Endothelium-denuded Rat Aorta Research Articles

Halichlorine is a novel L-type Ca 2+ channel inhibitor isolated from the marine sponge Halichondria okadai Kadota

Halichlorine, isolated from a marine sponge Halichondria okadai Kadota, has a unique structure and its physiological activity is virtually unknown. In the present study, we investigated the direct effect of halichlorine on vascular contractility. In endothelium-denuded rat aorta, while the treatment of halichlorine (0.01–10 μM) did not induce vascular contraction, halichlorine (0.01–10 μM) dose-dependently inhibited both the steady-state precontractions induced by high K + (65.4 mM) and phenylephrine (1 μM). The vasodilator effect of halichlorine (10 μM) on high K + (65.4 mM)-induced contraction was more potent than that on phenylephrine (1 μM)-induced contraction (65.4 mM high K +: 72.7 ± 3.4%; 1 μM phenylephrine: 34.7 ± 2.3%). To investigate the mechanism underlying the suppressive effect of halichlorine on vascular contractility, we examined the effect of halichlorine on intracellular Ca 2+ concentration in vascular smooth muscle with a fluorescent Ca 2+ indicator, fura-2. Treatment of halichlorine (10 μM) significantly inhibited the sustained [Ca 2+] i elevation induced by high K + (65.4 mM) (45.3 ± 5.5%). Furthermore, current measurements by whole-cell mode patch-clamp recording in rat aortic smooth muscle cells (A7r5 cells) demonstrated that halichlorine (10 μM) decreased the current density of the L-type Ca 2+ channel (peak Ca 2+-channel current densities: − 2.09 ± 0.27 pA/pF for control; − 0.58 ± 0.07 pA/pF for halichlorine). These results suggest that halichlorine inhibits L-type Ca 2+ channels in vascular smooth muscle cells, which inhibit intracellular Ca 2+ influx, and then reduce vascular contractions.

Unrepeatable extracellular Ca 2+-dependent contractile effects of cyclopiazonic acid in rat vascular smooth muscle

Cyclopiazonic acid (CPA), a specific reversible inhibitor of Ca 2+-pumps in sarcoplasmic reticulum, causes a slowly developing and subsequently diminishing characteristic contraction in endothelium-denuded rat vascular smooth muscle. We recently found that CPA-induced contractions were not completely repeatable in endothelium-denuded rat aorta and superior mesenteric artery. 10 µM CPA-induced contractions expressed as a percentage of 80 mM KCl-induced contraction were significantly decreased from 51.4 ± 5.7% to 11.8 ± 2.6% ( P < 0.0001) upon the second application in endothelium-denuded rat aorta, and this was not due to any irreversible cytotoxic effects of CPA. The decrease of CPA-induced contractile responses upon the second application was dependent on both types of blood vessels and doses of CPA upon the first application. CPA upon the second application in Ca 2+-containing solutions did induce its characteristic contractions in the rings pretreated with Ca 2+-free solutions or Ca 2+ entry blockers before and during its first application, suggesting that capacitative mode of Ca 2+ influx during the application of CPA might be responsible for the diminishment of contractions upon the second application. These data suggest that CPA by inducing a transient rise in cytosolic Ca 2+ level might cause a long-lasting upregulation of Ca 2+ extrusion across the plasma membrane in vascular smooth muscle cells and thus accelerate Ca 2+ efflux over a prolonged period, leading to unrepeatable contractile effects of CPA. Such long-lasting upregulation of Ca 2+ extrusion may contribute to the regulation of excitability of vascular smooth muscle cells and protect the cells against excitotoxic injury.

