Removal Of Endothelium Research Articles

Moderate hypothermia attenuates α1-adrenoceptor-mediated contraction in human varicose spermatic vein: The role of nitric oxide(Short communication).

The effects of moderate hypothermia (28°C) on the response of human varicose spermatic vein to α1-adrenoceptor agonist phenylephrine and the role of endothelial nitric oxide (NO) in these effects were studied. Concentration-response curves for phenylephrine (10-9 to 3 × 10-4 M) were recorded in rings with and without endothelium at 37 and 28°C. To further analyze the role of NO, in the response to phenylephrine during hypothermia, the effects of this agonist in the presence of NG-nitro-L-arginine methyl ester (10-4 M) were also determined. Under every condition tested, phenylephrine produced a marked, concentration-dependent contraction. Sensitivity of intact veins to the agonist was consistently lower at 28°C than at 37°C. There was no significant difference in phenylephrine response at 28 and 37°C in vessels without endothelium but at 28°C veins without endothelium showed a higher sensitivity than intact veins to phenylephrine. The sensitivity of veins with and without endothelium to nitroprusside (10-9 to 3 × 10-3 M) was significantly decreased during hypothermia, and endothelium removal did not affect the relaxation to this nitrovasodilator. These results suggest that moderate hypothermia decreases the sensitivity of human varicose spermatic vein to phenylephrine probably by increasing the availability of endothelial NO.

Lidocaine relaxation in isolated rat aortic rings is enhanced by endothelial removal: possible role of Kv, KATP channels and A2a receptor crosstalk.

BackgroundLidocaine is an approved local anesthetic and Class 1B antiarrhythmic with a number of ancillary properties. Our aim was to investigate lidocaine’s vasoreactivity properties in intact versus denuded rat thoracic aortic rings, and the effect of inhibitors of nitric oxide (NO), prostenoids, voltage-dependent Kv and KATP channels, membrane Na+/K+ pump, and A2a and A2b receptors.MethodsAortic rings were harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs-Henseleit solution, pH 7.4, 37 °C. The rings were pre-contracted sub-maximally with 0.3 μM norepinephrine (NE), and the effect of increasing lidocaine concentrations was examined. Rings were tested for viability after each experiment with maximally dilating 100 μM papaverine. The drugs 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate, ouabain, 8-(3-chlorostyryl) caffeine and PSB-0788 were examined.ResultsAll drugs tested had no significant effect on basal tension. Lidocaine relaxation in intact rings was biphasic between 1 and 10 μM (Phase 1) and 10 and 1000 μM (Phase 2). Mechanical removal of the endothelium resulted in further relaxation, and at lower concentrations ring sensitivity (% relaxation per μM lidocaine) significantly increased 3.5 times compared to intact rings. The relaxing factor(s) responsible for enhancing ring relaxation did not appear to be NO- or prostacyclin-dependent, as L-NAME and indomethacin had little or no effect on intact ring relaxation. In denuded rings, lidocaine relaxation was completely abolished by Kv channel inhibition and significantly reduced by antagonists of the MitoKATP channel, and to a lesser extent the SarcKATP channel. Curiously, A2a subtype receptor antagonism significantly inhibited lidocaine relaxation above 100 μM, but not the A2b receptor.ConclusionsWe show that lidocaine relaxation in rat thoracic aorta was biphasic and significantly enhanced by endothelial removal, which did not appear to be NO or prostacyclin dependent. The unknown factor(s) responsible for enhanced relaxation was significantly reduced by Kv inhibition, 5-HD inhibition, and A2a subtype inhibition indicating a potential role for crosstalk in lidocaine’s vasoreactivity.

Juvenile growth reduces the influence of epithelial sodium channels on myogenic tone in skeletal muscle arterioles.

Previous studies have documented that rapid juvenile growth is accompanied by functional changes in the arteriolar endothelium, but much less is known about functional changes in arteriolar smooth muscle over this period. In this study, we investigate the possible contribution of epithelial sodium channels (ENaC) to the myogenic behaviour of arterioles at two stages of juvenile growth. The effects of the ENaC inhibitor benzamil on different levels of myogenic tone were studied in isolated gracilis muscle arterioles from rats aged 21-28days ("weanlings") and 42-49days ("juveniles"). ENaC subunit expression in the arteriolar wall was also determined, and the interaction between ENaC and nitric oxide (NO) in regulating vascular tone was explored by combined use of benzamil and NG -monomethyl-l-arginine (l-NMMA). At physiological pressures, both steady-state myogenic tone and the dynamic adjustments in this tone triggered by acute pressure changes were less in juvenile arterioles than in weanling arterioles. α, β and γ ENaC protein was present in arterioles at both ages, but benzamil only had an effect on myogenic tone in weanling arterioles. In these vessels, benzamil increased, rather than decreased, myogenic tone, and this effect was prevented by l-NMMA or endothelial removal. These findings suggest that although ENaC is present in gracilis muscle arterioles of both weanling and juvenile rats, it is not obligatory for the genesis of myogenic activity in these vessels at either age. However, ENaC activity can significantly modulate the level of myogenic tone through stimulation of endothelial NO release at an early stage of growth.

