Relaxation Of Rat Aortic Rings Research Articles

The venom of the Lebanese viper, Montivipera bornmuelleri contains vasoactive compounds

Snake venoms are rich mixtures of polypeptides, which target, among other physiological networks, the cardiovascular system. Snake toxins such as phospholipase A2 (PLA2), natriuretic peptides (NPs), and bradykinin-potentiating peptides (BPP) exert various effects with vascular consequences (hypotension, vasorelaxation…). Some of these toxins have been developped as drugs with antihypertensive properties, such as Captopril™. We have previously shown that the venom of Montivipera bornmuelleri induces the relaxation of rat aortic rings. Here, we aim to identify and characterize the vasoactive compounds of this venom. The venom was fractionated by HPLC. The fractions were assayed on the endothelial cell line, Mile Sven 1 (MS1) and two different cell lines, which stably expressed muscarinic receptors M1 and M3. We screened for compounds able to inhibit acetylcholine-induced intracellular Ca 2+ rise, using the fluorescence probe FURA-2. Wire myography on isolated mesenteric arteries was used. Proteomic analysis was performed to characterize selected fractions. Among 23 fractions of the M. bornmuelleri venom, one fraction was selected for its ability to reduce acetylcholine-induced Ca 2+ rise in MS1 cells. The proteomic analysis led to identify a novel peptidyl toxin, homologous to a vascular endothelial growth factor toxin from Macrovipera lebetina . However, this novel toxin was unable to antagonize acetylcholine-induced Ca 2+ rise in cells expressing muscarinic receptors M1 or M3, suggesting that this peptide acts indirectly on the Ca 2+ signaling pathway in endothelial cells. In myography, the crude venom exhibited a vasorelaxant effect on mesenteric arteries, but the isolated peptide did not. The M. bornmuelleri venom exerts a vasorelaxant effect on mesenteric arteries. We found a novel toxin homologous to a vascular endothelial growth factor snake toxin, which modulates Ca 2+ signaling pathway in endothelial cells.

Antihypertensive potential of cis-[Ru(bpy)2(ImN)(NO)]3+, a ruthenium-based nitric oxide donor

The aim of this study was to investigate the antihypertensive properties of cis-[Ru(bpy)2ImN(NO)]3+ (FOR0811) in normotensive and in Nω–nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. Vasorelaxant effects were analyzed by performing concentration response curve to FOR0811 in rat aortic rings in the absence or presence of 1H-[1,2,4]-oxadiazolo-[4,3,-a]quinoxalin-1-one (ODQ), L-cysteine or hydroxocobalamin. Normotensive and L-NAME-hypertensive rats were treated with FOR0811 and the effects in blood pressure and heart rate variability in the frequency domain (HRV) were followed. FOR0811 induced relaxation in rat aortic rings. Neither endothelium removal nor L-cysteine altered the FOR0811 effects. However, the incubation with ODQ and hydroxocobalamin completely blunted FOR0811 effects. FOR0811 administered intravenously by bolus infusion (0.01–1 mg/bolus) or chronically by using subcutaneous implanted osmotic pumps significantly reduced the mean arterial blood pressure. The effect was long lasting and did not induce reflex tachycardia. FOR0811 prevented both LF and VLF increases in L-NAME hypertensive rats and has antihypertensive properties. This new ruthenium complex compound might be a promising nitric oxide donor to treat cardiovascular diseases.

Sodium nitroprusside encapsulated in Silica nanoparticles as a strategy for safer nitric oxide delivery

