Endothelium-denuded Aortic Rings Research Articles

STIM1/Orai1 contributes to sex differences in vascular responses to calcium in spontaneously hypertensive rats

Sex differences in Ca2+-dependent signalling and homoeostasis in the vasculature of hypertensive rats are well characterized. However, sex-related differences in SOCE (store-operated Ca2+ entry) have been minimally investigated. We hypothesized that vascular protection in females, compared with males, reflects decreased Ca2+ mobilization due to diminished activation of Orai1/STIM1 (stromal interaction molecule 1). In addition, we investigated whether ovariectomy in females affects the activation of the Orai1/STIM1 pathway. Endothelium-denuded aortic rings from male and female SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar-Kyoto) rats and from OVX (ovariectomized) or sham female SHRSP and WKY rats were used to functionally evaluate Ca2+ influx-induced contractions. Compared with females, aorta from male SHRSP displayed: (i) increased contraction during the Ca2+-loading period; (ii) similar transient contraction during Ca2+ release from the intracellular stores; (iii) increased activation of STIM1 and Orai1, as shown by the blockade of STIM1 and Orai1 with neutralizing antibodies, which reversed the sex differences in contraction during the Ca2+-loading period; and (iv) increased expression of STIM1 and Orai1. Additionally, we found that aortas from OVX-SHRSP showed increased contraction during the Ca2+-loading period and increased Orai1 expression, but no changes in the SR (sarcoplasmic reticulum)-buffering capacity or STIM1 expression. These findings suggest that augmented activation of STIM1/Orai1 in aortas from male SHRSP represents a mechanism that contributes to sex-related impaired control of intracellular Ca2+ levels. Furthermore, female sex hormones may negatively modulate the STIM/Orai1 pathway, contributing to vascular protection observed in female rats.

Glucosamine increases vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aorta

Diabetes is a well-known independent risk factor for vascular disease. However, its underlying mechanism remains unclear. It has been reported that increased influx of the hexosamine biosynthesis pathway (HBP) induces O-GlcNAcylation of proteins, leading to insulin resistance. In this study, we determined whether or not O-GlcNAc modification of proteins could increase vessel contraction. Using an endothelium-denuded aortic ring, we observed that glucosamine induced OGlcNAcylation of proteins and augmented vessel contraction stimulated by U46619, a thromboxane A(2) agonist, via augmentation of the phosphorylation of MLC(20), MYPT1(Thr855), and CPI17, but not phenylephrine. Pretreatment with OGT inhibitor significantly ameliorated glucosamine-induced vessel constriction. Glucosamine treatment also increased RhoA activity, which was also attenuated by OGT inhibitor. In conclusion, glucosamine, a product of glucose influx via the HBP in a diabetic state, increases vascular contraction, at least in part, through activation of the RhoA/Rho kinase pathway, which may be due to O-GlcNAcylation.

Potentiation of vasoconstriction and pressor response by low concentration of monomethylarsonous acid (MMA III)

A close link between arsenic exposure and hypertension has been well-established through many epidemiological reports, yet the mechanism underlying it remains unclear. Here we report that nanomolar concentrations of monomethylarsonous acid (MMA III), a toxic trivalent methylated arsenic metabolite, can potentiate agonist-induced vasoconstriction and pressor responses. In freshly isolated rat aortic ring, exposure to nanomolar MMA III (100–500 nM) potentiated phenylephrine (PE)-induced vasoconstriction while at higher concentrations (≥2.5 μM), suppression of vasoconstriction and apoptosis of vascular smooth muscle were observed. Potentiation of agonist-induced vasoconstriction was also observed with other contractile agonists and it was retained in endothelium-denuded aortic rings, suggesting that these events are agonist-independent and smooth muscle cell dependent. Interestingly, exposure to MMA III resulted in increased myosin light chain phosphorylation while PE-induced Ca 2+ influx was not affected, reflecting that Ca 2+ sensitization is involved. In line with this, MMA III enhanced agonist-induced activation of small GTPase RhoA, a key contributor to Ca 2+ sensitization. Of note, treatment of MMA III to rats induced significantly higher pressor responses in vivo, demonstrating that this event can occur in vivo indeed. We believe that RhoA-mediated Ca 2+ sensitization and the resultant potentiation of vasoconstriction by MMA III may shed light on arsenic-associated hypertension.

