Relaxation Of Rat Aorta Research Articles

A role for cyclooxygenase in aging-related changes of β-adrenoceptor-mediated relaxation in rat aortas

beta-Adrenoceptor-mediated vasorelaxation decreases with age in various vascular beds. The present study investigated the roles of cyclooxygenase (COX) on beta-adrenoceptor vasorelaxation by isoprenaline in 8- and 54-week-old rat aortas. The vasorelaxation responses by isoprenaline (0.03-3 microM) were significantly reduced in 54-week-old aortas compared to 8-week. Addition of the non-selective COX inhibitors indomethacin (10 microM) or aspirin (10 microM) restored isoprenaline vasorelaxation of 54-week-old aortas to levels found in 8-week-old aortas. This suggests the involvement of COX prostanoids in the age-related reduction of beta-adrenoceptor vasorelaxation. Immunohistochemistry revealed greater levels of COX-1 and COX-2 staining in 54-week-old aortas compared to 8-week with expression located mainly in medial smooth muscle. An age-linked increase in COX-1 and COX-2 protein was found in cremaster arterioles of 54-week-old rats (compared to 8-week) mainly in the endothelial layer. The age-related increase in COX-1 and COX-2 protein led to elevation of prostacyclin (measured as 6-keto prostaglandin F(1alpha)) and thromboxane A(2) (measured as thromboxane B(2)) in 54-week compared to 8-week-old aortas. Endothelium removal in 54-week aortas markedly reduced the 6-keto prostaglandin F(1alpha) level, thus suggesting an endothelial source for elevated prostacyclin. These findings in combination with the effects of COX inhibitors suggest that the age-related decrease in beta-adrenoceptor vasorelaxation by isoprenaline is due to an age-linked increase in COX expression, which elevates production of COX-derived vasoactive prostanoids.

NO‐donor melatonin derivatives: synthesis and in vitro pharmacological characterization

Numerous studies document that melatonin possesses a broad-spectrum antioxidant activity. It traps a number of reactive oxygen species (ROS) such as hydroxyl and peroxyl radicals, singlet oxygen and hypochlorous acid. It also inhibits peroxynitrite-induced reactions. It is known that atherosclerosis progression involves ROS-induced oxidation of low-density lipoproteins in sub-endothelial space and the depletion of nitric oxide (NO) in blood vessels, as well as a decreased sensitivity of the vessels to the actions of NO. Considering this, a series of new NO-donor antioxidants were designed and synthesized by joining melatonin with NO-donor nitrooxy and furoxan moieties as polyvalent agents potentially useful for the treatment of cardiovascular diseases involving atherosclerotic vascular changes. The in vitro antioxidant properties of the resulting products were assessed in the thiobarbituric acid reactive substances assay (TBARS), the ABTS(+.) as well as in the alkaline phosphatase (ALP) assay. The antioxidant capacities of NO-donor melatonins to inhibit lipoperoxidation (TBARS-IC(50)) was predominantly dependent on their lipophilicity, and therefore on their partitioning process into membranes. On the other hand, their comparable capacity to inhibit protein oxidation (ALP-IC(50)) was independent of their lipophilicity and was consistent with their similar ability to participate in electron transfer reactions. All the NO-donor melatonins were also evaluated for their ability to relax rat aorta strips precontracted with 1 microM phenylephrine. Finally, binding affinities and intrinsic activity studies, carried out at MT(1) and MT(2) receptor subtypes, showed a rather complex picture in need of further investigation.

Long-term fenofibrate treatment impairs endothelium-dependent dilation to acetylcholine by altering the cyclooxygenase pathway