Sodium nitroprusside (SNP) has been used as a nitric oxide (NO) donor in medicine for several decades, even before any knowledge of the biological role of NO. This medicine is used in cases of emergency of hypertension due to its fast release of NO causing fast drop in blood pressure. Unfortunately, this compound also releases cyanide (CN-) as a side product. Nanoencapsulated nitroprusside was synthesized by using silica nanoparticles functionalized with primary amine (MPSi-NH2) (the final compound was named APTES). The compound was bioassayed using vascular tissues from Wistar rats, albine guinea-pigs and New Zealand rabbits. All the protocols were approved by the Ethic committee of the Ceará State University under the protocol # 2897836/15. The thoracic aorta, pulmonary artery or corpora cavernosa were carefully removed and mounted in a 5 mL organ bath containing Krebs–Henseleit solution at 37°C, gassed with 95% O2: 5% CO2, attached to isometric transducers coupled to a powerlab data acquisition system. After equilibration, the tissues were precontracted with phenylephrine (PE, 10−6–10−5 M) and after a plateau was achieved increasing concentrations of SNP or APTES (10−10 to 10−5 M) were cumulatively added in intervals of 5 minutes. The relaxant effect was measured as percentage of the maximal contraction induced by phenylephrine. In addition, we also measured the level of phosphorylation of Ser239 of VASP, a useful marker for monitoring cGMP-dependent protein kinase activation and signaling. SNP APTEs relaxed rat aortic rings with similar potency than non-modified SNP with a concentration necessary to induce 50% vasodilation (CE50) of 26 nM [95% confidence interval=20–33 nM] compared to 14 nM [95% confidence interval=7,9–21 nM] for non-modified SNP. However, the maximal vasodilation induced by SNP APTEs (177.4±7.1%) was higher than the maximal relaxation induced by nonmodified SNP (143.8±9.6%). When comparing different vascular beds, i.e., guinea pig thoracic aorta vs. pulmonary arteries vs. rabbit corpora cavernosa, SNP APTEs was shown to have similar potency and efficacy (119.6±5.7% and 130nM vs. 108±6.3% and 275nM vs. 116±4.5% and 98 nM, respectively). Interestingly, SNP increase VASP phosphorylation by 3.8-fold and APTES increased by 4.13-fold (P<0.05 vs. control). Our synthetic strategy to make functionalized silica nanoparticles showed quite efficient to incorporate sodium nitroprusside, where we succeeded to have up to 320 umol g−1. Remarkably, we noticed a minor release of cyanide or even NP, despite full effect of NO was observed during a series of biological assays. Indeed, EC50 and maximal response for relaxation in aorta, pulmonary arteries and Rabbit corpora cavernosa were quite similar. Support or Funding Information CAPES, CNPq and FUNCAP Vasorelaxant Effect of Sodium nitroprusside encapsulated in Silica nanoparticles Original tracings depicting the strong and potent vascular relaxant effect of SNP APTES (panel A). Effect of SNP APTES on the phenylephrine precontracted aortic rings compared with non-modified sodium nitroprusside (Panel B). Panel C depicts the Effect of 1 μM SNP or SNP APTES compared with the addition of vehicle only in the level of phosphorylation of the residues Serine 239 of the protein Vasodilator-stimulated phosphoprotein (VASP) a substrate of activated cGMP-activated protein kinase (PKG) *p<0.05 vs SNP at the same concentration This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biotin improves relaxation of rat aortic rings in combination with antihypertensive drugs

Biotin at pharmacological concentrations has beneficial effects on hypertriglyceridaemia, hyperglycaemia and hypertension. We recently reported in a model of metabolic syndrome that biotin has an antihypertensive effect and decreases arterial contraction. In this study, we investigated the effect of biotin (10 nM) on arterial contraction in rat aortic rings with or without an endothelium induced by phenylephrine (1 × 10−9 to 1 × 10−5 M) or calcium (1 × 10−6 to 1 × 10−2.5 M) in the presence of antihypertensive drugs (BMY 7378, 100 nM; captopril, 1 μM; or nitrendipine, 100 nM). Biotin suppressed phenylephrine-induced vasoconstriction, which is partly modulated via endothelium-independent mechanisms. Biotin incubation with BMY 7378, captopril or nitrendipine improved the vasorelaxant effects of these drugs on aortic rings contracted by phenylephrine or calcium. Moreover, biotin decreased arterial contraction induced by calcium, suggesting the inhibition of extracellular influx and intracellular release of calcium. Our data demonstrate for the first time that biotin enhanced the vasorelaxant effect of some antihypertensive drug classes in rat aortic rings. Therefore, biotin could be used as a therapeutic strategy together with antihypertensive drugs for the treatment of hypertension.

Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension

ObjectiveNon-selective histone deacetylase (HDAC) inhibitors are known to improve hypertension. Here, we investigated the therapeutic effect and regulatory mechanism of the class I HDAC selective inhibitors, MS-275 and RGFP966, in angiotensin (Ang) II-induced hypertensive mice.Methods and resultsMS-275 inhibited the activity of HDAC1, HDAC2, and HDAC3, while RGFP966 weakly inhibited that of HDAC3 in a cell-free system. MS-275 and RGFP966 treatment reduced systolic blood pressure and thickness of the aorta wall in Ang II-induced hypertensive mice. MS-275 treatment reduced aorta collagen deposition, as determined by Masson’s trichrome staining. MS-275 decreased the components of the renin angiotensin system and increased vascular relaxation of rat aortic rings via the nitric oxide (NO) pathway. NO levels reduced by Ang II were restored by MS-275 treatment in vascular smooth muscle cells (VSMCs). However, MS-275 dose (3 mg·kg-1·day-1) was not enough to induce NO production in vivo. In addition, MS-275 did not prevent endothelial nitric oxide synthase (eNOS) uncoupling in the aorta of Ang II-induced mice. Treatment with MS-275 failed to inhibit Ang II-induced expression of NADPH oxidase (Nox)1, Nox2, and p47phox. MS-275 treatment reduced proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and monocyte chemoattractant protein (MCP)-1, as well as adhesion molecules. Histological analysis showed that Ang II-induced macrophage infiltration was reduced by MS-275 and RGFP966 administration.ConclusionsOur results indicate that class I HDAC selective inhibitors may be good therapeutic agents for the treatment of hypertension through the regulation of vascular remodeling and vasoconstriction, as well as inflammation.

VAS2870 Inhibits Histamine-Induced Calcium Signaling and vWF Secretion in Human Umbilical Vein Endothelial Cells.

In this study, we investigated the effects of NAD(P)H oxidase (NOX) inhibitor VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine) on the histamine-induced elevation of free cytoplasmic calcium concentration ([Ca2+]i) and the secretion of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) and on relaxation of rat aorta in response to histamine. At 10 μM concentration, VAS2870 suppressed the [Ca2+]i rise induced by histamine. Inhibition was not competitive, with IC50 3.64 and 3.22 μM at 1 and 100 μM concentrations of histamine, respectively. There was no inhibition of [Ca2+]i elevation by VAS2870 in HUVECs in response to the agonist of type 1 protease-activated receptor SFLLRN. VAS2870 attenuated histamine-induced secretion of vWF and did not inhibit basal secretion. VAS2870 did not change the degree of histamine-induced relaxation of rat aortic rings constricted by norepinephrine. We suggest that NOX inhibitors might be used as a tool for preventing thrombosis induced by histamine release from mast cells without affecting vasorelaxation.

Pomolic acid reduces contractility and modulates excitation-contraction coupling in rat cardiomyocytes

Pomolic acid (PA) isolated from Licania pittieri has hypotensive effects in rats, inhibits human platelet aggregation and elicits endothelium-dependent relaxation in rat aortic rings. The present study was designed to investigate the effects of PA on cardiomyocytes. Trabeculae and enzymatically isolated cardiomyocytes from rats were used to evaluate the concentration-dependent effects of PA on cardiac muscle tension and excitation-contraction coupling (ECC) by recording Ca2+ transients reported with Fluo-3 and Fura-2, as well as L-type Ca2+ currents (LTCC). PA reduced the contractile force in rat cardiac trabeculae with an EC50 = 14.3 ± 2.4 μM. PA also reduced the amplitude of Ca2+ transients in a concentration-dependent manner, with an EC50 = 10.5 ± 1.3 μM, without reducing sarcoplasmic reticulum (SR) Ca2+ loading. PA decreased the half width of the Ca2+ transient by 31.7 ± 3.3% and increased the decay time and decay time constant (τ) by 7.6 ± 2.7% and 75.6 ± 3.7%, respectively, which was associated with increased phospholamban (PLN) phosphorylation. PA also reversibly reduced the macroscopic LTCC in the cardiomyocyte membrane, but did not demonstrate any effects on skeletal muscle ECC. In conclusion, PA reduces LTCC, Ca2+ transients and cardiomyocyte force, which along with its vasorelaxant effects explain its hypotensive properties. Increased PLN phosphorylation protected the SR from Ca2+ depletion. Considering the effects of PA on platelet aggregation and the cardiovascular system, we propose it as a new potential, multitarget cardiovascular agent with a demonstrated safety profile.