Effects of .BETA.-estradiol on vasoconstriction in human umbilical artery and vein

Objective To determine the effects of β-estradiol on vasoconstriction in human umbilical artery and vein and its potential mechanisms. Methods Human umbilical cord samples were obtained from 96 term neonates of healthy singleton pregnant women born in the First Hospital of Soochow University between December 2013 and June 2015 (multiple pregnancy, pregnancy complications, cesarean delivery and low birth weight were excluded). Human umbilical arteries and veins were isolated and suspended in 37 ℃ organ baths containing 5 ml Krebs solution and exposed to β-estradiol followed by phenylephrine (PE) for vasoconstriction test. The subjects were divided into β-estradiol group and control group according to the presence or absence of β-estradiol incubation. To determine the effects and the possible underlying mechanisms of β-estradiol on PE-induced vasoconstriction, human umbilical artery and vein rings were pretreated with Nω-nitro-L-arginine (L-NMMA, nitric oxide synthesis inhibitor), fulvestrant (ICI182780, estradiol receptor antagonist), indomethacin (prostaglandin synthesis blocker), and removal of endothelium, then incubated with β-estradiol for 60 min followed by PE, and the concentration-response curves to PE were recorded. The concentration-response curves to phorbol 12,13-dibutyrate (PDBU, protein kinase C agonist) in Krebs solution in the presence or absence of β-estradiol were also obtained. Nonlinear regression and fitting curve were performed, and the two-sample ANOVA was used for analysis. Results (1) β-estradiol suppressed PE-induced vasoconstriction of human umbilical vein and artery. In human umbilical vein and artery of the control group, the maximum contraction intensity induced by PE was (59.17±5.98)% and (43.35±5.02)% of that induced by potassium chloride, respectively. The maximum contraction induced by PE in β-estradiol group was (5.87±1.32)% and (4.52±1.22)% of that induced by potassium chloride. (2) In both groups, incubation with L-NMMA or endothelium removal enhanced the vasoconstriction of human umbilical artery and vein, indicating that the inhibitory effect of β-estradiol was not influenced by the endothelium. (3) The suppression of β-estradiol on PE-induced vasoconstriction in human umbilical artery and vein was not significantly decreased by estrogen receptor antagonist. (4) β-estradiol did not affect human umbilical artery and vein vasoconstriction induced by PDBU. (5) In the control group, incubation with indomethacin did not affect human umbilical artery and vein vasoconstriction induced by PE. In the β-estradiol group, indomethacin significantly enhanced the contraction response induced by PE, suggesting that prostacycline synthesis was partly involved in β-estradiol-suppressed contractility in human umbilical artery and vein. The contractile response induced by phenylephrine was still lower in the β-estradiol group than in the control group, which was induced by indomethacin. Conclusions (1)β-estradiol can suppress vasoconstriction in human umbilical artery and vein, which is not dependent on endothelium and estrogen receptors, or protein kinase C activity. (2) Prostacycline synthesis is partly involved in β-estradiol-suppressed vasoconstriction in human umbilical artery and vein. Key words: Umbilical arteries; Umbilical veins; Vasoconstriction; Estradiol

Vasodilator activity of hydrogen sulfide (H2S) in human mesenteric arteries

The role of endogenous H2S has been highlighted as a gaseous transmitter. The vascular smooth muscle inhibitory effects of H2S have been characterized in isolated aorta and mesenteric arteries in rats and mice. Our study was aimed at investigating the vascular effects of H2S on human isolated mesenteric arteries and examining the underlying mechanisms involved.All experiments were performed on rings (4–8mm long) of human mesenteric arteries obtained from patients undergoing abdominal surgery. Ethical approval was obtained from the Ethics Committee of the University Hospital of the University of Florence (app. N. 2015/0024947). The effect of NaHS, an H2S donor, was determined using noradrenaline pre-contracted human isolated mesenteric rings.NaHS evoked a concentration-dependent relaxation (EC50 57μM). In contrast, homocysteine, an endogenous precursor of H2S, failed to affect human isolated mesenteric rings. Vasorelaxant response to NaHS was reduced by endothelium removal, application of the nitric oxide synthase inhibitor L-NAME and ODQ inhibitor of cyclic GMP. SQ 22536, an adenylate-cyclase inhibitor, failed to block NaHS-induced vasorelaxation. Inhibition of endogenous prostanoid production by indomethacin significantly reduced NaHS induced vasorelaxation. The role of potassium channels was also examined: blockers of the Ca2+-dependent potassium channel, charybdotoxin and apamin, failed to have any influence on the relaxant response to NaHS on this vascular tissue.In summary, H2S induced relaxation of isolated rings of human mesenteric arteries. Endothelium-dependent related mechanisms with the stimulation of ATP-sensitive potassium channels represents important cellular mechanisms for H2S effect on human mesenteric arteries.