Experimental studies and opinion articles emphasize that cardiovascular alterations associated with ageing can be improved by the long-term use of fenofibrates. We analyzed the effect of fenofibrate treatment on the acetylcholine-induced relaxation in rat aorta and the participation of nitric oxide (NO) and cyclooxygenase (COX)-derived factors in this effect. Acetylcholine relaxation in untreated and 6-week fenofibrate-treated Wistar rats was analyzed in the absence and presence of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), the specific inducible NO (iNOS) synthase inhibitor 1400W, the nonspecific COX inhibitor indomethacin, the specific COX-2 inhibitor NS-398, the specific thromboxane receptor antagonist SQ-29548, the thromboxane synthesis inhibitor furegrelate, the prostacyclin synthesis inhibitor tranylcypromine, or the 20-HETES synthesis inhibitor formamidine. eNOS, iNOS, COX-1, and COX-2 expression was studied by Western blotting. In addition, production of prostaglandin F(2alpha) (PGF(2alpha)), thromboxane A(2) (TxA(2)), prostaglandin E(2) (PGE(2)), isoprostanes, and prostacyclin (PGI(2)) was also measured. Fenofibrate treatment reduced acetylcholine relaxation. Indomethacin, NS-398, and tranylcypromine decreased acetylcholine relaxation in untreated rats but enhanced relaxation in treated rats. SQ-29548 increased acetylcholine responses in segments from treated rats but not in segments from untreated rats. L-NAME decreased vasodilator response to acetylcholine in both groups while furegrelate, NS-398, 1400W, and formamidine did not affect acetylcholine responses in either group. eNOS and COX-2 expression was higher in aorta from treated rats while COX-1 and iNOS remained unmodified. Basal and acetylcholine-stimulated NO and PGE(2) release were increased, and that of PGI(2) decreased in treated rats. TxA(2) release was similar, but PGF(2alpha) release was undetectable in both groups. Although it increases NO production through increases in eNOS expression, fenofibrate treatment induces endothelial dysfunction. This effect seems to be mediated by decreased PGI(2) and increased PGE(2) release, and it may help to explain the rise in thromboembolic events observed after long-term fenofibrate treatment in humans.

Celecoxib dilates guinea-pig coronaries and rat aortic rings and amplifies NO/cGMP signaling by PDE5 inhibition

Celecoxib carries a smaller cardiovascular risk for myocardial infarction and hypertension than other cyclooxygenase-2 (COX-2)-selective non-steroidal anti-inflammatory drugs NSAIDs ("coxibs") and may ameliorate endothelial dysfunction. We aimed to determine which mechanism possibly accounts for the beneficial effect by investigating its vascular action in different in vitro preparations in comparison with other coxibs and reference phosphodiesterase-5 (PDE5) inhibitors. To uncover potential effects on coronary flow, the effects of celecoxib in comparison with other NSAIDs and the PDE5 inhibitors, sildenafil and zaprinast, were investigated in guinea-pig Langendorff heart. This was supported by studies for vasorelaxation, interaction with the NO/cGMP pathway, and measurement of cyclic nucleotide amounts released from rat aortic rings, and inhibition of human PDE5 as well as PDE4 activity. Bolus injections of sildenafil, celecoxib, and zaprinast (at 100 nmol) into the Langendorff heart increased coronary flow by approximately 100, 65, and 25%, respectively, while rofecoxib, lumiracoxib, parecoxib, and diclofenac, except valdecoxib (>100 nmol), failed to increase coronary flow up to 300 nmol. In rat aorta, sildenafil, celecoxib and zaprinast caused endothelium-dependent relaxation with -log[EC(50)]M values of 8.90, 6.66 and 5.56, respectively; their rank order of potency corresponds to their coronary dilatory effect. Celecoxib-induced relaxation of aorta was attenuated by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 10(-5) M). In aortic rings, celecoxib (3x10(-5) M) caused a fivefold increase in the cGMP level and potentiated that induced by sodium nitroprusside (5x10(-7) M). Celecoxib and valdecoxib inhibited human PDE5A1 with an IC(50) of 1.6x10(-5) and 1x10(-4) M, respectively, whereas other coxibs were without inhibitory effect. Celecoxib caused coronary vasodilatation in guinea-pig hearts and relaxation of rat aorta and had a potentiating effect on the NO/cGMP signaling pathway in rat aorta through specific blockade of PDE5. These unexpected findings clearly support the notion that celecoxib possesses an as yet undisclosed molecule-specific property that possibly compensates a decrease of prostacyclin-dependent cAMP generation by concomitantly increasing cGMP levels resulting from inhibition of PDE5.