Vasorelaxant and Hypotensive Effects of Cheonwangbosimdan in SD and SHR Rats.

Historically, traditional herbal medicines (THMs) have been the conventional treatment strategy in the Korean medical system for treating many diseases. However, THMs have rarely been used to treat hypertension, and moreover few studies have investigated the interaction of blood pressure with the coadministration of synthetic antihypertensives. We aimed to evaluate the vasorelaxant and hypotensive effects of the traditional herbal prescription Cheonwangbosimdan (CWBSD; “Tianwangbuxindan” in Chinese) and the combination of CWBSD with amlodipine. CWBSD was extracted with distilled water at 100°C for 2 h. To investigate vasorelaxant activities, CWBSD with amlodipine (10 μg/ml) was added cumulatively (10–1,000 μg/ml) to isolated rat aortic rings precontracted using phenylephrine or potassium chloride in organ chambers. To investigate hypotensive effects, CWBSD (2,476 mg/kg) was orally administered with or without amlodipine (5 mg/kg) to spontaneously hypertensive rats (SHRs). CWBSD increased the relaxation of rat aortic rings induced by amlodipine (P < 0.01). In vivo, CWBSD coadministration with amlodipine also significantly decreased the blood pressure of SHRs compared to the amlodipine-treated group. These results suggested that CWBSD could be a useful herbal prescription to treat hypertension and we recommend establishing guidelines for the use of herbal medicines in conjunction with antihypertensive drugs, including amlodipine.

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.

The effect of urapidil, an alpha-1 adrenoceptor antagonist and a 5-HT1A agonist, on the vascular tone of the porcine coronary and pulmonary arteries, the rat aorta and the human pulmonary artery

Urapidil (Eupressyl®) an antihypertensive drug acting as an α1 antagonist and a 5-HT1A agonist, may be of special interest in the treatment of hypertension associated with preeclamptic toxaemia and hypoxia-induced pulmonary arterial vasoconstriction. However, the effect of urapidil on vascular tone has been poorly investigated. Vascular reactivity was evaluated using pulmonary and coronary arteries from 36 pigs, aortae from 22 rats and 9 human pulmonary artery samples suspended in organ chambers. Concentration-relaxation curves either to urapidil, 5-HT, or the 5-HT1A receptor agonist 8-OH-DPAT were constructed after pre-contraction of rings. Pig pulmonary and coronary artery rings were contracted with U46619, a thromboxane mimetic, rat aortic rings with either endothelin-1 or phenylephrine, and human pulmonary artery rings with U46619 or phenylephrine. Urapidil markedly inhibited phenylephrine-induced contractions in rat aortic rings with and without endothelium with a more pronounced effect observed in rings without endothelium. Both 5-HT and 8-OH-DPAT failed to induce relaxation in rat aortic rings with an intact endothelium. 5-HT, but not urapidil and 8-OH-DPAT, induced a concentration-dependent relaxation in the porcine coronary and pulmonary artery rings with an intact endothelium (P<0.05). 5-HT and phenylephrine but not urapidil caused concentration-dependent contractions in human pulmonary artery rings. The present findings, while confirming that urapidil is a potent inhibitor of α1-adrenoceptor-induced contraction, do not support the role of 5-HT1A receptor activation in the control of the vascular tone of the different types of arteries tested in response to urapidil. In addition, they indicate that urapidil seems to preferentially target arteries with endothelial dysfunction.

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.

