Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3 beta-ol in the isolated rat aorta

Pimarane-type diterpenes were described to exert antispasmodic and relaxant activities. Based on this observation we hypothesized that the diterpene ent-8(14),15-pimaradien-3 beta-ol (PA-3 beta-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3 beta-ol. Vascular reactivity experiments, using standard muscle bath procedures, were performed in isolated aortic rings from male Wistar rats. Cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3AM. PA-3 beta-ol (10, 50 and 100 mu mol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3 beta-ol also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 mu mol/l). PA-3 beta-ol (1-300 mu mol/l) concentration dependently relaxed phenylephrine-pre-contracted rings with intact or denuded endothelium. The diterpene also relaxed KCl-pre-contracted rings with intact or denuded endothelium. Moreover, Ca(2+) mobilization study showed that PA-3 beta-ol (100 mu mol/l) and verapamil (1 mu mol/l) inhibited the increase in Ca(2+)-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 mu mol/l) or KCl (60 mmol/l). Pre-incubation of intact or denuded aortic rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mu mol/l) and 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 1 mu mol/l) produced a rightward displacement of the PA-3 beta-ol concentration-response curves. On the other hand, 7-nitroindazole (100 mu mol/l), 1400 W (1 mu mol/l), indomethacin (10 mu mol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3 beta-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3 beta-ol involve extracellular Ca(2+) influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway. (C) 2009 Elsevier B.V. All rights reserved.

Relaxation evoked by extracellular Ca(2+) in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca(2+) channell

The perivascular nerve network expresses a Ca(2+) receptor that is activated by high extracellular Ca(2+) concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca(2+)-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca(2+) concentration. We also studied the role of the endothelium on the Ca(2+)-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenise-arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K(+) channels and L-type Ca(2+) channels on the Ca(2+)-induced relaxation were evaluated. We have observed that the Ca(2+)-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF(2 alpha) receptor antagonist) enhance the relaxing response to Ca(2+). This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca(2+)-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca(2+). The relaxing response to Call was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca(2+) channels and sarcoplasmic reticulum in this response. (c) 2008 Elsevier Inc. All rights reserved.

Role of endothelium in angiotensin II formation by the rat aorta and mesenteric arterial bed

We investigated the angiotensin II (Ang II)-generating system by analyzing the vasoconstrictor effect of Ang II, angiotensin I (Ang I), and tetradecapeptide (TDP) renin substrate in the absence and presence of inhibitors of the renin-angiotensin system in isolated rat aortic rings and mesenteric arterial beds with and without functional endothelium. Ang II, Ang I, and TDP elicited a dose-dependent vasoconstrictor effect in both vascular preparations that was completely blocked by the Ang II receptor antagonist saralasin (50 nM). The angiotensin converting enzyme (ACE) inhibitor captopril (36 µM) completely inhibited the vasoconstrictor effect elicited by Ang I and TDP in aortic rings without affecting that of Ang II. In contrast, captopril (36 µM) significantly reduced (80-90%) the response to bolus injection of Ang I, without affecting those to Ang II and TDP in mesenteric arteries. Mechanical removal of the endothelium greatly potentiated (70-95%) the vasoconstrictor response to Ang II, Ang I, and TDP in aortic rings while these responses were unaffected by the removal of the endothelium of mesenteric arteries with sodium deoxycholate infusion. In addition, endothelium disruption did not change the pattern of response elicited by these peptides in the presence of captopril. These findings indicate that the endothelium may not be essential for Ang II formation in rat mesenteric arteries and aorta, but it may modulate the response to Ang II. Although Ang II formation from Ang I is essentially dependent on ACE in both vessels, our results suggest the existence of an alternative pathway in the mesenteric arterial bed that may play an important role in Ang II generation from TDP in resistance but not in large vessels during ACE inhibition

Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication

Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of a1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal (»75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 µM), prostaglandin F2a (PGF2a; 1 µM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 µM and 500 µM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 ± 6% (N = 26 rings) to 57 ± 7% (N = 26 rings) and 34.9 ± 6% (N = 13 rings), respectively (P<0.05), and for PGF2a from a control response of 75 ± 10% (N = 16 rings) to 52 ± 8% (N = 19 rings) and 25.9 ± 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 µM and 500 µM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 ± 5.0% for the control response (N = 38 rings), 59 ± 6.0% in the presence of 200 µM heptanol (N = 17 rings), and 70 ± 6.0% in the presence of 500 µM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 ± 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent.

