L-arginine Methyl Ester Research Articles

Effects of commercial non-ionic alkyl oxyethylene and ionic biocompatible arginine-based surfactants on the photophysical behaviour of several poly(fluorene-1,4-phenylene)s

In this paper, the interaction between three water soluble conjugated polymers: sodium poly{9,9-bis[(4-butylsulfonate)phenoxy]-2,7-fluorene- alt-1,4-phenylene} (PBS-PFP), poly{9,9-bis[(6′-N,N,N-trimethylammonium) hexyl]-2,7-fluorene- alt-1,4-phenylene} dibromide (HTMA-PFP) and poly{9,9′-bis[(6″- N-acetyltyrosine)hexyl]-2,7-fluorene- alt-1,4-phenylene}, (Tyr–PFP) with neutral and charged surfactants has been studied. The optical spectroscopic properties of the three polymers in aqueous solution (PBS-PFP) and in DMSO–water mixtures (4% v/v, HTMA-PFP and Tyr–PFP) are also presented. Three different biocompatible surfactants were used: a single chain one, N α -lauroyl- l-arginine methyl ester hydrochloride (LAM), a gemini compound, N α ,N ω -bis( N α lauroylarginine) α, ω-dibutylamide dihydrochloride, (C 4(LA) 2) and an amino acid glyceride conjugate, 1-O-( l-arginyl)-2,3-O-di-lauroyl-sn-glycerol dichlorhydrate (1212R). In addition, the interaction of the same polymers with three commonly used commercial non-ionic surfactants: n-dodecyl pentaoxyethylene glycol ether (C 12E 5), 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100) and polyethylene glycol sorbitan monolaurate (Tween 20) was also studied. Electronic spectroscopy (absorption and steady-state emission) as well as molecular dynamics using the Gromacs package (for the interaction between HTMA-PFP and Triton X-100) and ionic conductivity measurements on HTMA-PFP/C 12E 5 are also reported.

Low Blood Pressure in Endothelial Cell–Specific Endothelin 1 Knockout Mice

Endothelin (ET) 1 is a potent vasoconstrictor peptide produced by vascular endothelial cells and implicated in various pathophysiologic states involving abnormal vascular tone. Homozygous ET-1 null mice have craniofacial and cardiac malformations that lead to neonatal death. To study the role of ET-1 in adult vascular physiology, we generated a mouse strain (ET-1(flox/flox);Tie2-Cre mice) in which ET-1 is disrupted specifically in endothelial cells. ET-1 peptide levels in plasma, heart, lung, kidney, and brain homogenates were reduced by 65% to 80% in these mice. mRNA levels for ET receptors were unaltered except that the ET(A) receptor mRNA was upregulated in the heart. ET-1(flox/flox);Tie2-Cre mice had mean blood pressures 10 to 12 mm Hg lower than genetic controls. In contrast, the blood pressure of mice systemically heterozygous for the ET-1 null allele (ET-1(dlox/+) mice) was unchanged compared with wild-type littermates. Despite the lower basal blood pressure, acute pharmacological responses to angiotensin II, captopril, phenylephrine, bradykinin, N(G)-nitro-L-arginine methyl ester, and exogenous ET-1 were normal in ET-1(flox/flox);Tie2-Cre mice. These results support an essential role of endothelial-derived ET-1 in the maintenance of basal vascular tone and blood pressure. Normal pharmacological responses of ET-1(flox/flox);Tie2-Cre mice suggest that the renin-angiotensin system, the adrenergic system, and NO are not significantly altered by endothelial ET-1. Taken in conjunction with other lines of genetically altered mice, our results provide evidence for a paracrine vasoregulatory pathway mediated by endothelial cell-derived ET-1 acting on the vascular smooth muscle ET(A) receptor.

Mechanisms underlying vascular effect of chronic resveratrol in streptozotocin‐diabetic rats