Endothelium-Dependent Vasorelaxant Effects of Dealcoholized Wine Powder of Wild Grape (Vitis coignetiae) in the Rat Thoracic Aorta.

The vasorelaxant effects of dealcoholized wild grape (Vitis coignetiae) wine were investigated with isolated rat thoracic aorta. In our present study, we demonstrate that wild grape wine powder (WGWP) induced relaxation of aortic rings preconstricted with norepinephrine in a dose-dependent manner (at concentrations ranging from 0.1 to 1 mg/mL). The vasorelaxant effect of WGWP was dependent on intact endothelia, which was attenuated by incubation with inhibitors of endothelium-derived relaxing factors, such as NG-nitro-L-arginine (nitric oxide synthase inhibitor), methylene blue (guanylate cyclase inhibitor), and indomethacin (cyclooxygenase inhibitor). Moreover, treatment with WGWP and atropine (muscarinic receptor antagonist) or diphenylhydramine (histamine receptor antagonist) significantly inhibited endothelium-dependent vasorelaxation. Our results suggest that WGWP induces relaxation in rat aortic rings in an endothelium-dependent manner. Results further indicate that this effect occurs via nitric oxide-cGMP pathway and prostacyclin-cAMP pathway through a muscarinic receptor and histamine receptor.

Hydrogen Sulfide: Biogenesis, Physiology, and Pathology

The seminal studies by Kimura's group in the late 1990s [1, 2] ushered in a new era of biological signaling mediated by hydrogen sulfide (H2S). H2S has been described as the third “gasotransmitter” along with nitric oxide (NO) and carbon monoxide (CO; [3]). While none of these molecules actually signal as a gas, their hydrophilicity and lipophilicity enable them to readily traverse intracellular compartments where they serve in a variety of autocrine and paracrine signals capacities.

The vasorelaxant effect of gallic acid involves endothelium-dependent and -independent mechanisms

The mechanisms of action involved in the vasorelaxant effect of gallic acid (GA) were examined in the isolated rat thoracic aorta. GA exerted a relaxant effect in the highest concentrations (0.4–10mM) in both endothelium-intact and endothelium-denuded aortic rings. Pre-incubation with L-NAME, ODQ, calmidazolium, TEA, 4-aminopyridine, and barium chloride significantly reduced the pEC50 values. Moreover, this effect was not modified by indomethacin, wortmannin, PP2, glibenclamide, or paxillin. Pre-incubation of GA (1, 3, and 10mM) in a Ca2+-free Krebs solution attenuated CaCl2-induced contractions and blocked BAY K8644-induced vascular contractions, but it did not inhibit a contraction induced by the release of Ca2+ from the sarcoplasmatic reticulum stores. In addition, a Western blot analysis showed that GA induces phosphorylation of eNOS in rat thoracic aorta. These results suggest that GA induces relaxation in rat aortic rings through an endothelium-dependent pathway, resulting in eNOS phosphorylation and opening potassium channels. Additionally, the relaxant effect by an endothelium-independent pathway involves the blockade of the Ca2+ influx via L-type Ca2+ channels.

Antihypertensive and cardioprotective effects of Cerebralcare granule® on spontaneously hypertensive rats from the perspective of the gaseous triumvirate NO-CO-H2S system.

Cerebralcare granule(®) (CG) has been reported to have hypotensive effect. However, several pathways involved in the mechanism of hypotension are still unclear. This study was designed to verify the antihypertensive effect of CG and to characterize its mechanism of action, especially from the perspective of gasotrasmmiter NO/cGMP, CO/HO and H2S/CSE systems. By using the widely used in vitro model of rat isolated thoracic aortic rings, the vasorelaxant effect of CG were studied. Furthermore, we assessed the chronic hypotensive effect of CG on spontaneously hypertensive rats (SHRs) and further to explore the potential mechanisms of its antihypertensive activity. Data in the present study demonstrated that oral treatment with CG could induce a potent antihypertensive effect. CG could reduce the intima-media thickness (IMT) of thoracic aorta significantly and increase the serum NO and H2S levels. In addition, the present results indicated that CG played a critical protective role against pressure overload-induced cardiac hypertrophy. CG not only inhibited the development of cardiac hypertrophy but also improved ventricular function. In vitro, the results showed that CG induced relaxation in rat aortic rings through an endothelium-dependent pathway mediated by NO/cGMP, CO/HO and H2S/CSE systems. Taken together, the present study demonstrated that CG could induce a potent antihypertensive effect that was partly due to the improvement of endothelial function. Also CG played a critical protective role against pressure overload-induced cardiac hypertrophy. In addition, CG could induce relaxation in rat aortic rings.