ChemInform Abstract: Ferrocenyl, Alkyl, and Aryl‐pyrido[2,3‐d]pyrimidines as Vasorelaxant of Smooth Muscle of Rat Aorta via cAMP Conservation Through Phosphodiesterase Inhibition.

New pyrido[2,3-d]pyrimidines , , , and have been synthesized. The vasorelaxant effect on smooth muscle isolated from rat aorta, via PDEs inhibition, of these compounds along with other pyrido[2,3-d]pyrimidines reported earlier by our group, has also been determined. These pyrido[2,3-d]pyrimidines were synthesized by the reaction of ferrocenyl-ethynyl ketones () or α-alkynyl ketones () with 6-amino-1,3-dimethyluracil using [Ni(CN)4]−4 as an active catalytic species, formed in situ in a Ni(CN)2/NaOH/H2O/CO/KCN aqueous system. Evaluation of the vasorelaxant effect of compounds demonstrated that all compounds relax the tissue in a concentration-dependent manner. The structural changes do not alter the effectiveness; however, there are differences related to potency expressed as EC50. Compounds (7-ferrocenyl-1,3-dimethyl-5-(m-tolyl)-pyrido[2,3-d]pyrimidine) and (7-ferrocenyl-1,3-dipropyl-5-(4-metoxyphenyl)-pyrido[2,3-d]pyrimidine) were the most potent compounds, even more than rolipram, reference drug; the EC50 was 0.41 ± 0.02 μM and 0.81 ± 0.11 μM for and , correspondingly. The EC50 of compounds (7-ferrocenyl-1,3-dimethyl-5-phenyl-pyrido[2,3-d]pyrimidine), (7-ferrocenyl-5-(3,5-dimethoxyphenyl)-1,3-dimethylpyrido[2,3-d]pyrimidine), and (5-n-butyl-7-ethyl-1,3-dimethylpyrido[2,3-d]pyrimidine) was similar to EC50 of rolipram. Compounds significantly induce concentration-dependent vasorelaxation in endothelium-intact aortic rings. In addition, the relaxation responses to each compound in either endothelium-intact or endothelium denuded aortic rings were comparable, suggesting that removal of the functional endothelium has no significant influence on its intrinsic vasorelaxant activity. In vitro capability of conserving cyclic-AMP or cyclic-GMP (adenosine and guanosine 3′, 5′-cyclic monophosphate) via PDE inhibition for compounds and was evaluated. Compounds and show the highest percent inhibition effect (94.83% and 83.98%, respectively) for the decomposition of c-AMP. Docking studies showed that the compound was selective for the inhibition of PDE-4.

Relaxant Effects of the Selective Estrogen Receptor Modulator, Bazedoxifene, and Estrogen Receptor Agonists in Isolated Rabbit Basilar Artery.

We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-β-estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-β-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERβ agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERβ (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).

Angiotensin-(1-7) Selectively Induces Relaxation and Modulates Endothelium-Dependent Dilation in Mesenteric Arteries of Salt-Fed Rats

This study investigated the acute effects of angiotensin-(1-7) and AVE0991 on active tone and vasodilator responses to bradykinin and acetylcholine in isolated mesenteric arteries from Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) versus a normal salt (NS; 0.4% NaCl) diet. Angiotensin-(1-7) and AVE0991 elicited relaxation, and angiotensin-(1-7) unmasked vasodilator responses to bradykinin in arteries from HS-fed rats. These effects of angiotensin-(1-7) and AVE0991 were inhibited by endothelium removal, A779, PD123319, HOE140 and <smlcap>L</smlcap>-NAME. Angiotensin-(1-7) also restored the acetylcholine-induced relaxation that was suppressed by the HS diet. Vasodilator responses to bradykinin and acetylcholine in the presence of angiotensin-(1-7) were mimicked by captopril and the AT₂ receptor agonist CGP42112 in arteries from HS-fed rats. Thus, in contrast to salt-induced impairment of vascular relaxation in response to vasodilator stimuli, angiotensin-(1-7) induces endothelium-dependent and NO-mediated relaxation, unmasks bradykinin responses via activation of mas and AT₂ receptors, and restores acetylcholine-induced vasodilation in HS-fed rats. AT₂ receptor activation and angiotensin-converting enzyme (ACE) inhibition shared the ability of angiotensin-(1-7) to enhance bradykinin and acetylcholine responses in HS-fed rats. These findings suggest a therapeutic potential for mas and/or AT₂ receptor activation and ACE inhibition in restoring endothelial function impaired by elevated dietary salt intake or other pathological conditions.

