Endothelium-denuded Aortic Rings Research Articles

Hydroalcoholic extract and pure compounds from Senecio nutans Sch. Bip (Compositae) induce vasodilation in rat aorta through endothelium-dependent and independent mechanisms

Ethnopharmacological relevanceSenecio nutans Sch. Bip. (Compositae) is an endemic plant of South America, and is used in herbal medicine in Andean communities for treating acute mountain sickness. Currently, the direct effects of hydroalcoholic extract of S. nutans (HAE S. nutans) or its isolated compounds on the vascular system are not well described. The aim of this study was to determine the effects and mechanism of action of S. nutans on vascular function in healthy rats. Material and methodsSeven compounds were isolated from the HAE S. nutans, and their structures were characterized using spectroscopic techniques as 1D and 2D NMR, and mass spectrometry. Vascular reactivity experiments were carried out in rat aorta. S. nutans-dependent vasodilation and phenylephrine-dependent contraction were measured in endothelium-intact and endothelium-denuded aortic rings of male rats. ResultsSeven pure compounds were isolate from HAE S. nutans, but two pure compounds showed significant vasodilation in rat aorta: 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone (compound E) and 5-acetyl-6-hydroxy-2-isopropenyl-2,3-dihydrobenzofurane (compound G). Although HAE S. nutans induced vasodilation in absence of endothelium, the vasodilation in intact aorta, via NO, was higher. HAE S. nutans reduced calcium-dependent contraction in endothelium-intact, but not in endothelium-denuded aortic rings. ConclusionHAE S. nutans and its isolated compounds caused vasodilation in rat aorta in absence of endothelium, suggesting its vasodilator properties is endothelium-dependent (NO) and or independent, and may involve a modulation of the calcium channels. This result is of clinical interest as potential therapy control of blood pressure.

NADPH Oxidase Plays a Role on Ethanol-Induced Hypertension and Reactive Oxygen Species Generation in the Vasculature.

Investigate the role of NADPH oxidase on ethanol-induced hypertension and vascular oxidative stress. Male Wistar rats were treated with ethanol (20% v/v). Apocynin (10 mg/kg/day, i.p.) prevented ethanol-induced hypertension. The increased contractility of endothelium-intact and endothelium-denuded aortic rings from ethanol-treated rats to phenylephrine was prevented by apocynin. Ethanol consumption increased superoxide anion (O2 (-)) generation and lipid peroxidation and apocynin prevented these responses. The decrease on plasma and vascular nitrate/nitrite (NOx) levels induced by ethanol was not prevented by apocynin. Treatment with ethanol did not affect aortic levels of hydrogen peroxide (H2O2) or reduced glutathione (GSH). Ethanol did not alter the activities of xanthine oxidase (XO), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Ethanol increased the expression of Nox1, PKCδ, nNOS, SAPK/JNK and SOD2 in the rat aorta and apocynin prevented these responses. No difference on aortic expression of Nox2, Nox4, p47phox, Nox organizer 1 (Noxo1), eNOS and iNOS was detected after treatment with ethanol. Ethanol treatment did not alter the phosphorylation of SAPK/JNK, p38MAPK, c-Src, Rac1 or PKCδ. The major new finding of our study is that the increased vascular generation of reactive oxygen species (ROS) induced by ethanol is related to increased vascular Nox1/NADPH oxidase expression. This mechanism is involved in vascular dysfunction and hypertension induced by ethanol. Additionally, we conclude that ethanol consumption induces the expression of different proteins that regulate vascular contraction and growth and that NADPH oxidase-derived ROS play a role in such response. The key findings of our study are that ethanol-induced hypertension is mediated by NADPH oxidase. Moreover, increased vascular Nox1 expression is related to the generation of reactive oxygen species (ROS) by ethanol. Finally, ROS induced by ethanol increase the expression of the regulatory vascular proteins.

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.

Effects of Acidification and Alkalinization on the Lipid Emulsion-Mediated Reversal of Toxic Dose Levobupivacaine-Induced Vasodilation in the Isolated Rat Aorta.