In this study, some underlying mechanisms responsible for the beneficial effect of chronic oral administration of resveratrol on aortic reactivity of streptozotocin (STZ)-diabetic rats were investigated. Male diabetic rats received resveratrol (10 mg/kg/day) for 8 weeks, 1 week after diabetes induction. Treatment of diabetic rats with resveratrol produced a hypoglycaemic effect and there were appropriate changes regarding serum lipids. Resveratrol also attenuated the increased malondialdehyde (MDA) content and reduced activity of superoxide dismutase (SOD) in liver and aortic tissues. Maximum contractile response of endothelium-intact aortic rings to KCl and phenylephrine (PE) was significantly lower in resveratrol-treated diabetic rats relative to untreated diabetics. Endothelium removal abolished the significant difference between resveratrol-treated and untreated diabetic groups regarding contractile response to KCl and PE. Meanwhile, endothelium-dependent relaxation to acetylcholine (ACh) was significantly higher in resveratrol-treated diabetic rats as compared to diabetic group and pretreatment with N(omega)-L-arginine methyl ester (L-NAME) and indomethacin (INDO) significantly attenuated these responses. Chronic treatment with resveratrol may prevent diabetes-related changes in vascular reactivity observed in diabetic rats directly and/or indirectly due to its hypoglycaemic and hypolipidaemic effects and attenuation of lipid peroxidation and through endothelial-derived factors.

Posttransplantation Therapeutic Rapamycin Concentration Protects Nitric Oxide–Related Vascular Endothelial Function: Comparative Effects in Rat Thoracic Aorta and Coronary Endothelial Cell Culture

Objective To investigate the potential effects of therapeutic dosages of the immunosuppression agent rapamycin on endothelial function with regard to nitric oxide (NO) synthesis in rat thoracic aorta in vivo and rat coronary endothelial cells in vitro. Materials and Methods Male Wistar rats were injected with rapamycin, 1.5 mg/kg/d intraperitoneally for 14 days. After the rats were sacrificed, the thoracic aortas were suspended in organ chambers and evaluated for endothelium-dependent and endothelium-independent vascular responses. Results Rapamycin administration resulted in increased relaxant responses to L-arginine and to greater concentrations of the calcium ionophore (A23187) in the aortas. However, potassium chloride, acetylcholine, sodium nitroprusside, and N G -nitro- l-arginine methyl ester responses remained unchanged. In addition, phenylephrine-induced contractions were significantly decreased in the aortas regardless of the presence of functional endothelium. In a series of in vitro experiments, isolated rat coronary endothelial cells were incubated with therapeutic concentrations of rapamycin (10 nmol/L). Nitrite accumulation in the supernatants revealed that rapamycin decreased nitrite release induced by interleukin-1β but did not affect basal or A23187-stimulated nitrite levels. Western blot analysis demonstrated that rapamycin decreased inducible NO synthase protein expression in coronary endothelial cells. Conclusion Posttransplantation therapeutic concentrations of rapamycin not only preserve vascular endothelial function mediated by NO synthesis but possibly interact in vivo with adrenergic receptors in favor of vasodilatory mechanisms.

CATIONIC TRYPSIN: A PREDOMINANT PROTEINASE IN PACIFIC SAURY (COLOLABIS SAIRA) PYLORIC CECA

ABSTRACT Cationic trypsin was purified from Pacific saury (Cololabis saira) pyloric ceca by the successive steps of ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The purification and yield were 90-fold and 8.9%, respectively. The molecular weight was determined to be 24 kDa using Sephacryl S-200 and SDS-PAGE. Cationic trypsin was stable at pH 7–11 for 30 min at 30C, and its maximal activity against L-arginine methyl ester hydrochloride (TAME) was at pH 8.5. Thermostability for cationic trypsin was up to 50C for 15 min and its temperature optimum was 60C. Cationic trypsin was stabilized by calcium ion. Activity decreased as NaCl concentration (0–30%) increased. Inhibitor susceptibility analysis revealed that the enzyme was inhibited effectively by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone. The Km and Kcat of the enzyme were 0.17 mM and 200 s−1, respectively. The N-terminal amino acid sequence of cationic trypsin was partially determined as IVGGYECQPH- and was very homologous to other trypsins. PRACTICAL APPLICATIONS Trypsin is a major member of the serine proteases, which constitute a large family of biologically important enzymes. Trypsins from various sources catalyze the hydrolysis of peptide bonds on the carboxyl sides of arginine and lysine. Hence, it is expected that like other trypsins, Pacific saury trypsin would also be used for processing aids in industry applications. Most trypsins are used for a variety of products in the food industry including baked foods, beer, wine, cereal, meat and fish products, and for production of protein hydrolysates and flavor extract. Trypsins are also used in mammalian cell culture to disaggregate adherent cells for research and the production of recombinant proteins, in diabetes diagnosis and therapy, in detergents and bating of leather.