Endothelium-independent vasodilation effect of hydroxysafflor yellow A in thoracic aorta of Wistar rats

Hydroxysafflor yellow A, the main active component of safflor yellow, in this study is used to investigate the vascular activity of hydroxysafflor yellow A (HSYA) on rat thoracic aorta and the underlying mechanism. The tension of the isolated thoracic aorta rings of rats incubated with different concentrations of HSYA was measured using organ bath technique. The effect of HSYA (10-4~10-6 mol/L) on the contraction induced by cumulative phenylephrine, KCl and CaCl2, was respectively examined by HSYA (10-4~10-6 mol/L) dose, which dependently inhibited the contraction induced by KCl (6×10-2 mol/L) or phenylephrine (PE, 10-6 mol/L) in both endothelium-intact and endothelium-denuded aortic rings. HSYA inhibited the CaCl2-induced contraction and downward shifted concentration-response curve of aortic rings precontracted with PE. Furthermore, HSYA-induced inhibition may be partially through the blockage of IP3 receptor. These results indicated that the role of HSYA may be involved in the reduction of Ca2+ influx and inhibition of IP3 receptor in vascular smooth muscle cells. Key words: Hydroxysafflor yellow A, vasodilation, Ca2+ channel, aorta.

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.

Effects induced by inhibitors of the phosphatidylinositol 3‐kinase/Akt and nitric oxide synthase/guanylyl cyclase pathways on the isometric contraction in rat aorta: a comparative study

This work was aimed to determine whether isometric contraction in Wistar rat aorta is related to the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent activation of endothelial nitric oxide synthase (eNOS). Basically, we hypothesized that additional increases in active tone occur after the pharmacological inhibition of a transduction pathway involved in NO synthesis or action. In intact aortic rings contracted with phenylephrine or high K(+), the cumulative administration of the PI3K inhibitor, LY294002, elicited significant decreases--but not supplementary increases--in tone. In endothelium-intact tissues, on the other hand, the Akt1/2 kinase inhibitor did not alter phenylephrine- and K(+)-induced isometric contractions. The PI3K inhibitor wortmannin (1 × 10(-7) m) produced a significant supplementary contraction only in endothelium-intact aortic rings precontracted with phenylephrine. Higher concentrations of this inhibitor produced relaxations of phenylephrine and high K(+)-constricted endothelium-intact and endothelium-denuded aortic rings. LY294002 and wortmannin did not cause any potentiating effect on phenylephrine- and angiotensin II-induced concentration-dependent contractile responses in endothelium-intact tissues. In intact aortic rings contracted with phenylephrine or high K(+), the addition of the NOS inhibitor, L-NAME, or the guanylyl cyclase inhibitor, ODQ, further augmented tone in a concentration-dependent manner, and these supplementary contractions were significantly reduced by endothelium removal. Taken together, our data suggest that the PI3K/Akt pathway is not counteracting aortic isometric contractions by activation of the eNOS. It appears, on the other hand, that the smooth muscle PI3K can stimulate contraction without activation of the protein kinase Akt in response to GPCR agonists and high K(+).

Mitochondrial aldehyde dehydrogenase prevents ROS-induced vascular contraction in angiotensin-II hypertensive mice

Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme that detoxifies aldehydes to carboxylic acids. ALDH2 deficiency is known to increase oxidative stress, which is the imbalance between reactive oxygen species (ROS) generation and antioxidant defense activity. Increased ROS contribute to vascular dysfunction and structural remodeling in hypertension. We hypothesized that ALDH2 plays a protective role to reduce vascular contraction in angiotensin-II (AngII) hypertensive mice. Endothelium-denuded aortic rings from C57BL6 mice, treated with AngII (3.6 μg/kg/min, 14 days), were used to measure isometric force development. Rings treated with daidzin (10 μmol/L), an ALDH2 inhibitor, potentiated contractile responses to phenylephrine (PE) in AngII mice. Tempol (1 mmol/L) and catalase (600 U/mL) attenuated the augmented contractile effect of daidzin. In normotensive mice, contraction to PE in the presence of the daidzin was not different from control, untreated values. AngII aortic rings transfected with ALDH2 recombinant protein decreased contractile responses to PE compared with control. These data suggest that ALDH2 reduces vascular contraction in AngII hypertensive mice. Because tempol and catalase blocked the contractile response of the ALDH2 inhibitor, ROS generation by AngII may be decreased by ALDH2, thereby preventing ROS-induced contraction.

