Endothelium-denuded Rat Aortic Rings Research Articles

Curcumane C and (±)-curcumane D, an unusual seco-cadinane sesquiterpenoid and a pair of unusual nor-bisabolane enantiomers with significant vasorelaxant activity from Curcuma longa

A new seco-cadinane sesquiterpenoid (curcumane C, 1) and a pair of new nor-bisabolene enantiomers [(+)- and (−)-curcumane D, 2a and 2b] were isolated from C. longa. Compound 1 possesses an unusual 4,5-seco-cadinane skeleton with a tetrahydrophthalide moiety, while 2a and 2b contain an unusual 15-nor-bisabolene skeleton with a chromone core. All compounds exhibited significant vasorelaxant effects against KCl-induced contraction of rat aortic rings. Compound 1 also exhibited a vasorelaxant effect against phenylephrine-induced contraction of rat aortic rings. Meanwhile, compound 1 showed a stronger vasorelaxant effect in endothelium-intact rat aortic rings compared with endothelium-denuded rat aortic rings, indicating that vasodilation by 1 involved both endothelium-dependent and endothelium-independent pathways. Furthermore, compound 1 increased the NO content in human umbilical vein endothelial cells and its vasorelaxant effect could be attenuated by treatment with L-NAME, an endothelium NO synthase inhibitor. Thus, the underlying vasodilatory mechanisms of 1 may be mediated via abrogation of extracellular Ca2+ influx and regulation of NO release in vascular endothelial cells.

Mechanisms underlying vascular hypocontractility induced by ethanol withdrawal: Role of cyclooxygenase 2-derived prostacyclin

The effects on the vasculature produced by ethanol withdrawal include both vasodilatation and hypocontractility, although a detailed biochemical understanding of these processes is yet to be accomplished. Here, we sought to investigate some of the mechanisms underlying vascular hypocontractility induced by ethanol withdrawal. Male Wistar rats were treated with increasing doses of 3–9% ethanol (v/v) for 21 days and the impact of ethanol withdrawal on the vascular function was assessed 48 h after immediate ethanol suspension. Endothelium-denuded rat aortic rings showed a reduced contractile response to phenylephrine, angiotensin II, serotonin and KCl after ethanol withdrawal, but the same phenomenon was not observed in endothelium-intact rings. Indomethacin, but not L-NAME, tiron, PEG-catalase and SC560, restored the contractile response to phenylephrine of endothelium-denuded aortas from abstinent rats. Hyporeactivity to phenylephrine induced by ethanol withdrawal was reversed by SC236, a selective cyclooxygenase (COX)-2 inhibitor. Similarly, Ro1138452, a selective prostacyclin IP receptor antagonist, reversed vascular hypocontractility induced by ethanol withdrawal. Increased concentrations of 6-keto-prostaglandin (PG)F1α, a stable product of PGI2, was detected in endothelium-denuded aortas from abstinent rats, and this response was prevented by indomethacin. However, no changes in aortic PGE2 levels were detected after ethanol withdrawal. In situ quantification of hydrogen peroxide (H2O2) and nitric oxide (NO) using fluorescent dyes revealed that ethanol withdrawal decreased the levels of these two compounds in the tunica media. Our studies show that the vascular hypocontractility induced by ethanol withdrawal is independent of the endothelium and it is mediated by PGI2 derived from COX-2.

Direct actions of the flavonoids naringenin, quercetin and genistein on rat cardiac and vascular muscles

Context: Flavonoids are natural polyphenolic compounds that are ubiquitous in plants. Numerous prospective epidemiological studies have found beneficial effects of flavonoid consumption on overall cardiovascular mortality. These effects have been attributed to nonspecific antioxidant, anti-atherosclerotic and antithrombotic properties of flavonoids. However, little is known about direct actions of flavonoids on cardiac and vascular smooth muscles. Aims: To evaluate the effects of three flavonoids (naringenin, quercetin and genistein) on electrical and contractile activities of isolated rat hearts and on contraction of rat abdominal aortic rings. Methods: Surface electrogram (ECG) and force of contraction (FC) were recorded in isolated rat hearts. The ventricular fibrillation threshold was evaluated with the flavonoids. The effects on aortic contraction were assessed in rat abdominal aortic rings pre-contracted with 60 mM of KCl. Results: Genistein tended to prolong the QT interval while naringenin tended to decrease it. The effects of all flavonoids on QRS and RR interval were mild and not statistically significant. Genistein and naringenin produced a negative inotropic effect in isolated rat hearts with an IC50 of 13.8 μM and 33.8 μM, respectively. Quercetin at low concentrations had a positive inotropic effect; at high concentrations, it exerted a negative inotropic effect. Naringenin and quercetin increased the threshold for ventricular fibrillation, whereas genistein decreased it. Naringenin, quercetin, and genistein induced concentration-dependent relaxation in endothelium-denuded rat aortic rings. Conclusions: These flavonoids have direct actions on rat cardiac and vascular smooth muscles.