Age-related changes in carotid vascular responses to adenosine and nitric oxide in the rat: in vitro and in vivo studies

We investigated how the ability of adenosine to release nitric oxide (NO) from carotid artery in vitro, and dilator responses evoked in carotid circulation in vivo by systemic infusion of adenosine, change with age in rats of 4-5, 10-12, and 42-44 wk (juvenile, mature, and middle aged). A secondary aim was to follow age-related changes in carotid/cerebral autoregulation. In opened carotid artery, graded doses of adenosine evoked graded increases in NO output measured with a NO sensor that were greater in mature and middle-aged than juvenile rats. Infusion of adenosine to reduce mean arterial pressure (ABP) to approximately 60 mmHg increased carotid vascular conductance (CVC) in all groups, but the increase was larger in mature rats; carotid blood flow (CBF) was unchanged in juvenile, increased in mature, but fell in 4/8 middle-aged rats. The NO synthase inhibitor nitro l-arginine methyl ester (l-NAME; 10 mg/kg iv) increased baseline ABP in all groups but caused larger percentage reductions in baseline CVC and CBF in mature and middle-aged than juvenile rats. Thereafter, the adenosine-evoked increase in CVC was unchanged in juvenile and middle-aged rats, yet CBF remained constant in juvenile but increased in middle-aged rats. In mature rats, the evoked increases in CVC and CBF were attenuated and further attenuated by l-NAME at 30 mg/kg. We propose that the ability of adenosine to release NO and cause vasodilation in the carotid artery and its circulation is greater in mature, than juvenile or middle-aged rats, but NO has greater tonic dilator influence in carotid circulation of mature and middle-aged than juvenile rats. By middle age, the lower limit of cerebral autoregulation has increased such that the tonic dilator influence of NO on ABP and CVC limits autoregulation of CBF to depressor responses. However, partial NO synthase inhibition overcomes this impairment, raising baseline ABP and allowing adenosine-evoked increases in CVC to increase CBF.

Evaluation of the antiulcerogenic activity profile of a flavonol diglucoside from Equisetum palustre L.

Ethnopharmacological relevance The aerial parts of Equisetum palustre L. are used to treat peptic ulcer disease in Turkey. In a previous study, a flavonol diglucoside i.e., kaempferol 3- O-1″-β- d-glucopyranosyl-3- O-1″′-β- d-glucopyranoside (KGG) was isolated as the major antiulcerogenic constituent from the plant. Aim of the study The present study was undertaken to evaluate the antiulcer activity profile of KGG using various in vivo experimental ulcer models as well as by assessing gastric biochemical parameters. Material and methods KGG was obtained from the ethanol extract of the aerial parts of the plant by successive chromatographical methods. The activity profile of the compound was investigated using several ulcerogenesis models such as indomethacin-, indomethacin plus HCl/EtOH-, cysteamine-, serotonin-, N G-nitro- l-arginine methyl ester plus EtOH-, diethyldithiocarbamate-, N-ethylmaleimide plus EtOH-, water immersion and restraint stress-, pyloric ligation-induced ulcers. In addition, effects of KGG on the biochemical parameters of gastric juice; i.e., inhibition of titratable gastric acidity, acid output, gastric pH, gastric secretion volume and peptic activity were studied. Results KGG exerted statistically significant gastroprotective activity against indomethacin-, indomethacin plus HCl/EtOH- and N-ethylmaleimide plus EtOH-induced ulcerogenesis. Moreover, KGG demonstrated weak activity against N G-nitro- l-arginine methyl ester plus EtOH, water immersion and immobilization-induced stress, pyloric ligation-induced and diethyldithiocarbamate-induced gastric ulcer models, and also it was ineffective in the prevention of ulcers induced by serotonin and cysteamine. On the other hand, among the gastric biochemical parameters studied, KGG was only found to increase the gastric acid pH from 2.03 to 3.35. Conclusion Results of this investigation have clearly demonstrated that KGG was found to improve the cytoprotective mechanisms of the gastric mucosa. On the other hand, a weak activity profile was observed on the parameters affecting the gastric acidity (water immersion and restraint-induced-, pyloric ligation-induced-ulcerogenesis and titratable acidity).

