Cross-talk with β2-adrenoceptors enhances ligand affinity properties from endothelial alpha1D-adrenoceptors that mediates carotid relaxation

Objectives Our main objectives were to investigate the affinity properties of endothelial and muscular α1D-adrenoceptors and to characterize the cross-talk between endothelial α1D- adrenoceptors and β2-adrenoceptors in rat carotid. Methods Relaxation and contraction concentration-response curves for phenylephrine (α1-adrenergic agonist) were obtained in carotid rings in absence or presence of increasing concentrations of BMY7378 (α 1D-adrenergic antagonist), combined or not with increasing concentration of ICI-118,551 (β2-adrenergic antagonist). Schild analysis was used to estimate the affinity constant from pA2 values of BMY7378. Key Findings BMY7378 produced an unsurmountable antagonism on phenylephrine-induced relaxation but a surmountable antagonism on phenylephrine-induced contraction. BMY7378 potency was higher in inhibiting the relaxation than the contraction induced by phenylephrine because the rightward shifts induced by BMY7378 were greater in the relaxation. The apparent pA 2 value for BMY7378 in phenylephrine-induced relaxation was greater than in contraction. When combined with ICI-118,551, BMY7378 yielded a surmountable antagonism on phenylephrine-induced relaxation and presented a pA2 value similar to that obtained in phenylephrine-induced contraction. Conclusions Endothelial α1D-adrenoceptors, which mediates rat carotid relaxation, present high ligand affinity because of the cross-talk with β2-adrenoceptors, which explains the higher potency of phenylephrine in inducing relaxation than contraction and the atypical unsurmountable antagonism produced by BMY7378 on phenylephrine-induced relaxation. © 2013 Royal Pharmaceutical Society.

Investigação da participação de adrenoceptores α1A e α1D na contração da cauda de epididímo de rato e sua androgênica e estrogênica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Renal and vascular effects of Crotalus durissus cumanensis venom and its crotoxin fraction

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The adipokine chemerin augments vascular reactivity to contractile stimuli via activation of the MEK-ERK1/2 pathway

Aims: Cytokines interfere with signaling pathways and mediators of vascular contraction. Endothelin-1 (ET-1) plays a major role on vascular dysfunction in conditions characterized by increased circulating levels of adipokines. In the present study we tested the hypothesis that the adipokine chemerin increases vascular contractile responses via activation of ET-1/ET-1 receptors-mediated pathways. Main methods: Male, 10-12 week-old Wistar rats were used. Endothelium-intact and endothelium-denuded aortic rings were incubated with chemerin (0.5 ng/mL or 5 ng/mL, for 1 or 24 h), and isometric contraction was recorded. Protein expression was determined by Western blotting. Key findings: Constrictor responses to phenylephrine (PE) and ET-1 were increased in vessels treated for 1 h with chemerin. Chemerin incubation for 24 h decreased PE contractile response whereas it increased the sensitivity to ET-1. Endothelium removal significantly potentiated chemerin effects on vascular contractile responses to PE and ET-1. Incubation with either an ERK1/2 inhibitor (PD98059) or ETA antagonist (BQ123) abolished chemerin effects on PE- and ET-1-induced vasoconstriction. Phosphorylation of MEK1/2 and ERK1/2 was significantly increased in vessels treated with chemerin for 1 and 24 h. Phosphorylation of these proteins was further increased in vessels incubated with ET-1 plus chemerin. ET-1 increased MEK1/2, ERK1/2 and MKP1 protein expression to values observed in vessels treated with chemerin. Significance: Chemerin increases contractile responses to PE and ET-1 via ERK1/2 activation. Our study contributes to a better understanding of the mechanisms by which the adipose tissue affects vascular function and, consequently, the vascular alterations present in obesity and related diseases. (c) 2012 Elsevier Inc. All rights reserved.