Pharmacological characterization of the mechanisms underlying the vascular effects of succinate

We investigated the mechanisms underlying the vascular effects of succinate. Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats and C57BL/6 wild type (WT) or GPR91−/– mice. Nitrate/nitrite (NOx) was measured colorimetrically whereas 6-keto-prostaglandin F1α (stable product of prostacyclin) was measured by enzyme immunoassay (EIA). Phosphorylation of endothelial nitric oxide synthase (eNOS) was assessed by western immunoblotting. Functional assays revealed that the direct effect of succinate in the vasculature is biphasic. At lower concentrations succinate induced relaxation while at higher concentrations succinate induced vascular contraction. Succinate concentration dependently relaxed rat aortic rings with intact endothelium. Endothelial removal reduced, but not abolished succinate-induced relaxation. Similarly, succinate relaxed endothelium-intact and endothelium-denuded aortas isolated from both C57BL/6 and GPR91−/– mice. Pre-incubation of endothelium-intact, but not endothelium-denuded rat aortic rings with l-NAME, indomethacin and tetraethylammonium (TEA) reduced succinate-induced relaxation. In endothelium-intact rings, succinate-induced relaxation was attenuated by ODQ, haemoglobin, Rp-8-Br-Pet-cGMPS, thapsigargin, wortmannin and SC-560. Blockade of K+ channels with 4-aminopyridine, apamin and charybdotoxin reduced succinate-induced relaxation. Succinate increased the concentration of NOx and 6-keto-prostaglandin F1α as well as eNOS phosphorylation at ser1177 residue. CaCl2-induced contraction of endothelium-intact or endothelium-denuded aortas was not affected by succinate. The major finding of our study is that it first demonstrates a direct effect of succinate in the vasculature. Succinate displays a biphasic and concentration-dependent effect. The vascular relaxation induced by succinate is partially mediated by endothelial GPR91 receptors via the NO-cGMP pathway, a vasodilator cyclooxygenase (COX) product(s) and the opening of K+ channels.

Sesquiterpene lactones from Hedyosmum brasiliense induce in vitro relaxation of rat aorta and corpus cavernosum

Hedyosmum brasiliense Miq., Chloranthaceae, has been used in Southern Brazil as a sedative, anti-inflammatory, and aphrodisiac. In this study, endothelium-intact and endothelium-denuded rat aortic rings and strips of corpus cavernosum were used to investigate the relaxant effects of an hexane fraction of leaves of H. brasiliense and its sesquiterpene lactones 13-hydroxy-8,9-dehydroshizukanolide, podoandin, and elemanolide 15-acetoxy-isogermafurenolide. The incubation of hexane fraction of leaves of H. brasiliense resulted in significant relaxation of endothelium-intact aortic rings previously contracted by phenylephrine. In addition, 13-hydroxy-8,9-dehydroshizukanolide and podoandin displayed a clear concentration-dependent ability to relax endothelium-intact (∼85 to 90%) and endothelium-denuded (∼45 to 55%) rat aortic rings. A less pronounced vascular relaxation was recorded when 15-hydroxy-isogermafurenolide was tested. Interestingly, in tissues previously incubated with the nitric oxide synthase inhibitor l-NAME (100μM), both 13-hydroxy-8,9-dehydroshizukanolide and podoandin had their effects in endothelium-intact vessels reduced to the same degree of relaxation observed in endothelium-denuded aortic rings. Podoandin, 13-hydroxy-8,9-dehydroshizukanolide, and 15-acetoxy-isogermafurenolide (100μM) were also able to relax precontracted corpus cavernosum strips by 49.5±3.9%, 65.9±7.3% and 57.9±5.5%, respectively. Our results demonstrated that 13-hydroxy-8,9-dehydroshizukanolide, podoandin and 15-acetoxy-isogermafurenolide, isolated from H. brasiliense, generate both endothelium-dependent and -independent relaxation of rat aortic rings, as well as being able to induce in vitro relaxation of rat corpus cavernosum. Importantly, the endothelium-dependent effect is fully dependent on nitric oxide production. Considering that penile erection depends on both relaxation of cavernosal smooth muscle and inflow of blood for the cavernous bodies, this is the first study reporting experimental evidence supporting the aphrodisiac properties of H. brasiliense.