The goal of this in vitro study was to examine the effects of pre-acidification and pre-akalinization on the lipid emulsion-mediated reversal of toxic dose levobupivacaine-induced vasodilation in isolated rat aorta. Isolated aortic rings with and without the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) were exposed to four types of Krebs solution (pH 7.0, 7.2, 7.4, and 7.6), followed by the addition of 60 mM potassium chloride. When the toxic dose of levobupivacaine (3 × 10-4 M) produced a stable and sustained vasodilation in the isolated aortic rings that were precontracted with 60 mM potassium chloride, increasing lipid emulsion concentrations (SMOFlipid®: 0.24, 0.48, 0.95 and 1.39%) were added to generate concentration-response curves. The effects of mild pre-acidification alone and mild pre-acidification in combination with a lipid emulsion on endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells were investigated by Western blotting. Mild pre-acidification caused by the pH 7.2 Krebs solution enhanced the lipid emulsion-mediated reversal of levobupivacaine-induced vasodilation in isolated endothelium-intact aortic rings, whereas mild pre-acidification caused by the pH 7.2 Krebs solution did not significantly alter the lipid emulsion-mediated reversal of the levobupivacaine-induced vasodilation in isolated endothelium-denuded aortic rings or endothelium-intact aortic rings with L-NAME. A lipid emulsion attenuated the increased eNOS phosphorylation induced by the pH 7.2 Krebs solution. Taken together, these results suggest that mild pre-acidification enhances the lipid emulsion-mediated reversal of toxic dose levobupivacaine-induced vasodilation in the endothelium-intact aorta via the inhibition of nitric oxide.

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.

Investigation of the mechanisms of Angelica dahurica root extract-induced vasorelaxation in isolated rat aortic rings.

BackgroundThe root of Angelica dahurica Bentham et Hooker (Umbelliferae) has been used as a traditional medicine for colds, headache, dizziness, toothache, supraorbital pain, nasal congestion, acne, ulcer, carbuncle, and rheumatism in China, Japan, and Korea. Interestingly, it has been used in the treatment of vascular diseases including hypertension. The aim of this study was to provide pharmacological evidence for the anti-hypertensive effect of A. dahurica by investigating the mechanism underlying its vasorelaxant effect.MethodsThe vasorelaxant effects of a 70 % methanol extract of the A. dahurica root (ADE) on rat thoracic aorta and its underlying mechanisms were assessed. Isolated rat aortic rings were suspended in organ chambers containing 10 ml Krebs-Henseleit (K-H) solution and placed between 2 tungsten stirrups and connected to an isometric force transducer. Changes in tension were recorded via isometric transducers connected to a data acquisition system.ResultsADE causes concentration-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings precontracted with phenylephrine (PE; 1 μM) or potassium (KCl; 60 mM) in K-H solution. And pre-treatment with ADE (1 mg/ml) inhibited calcium-induced vasocontraction of aortic rings induced by PE or KCl. However, ADE pre-treatment did not affect the contraction induced by PE or caffeine in Ca2+-free K-H solution.ConclusionsThese results suggested that the ADE has vasorelaxant effect and the vasorelaxant activity is mediated by endothelium-independent pathway that includes the blockade of extracellular calcium influx through the receptor-operated Ca2+ channel and voltage-dependent calcium channel pathways.

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.