Direct effects of lead (Pb 2+) on the relaxation of in vitro cultured rat aorta to acetylcholine

Lead (Pb 2+) exposure is related to increased blood pressure or hypertension of human or animals. Abnormal vascular relaxant responses of low level Pb 2+ exposed animals were reported by several studies. However, it is difficult to tell whether these effects were induced directly by Pb 2+ or not. In this study we hypothesized that Pb 2+ can directly affect the relaxation of vessels. Male Wistar rat aortae were removed and cultured in PMRI 1640 with 1 ppm Pb 2+ (4.8 μM lead acetate) for 0.5, 6, 12 and 24 h, and then their responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were examined. After incubated for 24 h, the relaxation induced by ACh was significantly decreased in Pb 2+ exposed aortic rings. However, there was not significant difference in relaxation induced by SNP between Pb 2+ exposed and control group. The nitrite in the culture media of aortic rings cultured for 24 h, measured with Griess method, was significantly decreased in the Pb 2+ exposed group. The expression of endothelial NOS (eNOS) and isoform NOS (iNOS) in the homogenate of aortic rings cultured for 24 h was measured by Western blot. The expression of eNOS of the Pb 2+ exposed group was significantly upregulated compared with that of the control group. However, there was no significant difference in the expression of iNOS in control and Pb 2+ exposed group. In conclusion, Pb 2+ was able to directly affect the relaxation of rat aorta. This effect may have some relation with the lower level of NO in the media, though the expression of eNOS was upregulated.

Endothelium-dependent relaxation of rat aorta induced by resveratrol

It has been noted that there are a number of biologically active phenolic compounds present in wine, particularly red wine. Such compounds include, for example, catechin, epicatechin, quercetin, rutin, trans-resveratrol and cis-resveratrol. The resveratrol isomers, in particular, have been found to promote vascular relaxation. The mechanisms by which resveratrol causes vasodilatation are uncertain. The aim of this study was to investigate the mechanism(s) of resveratrolinduced vasorelaxation in rat aorta with endothelium present. Rat aortic rings were precontracted with phenylephrine. Resveratrol induced relaxation of the rat aortic rings. L-NAME, an inhibitor of NO synthase, abolished relaxation of rat aorta, but methylene blue, an inhibitor of guanylate cyclase, did not abolish relaxation induced by resveratrol. Highly selective blocker of ATP-sensitive K+ channels, glibenclamide as well as a nonselective blocker of big Ca-sensitive K+ channels, charybdotoxin did not block resveratrol-induced relaxation of rat aorta. 4-Aminopiridine, which is a non selective blocker of voltage-gated K+ (Kv) channels, and margatoxin which inhibits Kv1 channels abolished relaxation of rat aortic rings induced by resveratrol. In conclusion, we have shown that resveratrol potently relaxed rat aortic rings with endothelium present. It seems that NO and smooth muscle KV channels are included in this relaxation. This finding is of clinical importance in both veterinary and human medicine.

The olive oil antioxidant hydroxytyrosol efficiently protects against the oxidative stress-induced impairment of the NO• response of isolated rat aorta

The Mediterranean diet, which is abundant in antioxidants, is associated with a relatively low incidence of coronary heart disease. Olive oil and olives, which contain the antioxidants hydroxytyrosol, oleuropein, and tyrosol, are important components of this diet. In this study, the effects of oxidative stress on the nitric oxide radical (NO(*))-mediated relaxation of rat aorta and the protection by these antioxidants were determined. Cumene hydroperoxide (CHP) was used to mimic oxidative stress induced by lipid hydroperoxides, which is mediated by the formation of hydroxyl radicals (OH(*)). CHP (300 microM) impaired the NO(*)-mediated relaxation of rat aorta by the acetylcholine receptor agonist carbachol (P < 0.05). This was due to a reduction in NO(*) production. A diminished NO(*)-mediated relaxation disturbs the vascular tone and leads to a rise in blood pressure, which is a well-established risk factor for coronary heart disease. Hydroxytyrosol (10 microM) efficiently protected the aorta against the CHP-induced impairment of the NO(*)-mediated relaxation (P < 0.05). Oleuropein, tyrosol, and homovanillic alcohol, a major metabolite of hydroxytyrosol, did not show protection. Moreover, hydroxytyrosol was found to be a potent OH(*) scavenger, which can be attributed to its catechol moiety. Because of its amphiphilic characteristics (octanol-water partitioning coefficient = 1.1), hydroxytyrosol will readily cross membranes and provide protection in the cytosol and membranes, including the water-lipid interface. The present study provides a molecular basis for the contribution of hydroxytyrosol to the benefits of the Mediterranean diet.