Antihypertensive Effect of the GaMiSamHwangSaSimTang in Spontaneous Hypertensive Rats.

The present study was designed to evaluate the antihypertensive effect of GaMiSamHwangSaSimTang (HVC1), a 30% ethanol extract of a mixture comprising Pruni Cortex, Scutellariae Radix, Coptidis Rhizoma, and Rhei Rhizoma, on spontaneous hypertensive rats (SHRs). The systolic blood pressure (SBP) was measured every 4 or 7 days using the noninvasive tail cuff system. The vasorelaxant effects on isolated aortic rings were evaluated. Aortic rings were contracted using phenylephrine (PE) or KCl, and the changes in tension were recorded via isometric transducers connected to a data acquisition system. In this study, oral administration of HVC1 decreased the SBP of SHRs over the experimental period. HVC1 induced concentration-dependent relaxation in the aortic rings that had been precontracted using PE or KCl. The vasorelaxant effects of HVC1 on endothelium-intact aortic rings were inhibited by pretreatment with Nω-Nitro-l-arginine methyl ester (L-NAME) or methylene blue. HVC1 inhibited the contraction induced by extracellular Ca2+ in endothelium-denuded rat aortic rings that had been precontracted using PE or KCl. In conclusion, HVC1 reduced the SBP of SHR and relaxed isolated SHR aortic rings by upregulating NO formation and the NO-cGMP pathway and blocking the entry of extracellular Ca2+ via receptor-operative Ca2+ channel and voltage-dependent Ca2+ channel.

Synthesis and evaluation of multi-functional NO-donor/insulin-secretagogue derivatives for the treatment of type II diabetes and its cardiovascular complications

Although there is a significant effort in the discovery of effective therapies to contrast both the pathological endocrine and metabolic aspects of diabetes and the endothelial dysfunction associated with this disease, no hypoglycemic drug has been proven to defeat the cardiovascular complications associated with type II diabetes. The aim of this research was to design new compounds exhibiting a double profile of hypoglycemic agents/NO-donors. The synthesis of molecules obtained by the conjunction of NO-donor moieties with two oral insulin-secretagogue drugs (repaglinide and nateglinide) was reported. NO-mediated vasorelaxing effects of the synthesized compounds were evaluated by functional tests on isolated endothelium-denuded rat aortic rings. The most potent molecule (4) was tested to evaluate the hypoglycemic and the anti-ischemic cardioprotective activities. This study indicates that 4 should represent a new insulin-secretagogue/NO-donor prodrug with an enhanced cardiovascular activity, which may contrast the pathological aspects of diabetes and endowed of cardioprotective activity.

Metformin exaggerates phenylephrine-induced AMPK phosphorylation independent of CaMKKβ and attenuates contractile response in endothelium-denuded rat aorta

Metformin, a widely prescribed antidiabetic drug, has been shown to reduce the risk of cardiovascular disease, including hypertension. Its beneficial effect toward improved vasodilation results from its ability to activate AMPK and enhance nitric oxide formation in the endothelium. To date, metformin regulation of AMPK has not been fully studied in intact arterial smooth muscle, especially during contraction evoked by G protein-coupled receptor (GPCR) agonists. In the present study, ex vivo incubation of endothelium-denuded rat aortic rings with 3mM metformin for 2h resulted in significant accumulation of metformin (∼600pmoles/mg tissue), as revealed by LC–MS/MS MRM analysis. However, metformin did not show significant increase in AMPK phosphorylation under these conditions. Exposure of aortic rings to a GPCR agonist (e.g., phenylephrine) resulted in enhanced AMPK phosphorylation by ∼2.5-fold. Importantly, in metformin-treated aortic rings, phenylephrine challenge showed an exaggerated increase in AMPK phosphorylation by ∼9.7-fold, which was associated with an increase in AMP/ATP ratio. Pretreatment with compound C (AMPK inhibitor) prevented AMPK phosphorylation induced by phenylephrine alone and also that induced by phenylephrine after metformin treatment. However, pretreatment with STO-609 (CaMKKβ inhibitor) diminished AMPK phosphorylation induced by phenylephrine alone but not that induced by phenylephrine after metformin treatment. Furthermore, attenuation of phenylephrine-induced contraction (observed after metformin treatment) was prevented by AMPK inhibition but not by CaMKKβ inhibition. Together, these findings suggest that, upon endothelial damage in the vessel wall, metformin uptake by the underlying vascular smooth muscle would accentuate AMPK phosphorylation by GPCR agonists independent of CaMKKβ to promote vasorelaxation.

