Endothelium-intact Rat Aortic Rings Research Articles

Effect of asiaticoside on systolic blood pressure and relaxation of isolated thoracic aorta of rats

To investigate the effect of asiaticoside on blood pressure and relaxation of thoracic aorta in rats and explore the underlying mechanism. SD rats treated with 50 and 100 mg/kg asiaticoside by daily gavage for 2 weeks were monitored for systolic blood pressure changes, and histological changes of the thoracic aorta were evaluated using HE staining. In isolated rat endothelium-intact and endothelium-denuded thoracic aorta rings, the effects of asiaticoside on relaxation of the aortic rings were tested at baseline and following norepinephrine (NE)- and KCl-induced constriction. The vascular relaxation effect of asiaticoside was further observed in NE-stimulated endothelium-intact rat aortic rings pretreated with L-nitroarginine methyl ester, indomethacin, zinc protoporphyrin Ⅸ, tetraethyl ammonium chloride, glibenclamide, barium chloride, Iberiotoxin, 4-aminopyridine, or TASK-1-IN-1. The aortic rings were treated with KCl and NE followed by increasing concentrations of CaCl2 to investigate the effect of asiaticoside on vasoconstriction induced by external calcium influx and internal calcium release. Asiaticoside at 50 and 100 mg/kg significantly lowered systolic blood pressure in rats without affecting the thoracic aorta histomorphology. While not obviously affecting resting aortic rings with intact endothelium, asiaticoside at 100 mg/kg induced significant relaxation of the rings constricted by KCl and NE, but its effects differed between endothelium-intact and endothelium-denuded rings. In endothelium-intact aortic rings pretreated with indomethacin, ZnPP Ⅸ, barium chloride, glyburide, TASK-1-IN-1 and 4-aminopyridine, asiaticoside did not produce significant effect on NE-induced vasoconstriction, and tetraethylammonium, Iberiotoxin and L-nitroarginine methyl ester all inhibited the relaxation effect of asiaticoside. In KCland NE-treated rings, asiaticoside obviously inhibited CaCl2-induced vascular contraction. Asiaticoside induces thoracic aorta relaxation by mediating high-conductance calcium-activated potassium channel opening, promoting nitric oxide release from endothelial cells and regulating Ca2+ influx and outflow, thereby reducing systolic blood pressure in rats.

Endothelium-dependent vasorelaxation effects of F5 fraction of Crinum amabile chloroform extract

BackgroundVascular dysfunction can lead to many health problems including hypertension and heart disease. The complexities of vascular dysfunction and vascular disorder-related diseases have prompted the search for many new biologically active compounds in the efforts of resolving the problems. Previous studies have shown that Amaryllidaceae alkaloids exert multiple biological activities, including the vasorelaxation effect. Crinum amabile, which is a family member of Amaryllidaceae, is believed to possess a promising pharmacological activity as a vasorelaxant.MethodThe vasorelaxation activities of Crinum amabile extracts and fractions were determined using in vitro models of phenylephrine pre-contracted intact and denuded rat aortic rings. The mechanistic pathways of vasorelaxation were investigated by pre-treatment of endothelium-intact rat aortic rings with L-NG Nitro Arginine Methyl Ester (L-NAME), methylene blue (MB), indomethacin, atropine and propranolol, respectively.ResultsThe results showed that chloroform extract (CE) of Crinum amabile exhibited the highest vasorelaxation activity, and further fractionation of CE found that its F5 fraction exerted the strongest activity. An in-depth study on the mechanistic pathway revealed that the endothelium-dependent vasorelaxation induced by F5 fraction was primarily achieved through stimulation of prostaglandin (PGI2) production and partially associated with NO/cGMP activation pathway.ConclusionFindings from this study suggest that Crinum amabile can serve as a promising candidate for the discovery and development of the new vasorelaxant drug.