Possible Involvement of Ca2+ Activated K+ Channels, SK Channel, in the Quercetin-Induced Vasodilatation

Effects of quercetin, a kind of flavonoids, on the vasodilating actions were investigated. Among the mechanisms for quercetin-induced vasodilatation in rat aorta, the involvement with the Ca(2+) activated K(+) (K(Ca)) channel was examined. Pretreatment with NE (5 microM) or KCl (60 mM) was carried out and then, the modulation by quercetin of the constriction was examined using rat aorta ring strips (3 mm) at 36.5. Quercetin (0.1 to 100 microM) relaxed the NE-induced vasoconstrictions in a concentration-dependent manner. NO synthesis (NOS) inhibitor, NG-monomethyl-L-arginine acetate (L-NMMA), at 100 microM reduced the quercetin (100 microM)-induced vasodilatation from 97.8+/-3.7% (n=10) to 78.0+/-11.6% (n=5, p<0.05). Another NOS inhibitor, L-NG-nitro arginine methyl ester (L-NAME), at 100 microM also had the similar effect. In the presence of both 100 microM L-NMMA and 10 microM indomethacin, the quercetin-induced vasodilatation was further attenuated by 100 microM tetraethylammonium (TEA, a K(Ca) channel inhibitor). Also TEA decreased the quercetin-induced vasodilatation in endothelium-denuded rat aorta. Used other K(Ca) channel inhibitors, the quercetin-induced vasodilatation was attenuated by 0.3 microM apamin (a SK channel inhibitor), but not by 30 nM charybdotoxin (a BK and IK channel inhibitor). Quercetin caused a concentration-dependent vasodilatation, due to the endothelium-dependent and -independent actions. Also quercetin contributes to the vasodilatation selectively with SK channel on smooth muscle.

Methylglyoxal Inhibits Smooth Muscle Contraction in Isolated Blood Vessels

Methylglyoxal (MGO) is a metabolite of glucose. In addition to evidence that increased plasma MGO level is associated with diabetic vascular complications, recent studies demonstrated that MGO accumulated in vascular tissues of hypertensive animals. We hypothesized that MGO could directly affect vascular reactivity. To test the hypothesis, we examined effects of MGO on contraction of isolated blood vessels. Treatment of endothelium-denuded rat aorta with MGO (420 μM, 30 min) shifted the concentration–response curve for noradrenaline (NA: 1nM–1 μM) to the right. The inhibitory effect was concentration-dependent (MGO: 42 – 420 μM). Indomethacin (10 μM) and cimetidine (30 μM) could not prevent the inhibitory effect of MGO. However, a non-selective K+-channel inhibitor, tetramethylammonium (10 mM), prevented it. Glibenclamide (3 μM), an ATP-sensitive K+-channel inhibitor or apamin (1 μM), a small conductance Ca2+-activated K+-channel inhibitor was ineffective, but iberiotoxin (100 nM), a large conductance Ca2+-activated K+ (BKCa)-channel inhibitor significantly prevented the effect of MGO. MGO (420 μM, 30 min) also inhibited the NA (1 nM – 1 μM)-induced contraction in mesenteric artery. The present results indicate that MGO has an inhibitory effect on contractility of isolated blood vessel, which is mediated via opening smooth muscle BKCa channel.

Pharmacology of Macitentan, an Orally Active Tissue-Targeting Dual Endothelin Receptor Antagonist

Macitentan, also called Actelion-1 or ACT-064992 [N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N'-propylaminosulfonamide], is a new dual ET(A)/ET(B) endothelin (ET) receptor antagonist designed for tissue targeting. Selection of macitentan was based on inhibitory potency on both ET receptors and optimization of physicochemical properties to achieve high affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major and pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonized the specific binding of ET-1 on membranes of cells overexpressing ET(A) and ET(B) receptors and blunted ET-1-induced calcium mobilization in various natural cell lines, with inhibitory constants within the nanomolar range. In functional assays, macitentan and ACT-132577 inhibited ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET(A) receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea (ET(B) receptors). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricle hypertrophy, and it markedly improved survival. In diabetic rats, chronic administration of macitentan decreased blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural injury). In conclusion, macitentan, by its tissue-targeting properties and dual antagonism of ET receptors, protects against end-organ damage in diabetes and improves survival in pulmonary hypertensive rats. This profile makes macitentan a new agent to treat cardiovascular disorders associated with chronic tissue ET system activation.