Distinct Activity of Peptide Mimetic Intracellular Ligands (Pepducins) for Proteinase‐Activated Receptor‐1 in Multiple Cells/Tissues

Proteinase-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, can be activated not only by PAR1-activating peptides (PAR1APs) based on the N-terminal cryptic tethered ligand sequence but also by an N-palmitoylated (Pal) peptide, Pal-RCLSSSAVANRSKKSRALF-amide (P1pal-19), based on the intracellular loop 3 of PAR1, designated pepducin, in human platelets or PAR1-transfected cells. The present article evaluated the actions of P1pal-19 and also the shorter peptide, Pal-RCLSSSAVANRS-amide (P1pal-12), known as a possible PAR1 antagonist, in multiple cells/tissues that naturally express PAR1. P1pal-19 as well as a PAR1AP, TFLLR-amide, evoked cytosolic Ca(2+) mobilization in cultured human lung epithelial cells (A549) and rat gastric mucosal epithelial cells (RGM1). P1pal-19 and TFLLR-amide, but not a PAR2-activating peptide, SLIGRL-amide, caused delayed prostaglandin E(2) formation in RGM1 cells. P1pal-19, like TFLLR-amide, produced endothelial NO-dependent relaxation in rat aorta and epithelial prostanoid-dependent relaxation in mouse bronchus. The P1pal-19-induced relaxation remained constant even after desensitization of PAR1 with TFLLR-amide in either tissue. P1pal-19 failed to mimic the contractile effects of TFLLR-amide in the endothelium-denuded preparations of rat aorta or superior mesenteric artery and the rat gastric longitudinal smooth muscle strips. P1pal-12 partially inhibited the vasorelaxation caused by TFLLR-amide and P1pal-19, but not SLIGRL-amide, in the rat aorta. Our data thus indicate that P1pal-19 is capable of mimicking the effects of PAR1APs in the endothelial and epithelial, but not smooth muscle, cells/tissues, and suggest that P1pal-12 may act as a PAR1 antagonist in the vascular endothelium.

Potassium Channels‐Mediated Vasorelaxation of Rat Aorta Induced by Resveratrol

Resveratrol, a phenolic substance present in grapes and a variety of medical plants, has been reported to induce vasorelaxation, however the mechanisms are uncertain. In this paper we investigate the possible participation of K(+) channels in the endothelium-independent vasodilatation of rat aorta induced by resveratrol. Resveratrol induced concentration-dependent relaxation of rings with endothelium and without endothelium. We used different potassium channel inhibitors to determine whether the K(+) channels mediated endothelium-independent relaxation of rat aorta induced by resveratrol. Highly selective blocker of ATP-sensitive K(+) channels, glibenclamide, as well as non-selective blockers of K(+) channels, tetraethylammonium, did not block resveratrol-induced relaxation of rat aortic rings. Charybdotoxin, a blocker of calcium-sensitive K(+) channels did not affect the resveratrol-induced relaxation. 4-Aminopiridine, non-selective blocker of voltage-gated K(+) (Kv) channels, and margatoxin that inhibits Kv1 channels abolished relaxation of rat aortic rings induced by resveratrol. In conclusion, we have shown that resveratrol potently relaxed rat aortic rings with denuded endothelium. It seems that 4-aminopiridine and margatoxin-sensitive K(+) channels located in the smooth muscle of rat aorta mediated this relaxation.

Comparison of the mechanisms underlying the relaxation induced by two nitric oxide donors: Sodium nitroprusside and a new ruthenium complex

We studied the mechanisms involved in the relaxation induced by nitric oxide (NO) donors, ruthenium complex ([Ru(terpy)(bdq)NO +] 3+-TERPY) and sodium nitroprusside (SNP) in denuded rat aorta. Both NO donors induced vascular relaxation independent of the agonist used in the pre-contraction. [Ru(terpy)(bdq)NO +] 3+ and SNP activated guanylyl cyclase (GC) and K + channels. The production of cGMP induced by [Ru(terpy)(bdq)NO +] 3+ — was higher than that obtained with SNP. The combination of GC inhibitor with K +channels blocker almost abolished the relaxation induced by the NO donors. The extracellular NO scavenger oxyhemoglobin reduced the potency without changing the maximum effect (Emax) of both NO donors. By using specific NO species scavengers, hydroxocobalamin and l-cysteine, we have identified the contribution of free radical NO (NO ) and nytroxil anion (NO −), respectively, to the rat aorta relaxation induced by both NO donors. The selective scavengers for NO and NO − reduced the potency but not the Emax of [Ru(terpy)(bdq)NO +] 3+. However, the NO − scavenger had no effect on the relaxation induced by SNP and NO scavenger reduced only the potency to SNP. The inhibition of sarcoplasmic reticulum Ca 2+-ATPase reduced only the potency of SNP without effect on the relaxation induced by [Ru(terpy)(bdq)NO +] 3+. Our results demonstrate that both NO donors induce relaxation by activating the GC and K + channels. The NO is the unique NO specie involved in the SNP-relaxation. On the other hand, the relaxant effect of [Ru(terpy)(bdq)NO +] 3+ involves both NO and NO −, that produce higher concentration of cGMP. The inhibition of sarcoplasmic reticulum Ca 2+-ATPase reduces the relaxation induced by SNP but it did not alter the relaxation induced by [Ru(terpy)(bdq)NO +] 3+.