VASORELAXANT MECHANISM(S) OF CLERODENDRUM VOLUBILE ETHANOL LEAF EXTRACT IN NORMAL AND DOXORUBICIN-TREATED ENDOTHELIUM INTACT AORTIC RINGS

Objectives: Doxorubicin (DOX) is a highly effective antibiotics anthracycline cytotoxic agent with a broad spectrum of activity in the treatment of solid and hematological malignancies. However, DOX is notorious for inducing cardiotoxicity and vascular dysfunction as its common off-target side effects. This study evaluated the possible vasorelaxant activity and mechanism(s) of action of Clerodendrum volubile ethanol leaf extract (CVE) in normal and DOX-pretreated endothelium intact aortic rings in Physiological Salt Solution (PSS) in vitro. Methods: The responses were recorded isometrically by an organ bath connected to Data Capsule Acquisition System. Effects of CVE on phenylephrine-precontracted endothelium intact rat aortic rings and the influence of the respective blockers for adrenergic, cholinergic, calcium channel, and prostacyclin receptors were investigated to unveil the possible underlying vasorelaxant mechanism(s) of CVE. Results: Our findings showed that CVE significantly induced vasorelaxation in phenylephrine hydrochloride (PE) and KCl precontracted endothelium intact aortic rings in a concentration-dependent manner. Furthermore, the CVE-induced vasorelaxation in PE- and KCl-precontracted aortic rings were inhibited by pre-incubation with atropine and indomethacin indicating that the vasorelaxant effect of CVE was profoundly mediated through cholinergic and prostacyclin mechanisms. Conclusion: Overall, results of this study report for the first time the vasorelaxant effect of CVE in isolated endothelium-intact doxorubicin-treated aortic rings of normotensive rats which was probably cholinergic and prostacyclin-mediated. Thus, results of this study provide further insight into the cardioprotective mechanism of CVE in doxorubicin-induced cardiotoxicity beyond the antioxidant and anti-apoptosis mechanisms that have been previously reported.

Standardized root extract of Withania somnifera and Withanolide A exert moderate vasorelaxant effect in the rat aortic rings by enhancing nitric oxide generation

Ethno-pharmacological relevanceWithania somnifera (L.) Dunal, commonly known as Ashwagandha, belongs to the family Solanaceae. In Ayurveda, Ashwagandha has been defined as one of the most important herb and is considered to be the best adaptogen. It is also an excellent rejuvenator, a general health tonic and cure for various disorders such as cerebrovascular, insomnia, asthma, ulcers, etc. Steroidal lactones (Withanolides: Withanolide A, Withaferin A, Withanolide D, Withanone, etc) isolated from this plant, possess promising medicinal properties such as anti-inflammatory, immune-stimulatory etc. Standardized root extract of the plant NMITLI-118R (NM) was prepared at CSIR-CIMAP, and was investigated for various biological activities at CSIR-CDRI. Among the notable medicinal properties, NM exhibited excellent neuroprotective activity in the middle cerebral artery occlusion (MCAO) rat model. Aim of the studyEndothelial dysfunction is the primary event in the cerebrovascular or cardiovascular disorders, present study was thus undertaken to evaluate vasoprotective potential of NM and its biomarker compound Withanolide A (WA) using rat aortic rings and EA.hy926 endothelial cells. Material and methodsTransverse aortic rings of 10 weeks old Wistar rats were used to evaluate effect of NM and WA on the vasoreactivity. While, mechanism of NM and WA mediated vasorelaxant was investigated in Ea.hy926 cell line by measuring NO generation, nitrite content, Serine 1177 phosphorylation of eNOS, reduced/oxidized biopterin levels and expression of endothelial nitric oxide synthase (eNOS) mRNA and protein. ResultsFingerprinting of NM using HPLC identified presence of WA in the extract. NM as well as WA exerted moderate vasorelaxant effect in the endothelium intact rat aortic rings which was lesser than acetylcholine (ACh). NM and WA augmented ACh induced relaxation in the rat aortic rings. NM and WA dependent vasorelaxation was blocked by N-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ), indicating role of NO/cGMP. Further Ea.hy926 cells treated with NM and WA showed accumulation of nitrite content, enhanced NO levels, eNOS expression and eNOS phosphorylation (Serine 1177). ConclusionAltogether NM and WA dependent improvement in the NO availability seems to be mediated by the enhanced eNOS phosphorylation. WA, seems to be one of the active constituent of NM, and presence of other vasoactive substances cannot be ruled out. The data obtained imply that the vasorelaxant property of NM is beneficial for its neuroprotective potential.