Mechanisms of Glucosamine‐Mediated Relaxing Responses in Isolated Rat Aortic Rings

Glucosamine (GlcN) has been shown to block capacitative Ca2+ entry, also referred to as store‐operated Ca2+ entry (SOCE), in rat cardiomyocytes in response to phenylephrine (PHE). We hypothesized that GlcN would suppress SOCE in smooth muscle cells and lower intracellular [Ca2+] in PHE‐contracted rat aortic rings leading to vasorelaxation. In the wire‐myograph, relaxing responses to GlcN (0.01 – 30 mM) were assessed in PHE‐contracted rat aortic rings. GlcN relaxed endothelium‐denuded (−E) aortic rings with a sensitivity (pEC50) of 2.73 ± 0.09. The cell impermeable and osmotic control L‐ glucose (L‐Glc; 0.01 – 30 mM) did not result in arterial relaxations. Inhibition of the sarcoendoplasmic reticulum (ER) Ca2+‐ATPase (SERCA) results in Ca2+ depletion in the ER, which activates SOCE. The selective SERCA inhibitors thapsigargin (10 μM) and cyclopiazonic acid (50 μM) both significantly suppressed relaxations to GlcN in −E aortic rings. In Ca2+ depleted –E aortic rings, re‐titration of CaCl2 (0.01 – 5 mM) resulted in a lower sensitivity to CaCl2 in the presence of 5 mM GlcN compared to L‐Glc. Pre‐incubation with the SOCE inhibitor SKF96365 (10 μM) reduced sensitivity to CaCl2to comparable levels for both GlcN and L‐Glc. The present findings demonstrate that GlcN acts via an endothelium‐independent and cGMP‐dependent mechanism that inhibits SOCE to lower smooth muscle cell [Ca2+]i with subsequent vasorelaxation.

The methyl ester of rosuvastatin elicited an endothelium-independent and 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent relaxant effect in rat aorta

The relaxant effect of the methyl ester of rosuvastatin was evaluated on aortic rings from male Wistar rats (250-300 g, 6 rats for each experimental group) with and without endothelium precontracted with 1.0 µM phenylephrine. The methyl ester presented a slightly greater potency than rosuvastatin in relaxing aortic rings, with log IC50 values of -6.88 and -6.07 M, respectively. Unlike rosuvastatin, the effect of its methyl ester was endothelium-independent. Pretreatment with 10 µM indomethacin did not inhibit, and pretreatment with 1 mM mevalonate only modestly inhibited the relaxant effect of the methyl ester. Nω-nitro-L-arginine methyl ester (L-NAME, 10 µM), the selective nitric oxide-2 (NO-2) inhibitor 1400 W (10 µM), tetraethylammonium (TEA, 10 mM), and cycloheximide (10 µM) partially inhibited the relaxant effect of the methyl ester on endothelium-denuded aortic rings. However, the combination of TEA plus either L-NAME or cycloheximide completely inhibited the relaxant effect. Inducible NO synthase (NOS-2) was only present in endothelium-denuded aortic rings, as demonstrated by immunoblot with methyl ester-treated rings. In conclusion, whereas rosuvastatin was associated with a relaxant effect dependent on endothelium and hydroxymethylglutaryl coenzyme A reductase in rat aorta, the methyl ester of rosuvastatin exhibited an endothelium-independent and only slightly hydroxymethylglutaryl coenzyme A reductase-dependent relaxant effect. Both NO produced by NOS-2 and K+ channels are involved in the relaxant effect of the methyl ester of rosuvastatin.

Endothelial Nitric Oxide-Dependent Vasorelaxant Effect of Isotirumalin, a Dihydroflavonol from Derris urucu, on the Rat Aorta

The present work aimed to investigate the vasorelaxant effect of isotirumalin, a dihydroflavonol isolated from Derris urucu (Leguminosae). The vasorelaxant effect of isotirumalin was investigated in the rat aorta, in the presence and in the absence of a functional endothelium. The production of nitric oxide (NO) induced by isotirumalin was measured simultaneously with its vasorelaxation using carbon microsensors. In endothelium-intact aortic rings, isotirumalin induced a concentration-dependent vasodilator effect the concentration required to produce 30% of relaxation (pIC₃₀=4.84±0.24) that was abolished in endothelium-denuded aortic rings or in the presence of Nω-nitro-L-arginine-methyl-ester (L-NAME; 300 µM). In addition, isotirumalin (100 µM) induced a simultaneous and significant increase on NO production, which was blunted in the presence of L-NAME. The present results demonstrate that isotirumalin is a vasodilator in the rat aorta and act by a mechanism dependent on the presence of a functional endothelium and on NO production.