Crocin-Induced Endothelium-Dependent Relaxation in Isolated Rat Aorta

Background: Crocin, an active ingredient of saffron, has been found to possess hypotensive effects. Objectives: The aim of this study was to evaluate the mechanism of vasodilatory effects induced by crocin on isolated rat aorta. Methods: To study the vasodilatory effects of crocin (0.1, 0.2, 0.4 and 0.8 M), isolated rat thoracic aorta rings were contracted by 10-6 M Phenylephrine (PE) or 80 mM of KCl. To assess the role of nitric oxide in mechanism of relaxation, induced by crocin, aortic rings were incubated by L-NG-Nitroarginine methyl ester (L-NAME: 10-6 M) for 20 minutes. Furthermore, the relaxant effect of crocin was evaluated both in intact and denuded endothelium aortic rings. Results: Crocin induced relaxation in endothelium-intact aortic rings precontracted with PE in a concentration dependent manner yet not in KCl precontracted aortic rings. The relaxant activity of crocin was abolished by incubation of aortic rings with L-NAME. In addition, the endothelium removal inhibited the relaxant activity of crocin. Conclusions: This study indicated that relaxant activity of crocin is mainly mediated through an endothelium-dependent mechanism.

Endothelial dysfunction of internal thoracic artery graft in patients with chronic kidney disease

ObjectivesThe present study was designed to evaluate the association between chronic kidney disease and the endothelial function of internal thoracic artery (ITA) grafts in patients undergoing coronary bypass surgery. An isometric tension study was performed in ITA strips obtained during surgery. Concentration-response curves for acetylcholine (ACh) and sodium nitroprusside were constructed in ITA strips partially precontracted with phenylephrine under the inhibition of cyclooxygenase. The integrity of the endothelium was verified histologically by en-face staining of the luminal surface with the use of silver nitrate solution. ResultsIn endothelium-intact ITA strips, ACh produced a concentration-dependent relaxation in patients with glomerular filtration rate (GFR, mL/min/1.73 m2) > 60. A concentration-dependent relaxation response also was observed in patients with GFR 30 to 60, but it was reduced significantly compared with those with GFR > 60. In both groups, removal of endothelium or treatment with nitric oxide (NO) synthase inhibitors almost abolished the ACh-induced relaxation. On the other hand, in patients with GFR < 30, mild contraction rather than relaxation was induced at a high concentration of ACh, which was modified neither by treatment with NO synthase inhibitors nor by removal of the endothelium. Vasodilator responses to sodium nitroprusside were comparable among the 3 groups. The relaxation of endothelium-intact strips to a peak ACh concentration correlated positively with GFR. This relationship held true in a multiple linear regression model, and interaction terms between GFR and other risk factors were not statistically significant. ConclusionsEndothelial function of ITA grafts to release NO is impaired at the time of surgery in patients with chronic kidney disease.

Endothelium-independent vasorelaxant effect of 20(S)-protopanaxadiol on isolated rat thoracic aorta.

Ginsenosides are considered to be the major pharmacologically active ginseng constituents, whereas 20(S)-protopanaxadiol [20(S)-PPD] is the active metabolite of ginsenosides in gut. In this study we investigated the effect of 20(S)-PPD on isolated rat thoracic aortas as well as its vasorelaxant mechanisms. Aortic rings with or without endothelium were prepared from Wistar rats and suspended in organ-chambers. The changes in tension of the preparations were recorded through isometric transducers connected to a data acquisition system. The aortic rings were precontracted with phenylephrine (PE, 1 μmol/L) or high-K+ (80 mmol/L). Application of 20(S)-PPD (21.5-108.5 μmol/L) caused concentration-dependent vasodilation of endothelium-intact aortic rings precontracted with PE or high-K+, which resulted in the EC50 values of 90.4 or 46.5 μmol/L, respectively. The removal of endothelium had no effect on 20(S)-PPD-induced relaxation. The vasorelaxant effect of 20(S)-PPD was also not influenced by the preincubation with β-adrenergic receptor antagonist propranolol, or with ATP-sensitive K+ channel blocker glibenclamide, voltage-dependent K+ channel blocker 4-AP and inward rectifier K+ channel blocker BaCl2, whereas it was significantly attenuated by the preincubation with Ca2+-activated K+ (BKCa) channel blocker TEA (1 mmol/L). Furthermore, the inhibition of NO synthesis, cGMP and prostacyclin pathways did not affect the vasorelaxant effect of 20(S)-PPD. In Ca2+-free solution, 20(S)-PPD (108.5 μmol/L) markedly decreased the extracellular Ca2+-induced contraction in aortic rings precontracted with PE or high-K+ and reduced PE-induced transient contraction. Voltage-dependent Ca2+ channel antagonist nifedipine inhibited PE-induced contraction; further inhibition was observed after the application of receptor-operated Ca2+ channel inhibitor SK&F 96365 or 20(S)-PPD. 20(S)-PPD induces vasorelaxation via an endothelium-independent pathway. The inhibition of voltage-dependent Ca2+ channels and receptor-operated Ca2+ channels and the activation of Ca2+-activated K+ channels are probably involved in the relaxation.