Brazilin isolated from the heartwood of Caesalpinia sappan L induces endothelium-dependent and -independent relaxation of rat aortic rings

Brazilin is one of the major constituents of Caesalpinia sappan L with various biological activities. This study sought to investigate the vasorelaxant effect of brazilin on isolated rat thoracic aorta and explore the underlying mechanisms. Endothelium-intact and -denuded aortic rings were prepared from rats. The tension of the preparations was recorded isometrically with a force displacement transducer connected to a polygraph. The phosphorylation levels of ERK1/2 and myosin light chain (MLC) were analyzed using Western blotting assay. Application of brazilin (10-100 μmol/L) dose-dependently relaxed the NE- or high K(+)-induced sustained contraction of endothelium-intact aortic rings (the EC50 was 83.51±5.6 and 79.79±4.57 μmol/L, respectively). The vasorelaxant effect of brazilin was significantly attenuated by endothelium removal or by pre-incubation with L-NAME, methylene blue or indomethacin. In addition, pre-incubation with brazilin dose-dependently attenuated the vasoconstriction induced by KCl, NE or Ang II. Pre-incubation with brazilin also markedly suppressed the high K(+)-induced extracellular Ca(2+) influx and NE-induced intracellular Ca(2+) release in endothelium-denuded aortic rings. Pre-incubation with brazilin dose-dependently inhibited the NE-stimulated phosphorylation of ERK1/2 and MLC in both endothelium-intact and -denuded aortic rings. Brazilin induces relaxation in rat aortic rings via both endothelium-dependent and -independent ways as well as inhibiting NE-stimulated phosphorylation of ERK1/2 and MLC. Brazilin also attenuates vasoconstriction via blocking voltage- and receptor-operated Ca(2+) channels.

Abstract 245: Prevention of Glyceryl Trinitrate Tolerance by Flavonoids

Background and propose: The anti-ischemic effects of glyceryl trinitrate (GTN) are rapidly blunted after continuous application. This phenomenon of GTN tolerance may be the result of an increased oxidative stress and/or a reduced bioactivation of GTN by the enzyme aldehyde dehydrogenase (ALDH). The polyphenolic compounds flavonoids are reported to potentiate vascular relaxation and inhibit oxidation. Therefore, in the present study, we assessed the hypothesis that flavonoids can prevent GTN tolerance. Whether or not this effect of flavonoids is related to their anti-oxidative property or modulation of the activity of ALDH was also examined. Experimental approach: Isolated rat aortic rings were incubated with modified Krebs-Henseleit solution containing GTN (30 μM) in organ chamber at 37°C for 1 hour to induce tolerance. Flavonoids (10 μM of apigenin, luteolin, kaempferol, quercetin, genistin, genistein, narigenin, catechin or epicatechin) or the vehicle (1 mM dimethyl sulfoxide) were added to the aortic rings 30 minutes before GTN treatment. Those rings without incubation with GTN were served as the respective control groups. After removing the treatment solution, rings were contracted with phenylephrine (1 μM) followed by cumulative addition of GTN (0.1 nM to 30 μM). GTN-induced relaxation was calculated as change in isometric tension of aortic rings from the contraction induced by phenylephrine. Vascular ALDH activity was assayed by spectrophotometry as the rate of NAD+ consumption. Key results: GTN-induced relaxation in rat aortic rings was reduced by prior incubation with GTN; this reduction was prevented by apigenin (10 μM), which did not affect the relaxations in the group without prior treatment with GTN. Apigenin (10 μM) also increased the vascular ALDH activity which was reduced by prior GTN incubation. Conclusion: Apigenin appears to have the potential to prevent the development of GTN tolerance, likely by restoring the bioactivation of GTN by ALDH.