Mechanisms underlying the vascular and hypotensive actions of the labdane ent-3-acetoxy-labda-8(17),13-dien-15-oic acid

We investigated the mechanisms underlying the vasorelaxant and hypotensive actions of the labdane-type diterpene ent-3-acetoxy-labda-8(17),13-dien-15-oic acid (labda-15-oic acid). Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats. cAMP and cGMP were measured by enzyme immunoassay (EIA) whereas nitrate measurement was performed by chemiluminescence. Nitric oxide (NO) concentration ([NO]c) was measured in endothelial cells by flow cytometry. The cytosolic calcium concentration ([Ca2+]c) in vascular smooth muscle cells (VSMC) was measured by confocal microscopy. Blood pressure measurements were performed in conscious rats. Labda-15-oic acid inhibited the contraction induced by phenylephrine and serotonin in either endothelium-intact or endothelium-denuded rat aortic rings. The labdane significantly reduced CaCl2-induced contraction in a Ca2+-free solution containing KCl or phenylephrine. Labda-15-oic acid (0.1–300μmol/l) concentration-dependently relaxed endothelium-intact and endothelium-denuded aortas pre-contracted with either phenylephrine or KCl. In endothelium-intact rings, the relaxation induced by labda-15-oic acid was affected by L-NAME, 7-nitroindazole, ODQ, hemoglobin, Rp-8-Br-Pet-cGMPS and thapsigargin. Blockade of K+ channels with 4-aminopyridine, apamin, charybdotoxin and glibenclamide affected the relaxation induced by labda-15-oic acid. The labdane increased cGMP and nitrate levels but did not affect cAMP levels in endothelium-intact aortas. Labda-15-oic acid increased [NO]c in endothelial cells and decreased [Ca2+]c in VSMC. The hypotension induced by intravenous administration of labda-15-oic acid (0.3–3mg/kg) was partially reduced by L-NAME. In conclusion, the mechanisms underlying the cardiovascular actions of the labdane involve the activation of the endothelial NO-cGMP pathway, the opening of K+ channels and the alteration on Ca2+ mobilization.

Role of calcium channels responsible for phenylephrine-induced contraction in rat aorta 3 days after acute myocardial infarction

BackgroundPhenylephrine (PE) produces tonic contraction through involvement of various calcium channels such as store-operated calcium channels (SOCCs) and voltage-operated calcium channels (VOCCs). However, the relative contribution of each calcium channel to PE-induced contraction has not been investigated in isolated rat aorta of early acute myocardial infarction (AMI).MethodsEndothelium-denuded rat aortic rings from rats 3 days after AMI or sham-operated (SHAM) rats were prepared in an organ chamber with Krebs-Ringer bicarbonate solution for isometric tension recording. We assessed the PE dose-response relationships in 2.5 mM calcium medium for both groups. The same procedure was repeated using rings pretreated with the SOCC inhibitor 2-aminoethoxydiphenyl borate, sarco/endoplasmic-reticulum calcium ATPase inhibitor thapsigargin (TG), diacyl glycerol lipase inhibitor RHC80267, and sodium-calcium exchanger inhibitor 3,4-dichlorobenzamil hydrochloride for 30 minutes before addition of calcium. When ongoing tonic contraction was sustained, dose-response curves to the VOCC inhibitor nifedipine were obtained to assess the relative contribution of each calcium channel under various conditions.ResultsThe effect of SOCC induction with TG pretreatment on PE-induced contraction was significantly lower in the AMI group compared to the SHAM group. In addition, there were significant decreases in the sensitivity and efficacy of the VOCC inhibitor nifedipine on PE-induced contraction in the AMI group.ConclusionsResults suggest that the change of vascular reactivity of PE in rat aorta 3 days after AMI is characterized by a decreased contribution of L-type VOCCs. The enhanced VOCC-independent calcium entry mechanisms after AMI can be mediated by enhanced capacitative calcium entry through the activation of SOCCs.