Mechanisms underlying the antihypertensive effect of Alstonia scholaris

Ethnopharmacological relevanceAlstonia scholaris has a long history of use in the Ayurveda traditional treatment of various ailments including hypertension. We have reported the blood pressure lowering activity of the extract of A. scholaris. The following research aim to delineate the pharmacological mechanism involve in the antihypertensive action. Materials and methodVasorelaxant effect of the n-butanol fraction of A. scholaris (NBF-ASME) was evaluated on rat aorta pre-contracted with phenyelphrine (PE, 1µM). Aortic rings preparation were pre-incubated with various antagonists like 1H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ 10μM), methylene blue (MB 10μM), Nω-nitro-L-arginine methyl ester hydrochloride (l-NAME 10μM), atropine (10μM), indomethacin (1μM), ML-9 and various K+ channel blockers such as glibenclamide (10μM) and tetraethyl ammonium (TEA 10μM) for mechanism study. ResultThe results showed that pre-incubation of aortic rings with the extract (0.5, 1 and 2mg/mL) significantly inhibit the contractile response of the rings to phenylephrine-induced contraction (p<0.05–0.001). Removal of endothelium, incubation with l-NAME, indomethacin, atropine and propranolol did not significantly affect the relaxation effect of NBF-ASME. Furthermore, the K+ channel blockers, TEA and glibenclamide showed no inhibitory effect. However, aortic rings pretreated with ODQ and ML-9 showed a significant suppression of the relaxation curve of NBF-ASME (p<0.01–0.001). In Ca2+-free solution, NBF-ASME inhibits the release of intracellular Ca2+ from the sarcoplasmic reticulum. NBF-ASME also inhibits calcium chloride (CaCl2)-induced contraction in endothelium-denuded aortic rings. ConclusionThe results from this study suggests that A. scholaris exerts vasodilation via calcium channels blockade, direct activation of soluble guanylate cyclase and possibly by also inhibiting the formation of inositol 1, 4, 5-triphosphate.

Vasorelaxant effects of Shunaoxin pill are mediated by NO/cGMP pathway, HO/CO pathway and calcium channel blockade in isolated rat thoracic aorta

Ethnopharmacological relevanceShunaoxin pill (SNX), one of the famous classical recipes in traditional Chinese medicine, is developed from the “Decoction of Xionggui”. It has been used for treatment of cerebrovascular related diseases. It is well known that vasodilatation plays a very important role in cerebrovascular diseases. The effect of SNX on vasorelaxant activity has not yet been explored. Therefore, we aimed to investigate the vasorelaxant effects of SNX on isolated rat thoracic aorta so as to assess some of the possible mechanisms. We also investigate the gasotransmitter signaling pathway involved which has been rarely reported in isolated rat thoracic aorta before. Aim of the studyThe present study was performed to examine the vasodilative activity of SNX and its mechanisms in isolated rat thoracic aorta. Materials and methodsSNX was studied on isolated rat thoracic aorta in vitro, including endothelium-intact and endothelium-denuded aortic rings. In present study, specific inhibitors including soluble guanylate cyclase (sGC) inhibitor 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), cyclooxygenase (COX) inhibitor indomethacin (INDO), NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME), heme oxygenase-1 (HO-1) inhibitor zinc-protoporphyrin (ZnPP), cystathionine γ-lyase (CSE) inhibitor DL-Propargylglycine (PAG), non-selective K+ channel inhibitor tetraethylammonium chloride (TEA), KV channel inhibitor 4-Aminopyridine (4-AP), and KATP channel inhibitor Glibenclamide (Gli) were used, they were added 20min before NE contraction and then added SNX to induce vasodilation. ResultsRemoval of endothelium or pretreatment of aortic rings (intact endothelium) with L-NAME, ODQ or ZnPP significantly blocked SNX-induced relaxation. Pretreatment with the non-selective K+ channel inhibitor TEA, KV channel inhibitor 4-AP or the KATP channel inhibitor Gli, none of them had influences on the SNX-induced response (p>0.05). Besides, SNX inhibited the contraction triggered by NE in endothelium-denuded rings in Ca2+-free medium. SNX also produced rightward parallel displacement of CaCl2 curves. ConclusionsThese results suggest that SNX can induce less endothelium-dependent and more endothelium-independent vascular relaxation. The NO/cGMP and HO/CO pathways, blockade of Ca2+ channels are inhibition of IP3R mediated Ca2+ mobilization from intracellular stores, are likely involved in this relaxation. Furthermore, the underlying mechanisms of combined compositions in SNX await further investigations.

Endothelium-Independent Vasorelaxant Effect of Ligusticum jeholense Root and Rhizoma on Rat Thoracic Aorta.