Histamine-Induced Dilation of Isolated Porcine Retinal Arterioles: Role of Endothelium-Derived Hyperpolarizing Factor.

Although endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles has been well described, the role of endothelium-derived hyperpolarizing factor (EDHF) in the retinal arteriolar response remains unclear. In the current study, we examined the contribution of EDHF to the retinal arteriolar dilation to the inflammatory agent histamine and investigated the signaling mechanisms underlying this vasomotor activity. Porcine retinal arterioles were isolated, cannulated, and pressurized without flow for functional study by using video microscopic techniques. The immunohistochemical staining was performed to determine histamine receptor subtypes. Histamine (0.1-30 μM) produced concentration-dependent dilation of retinal arterioles in a manner sensitive to H1- and H2-receptor antagonists chlorpheniramine and famotidine, respectively. Histamine-induced vasodilation was almost abolished after endothelial removal. In the intact vessels, vasodilation to histamine was partially inhibited by the inhibitors of cyclooxygenase (indomethacin), NO synthase (NG-nitro-L-arginine methyl ester, L-NAME), or Ca2+ -activated K+ (KCa) channels (apamin plus charybdotoxin). Combination of the above inhibitors abolished histamine-induced vasodilation. Residual vasodilation in the presence of indomethacin and L-NAME was further reduced by the cytochrome P450 enzyme inhibitor sulfaphenazole but not by the gap junction inhibitor carbenoxolone or the hydrogen peroxide scavenger catalase. Immunohistochemical signals for H1- and H2-receptor expression were found only in the endothelium. The endothelium plays an essential role in the dilation of porcine retinal arterioles to histamine via H1- and H2-receptor activation. The EDHF derived from cytochrome P450 contributed in part to this vasodilation via KCa channel activation, in addition to the endothelial release of NO and prostanoids.

Nitric oxide and Kir6.1 potassium channel mediate isoquercitrin-induced endothelium-dependent and independent vasodilation in the mesenteric arterial bed of rats

The vascular effect of flavonoid isoquercitrin was investigated in the perfused mesenteric vascular bed of rats. In preparations with functional endothelium isoquercitrin (100, 300 and 1000nmol) dose-dependently reduced the perfusion pressure by 13±2.2, 33±3.9, and 58±3.7mm Hg, respectively. Endothelium removal or inhibition of the nitric oxide synthase enzymes by l-NAME did not change the effects of 100 and 300 nmol isoquercitrin, but reduced by 30–40% the vasodilation induced by 1000 nmol isoquercitrin. Perfusion with nutritive solution containing 40mM KCl abolished the vasodilatory effect of all isoquercitrin doses. Treatment with glibenclamide, a Kir6.1 (ATP-sensitive) potassium channel blocker, inhibited vasodilation induced by 100 and 300 nmol isoquercitrin, but only partially reduced the effect of 1000 nmol isoquercitrin. The non-selective KCa (calcium-activated) potassium channel blocker tetraethylammonium, but not the selective KCa1.1 channel blocker iberiotoxin, reduced by around 60% vasodilation induced by all isoquercitrin doses. In addition, association of tetraethylammonium and glibenclamide, or l-NAME and glibenclamide, fully inhibited isoquercitrin-induced vasodilation. Our study shows that isoquercitrin induces vasodilation in resistance arteries, an effect mediated by K+ channel opening and endothelial nitric oxide production.

Increased vascular eNOS and cystathionine-γ-lyase protein after 6weeks oral administration of 3, 5, 7, 3', 4'-pentamethoxyflavone to middle-aged male rats.