Dietary effect on plasma lipid changes and pregnant outcomes in early-onset pre-eclampsia-like murine model

To investigate the effects of dietary factors on the pre-eclampsia-like mouse model development at multiple stages of gestation. Pre-eclampsia-like model was established in C57 wild-type (WT) and apoE(-/-) pregnant mice at early, middle and late gestational stages by injecting nitric oxide synthase (NOS) inhibitor L-arginine methyl ester (L-NAME) subcutaneously. Control groups received normal saline (NS) simultaneously. Each group was subdivided into standard chow subgroup (SC, n = 6) and high-fat diet subgroup (HF, n = 6). Blood pressure, urinary protein and plasma lipid were measured and fetal outcomes compared. Data were analyzed statistically. In early and middle L-NAME subgroups, the plasma concentrations of cholesterol (TC), triglyceride (TG) and free fatty acid (FFA) all increased. And the early L-NAME subgroup in apoE(-/-) mice was the most remarkable (P < 0.05). In apoE(-/-) groups (both L-NAME and NS groups), the plasma concentrations of total TC and TG were higher than those of WT groups (P < 0.05). But there was no significant difference in FFA between them. The plasma lipid levels of apoE(-/-) HF + L-NAME group were the highest among all the groups (P < 0.05). Fetal and placental weights significantly decreased in early and middle L-NAME groups in both apoE(-/-) and WT mice (P < 0.05). But no significant difference was found between late L-NAME subgroup and NS groups (P > 0.05). Compared with WT groups, the weights were lower in apoE(-/-) mice. The fetal and placental weights in HF groups were lower than SC groups and the changes in early HF + L-NAME subgroup in apoE(-/-) mice were the most remarkable (P < 0.05). A lower live fetal rate and a higher absorbed fetal rate were found in early L-NAME groups than those of the NS groups (P < 0.05). Pre-eclampsia occurring at an early stage is more likely to have abnormal plasma lipid levels and adverse feto-placental outcomes. High-fat dietary may aggravate the impact of L-NAME pre-eclampsia on pregnancy outcomes at an early gestational stage especially in ApoE(-/-) mice.

Effects of isosorbide mononitrate on the restoration of injured artery in mice in vivo

The pharmacological basis of isosorbide mononitrate (ISMN), a widely used drug for cardiovascular diseases, is that it is metabolized to nitric oxide (NO). However, NO is a double-edged sword that results in either beneficial or detrimental effect. Vascular injury is the common consequence of many cardiovascular diseases, but it is not determined whether ISMN influences the restoration of injured artery in vivo. Carotid artery injury was induced by electric stimulation in mice. Vasoconstriction and endothelium-dependent and -independent relaxation were recorded by a multichannel acquisition and analysis system. ISMN (10 mg/kg, p.o.) treatment for 1 week and 1 month had no effect on reendothelialization, histology and function of carotid artery injured by electric stimulation. l-arginine (500 mg/kg, p.o.) and Nω-nitro- l-arginine methyl ester (L-NAME) (50 mg/kg, p.o.) treatment for 1 week did not affect the reendothelialization process, but L-NAME treatment induced neointimal hyperplasia and inhibited endothelium-dependent relaxation in electrically injured artery. These results suggest that supplement of exogenous or endogenous NO has no effect on the restoration of injured artery, but inhibition of endogenous NO induces neointimal hyperplasia in injured artery. ISMN treatment does not affect the restoration of injured artery.

Inhibitory effect of caffeic acid phenethyl ester, a plant-derived polyphenolic compound, on rat intestinal contractility

Caffeic acid phenethyl ester (CAPE) exerts pharmacological actions (e.g. anti-inflammatory, chemopreventive) which are relevant for potential clinical application in the digestive tract. However, no study has been published on its possible effects on intestinal motility, to date. In the present study, we investigated the effect of this plant-derived polyphenolic compound on the spontaneous contractions of the rat isolated ileum. CAPE reduced (in a tetrodotoxin-insensitive manner) spontaneous ileal contractions and this effect was reduced by the l-type Ca 2+ channel blocker nifedipine and the chelant of calcium ethylenediaminetetraacetic acid. However, the effect of CAPE was not modified by a number of inhibitors/antagonists such as of phentolamine plus propranolol, atropine, tetrodotoxin, cyclopiazonic acid, ω-conotoxin, apamin, N G-nitro- l-arginine methyl ester, 3-isobutyl-1-methylxanthine, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or a combination of SR 140333, SR48968 and SR142801. In conclusion our study shows that (i) CAPE relaxed myogenic contractions of rat ileum and that (ii) this effect occurs, at least in part, throughout a mechanism involving l-type Ca 2+ channels.