In vivo hydroquinone exposure causes tracheal hyperresponsiveness due to TNF secretion by epithelial cells

Hydroquinone (HQ) is the main oxidative substance in cigarette smoke and a toxic product of benzene biotransformation. Although the respiratory tract is an inlet pathway of HQ exposure, its effect on airway muscle responsiveness has not been assessed. We thus investigated the effects of low dose in vivo HQ-exposure on tracheal responsiveness to a muscarinic receptor agonist. Male Swiss mice were exposed to aerosolised 5% ethanol/saline solution (HQ vehicle; control) or 0.04 ppm HQ (1 h/day for 5 days) and tracheal rings were collected 1 h after the last exposure. HQ exposure caused tracheal hyper-responsiveness to methacholine (MCh), which was abolished by mechanical removal of the epithelium. This hyperresponsiveness was not dependent on neutrophil infiltration, but on tumour necrosis factor (TNF) secretion by epithelial cells. This conclusion was based on the following data: (1) trachea from HQ-exposed mice presented a higher amount of TNF, which was abrogated following removal of the epithelium; (2) the trachea hyperresponsiveness and TNF levels were attenuated by in vivo chlorpromazine (CPZ) treatment, an inhibitor of TNF synthesis. The involvement of HQ-induced TNF secretion in trachea mast cell degranulation was also demonstrated by the partial reversion of tracheal hyperresponsiveness in sodium cromoglicate-treated animals, and the in vivo HQ-exposure-induced degranulation of trachea connective tissue and mucosal mast cells, which was reversed by CPZ treatment. Our data show that in vivo HQ exposure indirectly exacerbates the parasympathetic-induced contraction of airway smooth muscle cells, mediated by TNF secreted by tracheal epithelial cells, clearly showing the link between environmental HQ exposure and the reactivity of airways. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6 h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1 alpha) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS. (C) 2012 Elsevier Inc. All rights reserved.

Increased vascular contractility and oxidative stress in beta(2)-adrenoceptor knockout mice: the role of NADPH oxidase

Background/Aims: beta(2)-adrenoceptor (beta(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal beta(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional beta(2)-AR (beta 2KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from beta 2KO and wild-type mice (WT) were studied. beta 2KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between beta 2KO and WT mice. Basal NO availability was reduced in aortas from beta 2KO mice. Incubation of beta 2KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. beta 2KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in beta 2KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of beta 2KO mice. This study extends the knowledge of the relevance of the endogenous activity of beta(2)-AR to the maintenance of the vascular physiology. Copyright (C) 2012 S. Karger AG, Basel

Alterations in vasoconstrictor responses to the endothelium-derived contracting factor uridine adenosine tetraphosphate are region specific in DOCA-salt hypertensive rats

Uridine adenosine tetraphosphate (Up(4)A) has been recently identified as a novel and potent endothelium-derived contracting factor and contains both purine and pyrimidine moieties, which activate purinergic P2X and P2Y receptors. The present study was designed to compare contractile responses to Up(4)A and other nucleotides such as ATP (P2X/P2Y agonist), UTP (P2Y(2)/P2Y(4) agonist), UDP (P2Y(6) agonist), and alpha,beta-methylene ATP (P2X(1) agonist) in different vascular regions [thoracic aorta, basilar, small mesenteric, and femoral arteries] from deoxycorticosterone acetate-salt (DOCA-salt) and control rats. In DOCA-salt rats [vs. control uninephrectomized (Uni) rats]: (1) in thoracic aorta, Up(4)A-, ATP-, and UP-induced contractions were unchanged; (2) in basilar artery, Up(4)A-, ATP-, UTP- and UDP-induced contractions were increased, and expression for P2X(1), but not P2Y(2) or P2Y(6) was decreased; (3) in small mesenteric artery, Up(4)A-induced contraction was decreased and UDP-induced contraction was increased; expression of P2Y(2) and P2X(1) was decreased whereas P2Y(6) expression was increased; (4) in femoral artery, Up(4)A-. UTP-, and UDP-induced contractions were increased, but expression of P2Y(2), P2Y(6) and P2X(1) was unchanged. The alpha,beta-methylene ATP-induced contraction was bell-shaped and the maximal contraction was reached at a lower concentration in basilar and mesenteric arteries from Uni rats, compared to arteries from DOCA-salt rats. These results suggest that Up(4)A-induced contraction is heterogenously affected among various vascular beds in arterial hypertension. P2Y receptor activation may contribute to enhancement of Up(4)A-induced contraction in basilar and femoral arteries. These changes in vascular reactivity to Up(4)A may be adaptive to the vascular alterations produced by hypertension. (C) 2011 Elsevier Ltd. All rights reserved.

