependent vasorelaxation. Apigenin (0.5–72.0 μM) evoked a concentration-dependent relaxation in aortas (pD 2: 5.304 ± 0.049), which was weakened by l-NAME (the maximal relaxation fell from 87.6 ± 6.7% to 37.1 ± 8.8%, P < 0.01), but not by aminoguanidine and indomethacin. Apigenin markedly attenuated the inhibition of vasorelaxation induced by pyrogallol (the maximal relaxation elevated from 55.8% ± 6.6% to 69.5% ± 6.4%, and the pD 2 increased from 6.559 ± 0.119 to 7.057 ± 0.145, P < 0.01) and increased the inhibition of superoxide anion (from 94.6% to 74.5%), the NO level (from 77.1% to 94.4%), and the constitutive NOS activity (from 35.1% to 62.5%). These results indicate that pyrogallol decreased endothelium-dependent vasorelaxation in rat aortas via oxidative stress, which was markedly attenuated by apigenin. This may be mediated by weakening the oxidative stress and the NO reduction.