Electrochemical study of the intracellular transduction of vascular endothelial growth factor induced nitric oxide synthase activity using a multi-channel biocompatible microelectrode array

Background Nitric oxide (NO) plays a major role in physiology as a biological mediator. NO has been identified in nervous, immune and vascular systems and is a critical parameter in numerous pathologies, such as cancer. This article describes the electrochemical biomeasurements of NO synthase (NOS) activity from cultured endothelial cells using a multiple microelectrode array. Methods Firstly, the effect of biocompatible fibronectin coating on electrochemical measurements was investigated. Secondly, endothelial cells were deposited on the fibronectin coated sensor and NO release was triggered with vascular endothelial growth factor (VEGF). N G-nitro- l-arginine methyl ester (L-NAME) was used as an inhibitor of NO production, and different kinase blockers were investigated. Change in NOS activity was quantified using differential pulse voltammetry before and after addition of VEGF. Results Our results show that carefully applied layers of fibronectin have a very limited effect on electrochemistry and that VEGF induces an increase in NOS activity that is mainly mediated through the phosphatidylinositol 3 kinase (PI-3), and not by the extracellular signal-regulated kinases 1/2. Results obtained using electrochemical sensors were supported by wound healing assay demonstrating the critical role of phosphatidylinositol 3 kinase and extracellular signal-regulated kinases 1/2 for angiogenesis. Conclusion Electrochemical study of the intracellular transduction of the VEGF signal leading to NO synthesis was achieved, showing the critical role of PI-3 kinase. General significance This study presents an electrochemical sensor allowing measurements of NOS activity in cell cultures and tissue samples.

(±)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis

Praeruptorin A is a coumarin compound naturally occurring in the roots of Peucedanum praeruptorum Dunn., a commonly used traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. Although previous studies indicated the relaxant effects of (±)-praeruptorin A on tracheal and arterial preparations, little is known about the functional characteristics of the enantiomers. In the present study, the two enantiomers were successfully isolated and identified by using a preparative Daicel Chiralpak AD-H column, and their relaxant effects on aorta rings were observed and compared. (+)-Praeruptorin A showed more potent relaxation than (−)-praeruptorin A against KCl- and phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. Removal of the endothelium remarkably reduced the relaxant effect of (+)-praeruptorin A but not that of (−)-praeruptorin A. Pretreatment of aortic rings with N ω-nitro- l-arginine methyl ester ( l-NAME, an inhibitor of nitric oxide synthase) or methylene blue (MB, a soluble guanylyl cyclase inhibitor) resulted in similar changes of the relaxant effects of the two enantiomers to endothelium removal. Molecular docking studies also demonstrated that (+)-praeruptorin A was in more agreement to nitric oxide synthase pharmacophores than (−)-praeruptorin A. On the other hand, the two enantiomers of praeruptorin A could slightly attenuate the contraction of rat aortic rings induced by internal Ca 2+ release from sarcoplasmic reticulum (SR). These findings indicated that (+)-praeruptorin A and (−)-praeruptorin A exerted distinct relaxant effects on isolated rat aorta rings, which might be mainly attributed to nitric oxide synthesis catalyzed by endothelial nitric oxide synthase.

Expression pattern of thermogenesis-related factors in interscapular brown adipose tissue of alloxan-treated rats: Beneficial effect of l-arginine

Metabolic abnormalities underlying diabetes can be abrogated by l-arginine. Here we examined the molecular basis of disturbed interscapular brown adipose tissue (IBAT) thermogenesis and the possible role of nitric oxide (NO) in the IBAT of diabetic rats. To induce diabetes, adult Mill Hill hybrid hooded male rats were given a single alloxan dose (120 mg/kg). Both non-diabetic and diabetic groups were further divided into three subgroups receiving: (i) l-arginine·HCl (2.25%) or (ii) N ω-nitro- l-arginine methyl ester ( l-NAME·HCl, 0.01%) for 12 days in drinking water and (iii) untreated controls. Treatment of the diabetic animals started after diabetes induction (glucose level ⩾12 mmol/L). Diabetes led to a decrease in the mRNA levels of uncoupling protein 1 (UCP1), peroxisomal proliferator activator receptor gamma (PPARγ) and endothelial NO synthase (eNOS) as revealed by RT-PCR. The diabetic rats had reduced eNOS and inducible NOS (iNOS) protein contents accompanied by low tissue vascularization, a parameter directly related to tissue thermogenic state. Downregulation of glutathione peroxidase (GSH-Px) and catalase (CAT) transcripts were also observed in diabetes. In contrast, the expression level of PPARγ coactivator-1α (PGC-1α) mRNA was elevated. Supplementation with l-arginine not only restored diabetes-induced changes in the expressions of these molecules important for IBAT regulation, but also increased the vascularity. Interestingly, l-NAME induced similar patterns of changes in vascularity and PGC-1α mRNA level as did l-arginine. In summary, our results provide insight into the molecular basis underlying diabetes-induced metabolic and functional disturbances in the IBAT and suggest a beneficial role for the l-arginine–NO production pathway.