Crataegus Special Extract WS® 1442 Induces an Endothelium-Dependent, NO-mediated Vasorelaxation via eNOS-Phosphorylation at Serine 1177

This study investigates the influence of WS(R) 1442, a special extract of Crataegus leaves with flowers, on the relaxation of rat aorta and human mammarian artery (coronary bypass patients). Experiments were performed in the presence and absence (mechanical disruption) of endothelium. In addition, we investigated three fractions of WS(R) 1442 (fraction A: lipophilic, containing flavonoids and oligomeric procyanidins (OPC), fraction B: hydrophilic, containing flavonoids and low molecular weight OPC, fraction C: hydrophilic, essentially flavonoid-free and rich in high molecular weight OPC). WS 1442 induced a concentration-dependent vasodilation in isolated vessel rings that had been precontracted by 10 microM phenylephrine (concentration for halfmaximal relaxation (IC(50)): rat: 15.1 +/- 0.6 microg/ml (n = 7), human: 19.3 +/- 3.4 microg/ml (n = 6)). The maximal vasorelaxation induced after application of 100 microg of WS 1442 was 75.0 +/- 5.7% (rat) and 79.2 +/- 5.8% (human) of the papaverine (0.1 mM)-induced vasodilation. If the experiments were performed in the presence of L-nitroarginine methylester (10 microM, eNOS-inhibition) or after mechanical disruption of the endothelium, no vasorelaxation was observed in the presence of WS 1442. The vasorelaxant properties of WS 1442 were mediated by fraction C. WS 1442 induced an NO-liberation from human coronary artery endothelial cells as measured by diaminofluorescein. WS 1442 induced eNOS-activation was due to a phosphorylation at serine 1177. No eNOS-translocation or phosphorylation at serine 114 or threonine 495 was observed after application of WS 1442. It is concluded that WS 1442, induces an endothelium-dependent, NO-mediated vasorelaxation via eNOS phosphorylation at serine 1177.

Aqueous extract of Schizandra chinensis fruit causes endothelium-dependent and -independent relaxation of isolated rat thoracic aorta

An aqueous extract of Schizandra chinensis fruit (ScEx) has long been used to promote the vascular health of postmenopausal women in Korea. This study investigated the ability of ScEx to relax rat aorta constricted with norepinephrine (NE) and the mechanism(s) of such relaxation. ScEx induced partial, endothelium-dependent relaxation. In particular, the relaxation induced by lower concentrations of ScEx (0.1 and 0.3 mg/ml) was largely endothelium-dependent, and was essentially abolished by NG-nitro-L-arginine, methylene blue, 1H-[1,2,3] oxadiazole [4,4-a] quinoxalin-1-one, indomethacin, or ICI 182,780. The results indicate that the response to ScEx involves enhancement of the nitric oxide (NO)-cGMP system, and that it occurs via estrogen receptors. The magnitude of the inhibition with these treatments decreased with increasing ScEx concentration, however, indicating that other vasorelaxation mechanisms are involved, which depend on the ScEx concentration. Calcium concentration-dependent contraction curves in high potassium depolarization medium were shifted significantly to the right and downward after incubation with ScEx (0.3 and 1.0 mg/ml), implying that ScEx is also involved in inhibition of the extracellular calcium influx to vascular smooth muscle. These data demonstrate that ScEx caused both endothelium-dependent and -independent vasorelaxation, which may contribute to understanding the cardiovascular protective effect of ScEx.