Effects of treatment of middle-aged male rats with 3, 5, 7, 3', 4'-pentamethoxyflavone (PMF) on vascular and perivascular adipose tissue (PVAT) functions and blood chemistry were investigated. Rats received PMF (22mg/kg), orally or vehicle, twice a day for 6weeks. The PMF-treated rats had lower serum glucose, higher HDL-C levels, but no change in other parameters. Thoracic aortic and mesenteric rings of PMF treated rats produced lower maximal contraction to phenylephrine that was normalized by NG-nitro-L-arginine (L-NA) or endothelial removal. The aortic- and mesenteric rings of the PMF treated rats showed improved relaxation to acetylcholine, but not to glyceryl trinitrate, and had higher eNOS protein. DL-propargylglycine (PAG) caused greater increase in the baseline tension of the PMF-treated aortic ring and higher contraction to low concentrations of phenylephrine. PVAT lowered the contractile response of the L-NA pretreated aortic rings to phenylephrine for both groups, but PAG had no effect. The cystathionine-γ-lyase (CSE) protein of the thoracic rings, but not of the PVAT, shows increased expression after PMF treatment. Overall, PMF treatment of middle aged rats appeared to increase production of NO and H2S from the blood vessels by upregulating the expression of eNOS and CSE. PMF also decreased fasting serum glucose and increased HDL-C levels, with no toxicity to liver and kidney functions. Thus, PMF is a novel compound for possible use as a health product to prevent and/or to reduce the development of diabetes type II and/or cardiovascular disease.

Pharmacological characterization of the mechanisms underlying the vascular effects of succinate

We investigated the mechanisms underlying the vascular effects of succinate. Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats and C57BL/6 wild type (WT) or GPR91−/– mice. Nitrate/nitrite (NOx) was measured colorimetrically whereas 6-keto-prostaglandin F1α (stable product of prostacyclin) was measured by enzyme immunoassay (EIA). Phosphorylation of endothelial nitric oxide synthase (eNOS) was assessed by western immunoblotting. Functional assays revealed that the direct effect of succinate in the vasculature is biphasic. At lower concentrations succinate induced relaxation while at higher concentrations succinate induced vascular contraction. Succinate concentration dependently relaxed rat aortic rings with intact endothelium. Endothelial removal reduced, but not abolished succinate-induced relaxation. Similarly, succinate relaxed endothelium-intact and endothelium-denuded aortas isolated from both C57BL/6 and GPR91−/– mice. Pre-incubation of endothelium-intact, but not endothelium-denuded rat aortic rings with l-NAME, indomethacin and tetraethylammonium (TEA) reduced succinate-induced relaxation. In endothelium-intact rings, succinate-induced relaxation was attenuated by ODQ, haemoglobin, Rp-8-Br-Pet-cGMPS, thapsigargin, wortmannin and SC-560. Blockade of K+ channels with 4-aminopyridine, apamin and charybdotoxin reduced succinate-induced relaxation. Succinate increased the concentration of NOx and 6-keto-prostaglandin F1α as well as eNOS phosphorylation at ser1177 residue. CaCl2-induced contraction of endothelium-intact or endothelium-denuded aortas was not affected by succinate. The major finding of our study is that it first demonstrates a direct effect of succinate in the vasculature. Succinate displays a biphasic and concentration-dependent effect. The vascular relaxation induced by succinate is partially mediated by endothelial GPR91 receptors via the NO-cGMP pathway, a vasodilator cyclooxygenase (COX) product(s) and the opening of K+ channels.

Brain-derived neurotrophic factor of the cerebral microvasculature: a forgotten and nitric oxide-dependent contributor of brain-derived neurotrophic factor in the brain.

Evidence that brain-derived neurotrophic factor (BDNF), a neurotrophin largely involved in cognition, is expressed by cerebral endothelial cells led us to explore in rats the contribution of the cerebral microvasculature to BDNF found in brain tissue and the link between cerebrovascular nitric oxide (NO) and BDNF production. Brain BDNF protein levels were measured before and after insitu removal of the cerebral endothelium that was achieved by brain perfusion with a 0.2% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulphonate) solution. BDNF protein and mRNA levels as well as levels of endothelial NO synthase phosphorylated at serine 1177 (P-eNOSser1177 ) were measured in cerebral microvessel-enriched fractions. These fractions were also exposed to glycerol trinitrate. Hypertension (spontaneously hypertensive rats) and physical exercise training were used as experimental approaches to modulate cerebrovascular endothelial NO production. CHAPS perfusion resulted in a marked decrease in brain BDNF levels. Hypertension decreased and exercise increased P-eNOSser1177 and BDNF protein levels. However, BDNF mRNA levels that were increased by exercise did not change after hypertension. Finally, invitro exposure of cerebral microvessel-enriched fractions to glycerol trinitrate enhanced BDNF production. These data reveal that BDNF levels measured in brain homogenates correspond for a large part to BDNF present in cerebral endothelial cells and that cerebrovascular BDNF production is dependent on cerebrovascular endothelial eNOS activity. They provide a paradigm shift in the cellular source of brain BDNF and suggest a new approach to improve our understanding of the link between endothelial function and cognition.