PPARγ Activation Prevents Hypertensive Remodeling of Cerebral Arteries and Improves Vascular Function in Female Rats

Previous studies have shown that peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor expressed in vascular cells, is protective of the vasculature. We hypothesized that activation of PPARgamma could prevent hypertensive remodeling of cerebral arteries and improve vascular function. Ten female Sprague-Dawley rats were treated with the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) for 5 weeks, 8 were treated with l-NAME plus the PPARgamma activator rosiglitazone, and 8 received no treatment and served as controls. Blood pressure, myogenic activity, passive diameters and wall thickness of cerebral arteries, and brain capillary density were compared between the groups. Treatment with l-NAME caused an increase in arterial blood pressure that was sustained with rosiglitazone treatment. l-NAME also caused inward hypertrophic remodeling and enhanced myogenic reactivity of cerebral arteries that was reversed by rosiglitazone. In addition, l-NAME hypertension caused rarefaction of brain capillaries by approximately 12%, whereas treatment with rosiglitazone increased capillary density by approximately 20%. PPARgamma activation may be an effective and clinically relevant way to prevent hypertensive remodeling of cerebral arteries and capillary rarefaction as well as improving vascular function without affecting blood pressure.

Endothelium-dependent and -independent vasorelaxation induced by CIJ-3-2F, a novel benzyl-furoquinoline with antiarrhythmic action, in rat aorta

Aims This study was designed to examine the mechanism of relaxation induced by CIJ-3-2F, a benzyl-furoquinoline antiarrhythmic agent, in rat thoracic aorta at the tissue and cellular levels. Main methods Isometric tension of rat aortic ring was measured in response to drugs. Ionic channel activities in freshly dissociated aortic vascular smooth muscle cells (VSMCs) were investigated using a whole-cell patch-clamp technique. Key findings CIJ-3-2F relaxed both phenylephrine (PE) and high KCl (60 mM)-induced contractions with respective pEC 50 (-log EC 50) values of 6.91 ± 0.07 and 6.32 ± 0.06. Removal of endothelium or pretreatment with nitric oxide (NO)-pathway inhibitors N ω-nitro- l-arginine methyl ester (L-NAME), N G-monomethyl- l-arginine (L-NMMA), N 5-(1-iminoethyl)- l-ornithine (L-NIO), hemoglobin, methylene blue or 1 H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (ODQ) reduced the relaxant effect of CIJ-3-2F. Relaxation to CIJ-3-2F was also attenuated by K + channel blockers tetraethylammonium (TEA) or 4-aminopyridine (4-AP), but not by charybdotoxin plus apamin, iberiotoxin, glibenclamide, or BaCl 2. CIJ-3-2F non-competitively antagonized the contractions induced by PE, Ca 2+, and Bay K8644 in endothelium-denuded rings. In addition, CIJ-3-2F inhibited both the phasic and tonic contractions induced by PE but did not affect the transient contraction induced by caffeine. CIJ-3-2F reduced the Ba 2+ inward current through L-type Ca 2+ channel (IC 50 = 4.1 μM) and enhanced the voltage-dependent K + (K v) current in aortic VSMCs. Significance These results suggest that CIJ-3-2F induced both endothelium-dependent and -independent vasorelaxation; the former is likely mediated by the NO/cGMP pathway whereas the latter is probably mediated through inhibition of Ca 2+ influx or inositol 1,4,5-triphosphate (IP 3)-sensitive intracellular Ca 2+ release, or through activation of K v channels.

Characterization of vasoconstrictor-induced relaxation in the cerebral basilar artery