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

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

Comparative assessment of quality characteristics of Chungkookjang made from soybean seeds differing in oleic acid concentration

Chungkookjang is a beneficial fermented soybean food with anticancer, anti-inflammatory, and antioxidant activities. High oleic acid (HO)-containing diets are known to help in preventing cardiovascular disease (CVD). In the present study, we assessed functional quality characteristics of Chungkookjang made from soybean seeds differing in oleic acid concentration. Chungkookjang made from HO and normal oleic acid (NO) soybeans did not differ significantly in moisture content, pH, viable cell number, protease activity, amino type nitrogen, and polyphenol activity. However, the diphenylpicrylhydrazyl (DPPH)-scavenging activity and flavonoid contents of Chungkookjang made from HO soybeans were lower than those from NO soybeans. The oleic acid concentrations were not significantly altered by fermentation. Additionally, Chungkookjang from the HO soybean cultivar ‘Hosim’ affected the vascular relaxation of endothelium-intact rat aortic rings at concentrations as low as 0.03 mg/mL. The results showed that Chungkookjang from HO soybeans possesses characteristics that might be potentially helpful in reducing the risk of CVD.

In vitro vasorelaxation mechanisms of Isoapiole extracted from Lemonfragrant Angelica Root on rat thoracic aorta

Ethnopharmcological relevanceLemonfragrant Angelica (Ostericum citriodorum (Hance) C. Q. Yuan & Shan) is a traditional Chinese herb for treatment of angina pectoris, stomach pain and abdominal pain. However, its active components and mechanisms of action were not well understood. Aims of the studyIn this study, we investigated whether the isoapiole extracted from Lemonfragrant Angelica Root (LAR) could directly stimulate the production of nitric oxide (NO) in vascular endothelial cells (VECs) and lead to the vascular relaxation Materials and methodsVascular activity experiments were performed in aortic rings isolated from Wistar rats using standard muscle bath procedures. Isoapiole was added with different concentrations (0.75, 2.5, 5μg/mL), and vessel relaxation of rat aortic rings pre-contracted with norepinephrine (NE) or potassium chloride was recorded. NO release from aortic rings exposed to isoapiole (5μg/mL) was measured by Griess method. The endothelial nitric oxide synthase (eNOS) expression in primary human umbilical vein endothelial cells (HUVECs) incubated with isoapiole was determined using Western blot and microplate reader assay. Classical receptor antagonists, channel and enzymatic inhibitors were used to check the mechanisms involved. ResultsIsoapiole (0.75, 2.5, 5μg/mL) inhibited norepinephrine-induced contraction in endothelium-intact rat aortic rings. However, a very weak relaxation of aortic rings was obtained in endothelium-denuded preparations. Isoapiole (0.75, 2.5, 5μg/mL) did not have vascular relaxative effect on neither endothelium-intact nor endothelium-denuded aortas pre-contracted with KCl (60mmol/L). The vasorelaxation effect of isoapiole on rat aortic rings was attenuated by the eNOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). This result suggested that suggested that the isoapiole action was at least partially mediated by promoting eNOS expression. It was further found that isoapiole (5μg/mL) increased NO production in isolated rat thoracic aorta rings. Isoapiole increased eNOS expression leading to NO production in HUVECs. ConclusionIsoapiole stimulates NO production in the endothelium, leading to vascular dilatation.

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.