Effects of maternal PETN treatment of spontaneously hypertensive rats on blood pressure in the offspring

Pentaerithrityltetranitrat (PETN) ist ein organisches Nitrat und wird in der Klinik zur Behandlung der Angina Pectoris eingesetzt. PETN hat, wenn direkt verabreicht, kaum Wirkung auf den Blutdruck. Diese Arbeit wurde konzipiert, um einen potentiellen „perinatalen Programmierung“-Effekt von PETN in spontan-hypertensiven Ratten (SHR), einem Rattenmodel der genetischen Hypertonie, zu testen. Die F0-Elterntiere wurden mit PETN (50 mg/kg/Tag) während der Schwangerschaft und der Laktation behandelt; die F1-Nachkommen bekamen nach der Ablaktation normales Haltungsfutter. Der Blutdruck wurde an den Nachkommen vom 3. Monat bis zum 8. Monat nach der Geburt gemessen. Maternale PETN-Behandlung hatte kaum Wirkung auf den Blutdruck in den männlichen SHR-Nachkommen. Dagegen zeigten die weiblichen Nachkommen der PETN-Behandlungsgruppe eine persistente Reduktion des Blutdrucks. Der systolische Blutdruck war in den weiblichen Nachkommen in der PETN-Gruppe etwa 13 mmHg niedriger im 4. Monat und etwa 10 mmHg niedriger im 8. Monat als in den Kontrolltieren. Dieser lang-anhaltende Effekt ging mit einer substanziellen Änderung der Genexpression einher, die auch beim 8. Monat noch nachzuweisen war. In den Aorten der weiblichen F1-Nachkommen wurde Veränderungen an Genexpression der α-adrenergen Rezeptoren sowie Endothelin-Rezeptoren festgestellt, die aber funktionell von minimaler Bedeutung für die PETN-Wirkung waren. Hingegen war eine klare Rolle des StickstoffmoNOXid (NO) zu sehen. Maternale PETN-Behandlung führte zur Heraufregulation der endothelialen NO-Synthase (eNOS) und der GTP-Cyclohydrolase I (GCH-1). GCH-1 ist für die Biosynthese des Tetrahydrobiopterins, eines essentiellen eNOS-Kofaktors, entscheidend, und dadurch auch für die eNOS-Funktionalität. Zusätzlich wurden auch anti-oxidative Enzyme wie die mitochondriale Superoxid-Dismutase (SOD2), die Glutathion-Peroxidase 1 (GPx1) und die Hem-Oxygenase 1 (HO-1) heraufreguliert, und die Superoxid-produzierende NADPH-Oxidase NOX1 herunterreguliert. Dies kann zur Verminderung vom oxidativen Stress und Erhöhung der NO-Bioverfügbarkeit führen. Letztlich wurde auch ~ 74 ~ die Sirtuin 1 (SIRT1) durch maternale PETN-Behandlung heraufreguliert, die auch zur Heraufregulation der SOD2, GPx1 und eNOS beitragen kann. Im Organbad-Experiment wurde die Acetylcholin-induzierte, Endothel-abhängige Vasodilatation in der Aorta der weiblichen Nachkommen der PETN-Gruppe verstärkt. Diese verbesserte Endothelfunktion, was vermutlich aus der Genexpressionsänderung resultiert, stellt sehr wahrscheinlich einen Schlüsselmechanismus der Blutdrucksenkung in den Nachkommen der PETN-behandelten F0-Tiere dar.

Intraindividual comparison of human radial and internal thoracic arteries in vitro and the effect of preoperative calcium blocker therapy