Vasorelaxant and antihypertensive effects of methanolic fraction of the essential oil of Alpinia zerumbet

Alpinia zerumbet is used in folk medicine in Brazil to treat hypertension. However, several pathways involved in the mechanism of vasorelaxation are still unclear. This study was designed to verify the antihypertensive effect of the methanolic fraction of the essential oil of A. zerumbet (MFEOAz) and to characterize its mechanism of action. The thoracic aortic rings from the Wistar rats were perfused in the organ chambers filled with Kreb's solution, where the tension of each ring was measured. The antihypertensive effect of MFEOAz was assessed in rats submitted to chronic hypertension by inhibition of nitric oxide synthesis by indirect measurement of blood pressure with indirect tail cuff method. MFEOAz relaxed phenylephrine and KCl-induced contraction of either endothelium-intact or endothelium-denuded rat aortic rings in a concentration-dependent manner. Pre-incubation with MFEOAz (100 and 300μg/mL) in Ca2+-free Krebs solution attenuated phenylephrine- or caffeine-induced contraction. Pre-incubation with L-NAME, ODQ, wortmannin, atropine, indomethacin, catalase, SOD, TEA, 4-aminopyridine, glibenclamide, apamin, charybdotoxin, or iberiotoxin did not affect MFEOAz-induced relaxation. The intragastric administration of MFEOAz induced an antihypertensive effect. MFEOAz it seems inhibited the calcium influx via voltage-operated calcium channels and receptor-operated calcium channels, as well as inhibition of calcium mobilization from intracellular stores.

Vasorelaxation by hydrogen sulphide involves activation of Kv7 potassium channels

Hydrogen sulphide (H2S) has been recently hypothesized to be an endogenous adipocyte-derived relaxing factor, evoking vasorelaxation of conductance and resistance vessels. Although the activation of ATP-sensitive potassium channels is known to play a central role in H2S-induced vasorelaxation, activation of vascular Kv7 voltage-gated potassium channels has also been suggested. To investigate this possibility, the ability of selective activators and blockers of distinct classes of potassium channels to affect vasodilation induced by the H2S-donor NaHS, as well as NaHS-induced Rb+ efflux in endothelium-denuded rat aortic rings, was investigated. NaHS-induced changes of membrane potential were fluorimetrically assessed on human vascular smooth muscle (VSM) cells. Modulation of Kv7.4 channels by NaHS was assessed by electrophysiological studies, upon their heterologous expression in CHO cells. In isolated aortic rings, NaHS evoked vasorelaxing responses associated with an increase of Rb+-efflux. NaHS promoted membrane hyperpolarization of human VSM cells. These effects were antagonized by selective blockers of Kv7 channels. The H2S-donor caused a left-shift of current activation threshold of Kv7.4 channels expressed in CHO cells.Altogether, these results suggest that the activation of Kv7.4 channels is a key mechanism in the vascular effects of H2S. Given the relevant roles played by Kv7.4 channels in VSM contractility and by H2S in circulatory homeostasis regulation, these findings provide interesting insights to improve our understanding of H2S pathophysiology and to focus on Kv7.4 channels as novel targets for therapeutic approaches via the “H2S-system”.

The semi-synthetic kaurane ent-16α-methoxykauran-19-oic acid induces vascular relaxation and hypotension in rats