Insights on Possible Mechanism of Action of Vasorelaxant Activity Exhibited by Fungal Malbrancheamides

The alkaloids 1 – 3 biosynthesized by the coprophilous fungus Malbranchea aurantiaca displayed a significant vasorelaxant effect on endothelium-intact rat aortic rings pre-contracted with norepinephrine. This effect was not reduced when indomethacin, atropine or tetraethylamonium were added to the organ bath. Nevertheless, it was reduced in the presence of L-NAME, an inhibitor of the enzyme Nitric Oxide Synthase (NOS), suggesting that the pharmacological action was mediated by a nitrergic mechanism, as predicted by docking studies, were 1 showed a strong affinity by NOS.

Vasomodulatory effect of novel peroxovanadate compounds on rat aorta: Role of rho kinase and nitric oxide/cGMP pathway

The present study was undertaken to assess the role of reactive oxygen species (ROS) in rat aortic ring vasoreactivity and integrity by using various peroxovanadate (pV) compounds. All the pV compounds (1nM–300μM) used in the present study exerted concentration-dependent contractions on endothelium intact rat aortic rings. All compounds with an exception of DPV-asparagine (DPV-asn) significantly altered vascular integrity as shown by diminished KCl responses. Phenylephrine (PE)-mediated contractions (3nM–300μM) were unaltered in the presence of these compounds. Acetylcholine (Ach)-mediated relaxation in PE (1μM) pre-contracted rings was significantly reduced in presence of diperoxovanadate (DPV), poly (sodium styrene sulfonate-co-maleate)-pV (PSS-CoM-pV) and poly (sodium styrene 4-sulfonate)-pV (PSS-pV). However, no significant change in Ach-mediated responses was observed in the presence of poly (acrylate)-pV (PAA-pV) and DPV-asn. DPV-asn was thus chosen to further elucidate mechanism involved in peroxide mediated modulation of vasoreactivity. DPV-asn (30nM–300μM) exerted significantly more stable contractions, that was found to be catalase (100U/ml) resistant in comparison with H2O2 (30nM–300μM) in endothelium intact aortic rings. These contractile responses were found to be dependent on extracellular Ca2+ and were significantly inhibited in presence of ROS scavenger N-acetylcysteine (100μM). Intracellular calcium chelation by BAPTA-AM (10μM) had no significant effect on DPV-asn (30nM–300μM) mediated contraction. Pretreatment of aortic rings by rho-kinase inhibitor Y-27632 (10μM) significantly inhibited DPV-asn-mediated vasoconstriction indicating role of voltage-dependent Ca2+ influx and downstream activation of rho-kinase. The small initial relaxant effect obtained on addition of DPV-asn (30nM–1μM) in PE (1μM) pre-contracted endothelium intact rings, was prevented in the presence of guanylate cyclase inhibitor, methylene blue (10μM) and/or nitric oxide synthase (NOS) inhibitor, l-NAME (100μM) suggesting involvement of nitric oxide and cGMP. DPV-asn, like H2O2, exerted a response of vasoconstriction in normal arteries and vasodilation at low concentrations (30nM–1μM) in PE-pre contracted rings with overlapping mechanisms. These findings suggest usefulness of DPV-asn having low toxicity, in exploring the peroxide-mediated effects on various vascular beds. The present study also convincingly demonstrates role of H2O2 in the modulation of vasoreactivity by using stable peroxide DPV-asn and warrants future studies on peroxide mediated signaling from a newer perspective.

Endothelium-Dependent Vasodilator Effects of Peroxisome Proliferator-Activated Receptor β Agonists via the Phosphatidyl-Inositol-3 Kinase-Akt Pathway