Inhibitory effect of tramadol on vasorelaxation mediated by ATP-sensitive K+ channels in rat aorta

Tramadol produces a conduction block similar to lidocaine by exerting a local anesthetic-like effect. The aims of this in vitro study were to determine the effects of tramadol on the vasorelaxant response induced by the adenosine triphosphate-sensitive K(+) (K(ATP)) channel opener, levcromakalim, in an endothelium-denuded rat aorta, and to determine whether this effect of tramadol is stereoselective. The effects of tramadol (racemic, R(-) and S(+): 10(-6), 10(-5), 5 x 10(-5) M), and glibenclamide on the levcromakalim dose-response curve were assessed in aortic rings that had been pre-contracted with phenylephrine. In the rings pretreated independently with naloxone, and glibenclamide, the levcromakalim dose-response curves were generated in the presence or absence of tramadol. The effect of tramadol on the dose-response curve of diltiazem was assessed. Racemic, R(-) and S(+) tramadol (10(-5), 5 x 10(-5) M) attenuated (P < 0.0001) levcromakalim-induced relaxation in the ring with or without naloxone in a dose-dependent manner. The magnitude of the R(-)-tramadol-induced attenuation of vasorelaxant response induced by levcromakalim was greater (P < 0.05) than that induced by S(+)-tramadol. Glibenclamide almost abolished the levcromakalim-induced relaxation. Tramadol, 5 x 10(-5) M, did not significantly alter the diltiazem-induced relaxation. These results suggest that a supraclinical dose (10(-5) M) of tramadol [racemic, R(-) and S(+)] attenuates the vasorelaxation mediated by the K(ATP) channels in the rat aorta. The R(-) tramadol-induced attenuation of vasorelaxation induced by levcromaklim was more potent than that induced by S(+) tramadol. This attenuation is independent of opioid receptor activation.

Vascular activity of two silicon compounds, ALIS 409 and ALIS 421, novel multidrug-resistance reverting agents in cancer cells

The aim of this study was to investigate the effects of two novel multidrug-resistance reverting agents, ALIS 409 [1,3-dimethyl-1,3-p-fluorophenyl-1,3(3-morfolinopropyl)-1,3-disiloxan dihydrochloride] and ALIS 421 [1,3-dimethyl-1,3-(4-fluorophenyl)-1,3[3(4-buthyl)-(1-piperazinyl)-propyl]-1,3-disiloxan tetrahydrochloride], on vascular functions in vitro. A comparison of their mechanical and electrophysiological actions in rat aorta rings and single rat tail artery myocytes, respectively, was performed. In endothelium-denuded rat aorta rings, ALIS 409 and ALIS 421 antagonized 60 mM K(+)-induced contraction in a concentration-dependent manner with IC(50) values of 52.2 and 15.5 microM, respectively. ALIS 409 and ALIS 421 inhibited L-type Ca(2+) current recorded in artery myocytes in a concentration-dependent manner with IC(50) values of 6.4 and 5.6 microM, respectively. In rat aorta, ALIS 409 and ALIS 421 antagonized the sustained tonic contraction induced by phenylephrine with IC(50) values of 58.0 and 13.7 microM (endothelium-denuded rings) and of 73.9 and 31.9 microM (endothelium-intact rings), respectively. In endothelium-denuded rings, ryanodine reduced significantly the response to phenylephrine in the absence of extracellular Ca(2+) whereas nifedipine, ALIS 409 or ALIS 421 did not affect it. Phenylephrine-stimulated influx of extracellular Ca(2+) was markedly reduced when tissues were pretreated with ALIS 409, ALIS 421 or nifedipine, and stimulated when they were pretreated with ryanodine. Application of ALIS 409 (up to 100 microM) to intact rat aorta rings failed to induce mechanical responses. Our results provide functional evidence that the myorelaxing effect elicited either by ALIS 409 or by ALIS 421 involved mainly the direct blockade of extracellular Ca(2+) influx. This effect, however, took place at concentrations much higher than those effective as modifiers of multidrug resistance in cancer cells.

Ibudilast-Induced Decreases in Cytosolic Ca2+ Level and Contraction in Rat Aorta

The mechanism by which ibudilast induces vasodilation was examined in isolated endothelium-denuded rat aorta. Ibudilast inhibited the contractions induced by phenylephrine (PE) and high K+ with decrease of [Ca2+]i level in a concentration-dependent manner, to the same degree. 3-Isobutyl-1-methylxanthine (IBMX) inhibited PE-induced contraction and [Ca2+]i level in a concentration-dependent manner, but it inhibited high K+-induced contraction without decrease of [Ca2+]i level. In comparison with IBMX, the increases of cAMP and cGMP contents in ibudilast were much smaller than that of muscle tension. Ibudilast did not inhibit 12-deoxyphorbol 13-isobutyrate (DPB)-induced contraction in the presence of verapamil. Treatment with 30 µM ibudilast inhibited the extracellularly added Ca2+-induced muscle tension and increases in [Ca2+]i level during high K+ depolarization. These results suggested that ibudilast inhibited PE- and high K+-induced muscle contractions mainly by the inhibition of [Ca2+]i level in endothelium-denuded rat aorta.

Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition prevents endothelin A receptor antagonism in rat aorta

This study tested whether sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase regulates the ability of endothelin receptor antagonist to inhibit the endothelin-1 constriction. The endothelin A receptor antagonist BQ-123 (1 microM) completely relaxed constriction to 10 nM endothelin-1 in endothelium-denuded rat aorta. Challenge with cyclopiazonic acid (10 microM), a sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor, during the plateau of endothelin-1 constriction enhanced the constriction by approximately 30%. BQ-123 relaxed the endothelin-1 plus cyclopiazonic acid constriction by only approximately 10%. In contrast, prazosin (1 microM), an alpha-adrenergic receptor antagonist, still completely relaxed the 0.3 muM phenylephrine constriction in the presence of cyclopiazonic acid. Verapamil relaxed the endothelin-1 plus cyclopiazonic acid constriction by approximately 30%, whereas Ni(2+) and 2-aminoethoxydiphenyl borate, nonselective cation channel and store-operated channel blockers, respectively, completely relaxed the constriction. These results suggest that lowered sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase activity selectively decreases the ability of endothelin receptor antagonist to inhibit the endothelin A receptor. The decreased antagonism may be related to the opening of store-operated channels and subsequent greater internalization of endothelin A receptor.

Role of chloride transport proteins in the vasorelaxant action of nitroprusside in isolated rat aorta

Chloride ions play a key role in smooth muscle contraction, but little is known concerning their role in smooth muscle relaxation. Here we investigated the effect of chloride transport inhibitors on the vasorelaxant responses to nitroprusside in isolated and endothelium-denuded rat aorta, precontracted with phenylephrine 1 μM. Incubation of aortic rings in NO 3 − media strongly potentiated the vasorelaxant responses to nitroprusside. Bumetanide, DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) and acetazolamide strongly potentiated the vasorelaxant responses to nitroprusside (by 70–100%). EC 50 were 2.3 ± 0.5 μM for bumetanide, 26 ± 15 μM for DIDS and 510 ± 118 μM for acetazolamide ( n = 6 for condition). Niflumic acid, a selective inhibitor of ClCa (calcium-activated chloride channels), potentiated nitroprusside relaxation to a similar extent as chloride transport inhibitors, in a non-additive manner. Zinc and nickel ions, both modestly potentiated nitroprusside vasorelaxation (by 20–30%). Cobaltum had negligible effect on nitroprusside vasorelaxation. CPA ( p-chlorophenoxy-acetic acid), an inhibitor of volume-sensitive chloride channels (ClC), slightly potentiated nitroprusside vasorelaxation (by 15%), and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhibitors CFTR(inh)172 (5-[(4-Carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone), DPC (diphenylamine-2,2′-dicarboxylic acid) and glibenclamide were without significant effect. In conclusion, inhibition of chloride transport proteins strongly potentiates the vasorelaxant responses to nitroprusside in isolated rat aorta. This effect seems mediated by chloride depletion and inhibition of a chloride channel activated by both, calcium and cyclic GMP (cGMP).