Endothelium-Dependent Contractions of Isolated Arteries to Thymoquinone Require Biased Activity of Soluble Guanylyl Cyclase with Subsequent Cyclic IMP Production.

Preliminary experiments on isolated rat arteries demonstrated that thymoquinone, a compound widely used for its antioxidant properties and believed to facilitate endothelium-dependent relaxations, as a matter of fact caused endothelium-dependent contractions. The present experiments were designed to determine the mechanisms underlying this unexpected response. Isometric tension was measured in rings (with and without endothelium) of rat mesenteric arteries and aortae and of porcine coronary arteries. Precontracted preparations were exposed to increasing concentrations of thymoquinone, which caused concentration-dependent, sustained further increases in tension (augmentations) that were prevented by endothelium removal, Nω-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor], and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; soluble guanylyl cyclase [sGC] inhibitor). In L-NAME-treated rings, the NO-donor diethylenetriamine NONOate restored the thymoquinone-induced augmentations; 5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol (sGC activator) and cyclic IMP (cIMP) caused similar restorations. By contrast, in ODQ-treated preparations, the cell-permeable cGMP analog did not restore the augmentation by thymoquinone. The compound augmented the content (measured with ultra-high performance liquid chromatography-tandem mass spectrometry) of cIMP, but not that of cGMP; these increases in cIMP content were prevented by endothelium removal, L-NAME, and ODQ. The augmentation of contractions caused by thymoquinone was prevented in porcine arteries, but not in rat arteries, by 1-(5-isoquinolinylsulfonyl)homopiperazine dihydrochloride and trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Rho-kinase inhibitors); in the latter, but not in the former, it was reduced by 3,5-dichloro-N-[[(1α,5α,6-exo,6α)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-benzamide hydrochloride (T-type calcium channel inhibitor), demonstrating species/vascular bed differences in the impact of cIMP on calcium handling. Thymoquinone is the first pharmacological agent that causes endothelium-dependent augmentation of contractions of isolated arteries, which requires endothelium-derived NO and biased sGC activation, resulting in the augmented production of cIMP favoring the contractile process.

Restoring placental growth factor-soluble fms-like tyrosine kinase-1 balance reverses vascular hyper-reactivity and hypertension in pregnancy.

Preeclampsia (PE) is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanism. An imbalance between antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and angiogenic placental growth factor (PlGF) has been observed in PE, but the vascular targets and signaling pathways involved are unclear. We assessed the extent of sFlt-1/PlGF imbalance and vascular dysfunction in a rat model of HTN-Preg produced by reduction of uteroplacental perfusion pressure (RUPP), and tested whether inducing a comparable sFlt-1/PlGF imbalance by infusing sFlt-1 (10 μg·kg(-1)·day(-1)) in day 14 pregnant (Preg) rats cause similar increases in blood pressure (BP) and vascular reactivity. Using these guiding measurements, we then tested whether restoring sFlt-1/PlGF balance by infusing PIGF (20 μg·kg(-1)·day(-1)) in RUPP rats would improve BP and vascular function. On gestational day 19, BP was in Preg+sFlt-1 and RUPP > Preg, and in RUPP+PlGF < RUPP rats. Plasma sFlt-1/PlGF ratio was increased in Preg+sFlt-1, and RUPP and was reduced in RUPP+PlGF rats. In isolated endothelium-intact aorta, carotid, mesenteric, and renal artery, phenylephrine (Phe)- and high KCl-induced contraction was in Preg+sFlt-1 and RUPP > Preg, and in RUPP+PlGF < RUPP. The differences in vascular reactivity to Phe and KCl between groups were less apparent in vessels treated with the nitric oxide synthase (NOS) inhibitor l-NAME or guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or endothelium-denuded, suggesting changes in endothelial NO-cGMP pathway. In Phe precontracted vessels, ACh-induced relaxation was in Preg+sFlt-1 and RUPP < Preg, and in RUPP+PlGF > RUPP, and was blocked by N(ω)-nitro-l-arginine methyl ester (l-NAME) or ODQ treatment or endothelium removal. Western blots revealed that aortic total endothelial NOS (eNOS) and activated phosphorylated-eNOS were in Preg+sFlt-1 and RUPP < Preg and in RUPP+PlGF > RUPP. ACh-induced vascular nitrate/nitrite production was in Preg+sFlt-1 and RUPP < Preg, and in RUPP+PlGF > RUPP. Vascular relaxation to the exogenous NO donor sodium nitroprusside was not different among groups. Thus, a tilt in the angiogenic balance toward anti-angiogenic sFlt-1 is associated with decreased vascular relaxation and increased vasoconstriction and BP. Restoring the angiogenic/antiangiogenic balance using PlGF enhances endothelial NO-cGMP vascular relaxation and decreases vasoconstriction and BP in HTN-Preg rats and could offer a new approach in the management of PE.