Peroxisome proliferator-activated receptor beta/delta (PPAR-beta) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily that regulates the transcription of many target genes. More recently, acute, nongenomic effects of PPAR-beta agonists have also been described. In the present study, we hypothesized that PPAR-beta agonists might exert acute nongenomic effects on vascular tone. Here, we report that the structurally unrelated PPAR-beta ligands [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid (L-165041) and 4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl] methyl]thio]-2-methylphenoxy]acetic acid (GW0742) induced vascular relaxation in phenylephrine-precontracted endothelium-intact rat aortic rings, which was significantly inhibited by endothelial denudation or nitric-oxide synthase (NOS) inhibition with N(G)-nitro-l-arginine methylester. These relaxant effects reached steady state within 15 min. The relaxation induced by L-165041 and GW0742 in aortic rings precontracted with the thromboxane A(2) analog 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F2alpha (U-46619) was unaffected either by removal of extracellular calcium or by incubation with calcium-free solution containing the intracellular calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester. However, the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY-294002) inhibited the endothelium-dependent relaxant responses induced by both PPAR-beta agonists. Blockade of PPAR-beta with 3-[[[2-methoxy-4-(phenylamino)phenyl]amino]sulfonyl]-2-thiophenecarboxylic acid methyl ester (GSK0660) also partially inhibited these relaxant responses, although PPAR-gamma blockade with 2-chloro-5-nitro-N-phenylbenzamide (GW9662) had no effect. In human umbilical vein endothelial cells, L-165041 and GW0742 increased nitric oxide (NO) production and Akt and endothelial NOS (eNOS) phosphorylation, which were sensitive to PI3K inhibition and PPAR-beta blockade. In conclusion, the PPAR-beta agonists acutely caused vasodilatation, which was partially dependent on endothelial-derived NO. The eNOS activation is calcium-independent and seems to be related to activation of the PI3K-Akt-eNOS pathway.

P3. Postganglionic neurones and effectors: P3.1 Vasodilatation induced by nitric oxide donors is potentiated in endothelium intact rat aortic rings

P3. Postganglionic neurones and effectors: P3.1 Vasodilatation induced by nitric oxide donors is potentiated in endothelium intact rat aortic rings

Analysis of the mechanisms underlying the endothelium-dependent antivasoconstriction of puerarin in rat aorta

Puerarin, a major isoflavonoid compound from the Chinese herb, Ge-gen (Pueraria lobata), has effective treatment on myocardial and cerebral ischemia, glaucoma and sudden deafness in clinical setting in China. Our present work showed that puerarin (50, 150, 450 microM) concentration-dependently inhibited phenylephrine or KCl-induced contraction only in endothelium-intact rat aortic rings. In Ca(2+)-free solution, the antivasoconstriction of puerarin on phenylephrine was totally deprived. N (G)-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, indomethacin and the three K(+) channel blockers, glibenclamide, tetraethylammonium and Ba(2+) displayed significant inhibitory effects on the antivasoconstriction of puerarin. 8-bromo-cGMP significantly strengthened the action of puerarin. Puerarin (10-160 microM) concentration-dependently induced the NO production in the rat aortic cells. These findings suggested that the antivasoconstriction elicited by puerarin is endothelium-dependent. NO/NO-cGMP pathway, PGI(2) and the opening of K(+) channels sensitive to glibenclamide, tetraethylammonium, and Ba(2+), which might be triggered by the extracellular Ca(2+) influx in the endothelium, appear to contribute to the antivasoconstriction of puerarin.

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

Classically, nitric oxide (NO) formed by endothelial NO synthase (eNOS) freely diffuses from its generation site to smooth muscle cells where it activates soluble guanylyl cyclase (sGC), producing cGMP. Subsequently, cGMP activates both cGMP- and cAMP-dependent protein kinases [cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA), respectively], leading to smooth muscle relaxation. In endothelial cells, eNOS has been localized to caveolae, small invaginations of the plasma membrane rich in cholesterol. Membrane cholesterol depletion impairs acetylcholine (ACh)-induced relaxation due to alteration in caveolar structure. Given the nature of NO to be more soluble in a hydrophobic environment than in water, and assuming that colocalization of components in a signal transduction cascade seems to be a critical determinant of signaling efficiency by eNOS activation, we hypothesize that sGC, PKA, and PKG activation may occur at the plasma membrane caveolae. In endothelium-intact rat aortic rings, the relaxation induced by ACh, by the sGC activator 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), and by 8-bromo-cGMP was impaired in the presence of methyl-beta-cyclodextrin, a drug that disassembles caveolae by sequestering cholesterol from the membrane. sGC, PKG, and PKA were colocalized with caveolin-1 in aortic endothelium, and this colocalization was abolished by methyl-beta-cyclodextrin. Methyl-beta-cyclodextrin efficiently disassembled caveolae in endothelium. In summary, our results provide evidence of compartmentalization of sGC, PKG, and PKA in endothelial caveolae contributing to NO signaling cascade, giving new insights by which the endothelium mediates vascular smooth muscle relaxation.