Role of PKC in the novel synergistic action of urotensin II and angiotensin II and in urotensin II-induced vasoconstriction

The intracellular signaling of human urotensin II (hU-II) and its interaction with other vasoconstrictors such as ANG II are poorly understood. In endothelium-denuded rat aorta, coadministration of hU-II (1 nM) and ANG II (2 nM) exerted a significant contractile effect that was associated with increased protein kinase C (PKC) activity and phosphorylation of PKC-alpha/betaII and myosin light chain, whereas either hU-II or ANG II administered alone at these concentrations had no statistically significant effect. This synergistic effect was abrogated by the PKC inhibitor chelerythrine (10 and 30 microM), the selective PKC-alpha/betaII inhibitor Gö-6976 (0.1 and 1 microM), the hU-II receptor ligand urantide (30 nM and 1 microM), or the ANG II antagonist losartan (1 microM). Moreover, in endothelium-intact rat aorta, the synergistic effect of hU-II and ANG II was not exerted any longer, and this synergistic effect was unmasked by pretreatment of the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. hU-II (10 nM) alone caused a long-lasting increase in phospho-PKC-theta, phospho-myosin light chain, and PKC activity, which was associated with long-lasting vasoconstriction. These changes were prevented by chelerythrine. Methoxyverapamil-thapsigargin treatment reduced the hU-II-induced vasoconstriction by approximately 50%. The methoxyverapamil-thapsigargin-resistant component of hU-II-induced vasoconstriction was dose-dependently inhibited by chelerythrine. In conclusion, hU-II induces a novel PKC-dependent synergistic action with ANG II in inducing vasoconstriction. PKC-alpha/betaII is probably the PKC isoform involved in this synergistic action. Nitric oxide produced in the endothelium probably masks this synergistic action. The long-lasting vasoconstriction induced by hU-II alone is PKC dependent and associated with PKC-theta phosphorylation.

Vasodilator Effects of Bis-Dihydropyridines Structurally Related to Nifedipine

Calcium channel blockers are widely used in therapy for hypertension and angina pectoris, and among these blockers some 1,4-dihydropyridines (e.g. amlodipine, nitrendipine and nifedipine) have had widespread clinical use. In this work we investigated the vascular effects of four bis-1,4-dihydropyridines (bis-DHPs: 01-04), structurally related to nifedipine, in which a second 1,4-dihydropyridinic moiety was incorporated in the corresponding arylic moiety in para and meta position. Of these four bis-DHPs, the meta regioisomers (bis-DHP-03 and bis-DHP-04; 0.01-3.16 mg kg(-1)) and nifedipine induced a greater decrease on diastolic and systolic blood pressure than the para isomers (bis-DHP-01 and bis-DHP-02), as shown in two experimental models: normotensive and spontaneously hypertensive rats. Complementarily, bis-DHPs action was examined in intact and endothelium-denuded rat aorta, depolarized by KCl [80 mM] in one group and stimulated by noradrenaline (1 x 10(-7) M) in another and the corresponding IC(50) values were obtained (1.5 x 10(-6)-2. 4 x 10(-7)M). Later, the relaxing action of bis-DHP-03,04 and nifedipine on the contraction evoked by Ca(2+) in K(+)-depolarized rat aorta was analyzed and the corresponding EC(50) values for the meta isomers and nifedipine were obtained. The results showed a concentration dependent vasodilating activity in both KCl precontracted and noradrenaline stimulated aorta rings. The apparent order of potency with and without endothelium in both experimental models was nifedipine >bis-DHP-04 >bis-DHP-03. The cumulative concentration-effect curves for Ca(2+) in the presence of the bis-DHPs tested show the same potency order. Unlike nifedipine, the tested compounds are not photosensitive, which makes them more attractive in therapy for hypertension related diseases.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and Na–K–Cl cotransporter NKCC1 isoform mediate the vasorelaxant action of genistein in isolated rat aorta

The soy phytoestrogen genistein is a potent vasorelaxant, but its mechanism of action is poorly understood. Here, we used endothelium-denuded rat aorta to investigate the role of the cyclic AMP(cAMP)-activated, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, and its associated Na–K–Cl cotransporter NKCC1. Isolated, endothelium-denuded rat aorta was contracted with phenylephrine 1 μM, and the vasorelaxant responses to genistein were investigated under conditions where CFTR was inhibited by DPC (diphenylamine-2-carboxylic acid) or glibenclamide ( n = 6 for compound). Both compounds fully antagonized the vasorelaxant responses to genistein, with IC 50 = 57 ± 18 μM and 42 ± 11 μM for DPC and glibenclamide respectively. H-89, a selective protein kinase A (PKA) inhibitor, blocked the vasorelaxant responses to genistein. Finally, the NKCC1 inhibitor, bumetanide fully antagonized the vasorelaxant responses to genistein against phenylephrine- or KCl-induced contractions, with IC 50 = 2.0 ± 0.2 μM and 1.6 ± 0.5 μM, respectively ( n = 6 for condition). These results strongly suggest that CFTR opening is involved in the vasorelaxant action of genistein, and that cAMP-dependent CFTR phosphorylation and chloride entry via the NKCC1 cotransporter are required for genistein action.