Vasoactive intestinal peptide‐induced relaxation is mediated by nitric oxide activation in the endothelial caveolae of rat aorta

Vasoactive intestinal peptide (VIP) a non-adrenergic non-cholinergic (NANC) transmitter widely distributed in both the central and peripheral nervous system is an endothelium-dependent vasodilator. The contribution of endothelium derived relaxing factors to VIP-induced relaxation remains unclear. We hypothesized that nitric oxide (NO) produced by endothelial NO synthase (eNOS) at the caveolae plays a role in this relaxation. We assessed the effect of VIP on isolated rat aorta rings in vitro which was incubated with either N-nitro-L-Arginine, L-NNA, (a NOS inhibitor), indomethacin (a cycloxygenase inhibitor), or methyl-beta-cyclodextrin (a membrane sequester that disassembles caveolae). In intact and denuded aortic rings precontracted with phenylephrine (PE, 0.1 μmol/L), the addition of VIP (1nmol/L to 1 μmol/L) produced relaxation in a dose-dependent manner. The maximal relaxation induced by VIP in endothelium-intact aortic rings (53 ± 9%, n=8) was impaired after removal of the endothelium (11 ± 2%, n=3, P<0.05), or in the presence of L-NNA (20 ± 11%, n=5, P <0.05) and methyl-β-cyclodextrin (20 ± 8%, n=6, P<0.05). In contrast, VIP-induced relaxation was enhanced in the presence of indomethacin (83 ± 7%, n=5, P<0.05). These data suggest that NO produced by eNOS at the caveolae plays a stimulatory role in VIP-induced relaxation in rat aorta, whereas prostaglandins play an inhibitory role.

The nitric oxide donor cis‐[Ru(Cl)(bpy)2(NO)](PF6) induces rat aorta relaxation and Ca2+ reduction under visible light irradiation.

The relaxant effect of the nitric oxide (NO) donors cis-[Ru(Cl)(bpy)2(NO)](PF6) (RUNOCL) and sodium nitroprusside (SNP) was investigated in the rat denuded aortic rings and on the cytosolic Ca2+concentration ([Ca2+]c) in aorta smooth muscle cells. RUNOCL and SNP induced relaxation in a concentration dependent way. RUNOCL was studied by photo-induction using a visible light emission source (3.4x1017 einstein s−1) system with excitation range up to ?? 300 nm to release NO from the complex. The maximum effect induced by RUNOCL was 101.16±3.74% and pD2 6.62±0.16, n=7 and SNP 99.39±4.37% and pD2 8.57±0.22, n=5. Cells loaded with Fluo-3AM were imaged by a confocal scanning laser microscope excited at 488 nm line of argon ion laser and the emission was measured at 510 nm. ([Ca2+]c was calculated in fluorescence intensities (IF) from control IF (100% phenylephrine 0.1μmol/L). RUNOCL (100 μmol/L) or SNP (100 μmol/L) was added to the chamber and the %IF was measured after 240 seconds in the absence or after incubation for 30 min with the given inhibitor. RUNOCL reduced ([Ca2+]c to 60.0±10.0%, n=4 and in presence of guanylyl cyclase inhibitor (10 μmol/L ODQ) IF was 81.0±5.0% and with K+channel blocker (1mmol/L TEA) IF was 79.0±6.43%. The combination of ODQ and TEA increased the IF (97.0±3.5%). SNP reduced IF to 81.4±4.7%, n=4 that was inhibited by ODQ (94.0±3.6%) and by TEA (88.0±2.1%). Combination of ODQ and TEA increased the IF (92.0±2.8%). In conclusion, RUNOCL and SNP reduce [Ca2+]c and this effect involves guanylyl cyclase and K+ channels sensitive to TEA. Supported by FAPESP and CNPq.

Endothelium-Dependent Induction of Vasorelaxation by the Butanol Extract of Phellinus igniarius in Isolated Rat Aorta

The butanol extract of Phellinus igniarius (BPI) induced relaxation of the phenylephrin e-precontracted rat aorta in a dose-dependent manner, and its effect was abolished by the removal of functional endothelium. Pretreatment of the aortic tissues with N(G)-nitro-L-arginine methyl ester (L-NAME), methylene blue, or 1H-[1,2,4]-oxadiazole-[4,3-alpha]-quinoxalin1-one (ODQ) inhibited the vascular relaxation induced by BPI. BPI-induced vascular relaxations were also markedly attenuated by the addition of verapamil or diltiazem, while the relaxant effect of BPI was not blocked by pretreatment with indomethacine, glibenclamide, tetraethylammonium (TEA), atropine, or propranolol. Incubation of endothelium-intact rat aorta with BPI increased the production of cGMP in a dose-dependent manner. These results suggest that BPI dilates vascular smooth muscle via endothelium-dependent nitric oxide-cGMP signaling pathway, with the possible involvement of L-type Ca(2+) channels.