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation.

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.

Role of endogenous reactive oxygen derived species and cyclooxygenase mediators in 5-hydroxytryptamine-induced contractions in rat aorta: relationship to nitric oxide

Endogenous reactive oxygen species (superoxide anion, hydroxyl radical and hydrogen peroxide), endothelium-derived nitric oxide and cyclooxygenase mediators are involved in the regulation of vascular smooth muscle tone. An imbalance of these mediators can have profound implications in various cardiovascular disorders. Involvement of endogenous reactive oxygen species, endothelium-derived nitric oxide (NO) and cyclooxygenase mediators in 5-hydroxytryptamine- (5-HT-) induced contractions of endothelium intact rat aortic rings have been investigated in the present study. The contribution of each of the endogenous reactive oxygen species in mediating 5-HT-induced contractions was studied by pretreating the rings with their respective scavengers. Pretreatment of the rings with superoxide dismutase (superoxide radical scavenger), catalase (H 2O 2inactivator), mannitol (extracellular OH · scavenger), or thiourea (intracellular OH · radical scavenger) significantly depressed the 5-HT-induced contractions in the aortic rings. The responses to 5-HT in the presence of SOD or catalase were augmented byl -NAME pretreatment. Though aminotriazole partially inhibited the catalase activity, it inhibited 5-HT-induced contractions significantly. The results obtained thus suggest that endogenous generation of ROS (O2·− , H 2O 2and OH ·) modulates 5-HT-induced rat aortic ring contractions. In addition, H 2O 2generated in the endothelium seems to regulate the vascular response and also act as a mediator to release other vasoactive substances. Basal production of NO by the endothelium seems to affect the vascular response due to its interaction with ROS mediators.

L-NAME inhibits Mg(2+)-induced rat aortic relaxation in the absence of endothelium.

1 L-NG-nitro-arginine methyl ester (L-NAME; 100 microM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 microM acetylcholine (ACh) and 2-10 mM Mg2+ in endothelium-intact (+E) rat aortic rings precontracted with 1 microM phenylephrine (PE). In PE-precontracted endothelium-denuded (-E) rat aorta, 3 microM ACh did not, but Mg2+ caused relaxation which was reversed by L-NAME, but not by D-NAME. 2 The concentration response profiles of L-NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 microM ACh were not significantly different. 3 L-NAME (100 microM) also reversed Mg(2+)-relaxation of -E aorta pre-contracted with 20 mM KCl or 10 microM prostaglandin F2alpha (PGF2alpha). L-NG-monomethyl-arginine (L-NMMA; 100 microM) was also effective in reversing the Mg(2+)-relaxation. 4 Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE-pre-contracted -E aorta, which was subsequently reversed by 100 microM L-NAME. 5 Reversal of the Mg(2+)-relaxation by 100 microM L-NAME in PE-precontracted -E aorta persisted following pre-incubation with 1 microM dexamethasone or 300 microM aminoguanidine (to inhibit the inducible form of NOS, iNOS). 6 Pretreatment of either +E or -E aortic rings with 100 microM L-NAME caused elevation of contractile responses to Ca2+ in the presence of 1 microM PE. 7 Our results suggest that L-NAME exerts a direct action on, as yet, unidentified vascular smooth muscle plasma membrane protein(s), thus affecting its reactivity to divalent cations leading to the reversal of relaxation. Such an effect of L-NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.