Ligusticum jeholense has been used as the traditional medicine ‘Go-Bon’ (Chinese name, Gao-ben) in China and Korea. Considering the increased use of medicinal herbs to treat hypertension, in this study, we aimed to investigate the mechanisms of the vasorelaxation effect caused by L. jeholense. We tested the methanol (MeOH) extract of L. jeholense root and rhizoma for vasorelaxant effects; while using an isolated organ-chamber technique, L. jeholense extract (LJE) induced relaxation in the rat aortic rings by stimulating vascular endothelial and smooth muscle cells. LJE showed concentration-dependent relaxant effects on endothelium-intact and endothelium-denuded aortic rings pre-contracted with both phenylephrine (PE) and potassium chloride (KCl) in Krebs-Henseleit (KH) buffer. The vasorelaxant effect of LJE was partly attenuated by pre-treatment with glibenclamide or 4-aminopyridine (4-AP) as K+ channel blockers. Moreover, LJE showed concentration-dependent inhibition of vasoconstriction by Ca2+ supplementation in the aortic rings that were pre-contracted with PE or KCl in Ca2+-free KH buffer. In addition, a combination of LJE and nifedipine, pre-incubated further, decreased PE-induced contractions. The results suggested that LJE-induced vasorelaxation were related to blocking K+ channels and inhibiting entry of extracellular Ca2+ via receptor-operative Ca2+ channels (ROCCs) or voltage-dependent Ca2+ channels (VDCCs).

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.

Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas

Elsholtzia splendens (ES) is, rich in flavonoids, used to repair copper contaminated soil in China, which has been reported to benefit cardiovascular systems as folk medicine. However, few direct evidences have been found to clarify the vasorelaxation effect of total flavonoids of ES (TFES). The vasoactive effect of TFES and its underlying mechanisms in rat thoracic aortas were investigated using the organ bath system. TFES (5–200mg/L) caused a concentration-dependent vasorelaxation in endothelium-intact rings, which was not abolished but significantly reduced by the removal of endothelium. The nitric oxide synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (100μM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (30μM) significantly blocked the endothelium-dependent vasorelaxation of TFES. Meanwhile, NOS activity in endothelium-intact aortas was concentration-dependently elevated by TFES. However, indomethacin (10μM) did not affect TFES-induced vasorelaxation. Endothelium-independent vasorelaxation of TFES was significantly attenuated by KATP channel blocker glibenclamide. The accumulative Ca2+-induced contraction in endothelium-denuded aortic rings primed with KCl or phenylephrine was markedly weakened by TFES. These results revealed that the NOS/NO/cGMP pathway is likely involved in the endothelium-dependent vasorelaxation induced by TFES, while activating KATP channel, inhibiting intracellular Ca2+ release, blocking Ca2+ channels and decreasing Ca2+ influx into vascular smooth muscle cells might contribute to the endothelium-independent vasorelaxation conferred by TFES.

Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas.

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.

Vasorelaxation induced by a new naphthoquinone-oxime is mediated by NO-sGC-cGMP pathway.

It has been established that oximes cause endothelium-independent relaxation in blood vessels. In the present study, the cardiovascular effects of the new oxime 3-hydroxy-4–(hydroxyimino)-2-(3-methylbut-2-enylnaphtalen-1(4H)-one (OximeS1) derived from lapachol were evaluated. In normotensive rats, administration of Oxime S1 (10, 15, 20 and 30 mg/Kg, i.v.) produced dose-dependent reduction in blood pressure. In isolated aorta and superior mesenteric artery rings, Oxime S1 induced endothelium-independent and concentration-dependent relaxations (10−8 M to 10−4 M). In addition, Oxime S1-induced vasorelaxations were attenuated by hydroxocobalamin or methylene blue in aorta and by PTIO or ODQ in mesenteric artery rings, suggesting a role for the nitric oxide (NO) pathway. Additionally, Oxime S1 (30 and 100 µM) significantly increased NO concentrations (13.9 ± 1.6 nM and 17.9 ± 4.1 nM, respectively) measured by nitric oxide microsensors. Furthermore, pre-contraction with KCl (80 mM) prevented Oxime S1-derived vasorelaxation in endothelium-denuded aortic rings. Of note, combined treatment with potassium channel inhibitors also reduced Oxime S1-mediated vasorelaxation suggesting a role for potassium channels, more precisely Kir, Kv and KATP channels. We observed the involvement of BKCa channels in Oxime S1-induced relaxation in mesenteric artery rings. In conclusion, these data suggest that the Oxime S1 induces hypotension and vasorelaxation via NO pathway by activating soluble guanylate cyclase (sGC) and K+ channels.