Lack of heterologous receptor desensitization induced by angiotensin II type 1 receptor activation in isolated normal rat thoracic aorta

We tested whether heterologous receptor desensitization induced by activation of AT1 receptors may explain the purported relaxation produced by angiotensin II in normal rat aorta. Also, the role for AT2 receptors in the promotion of vasodilation was studied. In endothelium-intact and endothelium-denuded aortic rings, angiotensin II elicited biphasic contractions, which were significantly depressed when repeated in each tissue. Angiotensin II produced biphasic responses on phenylephrine preconstricted endothelium-intact and endothelium-denuded tissues, without reducing precontractile tone. These responses were abolished in the presence of the AT1 receptor antagonist losartan, but no relaxing responses to angiotensin II were uncovered. PD123319 did not influence angiotensin II responses in endothelium-intact tissues precontracted with phenylephrine; thus, under AT2 receptors blockade the contractile effects of angiotensin II were not overexposed. In conclusion, angiotensin II-induced biphasic responses can be attributed to AT1 receptors activation and rapid desensitization with time. Desensitization proved to be homologous in nature, since precontractile tone induced by phenylephrine was not depressed by angiotensin II (i.e., angiotensin II did not induce heterologous α1-adrenergic receptors desensitization). We found no functional evidence of the participation of AT2 receptors in angiotensin II elicited biphasic contractions. Angiotensin II does not exert relaxant effects in normal rat aorta.

Hemodynamics and aortic tension induced by two septic shock models in rats

To compare the ventricular-dynamic parameters and thoracic aorta tension induced by two septic shock models in rats. Septic shock models were induced by cecal ligation or puncture (CLP) and intraperitoneal injection of lipopolysaccharide (LPS) in rats. The carotid artery was cannulated and connected to a pressure transducer to determine mean arterial blood pressure (MABP). Ventricular dynamic parameters, including heart rate (HR), left ventricular developed pressure (LVDP) and maximal rise/fall velocity of ventricular pressure (± dP/dtmax) were determined. Isolated thoracic rings were mounted on an organ bath and the tension of the vessel was recorded. The mortality was 65.2% in CLP shock rats, but no death in LPS shock rats. The MABP and HR of CLP rats were decreased more prominently than those of LPS rats (P < 0.01). Contraction induced by high K(+) (60 mmol/L) or 10⁻⁶ mol/L phenylephrine (PE) in endothelium-intact and endothelium-denuded aortic rings was all attenuated, but in LPS rats it was more prominent (P < 0.01). Two rat septic shock models can decrease ventricular-dynamic parameters and vasoconstriction responsiveness of aorta. The ventricular-dynamic parameters decrease more prominently in CLP model, while vasoconstriction responsiveness of aorta changes more in LPS model.

α1‐Adrenoceptor‐mediated contraction of rat aorta is partly mediated via transactivation of the epidermal growth factor receptor

High level of plasma catecholamines is a risk factor for vascular diseases such as hypertension and atherosclerosis. Catecholamines induce hypertrophy of vascular smooth muscle through α(1) -adrenoceptors, which in cell culture involves the transactivation of epidermal growth factor receptor (EGFR). We hypothesized that EGFR transactivation was also involved in contractions of rat aorta mediated by α(1) -adrenoceptors. Thoracic aorta was isolated from 12-14 week old male Wistar rats. In vitro aortic contractile responses to cumulative doses of phenylephrine were characterized in the absence and presence of the EGFR kinase inhibitors, AG1478 and DAPH, in intact and endothelium-denuded rings. Involvement of signal transduction pathways was investigated by using heparin and inhibitors of Src, matrix metalloproteinase (MMP), extracellular signal-regulated kinase (ERK)1/2 and phosphatidyl inositol 3-kinase (PI3K). Phosphorylation of EGFR and ERK1/2 was measured after short-term phenylephrine or EGF stimulation in aorta segments in the presence of AG1478 and the PI3K inhibitor, wortmannin. AG1478 and DAPH concentration dependently attenuated phenylephrine-induced contractile responses in intact or endothelium-denuded aortic rings. Inhibition of PI3K (wortmannin and LY294002) but not heparin or inhibitors of Src or MMP, prevented the effect of AG1478 on the responses to phenylephrine. Phenylephrine induced phosphorylation of EGFR, which was partially blocked by AG1478. Phenylephrine also increased phosphorylation of ERK1/2, time-dependently and was blocked by AG1478 and wortmannin. Contractions of rat thoracic aorta mediated by α(1) -adrenoceptors involved transactivation of EGFR, mediated via a PI3K and ERK1/2 dependent pathway.