The patency rate of radial artery (RA) conduits is considerably lower than that of internal thoracic artery (ITA) grafts and the evidence suggests that this is due to a clinically suspected higher incidence of vasospasm. The aim of this study was, therefore, to compare intraindividually the pharmacological reactivity of RA with that of ITA. Both RA and ITA were taken from the same patients and investigated in parallel. Changes in tone were monitored isometrically on ring preparations from both arteries in organ baths with modified Krebs-Henseleit solution containing 1.25 mm calcium chloride at 1 g passive preload. In intraindividual comparisons maximal receptor-mediated contractile responses to noradrenaline and endothelin-1 and endothelium-dependent acetylcholine-induced relaxant responses revealed no differences between both arteries. By contrast, depolarization-induced contractions to potassium chloride (KCl) appeared to be significantly higher in RA than in ITA. Further analysis, however, revealed that this difference was due to preoperative calcium entry blocker (Ca(2+)eB) therapy. Compared with control tissues, maximal responses to KCl were significantly attenuated in the ITA but unchanged in RA when arteries were obtained from patients with preoperative Ca(2+)eB therapy. The present results suggested that functional responses to pharmacological stimuli of both RA and ITA were quite similar. Preoperative Ca(2+)eB therapy, however, attenuated markedly responses to KCl of the ITA leaving those of RA unchanged. These results, demonstrating a lower sensitivity to Ca(2+)eB of RA, therefore suggested that in addition to Ca(2+)eB other classes of drug may be more effective at reducing the propensity of RA conduits to vasospasm.

Rapamycin impairs endothelial cell function in human internal thoracic arteries.

BACKGROUND Definitive fate of the coronary endothelium after implantation of a drug-eluting stent remains unclear, but evidence has accumulated that treatment with rapamycin-eluting stents impairs endothelial function in human coronary arteries. The aim of our study was to demonstrate this phenomenon on functional, morphological and biochemical level in human internal thoracic arteries (ITA) serving as coronary artery model. METHODS After exposure to rapamycin for 20 h, functional activity of ITA rings was investigated using the organ bath technique. Morphological analysis was performed by scanning electron microscopy and evaluated by two independent observers in blinded fashion. For measurement of endothelial nitric oxide synthase (eNOS) release, mammalian target of rapamycin (mTOR) and protein kinase B (PKB) (Akt) activation, Western blotting on human mammary epithelial cells-1 and on ITA homogenates was performed. RESULTS Comparison of the acetylcholine-induced relaxation revealed a significant concentration-dependent decrease to 66 ± 7 % and 36 ± 7 % (mean ± SEM) after 20-h incubation with 1 and 10 μM rapamycin. Electron microscopic evaluation of the endothelial layer showed no differences between controls and samples exposed to 10 μM rapamycin. Western blots after 20-h incubation with rapamycin (10 nM-1 μM) revealed a significant and concentration-dependent reduction of p (Ser 1177)-eNOS (down to 38 ± 8 %) in human mammary epithelial cells (Hmec)-1. Furthermore, 1 μM rapamycin significantly reduced activation of p (Ser2481)-mTOR (58 ± 11 %), p (Ser2481)-mTOR (23 ± 4 %) and p (Ser473)-Akt (38 ± 6 %) in ITA homogenates leaving Akt protein levels unchanged. CONCLUSIONS The present data suggests that 20-h exposure of ITA rings to rapamycin reduces endothelium-mediated relaxation through down-regulation of Akt-phosphorylation via the mTOR signalling axis within the ITA tissue without injuring the endothelial cell layer.

Prevention of vascular dysfunction and arterial hypertension in mice generated by assisted reproductive technologies by addition of melatonin to culture media.

Assisted reproductive technologies (ART) induce vascular dysfunction in humans and mice. In mice, ART-induced vascular dysfunction is related to epigenetic alteration of the endothelial nitric oxide synthase (eNOS) gene, resulting in decreased vascular eNOS expression and nitrite/nitrate synthesis. Melatonin is involved in epigenetic regulation, and its administration to sterile women improves the success rate of ART. We hypothesized that addition of melatonin to culture media may prevent ART-induced epigenetic and cardiovascular alterations in mice. We, therefore, assessed mesenteric-artery responses to acetylcholine and arterial blood pressure, together with DNA methylation of the eNOS gene promoter in vascular tissue and nitric oxide plasma concentration in 12-wk-old ART mice generated with and without addition of melatonin to culture media and in control mice. As expected, acetylcholine-induced mesenteric-artery dilation was impaired (P = 0.008 vs. control) and mean arterial blood pressure increased (109.5 ± 3.8 vs. 104.0 ± 4.7 mmHg, P = 0.002, ART vs. control) in ART compared with control mice. These alterations were associated with altered DNA methylation of the eNOS gene promoter (P < 0.001 vs. control) and decreased plasma nitric oxide concentration (10.1 ± 11.1 vs. 29.5 ± 8.0 μM) (P < 0.001 ART vs. control). Addition of melatonin (10(-6) M) to culture media prevented eNOS dysmethylation (P = 0.005, vs. ART + vehicle), normalized nitric oxide plasma concentration (23.1 ± 14.6 μM, P = 0.002 vs. ART + vehicle) and mesentery-artery responsiveness to acetylcholine (P < 0.008 vs. ART + vehicle), and prevented arterial hypertension (104.6 ± 3.4 mmHg, P < 0.003 vs. ART + vehicle). These findings provide proof of principle that modification of culture media prevents ART-induced vascular dysfunction. We speculate that this approach will also allow preventing ART-induced premature atherosclerosis in humans.