Resveratrol decreases calcium sensitivity of vascular smooth muscle and enhances cytosolic calcium increase in endothelium

Resveratrol causes endothelium dependent and independent relaxation of vascular smooth muscle. This study investigated the mechanisms behind the effect of resveratrol on vascular tone. Resveratrol (0.1 mM) inhibited KCl-stimulated contractions in endothelium-denuded rat aorta and this inhibition was not reversed by tetraethylammonium (TEA) (5 mM), glyburide (3 μM), ouabain (0.1 mM), thapsigargin (1 μM), or indomethacin (10 μM). KCl (90 mM) increased the intracellular free calcium concentration ([Ca 2+] i) in the isolated smooth muscle cells from the rat aorta and resveratrol (0.1 mM) did not inhibit the KCl-stimulated [Ca 2+] i increase. The CaCl 2 (0.1–100 μM) stimulated contractions were inhibited by resveratrol (0.1 mM) in the Triton X-100 skinned smooth muscle of the aorta. In heart valve endothelium, resveratrol (0.1 mM) augmented the acetylcholine (10 μM) stimulated [Ca 2+] i increase. Resveratrol-induced augmentation of the acetylcholine-stimulated [Ca 2+] i elevation was reversed by glyburide (3 μM), but not by TEA (5 mM). The present study indicated that resveratrol affected vascular smooth muscle and endothelium in different ways. Resveratrol decreased the Ca 2+ sensitivity but did not affect the KCl-stimulated [Ca 2+] i increase in the vascular smooth muscle. In the endothelial cells, resveratrol enhanced the agonist-stimulated [Ca 2+] i increase that might trigger nitric oxide synthesis from endothelial cells.

Biochemistry and Pharmacology of Novel Anthranilic Acid Derivatives Activating Heme-Oxidized Soluble Guanylyl Cyclase

The heme-enzyme soluble guanylyl cyclase (sGC) is an ubiquitous NO receptor, which mediates NO downstream signaling by the generation of cGMP. We studied the mechanism of action of the anthranilic acid derivatives 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (HMR1766) (proposed international nonproprietary name, ataciguat sodium) and 2-(4-chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benzamide (S3448) as a new class of sGC agonists. Both compounds activated different sGC preparations (purified from bovine lung, or crude from human corpus cavernosum) in a concentration-dependent and quickly reversible fashion (EC50 = 0.5-10 microM), with mixed-type activation kinetics. Activation of sGC by these compounds was additive to activation by NO donors, but instead of being inhibited, it was potentiated by the heme-iron oxidants 1H-[1,2,4]-oxdiazolo[3,4-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), suggesting that the new compounds target the ferric heme sGC isoform. Protoporphyrin IX acted as a competitive activator, and zinc-protoporphyrin IX inhibited activation of heme-oxidized sGC by HMR1766 and S3448, whereas heme depletion of sGC by Tween 20 treatment reduced activation. Both compounds increased cGMP levels in cultured rat aortic smooth muscle cells; induced vasorelaxation of isolated endothelium-denuded rat aorta, porcine coronary arteries, and human corpus cavernosum (EC50 1 to 10 microM); and elicited phosphorylation of the cGMP kinase substrate vasodilator-stimulated phosphoprotein at Ser239. HMR1766 intravenous bolus injection decreased arterial blood pressure in anesthetized pigs. All of these pharmacological responses to the new compounds were enhanced by ODQ and NS2028. Our findings suggest that HMR1766 and S3448 preferentially activate the NO-insensitive heme-oxidized form of sGC, which exists to a variable extent in vascular tissues, and is a pharmacological target for these new vasodilator drugs.