Vasorelaxant effects of the potassium channel opener SR 47063 on the isolated human saphenous vein and rat aorta

The vasorelaxant effects of SR 47063 (4-(2-cyanimino-1,2-dihydropyrid-1-yl)-2,2-dimethyl-6-nitrochromene), a new K+-channel opener structurally related to levcromakalim, were examined in isolated human saphenous vein (HSV) and rat aorta (RA). HSV or RA rings were precontracted with either KCl or noradrenaline and cumulative relaxant concentration-response curves were obtained for SR 47063 (0.1 nM to 1 µM) in the presence or absence of 3 µM glibenclamide. SR 47063 potently relaxed HSV and RA precontracted with 20 mM (but not 60 mM) KCl or 10 µM noradrenaline in a concentration-dependent manner, showing slightly greater activity in the aorta. The potency of the effect of SR 47063 on HSV and RA was 12- and 58-fold greater, respectively, than that reported for the structurally related K+-channel opener levcromakalim. The vasorelaxant action of SR 47063 in both blood vessels was strongly inhibited by 3 µM glibenclamide, consistent with a mechanism of action involving ATP-dependent K+-channels.

Calcium antagonism and the vasorelaxation of the rat aorta induced by rotundifolone

The vasorelaxing activity of rotundifolone (ROT), a major constituent (63.5%) of the essential oil of Mentha x villosa, was tested in male Wistar rats (300-350 g). In isolated rat aortic rings, increasing ROT concentrations (0.3, 1, 10, 100, 300, and 500 µg/ml) inhibited the contractile effects of 1 µM phenylephrine and of 80 or 30 mM KCl (IC50 values, reported as means ± SEM = 184 ± 6, 185 ± 3 and 188 ± 19 µg/ml, N = 6, respectively). In aortic rings pre-contracted with 1 µM phenylephrine, the smooth muscle-relaxant activity of ROT was inhibited by removal of the vascular endothelium (IC50 value = 235 ± 7 µg/ml, N = 6). Furthermore, ROT inhibited (pD2 = 6.04, N = 6) the CaCl2-induced contraction in depolarizing medium in a concentration-dependent manner. In Ca2+-free solution, ROT inhibited 1 µM phenylephrine-induced contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (20 mM). In conclusion, in the present study we have shown that ROT produces an endothelium-independent vasorelaxing effect in the rat aorta. The results further indicated that in the rat aorta ROT is able to induce vasorelaxation, at least in part, by inhibiting both: a) voltage-dependent Ca² channels, and b) intracellular Ca2+ release selectively due to inositol 1,4,5-triphosphate activation. Additional studies are required to elucidate the mechanisms underlying ROT-induced relaxation.

The methyl ester of rosuvastatin elicited an endothelium-independent and 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent relaxant effect in rat aorta

The relaxant effect of the methyl ester of rosuvastatin was evaluated on aortic rings from male Wistar rats (250-300 g, 6 rats for each experimental group) with and without endothelium precontracted with 1.0 µM phenylephrine. The methyl ester presented a slightly greater potency than rosuvastatin in relaxing aortic rings, with log IC50 values of -6.88 and -6.07 M, respectively. Unlike rosuvastatin, the effect of its methyl ester was endothelium-independent. Pretreatment with 10 µM indomethacin did not inhibit, and pretreatment with 1 mM mevalonate only modestly inhibited the relaxant effect of the methyl ester. Nω-nitro-L-arginine methyl ester (L-NAME, 10 µM), the selective nitric oxide-2 (NO-2) inhibitor 1400 W (10 µM), tetraethylammonium (TEA, 10 mM), and cycloheximide (10 µM) partially inhibited the relaxant effect of the methyl ester on endothelium-denuded aortic rings. However, the combination of TEA plus either L-NAME or cycloheximide completely inhibited the relaxant effect. Inducible NO synthase (NOS-2) was only present in endothelium-denuded aortic rings, as demonstrated by immunoblot with methyl ester-treated rings. In conclusion, whereas rosuvastatin was associated with a relaxant effect dependent on endothelium and hydroxymethylglutaryl coenzyme A reductase in rat aorta, the methyl ester of rosuvastatin exhibited an endothelium-independent and only slightly hydroxymethylglutaryl coenzyme A reductase-dependent relaxant effect. Both NO produced by NOS-2 and K+ channels are involved in the relaxant effect of the methyl ester of rosuvastatin.

Effects of isoproterenol treatment for 7 days on inflammatory mediators in the rat aorta