The present work investigates the mechanisms involved in the vasorelaxant effect of ent-16α-methoxykauran-19-oic acid (KA–OCH3), a semi-synthetic derivative obtained from the kaurane-type diterpene ent-kaur-16-en-19-oic acid (kaurenoic acid). Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats using standard muscle bath procedures. The cytosolic calcium concentration ([Ca2+]c) was measured by confocal microscopy using the fluorescent probe Fluo-3 AM. Blood pressure measurements were performed in conscious rats. KA–OCH3 (10, 50 and 100μmol/l) inhibited phenylephrine-induced contraction in either endothelium-intact or endothelium-denuded rat aortic rings. KA–OCH3 also reduced CaCl2-induced contraction in a Ca2+-free solution containing KCl (30mmol/l) or phenylephrine (0.1μmol/l). KA–OCH3 (0.1–300μmol/l) concentration-dependently relaxed endothelium-intact and endothelium-denuded aortas pre-contracted with either phenylephrine or KCl, to a greater extent than kaurenoic acid. Moreover, a Ca2+ mobilisation study showed that KA–OCH3 (100μmol/l) inhibited the increase in Ca2+ concentration in smooth muscle and endothelial cells induced by phenylephrine or KCl. Pre-incubation of intact or denuded aortic rings with NG-nitro-l-arginine methyl ester (L-NAME, 100μmol/l), 7-nitroindazole (100μmol/l), wortmannin (0.5μmol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1μmol/l) produced a rightward displacement of the KA–OCH3 concentration–response curve. Intravenous administration of KA–OCH3 (1–10mg/kg) reduced mean arterial blood pressure in normotensive rats. Collectively, our results show that KA–OCH3 induces vascular relaxation and hypotension. The mechanisms underlying the cardiovascular actions of KA–OCH3 involve blockade of Ca2+ influx and activation of the NO-cGMP pathway.

Heat shock augments angiotensin II-induced vascular contraction through increased production of reactive oxygen species

A temporal increase in temperature triggers a series of stress responses and alters vascular smooth muscle (VSM) contraction induced by agonist stimulation. Here we examined the role of reactive oxygen species (ROS) in heat shock-dependent augmentation of angiotensin II (AngII)-induced VSM contraction. Endothelium-denuded rat aortic rings were treated with heat shock for 45 min at 42 °C and then subjected to assays for the production of force, ROS, and the expression of ROS-related enzymes. AngII-induced contraction was enhanced in heat shock-treated aorta. AngII-induced production of hydrogen peroxide and superoxide were elevated in response to the heat shock treatment. Pre-treatment with superoxide dismutases (SOD) mimetic and inhibitors for glutathione peroxidase and NADPH oxidase but not for xanthine oxidase eliminated an increase in the AngII-induced contraction in the heat shock-treated aorta. Heat shock increased the expression of p47phox, a cytosolic subunit of NADPH oxidase, but not Cu–Zn–SOD and Mn–SOD. In addition, heat shock increased contraction that was evoked by hydrogen peroxide and pyrogallol. These results suggest that heat shock causes an elevation of ROS as well as a sensitization of ROS signal resulting in an augmentation of VSM contraction in response to agonist.

Darusentan is a potent inhibitor of endothelin signaling and function in both large and small arteriesThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

Endothelin is a potent vasoconstrictor often up-regulated in hypertension. Endothelin vasoconstriction is mediated via the G-protein coupled endothelin A (ETA) receptor present on vascular smooth muscle. Endothelin receptor antagonists (ERAs) have been shown to antagonize ET-induced vasoconstriction. We describe the primary pharmacology of darusentan, a propanoic acid based ERA currently in phase 3 clinical trials for resistant hypertension. Darusentan was tested in membrane-, cell-, and tissue-based assays to determine its biochemical and functional potency. Rat aortic vascular smooth muscle cells (RAVSMs) were characterized using flow cytometry. RAVSM membrane fractions tested in saturation experiments exhibited moderate endothelin receptor density. Receptor counting revealed that >95% of the endothelin receptors in these fractions were the ETA subtype. (S)-Darusentan competed for radiolabeled endothelin binding in RAVSM membranes with single-site kinetics, exhibiting a Ki = 13 nmol/L. (R)-Darusentan exhibited no binding activity. In cultured RAVSMs, endothelin induced increases in inositol phosphate and Ca2+ signaling, both of which were attenuated by (S)-darusentan in a concentration-dependent manner. In isolated endothelium-denuded rat aortic rings, (S)-darusentan inhibited endothelin-induced vascular contractility with a pA2 = 8.1 +/- 0.14 (n = 4 animals; mean +/- SD). (R)-Darusentan had no effect. The vasorelaxant potency of (S)-darusentan did not change when determined in isolated denuded rat mesenteric arterioles, suggesting a similar mode of action in both conductance and resistance arteries. In vascular smooth muscle, (S)-darusentan is an ERA with high affinity for the ET receptor, which in this preparation is predominantly ETA receptors. (S)-Darusentan inhibits endothelin-induced signaling related to pro-contractile activity and is a potent inhibitor of vasoconstriction in large and small arteries.