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.

Thyroid hormone exacerbates vasoconstriction in insulin resistance: The role of ONOO−

Insulin resistance has been proposed to play a pivotal role in vasoconstriction due to increased oxidative stress. Hyperthyroidism would amplify cardiovascular disease risk in diabetic patients, though thyroid hormone advance vascular relaxation and reduce vascular contraction by virtue of NO production in vascular smooth muscle cells (VSMCs). Thus, we aimed to investigate the vascular tone and its underlying mechanism in insulin resistance accompanied by hyperthyroidism. Vascular reactivity studies showed that endothelium-denuded thoracic aortic rings from rats fed with high-fat high-sucrose (HFHS) diet and L-T4 (HFHS+L-T4) exhibited a stronger contractile response to noradrenaline than HFHS rats, which was reversed by L-NAME and GSH. Furthermore, rat thoracic aortic smooth muscle cells (A10) simultaneously stimulated with high glucose insulin (high Glc/Ins) and T3 demonstrated lower NO, superoxide anion (O2−) levels, and higher iNOS, nitrite (NO2−), peroxynitrite (ONOO−) levels than that treated with T3 only. Excessive ONOO− markedly aggravated oxidative stress. Thus, we hypothesized that the elevated concentration of ONOO− which is generated by NO and O2− could be critically instrumental in the progression of vasoconstriction by increasing oxidative stress.

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.

Dysfunction of Vascular Smooth Muscle and Vascular Remodeling by Simvastatin

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are widely prescribed for hypercholesterolemia. With the increasing use of statins, numerous reports demonstrated that statins can cause damage to skeletal muscles. However, the toxicities of statins on vascular smooth muscle, which are essential to cardiovascular homeostasis, have not been previously described. Here, we examined the effects of simvastatin on the contractile function and the integrity of vascular smooth muscle in isolated rat thoracic aortic rings, primary cultured vascular smooth muscle cells (VSMCs) in vitro and rats in vivo. In aortic rings, simvastatin suppressed the normal agonist-induced contractile responses in time- and concentration-dependent manners (0.86 g ± 0.11 at 10 μM simvastatin for 24 h compared with 1.89 g ± 0.11 at control). The suppression persisted in the endothelium-denuded aortic rings and was irreversible even after wash-out of simvastatin. Simvastatin suppressed the contraction induced by Bay K8644, an activator of voltage-operated Ca²⁺ channel (VOCC) in rat aortic rings and abolished agonist-induced intracellular Ca²⁺ increase in VSMCs. The simvastatin-induced contractile dysfunction was reversed by the supplementation of mevalonate and geranylgeranylpyrophosphate, precursors for protein isoprenylation. Consistently, activation of RhoA, a representative isoprenylated protein, was disrupted by simvastatin in VSMCs and RhoA-mediated phosphorylation of MYPT1 and CPI-17, and tonic tension were also suppressed. Notably, prolonged treatment of simvastatin up to 48 h induced apoptosis of vascular smooth muscle in aortic rings. Most importantly, simvastatin treatment in vivo significantly attenuated the agonist-induced vasoconstriction in rats ex vivo and induced a decrease in luminal area of the vascular wall. Collectively, these results demonstrate that simvastatin can impair the normal vascular contractility by disturbing Ca²⁺ influx and RhoA activity, ultimately leading to apoptosis and structural remodeling.