Inhibition of RhoA Translocation and Calcium Sensitization by In Vivo ADP-Ribosylation with the Chimeric Toxin DC3B

Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10−6 M, 48 h; Aullo et al., 1993; Boquet et al. 1995) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)γS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPγS-induced translocation of cytosolic RhoA (Gong et al., 1997a) to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPγS, which prevents immunoprecipitation of non-ADP–ribosylated RhoA. Dissociation of cytosolic RhoA–rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.

What determines the folding of the chromatin fiber?

In this review, we attempt to summarize, in a critical manner, what is currently known about the processes of condensation and decondensation of chromatin fibers. We begin with a critical analysis of the possible mechanisms for condensation, considering both old and new evidence as to whether the linker DNA between nucleosomes bends or remains straight in the condensed structure. Concluding that the preponderance of evidence is for straight linkers, we ask what other fundamental process might allow condensation, and argue that there is evidence for linker histone-induced contraction of the internucleosome angle, as salt concentration is raised toward physiological levels. We also ask how certain specific regions of chromatin can become decondensed, even at physiological salt concentration, to allow transcription. We consider linker histone depletion and acetylation of the core histone tails, as possible mechanisms. On the basis of recent evidence, we suggest a unified model linking targeted acetylation of specific genomic regions to linker histone depletion, with unfolding of the condensed fiber as a consequence.

Substance P preferentially inhibits large conductance nicotinic ACh receptor channels in rat intracardiac ganglion neurons

The effects of substance P (SP) on nicotinic acetylcholine (ACh)-evoked currents were investigated in parasympathetic neurons dissociated from neonatal rat intracardiac ganglia using standard whole cell, perforated patch, and outside-out recording configurations of the patch-clamp technique. Focal application of SP onto the soma reversibly decreased the peak amplitude of the ACh-evoked current with half-maximal inhibition occurring at 45 mu M and complete block at 300 mu M SP. Whole cell current-voltage (I-V) relationships obtained in the absence and presence of SP indicate that the block of ACh-evoked currents by SP is voltage independent. The rate of decay of ACh-evoked currents was increased sixfold in the presence of SP (100 mu M), suggesting that SP may increase the rate of receptor desensitization. SP-induced inhibition of ACh-evoked currents was observed following cell dialysis and in the presence of either 1 mM 8-Br-cAMP, a membrane-permeant cAMP analogue, 5 mu M H-7, a protein kinase C inhibitor, or 2 mM intracellular AMP-PNP, a nonhydrolyzable ATP analogue. These data suggest that a diffusible cytosolic second messenger is unlikely to mediate SP inhibition of neuronal nicotinic ACh receptor (nAChR) channels. Activation of nAChR channels in outside-out membrane patches by either ACh (3 mu M) or cytisine (3 mu M) indicates the presence of at least three distinct conductances (20, 35, and 47 pS) in rat intracardiac neurons. In the presence of 3 mu M SP, the large conductance nAChR channels are preferentially inhibited. The open probabilities of the large conductance classes activated by either ACh or cytisine were reversibly decreased by 10- to 30-fold in the presence of SP. The single-channel conductances were unchanged, and mean apparent channel open times for the large conductance nAChR channels only were slightly decreased by SP. Given that individual parasympathetic neurons of rat intracardiac ganglia express a heterogeneous population of nAChR subunits represented by the different conductance levels, SP appears to preferentially inhibit those combinations of nAChR subunits that form the large conductance nAChR channels. Since ACh is the principal neurotransmitter of extrinsic (vagal) innervation of the mammalian heart, SP may play an important role in modulating autonomic control of the heart.