The aim of the present study was to evaluate the effect of overstimulation of beta-adrenoceptors on vascular inflammatory mediators. Wistar rats were treated with the beta-adrenoceptor agonist isoproterenol (0.3 mg(.)kg(-1.)day(-1) sc) or vehicle (control) for 7 days. At the end of treatment, the right carotid artery was catheterized for arterial and left ventricular (LV) hemodynamic evaluation. Isoproterenol treatment increased LV weight but did not change hemodynamic parameters. Aortic mRNA and protein expression were quantified by real-time RT-PCR and Western blot analysis, respectively. Isoproterenol enhanced aortic mRNA and protein expression of IL-1 beta (124% and 125%) and IL-6 (231% and 40%) compared with controls but did not change TNF-alpha expression. The nuclear-to-cytoplasmatic protein expression ration of the NF-beta B p65 subunit was increased by isoproterenol treatment (51%); in addition, it reduced the cytoplasmatic expression of I kappa B-alpha (52%) in aortas. An electrophoretic mobility shift assay was performed using the aorta, and increased NF-kappa B DNA binding (31%) was observed in isoproterenol-treated rats compared with controls (P < 0.05). Isoproterenol treatment increased phenylephrine-induced contraction in aortic rigs (P < 0.05), which was significantly reduced by superoxide dismutase (150 U/ml) and sodium salicylate (5 mM). Cotreatment with thalidomide (150 mg(.)kg(-1.)day(-1) for 7 days) also reduced hyperreactivity to phenylephrine induced by isoproterenol. In conclusion, overstimulation of beta-adrenoceptors increased proinflammatory cytokines and upregulated NF-kappa B in the rat aorta. Moreover, local oxidative stress and the proinflammatory state seem to play key roles in the altered vascular reactivity of the rat aorta induced by chronic beta-adrenergic stimulation.

Effects of castration on alpha(1)-adrenoceptor subtypes in the rat aorta

The expression of alpha(1)-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the alpha(1)-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the alpha(1B)/alpha(1D)-adrenoceptor alkylating agent chloroethylclonidine resulted in approximate to1600-fold rightward shift in the concentration-response curves to noradrenaline. The pA(2) values found for WB 4101, benoxathian (alpha(1A)-selective) and BMY 7378 (alpha(1D)-selective) indicate that alpha(1D)-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pK(B) estimated through the lowest effective concentrations of the alpha(1A)-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pK(B) estimated in aorta from control rats is consistent with the interaction with alpha(1D)-adrenoceptors (7.58 +/- 0.06), while that calculated in organs from control rats is consistent with alpha(1A)-adrenoceptors (8.76 +/- 0.09). These results suggest that castration induces plasticity in the alpha(1)-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline. (C) 2003 Elsevier B.V. All rights reserved.

Effect of nωnla, an inhibitor of No-synthase, on the release of tissue-plasminogem activator from rat aorta

Alterations in the synthesis or enhanced inactivation of nitric oxide (NO) and increase in fibrin deposition in the vascular bed lead to an imbalance that can induced intravascular coagulation. NO is produced through L-arginine pathway by constitutive and inducible nitric oxide synthase (NOS). The inducible isoform can be activated by cytokines such as tumor necrosis factor alfa. We evaluated NO-induced tissue-plasminogen activator (t-PA) release from isolated aortic segments of Wistar rats measuring the fibrinolytic activity in the fibrin plate. Inhibition of NO biossynthesis with Nω-nitro-L-arginine (NωNLA) significantly attenuated the fibrinolytic activity (FA) evoked by aortic segments of this group (GII) compared to the saline group (GI). The administration of L-arginine produced restoration of FA in this group (GIII) treated with NωNLA suggesting that t-PA arising from segments of rat aorta is influenced by NO.

Vasoconstrictor effect of Africanized honeybee (Apis mellifera L.) venom on rat aorta

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ayahuasca alters structural parameters of the rat aorta

Ayahuasca is a hallucinogenic brew traditionally used by Northwestern Amazonian indigenous groups for therapeutic purposes. It is prepared by the decoction of Banisteriopsis caapi with the leaves of Psychotria viridis. Banisteriopsis caapi contains β-carbolines that are inhibitors of monoamine oxidase and P. viris is rich in dimethyltryptamine, a 5-HT(1A/2A/2C) agonist. Acute ayahuasca administration produces moderate cardiovascular effects in healthy volunteers, but information regarding long-term use is lacking. This study investigated the effects of ayahuasca (2-4 mL/kg) in the rat aorta after acute and chronic (14 days) administration. Ayahuasca caused flattening and stretching of vascular smooth muscle cells and changes in the arrangement and distribution of collagen and elastic fibers. Chronic treatment with the higher dose significantly increased media thickness and the ratio of media thickness to lumen diameter. More research is needed on the cardiovascular function of long-term ayahuasca consumers.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Neural differentiation of rat aorta pericyte cells

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins beta 3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain. (C) 2011 International Society for Advancement of Cytometry

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

Aim: To investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta. Methods: The relaxation response to NaOH-induced extracellular alkalinization (7.4-8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10(-5) M), NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10(-7) M), 2,5-dimethylbenzimidazole (DMB, 2 x 10(-5) M) and methyl-B-cyclodextrin (10(-2) M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 mu M), in the presence and absence of DMB (2 x 10(-5) M). Results: The extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca(2+)/calmodulin and Na(+)/Ca(2+). exchanger (NCX), and partially blunted by the caveolae disassembly. Conclusions: These results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca(2+) concentration and activating the Ca(2+)/calmodulin-dependent NOS. In turn, NO is released promoting relaxation. (C) 2010 Elsevier Inc. All rights reserved.