Vasorelaxant effects of Cerebralcare Granule® are mediated by NO/cGMP pathway, potassium channel opening and calcium channel blockade in isolated rat thoracic aorta

Ethnopharmacological relevanceCerebralcare Granule (CG), one of the famous classical recipes in traditional Chinese medicine, is developed from the “Decoction of Four Drugs”. It has been used for treatment of cerebrovascular related diseases, such as hypertension. It is well known that vasodilatation plays a very important role in hypertensive. Despite the popular medicinal use of CG, little data was available to its activity and mechanism involved in vasodilatation. Therefore, we aimed to investigate the vasorelaxant effects of CG on isolated rat thoracic aorta so as to assess some of the possible mechanisms. The present study was performed to examine the vasodilative activity of CG and its mechanisms in isolated rat thoracic aorta. Materials and methodsCG was studied on isolated rat thoracic aorta in vitro, including endothelium-intact and endothelium-denuded aortic rings. In present study, specific inhibitors including NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME), cyclooxygenase (COX) inhibitor indomethacin (INDO), non-selective K+ channel inhibitor tetraethylammonium chloride (TEA), Kir channel inhibitor BaCl2, KATP channel inhibitor Glibenclamide (Gli) and cholinergic receptor antagonist atropine were used, they were added 20min before NE contraction and then added CG-induced vasodilation. ResultsRemoval of endothelium or pretreatment of aortic rings (intact endothelium) with l-NAME (0.1mM) or INDO (0.01mM) significantly blocked the CG induced relaxation. Pretreatment with the non-selective K+ channel inhibitor TEA (1mM), or the Kir channel inhibitor BaCl2 (0.1mM), neither of them had no influence on the CG-induced response (p>0.05). However, pretreatment with the KATP channel inhibitor Gli (0.01mM) produced significant inhibition on the CG-induced response (p<0.01). Besides, CG also inhibited the contraction triggered by NE in endothelium-denuded rings in Ca2+-free medium. CG (0.4, 0.8 and 3.2mg/mL) produced rightward parallel displacement of CaCl2 curves and reduced the maximum contraction induced by 30mM CaCl2 to 31.1±9.3%, 18.8±6.9% and 9.4±4.5%, respectively. The relaxation, induced by CG on endothelium-intact rat aortic rings pre-contracted with NE, was significantly attenuated in the presence of atropine (EC50=3.7mg/mL, p<0.01). ConclusionsOur results suggest that CG induces relaxation in rat aortic rings through an endothelium-dependent pathway mediated by NO/cGMP pathway and an endothelium-independent pathway involving blockade of Ca2+ channels, inhibition of Ca2+ mobilization from intracellular stores, opening of KATP channel. In addition, the muscarinic receptor stimulation is also one of the vasorelaxant mechanisms.

Potent inhibition of nitroglycerin bioactivation by diphenyleneiodonium (DIP)

Background Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMPmediated vasodilation [1]. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI) [2], a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2.

Potent Inhibition of Aldehyde Dehydrogenase-2 by Diphenyleneiodonium: Focus on Nitroglycerin Bioactivation

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI), a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2. DPI (0.3 µM) inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. Denitration and bioactivation of 1-2 µM GTN, assayed as 1,2-glycerol dinitrate formation and activation of purified sGC, respectively, were inhibited by DPI with a half-maximally active concentration of about 0.2 µM in a GTN-competitive manner. Molecular modeling indicated that DPI binds to the catalytic site of ALDH2, and this was confirmed by experiments showing substrate-competitive inhibition of the dehydrogenase and esterase activities of the enzyme. Our data identify ALDH2 as highly sensitive target of DPI and explain inhibition of GTN-induced relaxation by this drug observed previously. In addition, the data provide new evidence for the essential role of ALDH2 in GTN bioactivation and may have implications to other fields of ALDH2 research, such as hepatic ethanol metabolism and cardiac ischemia/reperfusion injury.