Role of the Adventitia in the Cyclic GMP-Mediated Relaxant Effect of Nω-Hydroxy-L-Arginine in Rat Aorta

N^ω-hydroxy-L-arginine (L-NOHA), the stable intermediate of the nitric oxide synthase (NOS)-catalyzed reaction, can induce NO/cyclic GMP-dependent relaxation in the rat aorta, in an endothelium- and NOS-independent manner. In this study, the role of the adventitia in the endothelium-independent effect of L-NOHA was investigated. Despite a decrease in norepinephrine (NE)-induced precontraction, adventitia removal in the rat aorta did not markedly alter the relaxant effect of forskolin, S-nitroso-N-acetylpenicillamine or glyceryl trinitrate. In contrast, both inhibition of NE-induced contraction and relaxation of NE-precontracted rings produced by L-NOHA were diminished in the absence of adventitia. Moreover, exposure to L-NOHA significantly enhanced the cyclic GMP level in the media of the aorta with, but not without adventitia. These findings demonstrate the role of the adventitia in the L-NOHA-induced decrease in tone and increase in cyclic GMP in the endothelium-denuded rat aorta. They suggest that NO or an NO-related compound formed from L-NOHA in the adventitia may produce paracrine effects.

Implication of Cyclic Nucleotide Phosphodiesterase Inhibition in the Vasorelaxant Activity of the Citrus-Fruits Flavonoid (±)-Naringenin

The potential vasorelaxant, antioxidant and cyclic nucleotide phosphodiesterase (PDE) inhibitory effects of the citrus-fruit flavonoids naringin and (+/-)-naringenin were comparatively studied for the first time in this work. (+/-)-Naringenin (1 microM - 0.3 mM) did not affect the contractile response induced by okadaic acid (OA, 1 microM). However, (+/-)-naringenin relaxed, in a concentration-dependent manner, the contractions elicited by phenylephrine (PHE, 1 microM) or by a high extracellular KCl concentration (60 mM) in intact rat aortic rings. Mechanical removal of endothelium and/or pretreatment of aorta rings with glibenclamide (GB, 10 microM) or tetraethylammonium (TEA, 2 mM) did not significantly modify the vasorelaxant effects of this flavanone. (+/-)-Naringenin (10 microM - 0.1 mM) did not alter the basal uptake of 4) Ca2+ but decreased the influx of 45Ca2+ induced by PHE and KCl in endothelium-containing and endothelium-denuded rat aorta. (+/-)-Naringenin (10 microM - 0.1 mM) was ineffective to scavenge superoxide radicals (O*2-) generated by the hypoxanthine (HX)-xanthine oxidase (XO) system and/or to inhibit XO activity. (+/-)-Naringenin (0.1 mM) significantly increased the production of cGMP and cAMP decreased by PHE (1 microM) and high KCl (60 mM) in cultured rat aortic myocytes. (+/-)-Naringenin preferentially inhibited calmodulin (CaM)-activated PDE1, PDE4 and PDE5 isolated from bovine aorta with IC50 values of about 45 microM, 60 microM and 68 microM, respectively. In contrast, the 7-rhamnoglucoside of (+/-)-naringenin, naringin (1 microM - 0.3 mM), was totally inactive in all experiments. These results indicate that the vasorelaxant effects of (+/-)-naringenin seem to be basically related to the inhibition of PDE1, PDE4 and PDE5 activities.

Vasorelaxant Effect of Mexican Medicinal Plants on Isolated Rat Aorta

The vasorelaxant effect of methanol extracts (0.86–50 µg/ml) of Iresine calea, Psyttacanthus calyculathus. (DC.) G. Don, Laelia autumnalis. (Lex.) Lindley, Brickellia cavanillesii. (Cass.) Gray, and Lepechinia caulescens. (Ortega) Epling, plant species used in Mexican folk medicine for the treatment of hypertension and related diseases, were evaluated in isolated rat aortic rings. The extracts of I. calea. and P. calyculathus. did not show a vasorelaxant activity on norepinephrine-evoked contraction (NE, 1 × 10−7.5 M) in endothelium-intact (+ E) and endothelium-denuded (− E) rat aorta rings. On the other hand, L. autumnalis. and B. cavanillesii. induced a concentration-dependent and endothelium-independent relaxation on rat aorta. However, the methanol extract of L. caulescens. produced a significant vasodilator effect in a concentration-dependent and endothelium-dependent manner. In order to determine the mode of the vasorelaxant action evoked by L. caulescens., the extract was evaluated in the presence of L-NAME (inhibitor of nitric oxide synthase at 1 × 10−4 M) and indomethacin (inhibitor of cyclooxygenases at 1 × 10−5 M). Relaxation was blocked by L-NAME, indicating the extract vasodilating properties are endothelium mediated due to liberation of nitric oxide.

Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin

This study investigated the vasorelaxant activity, superoxide radicals (O2(*-))-scavenging capacity and cyclic nucleotide phosphodiesterase (PDE)-inhibitory effects of hesperidin and hesperetin, two flavonoids mainly isolated from citrus fruits. Hesperetin concentration-dependently relaxed the isometric contractions induced by noradrenaline (NA, 1 microM) or by a high extracellular KCl concentration (60 mM) in intact rat isolated thoracic aorta rings. However, hesperetin (10 microM-0.3 mM) did not affect the contractile response induced by okadaic acid (OA, 1 microM). Mechanical removal of endothelium and/or pretreatment of aorta rings with glibenclamide (GB, 10 microM), tetraethylammonium (TEA, 2 mM) or nifedipine (0.1 microM) did not significantly modify the vasorelaxant effects of this flavonoid. Hesperetin (10 microM-0.1 mM) did not affect the basal uptake of (45)Ca(2+) but decreased the influx of (45)Ca(2+) induced by NA and KCl in endothelium-containing and endothelium-denuded rat aorta. Hesperetin (10 microM-0.1 mM) did not scavenge O2(*-) generated by the phenazine methosulfate (PMS)-reduced beta-nicotinamide adenine dinucleotide (NADH) system. Hesperetin (0.1 mM) significantly reversed the inhibitory effects of NA (1 microM) and high KCl (60 mM) on cyclic nucleotide (cAMP and cGMP) production in cultured rat aortic myocytes. Hesperetin preferentially inhibited calmodulin (CaM)-activated PDE1 and PDE4 isolated from bovine aorta with IC(50) values of about 74 microM and 70 microM respectively. In contrast, the 7-rhamnoglucoside of hesperetin, hesperidin (10 microM-0.1 mM), was inactive in practically all experiments, although it inhibited basal and cGMP-activated PDE2 isolated from platelets (IC(50) values of 32+/-4 microM and 137+/-34 microM respectively). These results suggest that the vasorelaxant effects of hesperetin are basically due to the inhibition of PDE1 and PDE4 activities.

Functional characterization of large conductance calcium-activated K+ channel openers in bladder and vascular smooth muscle.

Calcium activated K(+) channels (K(Ca) channels) are found in a variety of smooth muscle tissues, the most characterized of which are the large conductance K(Ca) channels (BK(Ca) or maxi-K(+) channels). Recent medicinal chemistry efforts have identified novel BK(Ca) openers including 2-amino-5-(2-fluoro-phenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BMS-204352 and its analog 3-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-6-trifluoromethyl-1,3-dihydro-indol-2-one (compound 1), and 5,7-dichloro-4-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-1H-quinolin-2-one (compound 2). Although these compounds are effective BK(Ca) openers as shown by electrophysiological methods, little is known about their effects on smooth muscle contractility. In this study, the responsiveness of structurally diverse BK(Ca) openers-NS-8, compounds 1 and 2 and the well characterized nonselective NS-1619-was assessed using segments of endothelium denuded rat aorta, rat and guinea pig detrusor precontracted with extracellular K(+), and Landrace pig detrusor stimulated by electrical field. In all preparations, the compounds tested inhibited or completely abolished contractions with similar potencies (-logIC(50) values: 3.8 to 5.1). In rat aorta, in the presence of 80 mM K(+), the compounds significantly shifted the concentration-response curve to the right compared with those obtained in 30 mM K(+). These data are consistent with K(+) channel (BK(Ca) channel) activation as the underlying mechanism of relaxation by compounds that share the electrophysiological property of BK(Ca) current activation. The similar potencies at detrusor and vascular smooth muscle suggest that the achievement of smooth muscle selectivity in vitro with the representative compounds examined in this study may prove to be a challenge when targeting BK(Ca) channels for smooth muscle indications such as overactive bladder.