The vascular endothelium regulates vascular smooth muscle functions by releasing endothelium-derived vasoactive substances. To identify physiological mechanisms mediating the inhibitory effect of the endothelium on vasoconstrictors, the basilar arteries isolated from Wistar rats were used in an organ bath study. In the intact basilar artery (with endothelium), 100 nM serotonin (5-HT) induced phasic contraction (28.7 ± 4.1% of 60 mM KCl-induced contraction) followed by profound time-dependent relaxation at 3 min (3.8 ± 0.4%). In the denuded artery (without endothelium), the 5-HT-induced contraction was enhanced (51.7 ± 16.1%), while the relaxation was abolished. In the intact basilar artery, the contraction was facilitated and the amplitude of the phasic contraction was significantly enhanced (70.1 ± 10.3%), but time-dependent relaxation was still manifested at 3 min (25.7 ± 10.0%) in the presence of Nω-nitro- l-arginine methyl ester hydrochloride (L-NAME) and indomethacin. Time-dependent relaxation induced by 5-HT was abolished in Ca 2+-free and in K +-free Krebs–Henseleit buffer (KHB). Furthermore, the 5-HT-induced contraction was enhanced by treatment with ouabain (105.6 ± 11.8%), tetraethylammonium chloride (133.2 ± 7.9%), charybdotoxin with apamin (145.4 ± 6.4%) or BaCl 2 (72.2 ± 13.8%) at 3 min; also, time-dependent relaxation was abolished by these blockers in the presence of L-NAME and indomethacin. U46619 (100 nM) induced sustained contraction without time-dependent relaxation in normal KHB, but charybdotoxin with apamin did not affect the contraction. The results suggest that time-dependent relaxation is modulated by endothelial sodium-potassium pump (Na +/K +-ATPase) and Ca 2+-activated K + channel (K Ca) activity, especially small- and intermediate-conductance K Ca-prominent ionic mechanisms of the so-called endothelium-derived hyperpolarizing factor.

Water extract of Zanthoxylum piperitum induces vascular relaxation via endothelium-dependent NO-cGMP signaling

Aim of the study The aim of the present study was to define the effects of extracts of leaves of Zanthoxylum piperitum (ZP) on the vascular tension and its mechanisms responsible in rat thoracic aortic rings. Materials and methods Methanol extract of ZP and aqueous fraction of the methanol extract (AZP) were examined for their vascular relaxant effects in isolated phenylephrine-precontracted aortic rings. Results Methanol extract of ZP and aqueous fraction of the methanol extract (AZP) induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Endothelium-denudation abolished the AZP-induced vasorelaxation. Pretreatment of the endothelium-intact aortic rings with N G-nitro- l-arginine methylester (L-NAME) and 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ) inhibited the AZP-induced vasorelaxation. Inhibition of Ca 2+ entry via L-type Ca 2+ channels failed to block the AZP-induced vasorelaxation. Extracellular Ca 2+ depletion slightly but significantly attenuated the AZP-induced vasorelaxation. Thapsigargin significantly attenuated the AZP-induced vasorelaxation. Further, Gd 3+ and 2-aminoethyl diphenylborinate (2-APB), inhibitors of store-operated Ca 2+ entry (SOCE), markedly attenuated the AZP-induced vasorelaxation. Also, wortmannin, an inhibitor of Akt, an upstream signaling molecule of eNOS, attenuated the AZP-induced vasorelaxation. AZP increased cGMP levels of the aortic rings in a concentration-dependent manner and the effect was blocked by L-NAME, ODQ, thapsigargin, Gd 3+, 2-APB, and wortmannin. K + channel inhibition with glibenclamide and tetraethylammonium, cyclooxygenase inhibition with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on the AZP-induced vasorelaxation. Conclusion Taken together, the present study suggests that AZP relaxes vascular smooth muscle via endothelium-dependent activation of NO-cGMP signaling through the Akt- and SOCE-eNOS pathways.

Choroidal blood flow compensation in rats for arterial blood pressure decreases is neuronal nitric oxide-dependent but compensation for arterial blood pressure increases is not

Choroidal blood flow (ChBF) compensates for changes in arterial blood pressure (ABP) and thereby remains relatively stable within a ±40 mmHg range of basal ABP in rabbits, humans and pigeons. In the present study, we investigated if ChBF can compensate for increases and decreases in ABP in rats. ChBF was continuously monitored using laser Doppler flowmetry in anesthetized rats, and ABP measured via the femoral artery. At multiple intervals over a 2–4 h period during which ABP varied freely, ChBF and ABP were sampled and the results compiled across rats. We found that ChBF remained near baseline over an ABP range from 40 mmHg above basal ABP (90–100 mmHg) to 40 mmHg below basal ABP, but largely followed ABP linearly below 60 mmHg. Choroidal vascular resistance increased linearly as BP increased above 100 mmHg, and decreased linearly as BP declined from basal to 60 mmHg, but resistance declined no further below 60 mmHg. Inhibition of nitric oxide (NO) formation by either a selective inhibitor of neuronal nitric oxide synthase (NOS) (N ω-propyl- l-arginine) or a nonselective inhibitor of both neuronal NOS and endothelial NOS (N ω-nitro- l-arginine methyl ester) did not affect compensation above 100 mmHg ABP, but did cause ChBF to linearly follow declines in BP below 90 mmHg. In NOS-inhibited rats, vascular resistance increased linearly with BP above 100 mmHg, but remained at baseline below 90 mmHg. These findings reveal that ChBF in rats, as in rabbits, humans and pigeons, compensates for rises and/or declines in arterial blood pressure so as to remain relatively stable within a physiological range of ABPs. The ChBF compensation for low ABP in rats is dependent on choroidal vasodilation caused by neuronal NO formation but not the compensation for elevated BP, implicating parasympathetic nervous system vasodilation in the ChBF compensation to low ABP.