Pharmacological comparison of the vasorelaxant action displayed by kaurenoic acid and pimaradienoic acid

The vascular effects of two natural occurring diterpenes from the kaurane and pimarane classes were compared. The diterpenes ent-kaur-16-en-19-oic acid (kaurenoic acid; KA) and ent-pimara-8(14), 15-dien-19-oic acid (pimaradienoic acid; PA) were tested for their antispasmodic activity on isolated rat aorta. Vascular reactivity experiments, using standard muscle bath procedures, showed that KA and PA (both at 50 and 100 microM) inhibited phenylephrine and KCl-induced contraction in both endothelium-intact and endothelium-denuded rat aortic rings, with PA being more effective than KA. These compounds also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mm). Again, PA produced a greater reduction in CaCl(2)-induced contraction than KA. PA (1-300 microM) and KA (1-450 microM) concentration dependently relaxed endothelium-denuded aortic rings pre-contracted with KCl (maximum relaxation 102.31+/-6.94% and 82.71+/-1.40%, respectively). Similarly, the relaxation induced by KA on aortic rings pre-contracted with phenylephrine (73.06+/-3.68%) was less pronounced than that found for PA (102.21+/-3.64%). Incubation of endothelium-denuded rings for different periods showed that at 50 microM, KA and PA achieved maximum inhibitory activity on KCl-induced contraction after incubation for 60 (53.48+/-5.83%) and 30 min (83.89+/-2.12%), respectively. At 100 microM, KA and PA inhibited KCl-induced contraction, with a maximum after incubation for 30 min (73.58+/-5.30% and 92.07+/-1.20%, respectively). The maximum inhibition induced by PA at both concentrations tested was greater than that induced by KA. The results provide evidence that structural differences between diterpenes, independent of the C-19 carboxylic acid site, influence selectivity for voltage-operated Ca2+ channels and rate of equilibrium with the target site for their vasorelaxant action in rat aortic rings.

Pimaradienoic acid inhibits vascular contraction and induces hypotension in normotensive rats

The present investigation was designed to investigate the effect of the diterpene ent-pimara-8(14),15-dien-19-oic acid (pimaradienoic acid, PA) on smooth muscle extracellular Ca(2+) influx. To this end, the effect of PA on phenylephrine- and KCl-induced increases in cytosolic calcium concentration ([Ca(2+)](c)), measured by the variation in the ratio of fluorescence intensities (R340/380 nm) of Fura-2, was analysed. Whether bolus injection of PA could induce hypotensive responses in conscious normotensive rats was also evaluated. PA inhibited the contraction induced by phenylephrine (0.03 or 10 micromol L(-1)) and KCl (30 or 90 mmol L(-1)) in endothelium-denuded rat aortic rings in a concentration dependent manner. Pre-treatment with PA (10, 100, 200 micromol L(-1)) attenuated the contraction induced by CaCl(2) (0.5 nmol L(-1) or 2.5 mmol L(-1)) in denuded rat aorta exposed to Ca(2+)-free medium containing phenylephrine (0.1 micro mol L(-1)) or KCl (30 mmol L(-1)). Interestingly, the inhibitory effect displayed by PA on CaCl(2)-induced contraction was more pronounced when KCl was used as the stimulant. Phenylephrine- and KCl-induced increases in [Ca(2+)](c) were inhibited by PA. Similarly, verapamil, a Ca(2+)-channel blocker, also inhibited the increase in [Ca(2+)](c) induced by either phenylephrine or KCl. Finally, bolus injection of PA (1-15 mg kg(-1)) produced a dose-dependent decrease in mean arterial pressure in conscious normotensive rats. The results provide the first direct evidence that PA reduces vascular contractility by reducing extracellular Ca(2+) influx through smooth muscle cellular membrane, a mechanism that could mediate the hypotensive response induced by this diterpene in normotensive rats.