Characterization and Antihypertensive Effect of the Complex of (-)-β- pinene in β-cyclodextrin.

This work aimed to characterize and evaluate the antihypertensive effect of the (-)-β-pinene/β-cyclodextrin (βP/β-CD) complex. The complex was prepared through physical mixture and slurry complexation methods and was analyzed through differential scanning calorimetry, thermogravimetry/derivative thermogravimetry, fourier transform infrared spectroscopy, diffraction X-ray, docking and scanning electron microscopy. Normotensive or L-NAME-induced hypertensive rats were used in pharmacological experiments. Mean arterial pressure (MAP) was determined with direct blood pressure measurements from the abdominal aorta. The drugs were orally administrated and their effects were recorded during 48 hours. Vascular effects of βP were evaluated in isolated ring of mesenteric artery. The physicochemical characterization showed βP/β-CD complex formation. In hypertensive rats (MAP = 156±16 mmHg), the complex, but not βP alone, promoted hypotension at 36 and 48 hours after administration (MAP = 124±3 and 110±5 mmHg, respectively). In arterial rings, βP vasorelaxed rings precontracted with phenylephrine (Emax = 105±6%), which was not changed after the removal of the vascular endothelium (Emax = 108±4%), after the pre-contraction with KCl 80 mM (Emax = 107±8%) or S(-)-BayK8644 (Emax = 107±5%), or after incubation with TEA (Emax = 113±4%). Finally, βP inhibited CaCl2- and sodium-orthovanadate-induced contractions. In conclusion, the slurry complexation method was the best among them. Pharmacological results demonstrated that the complex promoted antihypertensive effect. Furthermore, βP induced endothelium- independent vasorelaxation possibly caused by the inhibition of the Ca2+ influx through L-type Ca2+ channel associated to a decrease in calcium sensitivity.

Adenosine relaxation in isolated rat aortic rings and possible roles of smooth muscle Kv channels, KATP channels and A2a receptors.

BackgroundAn area of ongoing controversy is the role adenosine to regulate vascular tone in conduit vessels that regulate compliance, and the role of nitric oxide (NO), potassium channels and receptor subtypes involved. The aim of our study was to investigate adenosine relaxation in rat thoracic aortic rings, and the effect of inhibitors of NO, prostanoids, Kv, KATP channels, and A2a and A2b receptors.MethodsAortic rings were freshly harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs-Henseleit solution, 11 mM glucose, pH 7.4, 37 °C. Isolated rings were pre-contracted sub-maximally with 0.3 μM norepinephrine (NE), and the effect of increasing concentrations of adenosine (1 to 1000 μM) were examined. The drugs L-NAME, indomethacin, 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate, ouabain, 8-(3-chlorostyryl) caffeine and PSB-0788 were examined in intact and denuded rings. Rings were tested for viability after each experiment.ResultsAdenosine induced a dose-dependent, triphasic relaxation response, and the mechanical removal of the endothelium significantly deceased adenosine relaxation above 10 μM. Interestingly, endothelial removal significantly decreased the responsiveness (defined as % relaxation per μM adenosine) by two-thirds between 10 and 100 μM, but not in the lower (1–10 μM) or higher (>100 μM) ranges. In intact rings, L-NAME significantly reduced relaxation, but not indomethacin. Antagonists of voltage-dependent Kv (4-AP), sarcolemma KATP (glibenclamide) and mitochondrial KATP channels (5-HD) led to significant reductions in relaxation in both intact and denuded rings, with ouabain having little or no effect. Adenosine-induced relaxation appeared to involve the A2a receptor, but not the A2b subtype.ConclusionsIt was concluded that adenosine relaxation in NE-precontracted rat aortic rings was triphasic and endothelium-dependent above 10 μM, and relaxation involved endothelial nitric oxide (not prostanoids) and a complex interplay between smooth muscle A2a subtype and voltage-dependent Kv, SarcKATP and MitoKATP channels. The possible in vivo significance of the regulation of arterial compliance to left ventricular function coupling is discussed.