HMC05 attenuates vascular contraction through inhibition of RhoA/Rho-kinase signaling pathway

HMC05, an extract from eight different herbal mixtures, has been developed to treat cardiovascular disease. This extract has a vasorelaxant and anti-atherosclerotic action. We hypothesized that HMC05 attenuates vascular contraction through inhibition of the RhoA/Rho-kinase signaling pathway. Rat aortic ring preparations were mounted in organ baths and subjected to contraction and relaxation. Phosphorylation of 20 kDa myosin light chains (MLC(20)) and myosin phosphatase targeting subunit 1 (MYPT1) were examined by immunoblot. We also measured the amount of GTP RhoA as a marker for RhoA activation. In endothelium-denuded aortic ring preparations, HMC05 relaxed vascular contraction induced by 6.0 mM NaF, 100 nM phenylephrine, 30 nM thromboxane A(2) agonist U46619 or 1.0 μM protein kinase C (PKC) activator phorbol-12,13-dibutyrate (PDBu) in a decreasing order. HMC05 relaxed aortic ring preparations precontracted with sodium fluoride (NaF) whether endothelium was intact or denuded. Pre-incubation with HMC05 for 30 min dose-dependently inhibited the NaF-induced contractile response. In vascular strips, HMC05 decreased the phosphorylation level of both MLC(20) and MYPT1(Thr855) induced by 6.0 mM NaF. Furthermore, HMC05 decreased the amount of GTP RhoA activated by NaF. HMC05 attenuates vascular contraction through inhibition of the RhoA/Rho-kinase signaling pathway. HMC05 may be useful for the treatment and/or prevention of cardiovascular diseases associated with activation of RhoA/Rho-kinase signaling pathway.

Hypotensive activity of an n-butanol extract and their purified compounds from leaves of Phyllanthus acidus (L.) Skeels in rats

We aimed to investigate the effects, identify the active substances and establish the mechanisms involved in the hypotensive activity of an n-butanol extract from leaves of Phyllanthus acidus (PA extract). PA extract caused a decrease in blood pressure of anesthetized rats that was not modified by atropine or propranolol. PA extract caused a persistent dilatation of thoracic aortic rings preconstricted with either phenylephrine or KCl, and these effects were not modified by LNA or removal of the vascular endothelium. For phenylephrine-preconstricted aortic rings, the dilatory activity of the PA extract was not modified by atropine, propranolol or indomethacin. TEA, glybenclamide or ODQ significantly inhibited the dilatory activity of the PA extract on endothelium-denuded aortic rings. Nifedipine or a Ca 2+-free medium depressed the aortic rings constrictor response to phenylephrine, and that was further augmented by the PA extract. Adenosine, 4-hydroxybenzoic acid, caffeic acid, hypogallic acid, and kaempferol were isolated from the PA extract. Each caused a decrease in blood pressure and dilatation of the aortic rings. LNA or removal of the endothelium reduced this activity. ODQ and TEA attenuated the vasodilatory activity of adenosine whereas glybenclamide and ODQ attenuated the effect of hypogallic acid. These results suggest that the hypotensive activities of the PA extract is likely the result of the direct action of these five compounds on the blood vessels by stimulating release of nitric oxide from the vascular endothelium, in part through stimulation of soluble guanylate cyclase, and opening of K ATP and K Ca channels in the vascular smooth muscle.