The Novel Imidazopyridine 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) Is a Highly Selective Inhibitor of the Inducible Nitric-Oxide Synthase

We have identified imidazopyridine derivatives as a novel class of NO synthase inhibitors with high selectivity for the inducible isoform. 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) showed half-maximal inhibition of crudely purified human inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NO synthases at 86 nM, 17 microM, and 162 microM, respectively. Inhibition of inducible NO synthase was competitive with l-arginine, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. In radioligand and surface plasmon resonance experiments, BYK191023 exhibited an affinity for iNOS, nNOS, and eNOS of 450 nM, 30 microM, and >500 microM, respectively. Inhibition of cellular nitrate/nitrite synthesis in RAW, rat mesangium, and human embryonic kidney 293 cells after iNOS induction showed 40- to 100-fold higher IC(50) values than at the isolated enzyme, in agreement with the much higher l-arginine concentrations in cell culture media and inside intact cells. BYK191023 did not show any toxicity in various rodent and human cell lines up to high micromolar concentrations. The inhibitory potency of BYK191023 was tested in isolated organ models of iNOS (lipopolysaccharide-treated and phenylephrine-precontracted rat aorta; IC(50) = 7 microM), eNOS (arecaidine propargyl ester-induced relaxation of phenylephrine-precontracted rat aorta; IC(50) > 100 microM), and nNOS (field-stimulated relaxation of phenylephrine-precontracted rabbit corpus cavernosum; IC(50) > 100 microM). These data confirm the high selectivity of BYK191023 for iNOS over eNOS and nNOS found at isolated enzymes. In summary, we have identified a new highly selective iNOS inhibitor structurally unrelated to known compounds and l-arginine. BYK191023 is a valuable tool for the investigation of iNOS-mediated effects in vitro and in vivo.

Signal transduction pathways involved in particulate matter induced relaxation in rat aorta—Spontaneous hypertensive versus Wistar Kyoto rats

Previously we reported that in vivo exposure to ambient particulate matter (PM) induces vasodilatation in rat aorta. The purpose of the current study was to investigate the intracellular messengers involved in PM-elicited vasodilatation in aortas from spontaneous hypertensive (SHR) and normotensive (WKY) rats. MethodsThe contribution of three different intracellular pathways, i.e. (1) the NO-cGMP pathway, (2) prostanoids signaling and (3) endothelial hyperpolarisation factors were evaluated by using specific inhibitors (NS2028, Diclofenac and high K-concentration/17-ODYA, respectively). Using antagonists of capsaicin- or histamine receptors we tested potential interactions of PM with these receptors. Particle suspensions (EHC-93), particle filtrates (particle-free) and Cu2+- or Zn2+-containing solutions were used to obtain cumulative dose–response curves of relaxation in normal and endothelium-denuded rings. ResultsOur present data confirm that PM and its soluble components elicit an endothelium-independent vasodilatation in rat aorta rings. The response is mainly linked to the activation of soluble guanylate cyclase (sGC), since its inhibition by NS2028 almost abolished relaxation. Indeed PM suspensions stimulated cGMP production in purified isolated sGC. Neither the receptor nor their signaling pathways played a significant role in the direct relaxation by PM or metals. Vasodilatation responses were significantly higher in SHR than WKY control rats. ConclusionOur data demonstrate that PM elicits a dose-dependent vasodilatation via activation of sGC in vascular smooth muscles. PM components, including soluble transition metals play a major role in this response. The stronger effect in SHR rats is in accordance with the observation that acute effects of PM are mainly seen in patients with underlying cardiovascular diseases.

Pravastatin Restores DDAH Activity and Endothelium-Dependent Relaxation of Rat Aorta After Exposure to Glycated Protein