Endothelium-independent and -dependent vasoactivity of 6-nitronorepinephrine

Vasoactivities of 6-nitronorepinephrine were investigated using rat aorta. 6-Nitronorepinephrine (>100 μM) caused dose-dependent contraction in both endothelium-intact and -denuded aorta, although the latter showed greater contraction than the former. Prazosin (>3 nM), an α 1-adrenoceptor antagonist, attenuated significantly the 6-nitronorepinephrine-induced contractions, thereby suggesting the α 1-adrenoceptor involvement. Aortic rings prepared from reserpine-pretreated rats showed the 6-nitronorepinephrine-induced a contraction to the extent similar to those from untreated rats, suggesting that endogenous norepinephrine does not play a role in the 6-nitronorepinephrine-induced contraction. 6-Nitronorepinephrine (>10 μM) potentiated norepinephrine-induced contraction only in the presence of endothelium. The augmentation was attenuated by catalase (1200 U/ml). H 2O 2 (10–300 μM) augmented the norepinephrine-induced contraction only in the endothelium-intact rat aortic rings. 6-Nitronorepinephrine attenuated significantly acetylcholine-induced relaxation. Catalase prevented the 6-nitronorepinephrine-induced inhibition of the acetylcholine-induced relaxation. These results suggest that 6-nitronorepinephrine has a weak α 1-adrenoceptor agonistic property and that the endothelium-dependent potentiation by 6-nitronorepinephrine of the norepinephrine-induced contraction is mediated through production of H 2O 2.

Localization of 2 11beta-OH steroid dehydrogenase isoforms in aortic endothelial cells.

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) is expressed in vascular smooth muscle cells (VSMC) but has not been reported to be present in vascular endothelial cells. This enzyme assists in regulating the cellular concentration of active endogenous glucocorticoids (GCs). We have observed that endothelium intact rat aortic rings express message for both Type 1 and Type 2 11beta-HSD whereas primary cultures of VSMC express only mRNA for the Type I isoform. Since GCs diminish prostacyclin synthesis in endothelial cells, we hypothesized that 11beta-HSD is present in vascular endothelial cells. In primary cultures of rat aortic endothelial (RAE) cells, mRNA from both isoforms of 11beta-HSD could be detected by RT-PCR with higher levels of the Type 1 isoform. The oxo-reductase reaction "activating" 11-dehydro metabolites back to the parent steroid is the preferred enzyme direction (12:1 after a 120 minutes steroid incubation) in intact RAE cells. When RAE cells are grown in the presence of antisense oligonucleotides specific for Type 1 11beta-HSD, oxo-reductase activity is decreased by approximately 50% but the dehydrogenase reaction, which inactivates endogenous GCs and is characteristic of the Type 2 isoform, is unaffected. Thus endothelial cells appear to express both isoforms of 11beta-HSD; the Type 1 isoform dominates functioning in the oxo-reductase mode. Inhibition of the oxo-reductase reaction may lower the local concentrations of GC and indirectly allow for increased production of prostacyclin in endothelial cells.

Attenuation of Platelet-Induced Vasorelaxation by Pretreatment of Platelets with Oxidized Low Density Lipoproteins: Important Role of Serotonin

Aggregating platelets cause relaxation of precontracted vascular tissues with intact endothelium. To determine the modulation of vasorelaxation by lipoprotein-rich platelets, rat platelets were preincubated with buffer, native low density lipoprotein (n-LDL, 100 μg protein/ml), oxidized LDL (ox-LDL, 100 μg protein/ml) or high density lipoprotein (HDL, 100 μg protein/ml) at 37°C for 1 hour, washed twice, and then suspended into organ baths containing precontracted endothelium-intact rat aortic rings. While all platelet preparations caused relaxation of rat aortic rings, the magnitude of relaxation induced by ox-LDL-treated platelets was less than that by buffer-treated platelets (p < 0.05). Pretreatment of platelets with native LDL or HDL did not affect the magnitude of vasorelaxation. Treatment of platelets with the cyclooxygenase inhibitor indomethacin (5 × 10−5 M) did not affect the attenuated vasorelaxation in response to ox-LDL-treated platelets. Pretreatment of aortic rings with the thromboxane (TX) ...