Impaired 3′,5′-Cyclic Adenosine Monophosphate–Mediated Signaling in Immediate Early Responsive Gene X-1–Deficient Vascular Smooth Muscle Cells

Gene-targeted deletion of the immediate early responsive gene X-1 (IEX-1) results in a significant increase in systemic arterial blood pressure, but the underlying mechanism is not understood. Studies of arterial reactivity in isolated aortas revealed normal endothelium-dependent and -independent vasorelaxation and vasoconstriction but reduced cAMP-dependent vasorelaxation in the absence of IEX-1. This defect in cAMP signaling was also evident in endothelium-denuded aortic rings, consistent with the enhancement of mitochondrial O2·- production only in IEX-1-deficient vascular smooth muscle cells, not in endothelial cells. Excessive production of reactive oxygen species at mitochondria augmented the expression of Gα(i2), suppressing cAMP production in vascular smooth muscle cells. The role of mitochondrial reactive oxygen species in the upregulation of Gα(i2) leading to the development of hypertension was supported by the ability of antioxidant or pertussis toxin to restore the cAMP-dependent vasorelaxation to a normal level and reverse established hypertension in IEX-1 homozygous knockout mice. Our results suggest that hypertension in IEX-1 knockout mice may arise primarily from impaired cAMP signaling induced by overproduction of mitochondrial reactive oxygen species in vascular smooth muscle cells and demonstrate a causal relationship between mitochondrial dysfunction and cAMP-dependent vasorelaxation.

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.

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.

Effects of 3′,4′‐dihydroxyflavonol on vascular contractions of rat aortic rings

1. 3',4'-Dihydroxyflavonol (DiOHF) is an effective vasodilator with anti-oxidant activity. The aim of the present study was to elucidate the effects of DiOHF on vascular contractions. 2. Contractile and relaxation responses were determined in rat endothelium-denuded aortic rings mounted in organ baths. In addition, the phosphorylation of myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17) was determined using western blot analysis. Levels of GTP RhoA, as a marker of RhoA activation, were also measured. 3. Cumulative addition of increasing concentrations of NaF (3.0-12.0 mmol/L) or U46619 (1.0-1000 nmol/L) concentration-dependently increased vascular tension. These responses were inhibited by pretreatment of aortic rings with DiOHF (10, 30 or 100 micromol/L), which dose-dependently decreased vascular contractions induced by 8.0 mmol/L NaF, 30 nmol/L U46619, 0.1 micromol/L phenylephrine and 50 mmol/L KCl. 4. The K(+) channel blockers 4-aminopyridine (3 mmol/L), charybdotoxin (10 nmol/L), apamin (500 nmol/L) and glibenclamide (10 micromol/L) had no effect on vascular relaxation induced by DiOHF (1-30 micromol/L). 5. At 30 micromol/L, DiOHF decreased the activation of RhoA and subsequent phosphorylation of MYPT1, CPI-17 and MLC(20) to almost basal levels. 6. In conclusion, DiOHF decreases vascular contraction at least partly by inhibition of the RhoA/Rho-kinase pathway in rat endothelium-denuded aorta. These results suggest that DiOHF may have therapeutic potential in the treatment of cardiovascular diseases.

Vascular Smooth Muscle Dysfunction and Remodeling Induced by Ginsenoside Rg3, a Bioactive Component of Ginseng

Ginseng, one of most well-known herbal medicines, is widely and indiscreetly used among the patients with cardiovascular disorders, raising concern over abuse of this medicine and unwanted effects. In this study, we investigated the effects of ginsenoside Rg3 (Rg3), an active ingredient of ginseng, on vascular contractility and structural integrity to explore its potential vascular toxicity. In isolated rat aorta, Rg3 suppressed the normal agonist-induced contractile response. This suppression persisted even after a rigorous washout. In the endothelium-denuded aortic ring, impairment of vascular contractility by Rg3 was retained, suggesting that vascular smooth muscle was affected. In primary vascular smooth muscle cells, Rg3 abolished agonist-induced Ca(2+) increase, indicating that Ca(2+) regulation was disrupted. Rg3 suppressed the contraction induced by Bay K8644, an L-type Ca(2+) channel activator, whereas store-operated Ca(2+) channel or intracellular Ca(2+) store-mediated contraction was not affected, suggesting that the L-type Ca(2+) channel was selectively impaired by Rg3. These in vitro results were further confirmed in vivo where Rg3 treatment significantly attenuated the agonist-induced pressor response. More importantly, 4-week repeated treatment with Rg3 in normal animals induced eutrophic outward remodeling in the thoracic aorta, that is, it brought about an increased luminal area without changes in the wall area. These results suggest that Rg3 can induce the vascular smooth muscle dysfunction by disturbing Ca(2+) influx from the L-type Ca(2+) channel, ultimately leading to impaired vascular contractility and structural remodeling.