Endothelium-independent vasorelaxation by the selective alpha estrogen receptor agonist propyl pyrazole triol in rat aortic smooth muscle

Abstract Objectives This study investigated the signalling mechanism of the relaxant responses to the estrogen receptor alpha (ERα) agonist PPT (propyl pyrazole triol) in endothelium-denuded rat aortic rings. Methods Several compounds, including protein kinase G (PKG) inhibitors and potassium channel inhibitors, were tested against PPT-dependent rat aortic relaxation. Cyclic GMP and cytosolic calcium responses to PPT in isolated aortic smooth muscle were investigated in parallel. Key findings PPT vasorelaxation was largely reduced by the selective ERα antagonist methyl-piperidinopyrazole (MPP; −91.6 ± 2.5%), by the selective PKG inhibitor Rp-8-Br-cGMP (−78.6 ± 4.9%), by the specific soluble guanylyl cyclase inhibitor ODQ (1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; −85.3 ± 5.2%) and to a lesser extent by the selective BKCa (large-conductance calcium- and voltage-activated potassium channel) inhibitor iberiotoxin (−59.3%), the selective IKCa (intermediate-conductance calcium-activated potassium channel) inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; −50.7%) and the voltage-gated potassium channel inhibitor 4-aminopyridine (−40.8%). In isolated aortic smooth muscle, PPT strongly enhanced the cyclic GMP content (+144%) and Rp-8-Br-cGMP largely reduced the PPT-dependent calcium signal (−80.8%). Conclusions ERα receptor stimulation in rat aortic smooth muscle evokes a PKG-signalling pathway, likely triggering relaxation by BKCa and IKCa channel opening.

Endothelium-independent vasorelaxation by the selective alpha estrogen receptor agonist propyl pyrazole triol in rat aortic smooth muscle

This study investigated the signalling mechanism of the relaxant responses to the estrogen receptor (ERalpha) agonist PPT (propyl pyrazole triol) in endothelium-denuded rat aortic rings. Several compounds, including protein kinase G (PKG) inhibitors and potassium channel inhibitors, were tested against PPT-dependent rat aortic relaxation. Cyclic GMP and cytosolic calcium responses to PPT in isolated aortic smooth muscle were investigated in parallel. PPT vasorelaxation was largely reduced by the selective ERalpha antagonist methyl-piperidinopyrazole (MPP; -91.6+/-2.5%), by the selective PKG inhibitor Rp-8-Br-cGMP (-78.6+/-4.9%), by the specific soluble guanylyl cyclase inhibitor ODQ (1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; -85.3+/-5.2%) and to a lesser extent by the selective BKCa (large-conductance calcium- and voltage-activated potassium channel) inhibitor iberiotoxin (-59.3%), the selective IKCa (intermediate-conductance calcium-activated potassium channel) inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; -50.7%) and the voltage-gated potassium channel inhibitor 4-aminopyridine (-40.8%). In isolated aortic smooth muscle, PPT strongly enhanced the cyclic GMP content (+144%) and Rp-8-Br-cGMP largely reduced the PPT-dependent calcium signal (-80.8%). ERalpha receptor stimulation in rat aortic smooth muscle evokes a PKG-signalling pathway, likely triggering relaxation by BKCa and IKCa channel opening.

Scutellarin‐induced endothelium‐independent relaxation in rat aorta

Scutellarin is a flavonoid extracted from the traditional Chinese herb, Erigeron breviscapus Hand Mazz. In the present study, the vasorelaxant effects of scutellarin and the underlying mechanism were investigated in isolated rat aorta. Scutellarin (3, 10, 30, 100 microm) caused a dose-dependent relaxation in both endothelium-intact and endothelium-denuded rat aortic rings precontracted with noradrenaline bitartrate (IC(50) = 7.7 +/- 0.6 microm), but not with potassium chloride. Tetraethylammonium, glibenclamide, atropine, propranolol, indomethacin and N(G)-nitro-l-arginine methyl ester had no influence on the vasorelaxant effect of scutellarin, which further excluded the involvement of potassium channels, muscarinic receptor, nitric oxide pathway and prostaglandin in this effect. Pretreatment with scutellarin decreased the tonic phase, but not the phasic phase of the noradrenaline bitartrate induced tension increment. Scutellarin also alleviated Ca(2+)-induced vasoconstriction in Ca(2+)-depleted/noradrenaline bitartrate pretreated rings in the presence of voltage-dependent calcium channel blocker verapamil. The noradrenaline bitartrate evoked intracellular calcium increase was inhibited by scutellarin. Scutellarin had no effect on phorbol-12,13-diacetate induced contraction in a calcium-free bath solution. These results showed that scutellarin could relax thoracic artery rings in an endothelium-independent manner. The mechanism seems to be the inhibition of extracellular calcium influx independent of the voltage-dependent calcium channel.