This study was designed to investigate whether glycated bovine serum albumin (AGE-BSA) inhibits dimethylarginine dimethylaminohydrolase (DDAH) activity to contribute to its adverse effect on endothelium-dependent relaxation in rat aorta, and whether pravastatin reverses the inhibition of DDAH activity and endothelial dysfunction induced by AGE-BSA. Endothelium-dependent relaxation of aortic rings was measured by isometric tension recording, and DDAH activity, and the contents of nitrite/nitrate as well as malondialdehyde (MDA) in aortic tissue were determined after exposure of Sprague-Dawley rat aorta to AGE-BSA (1.70 mmol/L) for 60 minutes in the presence or absence of pravastatin. In comparison with control, both endothelium-dependent relaxation and DDAH activity (0.032 +/- 0.002 versus 0.095 +/- 0.003 U/g protein, n = 5, P < 0.01) were significantly inhibited in isolated rat aorta after exposure to AGE-BSA, which was accompanied by decreases of nitrite/nitrate contents and elevations of MDA levels in aorta. Treatment with pravastatin (1 mmol/L) not only prevented the inhibition of endothelial function but also reversed the decrease of DDAH activity induced by AGE-BSA and normalized the alterations in nitrite/nitrate and MDA contents. Similar effects were observed when rat aorta exposed to AGE-BSA in the presence of antioxidant pyrrolidine dithiocharbamate (PDTC, 30 micromol/L) or protein kinase C inhibitor chelerythrine (1 micromol/L). These results suggested that decreased DDAH activity may be involved in endothelial dysfunction of rat aorta induced by AGE-BSA, and that pravastatin restores DDAH activity and endothelium-dependent relaxation after aorta exposure to AGE-BSA, which may be secondary to its antioxidative effects.

Pharmacological Profile and Therapeutic Potential of BM‐573, a Combined Thromboxane Receptor Antagonist and Synthase Inhibitor

BM-573 (N-terbutyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl]urea), a torsemide derivative, is a novel non-carboxylic dual TXA2 synthase inhibitor and receptor antagonist. The pharmacological profile of the drug is characterized by a higher affinity for the thromboxane receptor than that of SQ-29548, one of the most powerful antagonists described to date, by a complete prevention of human platelet aggregation induced by arachidonic acid at a lower dose than either torsemide or sulotroban, and by a significantly prolonged closure time measured by the platelet function analyser (PFA-100). Moreover, at the concentrations of 1 and 10 microM, BM-573 completely prevented production of TXB2 by human platelets activated by 0.6 mM of arachidonic acid. BM-573 prevents rat fundus contraction induced by U-46619 but not by prostacyclin or other prostaglandins. Despite possessing a chemical structure very similar to that of a diuretic torsemide, BM-573 has no diuretic activity. BM-573 does not prolong bleeding time and, unlike some of the other sulfonylureas, has no effect on blood glucose levels. In vivo, BM-573 appears to have antiplatelet and antithrombotic activities since it reduced thrombus weight and prolonged the time to abdominal aorta occlusion induced by ferric chloride. BM-573 also relaxed rat aorta and guinea pig trachea precontracted with U-46619. In pigs, BM-573 completely antagonized pulmonary hypertensive effects of U-46619 and reduced the early phase of pulmonary hypertension in models of endotoxic shock and pulmonary embolism. Finally, BM-573 protected pigs from myocardial infarction induced by coronary thrombosis. These results suggest that BM-573 should be viewed as a promising therapeutic agent in the treatment of pulmonary hypertension and syndromes associated with platelet activation.

Effects of dual-action genistein derivatives on relaxation in rat aorta

Protein tyrosine kinases and nitric oxide (NO) play important roles in several cardiovascular diseases. In this study, we examined the actions of two compounds, each has structure of genistein (a tyrosine kinase inhibitor) and an NO donor, on endothelium-independent relaxation responses in the isolated rat aorta. By rational drug design, genistein was modified to acquire an NO donor, and we synthesized two such compounds (G-II, G-VI). These compounds and genistein induced dose-dependent relaxation responses in endothelium-denuded aortic strips, the rank order of potencies being G-VI > G-II > genistein. Incubation of endothelium-denuded strips with 1H-[1,2,4] oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 10 microM), a guanylyl cyclase inhibitor, inhibited both the G-II- and G-VI-induced relaxations, but not the genistein-induced relaxation. The residual relaxations induced by these two compounds were similar to the genistein-induced relaxation. Incubation of endothelium-denuded strips with lysophosphatidylcholine (LPC, 20 microM)-which is a major atherogenic lysophospholipid component of oxidized low-density lipoprotein and is known to activate tyrosine kinase-caused a significant rightward shift in the dose-response curve for genistein. LPC also shifted the G-II- and G-VI-induced relaxation curves to the right; however, these relaxations in the presence of LPC were greater than that induced by genistein. The sodium nitroprusside-induced relaxation in endothelium-denuded strips was similar between in the absence and presence of LPC. These results suggest that each of our newly developed G-II and G-VI compounds has a dual action, as an NO donor and a tyrosine kinase inhibitor. These compounds may be useful against certain cardiovascular diseases.