Sustained delivery of sphingosine-1-phosphate using poly(lactic-co-glycolic acid)-based microparticles stimulates Akt/ERK-eNOS mediated angiogenesis and vascular maturation restoring blood flow in ischemic limbs of mice

Therapeutic angiogenesis is a promising strategy for treating ischemia. The lysophospholipid mediator sphingosine-1-phosphate (S1P) acts on vascular endothelial cells to stimulate migration and tube formation, and plays the critical role in developmental angiogenesis. We developed poly(lactic-co-glycolic-acid) (PLGA)-based S1P-containing microparticles (PLGA-S1P), which are biodegradable and continuously release S1P, and studied the effects of PLGA-S1P on neovascularization in murine ischemic hindlimbs. Intramuscular injections of PLGA-S1P stimulated blood flow in C57BL/6 mice dose-dependently, with repeated administrations at a 3-day interval, rather than a single bolus or 6-day interval, over 28 days conferring the optimal stimulating effect. In Balb/c mice that exhibit limb necrosis and dysfunction due to retarded blood flow recovery, injections of PLGA-S1P stimulated blood flow with alleviation of limb necrosis and dysfunction. PLGA-S1P alone did not induce edema in ischemic limbs, and rather blocked vascular endothelial growth factor-induced edema. PLGA-S1P not only increased the microvessel densities in ischemic muscle, but promoted coverage of vessels with smooth muscle cells and pericytes, thus stabilizing vessels. PLGA-S1P stimulated Akt and ERK with increased phosphorylation of endothelial nitric oxide synthase in ischemic muscle. The effects of the nitric oxide synthase inhibitor, Nω-nitro- l-arginine methylester, showed that PLGA-S1P-induced blood flow stimulation was partially dependent on nitric oxide. Injections of PLGA-S1P also increased the expression of angiogenic factors and the recruitment of CD45-, CD11b- and Gr-1-positive myeloid cells, which are implicated in post-ischemic angiogenesis, into ischemic muscle. These results indicate that PLGA-based, sustained local delivery of S1P is a potentially useful therapeutic modality for stimulating post-ischemic angiogenesis.

Effects of preeclampsia-like symptoms at early gestational stage on feto-placental outcomes in a mouse model

Background Early and late-onset preeclampsia is thought to be different disease entities. This study aimed to determine the effects of early-onset preeclampsia-like symptoms on feto-placental outcomes and the adverse impacts of various factors on placental and fetal growth and development at different gestational stages in a mouse model. Methods Pregnant C57BL/6J mice were divided into control and preeclampsia (PE) groups, and injected subcutaneously with the nitric oxide synthase inhibitor L-arginine methyl ester (L-NAME) 50 mg kg-1d-1. The PE group was divided into early-, mid- and late-PE groups with L-NAME injections starting on days 7, 11 and 16 of pregnancy, respectively. Corresponding control groups were injected with saline at the same time points. Blood pressure was measured until days 14 and 18, when the fetuses and placentas were removed under anesthesia. Blood pressure, urinary protein, and fetal and placental conditions were analyzed. Results Blood pressure and urinary protein increased following L-NAME injection. The fetal survival rate and fetal weight were reduced and the fetal absorption rate was increased in the early-PE group on days 14 and 18 of pregnancy, compared with the control group. There were no significant differences in these parameters between the late-PE group and the respective control group. Placental weights in the early- and mid-PE groups were significantly reduced at days 14 and 18 of pregnancy compared with the control groups, but there was no significant difference in placental weight between the late-PE group and the respective control group. Morphologic examination of placentas from the early- and mid-PE groups showed varying degrees of fibrinoid necrosis and villous interstitial edema, but no significant pathologic changes were found in the placentas from the late-PE or control groups. Conclusion Preeclampsia-like symptoms occurring during the early stage of pregnancy are more likely to affect placental and fetal development, whereas late onset preeclampsia-like symptoms have a direct impact on the mothers. Chin Med J 2010;123(6):707-712