Daniella Bonaventura

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca2+ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca2+ mobilization study shows that l-NAME inhibited the reduction of Ca2+-concentration in VSM cells and reduced the increase in Ca2+-concentration in endothelial cells induced by SNP. This enhancement in Ca2+-concentration in the endothelial cells is due to a voltage-dependent Ca2+ channels activation. The present findings indicate that the relaxation and [Ca2+]i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.

Pimaradienoic acid inhibits vascular contraction and induces hypotension in normotensive rats.

The present investigation was designed to investigate the effect of the diterpene ent‐pimara‐8(14),15‐dien‐19‐oic acid (pimaradienoic acid, PA) on smooth muscle extracellular Ca2+ influx. To this end, the effect of PA on phenylephrine‐ and KCl‐induced increases in cytosolic calcium concentration ([Ca2+]c), measured by the variation in the ratio of fluorescence intensities (R340/380 nm) of Fura‐2, was analysed. Whether bolus injection of PA could induce hypotensive responses in conscious normotensive rats was also evaluated. PA inhibited the contraction induced by phenylephrine (0.03 or 10 μmol L−1) and KCl (30 or 90 μmol L−1) in endothelium‐denuded rat aortic rings in a concentration dependent manner. Pre‐treatment with PA (10, 100, 200 μmol L−1) attenuated the contraction induced by CaCl2 (0.5 nmol L−1 or 2.5 μmol L−1) in denuded rat aorta exposed to Ca2+‐free medium containing phenylephrine (0.1 μmol L−1) or KCl (30 μmol L−1). Interestingly, the inhibitory effect displayed by PA on CaCl2‐induced contraction was more pronounced when KCl was used as the stimulant. Phenylephrine‐ and KCl‐induced increases in [Ca2+]c were inhibited by PA. Similarly, verapamil, a Ca2+‐channel blocker, also inhibited the increase in [Ca2+]c induced by either phenylephrine or KCl. Finally, bolus injection of PA (1–15 mg kg−1) produced a dose‐dependent decrease in mean arterial pressure in conscious normotensive rats. The results provide the first direct evidence that PA reduces vascular contractility by reducing extracellular Ca2+ influx through smooth muscle cellular membrane, a mechanism that could mediate the hypotensive response induced by this diterpene in normotensive rats.

Chronic Methionine Load-Induced Hyperhomocysteinemia Enhances Rat Carotid Responsiveness for Angiotensin II

The purpose of the present study was to investigate the effects of chronic methionine treatment on vascular smooth muscle contractility for angiotensin II (Ang II). Methionine at 0.1, 1 and 2 g/kg body weight was administered daily in the drinking water for 2, 4, 8 and 16 weeks. Rat carotid rings from control and treated animals were placed in an organ chamber containing Krebs solution. Concentration-response curves for Ang II and potassium chloride (KCl) were determined. Methionine-rich diet enhanced the plasmatic homocysteine concentration, and the magnitude of the contractile response to Ang II was increased in carotid rings from treated animals after 8 and 16 weeks. However, the treatment did not alter KCl-induced contraction. In another set of experiments, the rings were incubated with indomethacin and curves for Ang II were obtained. Exposure of the rings to indomethacin inhibited the enhancement in the contractile response to Ang II. The present findings show that chronic methionine treatment enhances homocysteine plasmatic concentration leading to an enhanced Ang-II-induced contraction, which appears to be related to the release of vasoconstrictor prostanoid(s).

Endothelium negatively modulates the vascular relaxation induced by nitric oxide donor, due to uncoupling NO synthase.

Nitrosyl ruthenium complexes have been characterized as nitric oxide (NO) donors that induce relaxation in the denuded rat aorta. There are some differences in their vascular relaxation mechanisms compared with sodium nitroprusside. This study investigates whether the endothelium could interfere with the [Ru(terpy)(bdq)NO](3+)-TERPY-induced vascular relaxation, by analyzing the maximal relaxation (Emax) and potency (pD(2)) of TERPY. Vascular reactivity experiments showed that the endothelium negatively modulates (pD(2): 6.17+/-0.07) the TERPY relaxation in intact rat aortic rings compared with the denuded rat aorta (pD(2): 6.65+/-0.07). This effect is abolished by a non-selective NO-synthase (NOS) inhibitor L-NAME (pD(2): 6.46+/-0.10), by the superoxide anion (O(2)(-)) scavenger TIRON (pD(2): 6.49+/-0.08), and by an NOS cofactor BH(4) (pD(2): 6.80+/-0.10). The selective dye for O(2)(-) (DHE) shows that TERPY enhances O(2)(-) concentration in isolated endothelial cells (intensity of fluorescence (IF):11258.00+/-317.75) compared with the basal concentration (IF: 7760.67+/-381.50), and this enhancement is blocked by L-NAME (IF: 8892.33+/-1074.41). Similar results were observed in vascular smooth muscle cells (concentration of superoxide after TERPY: 2.63+/-0.17% and after TERPY+L-NAME: -4.63+/-0.14%). Considering that TERPY could induce uncoupling NOS, thus producing O(2)(-), we have also investigated the involvement of prostanoids in the negative modulation of the endothelium. The non-selective cyclooxygenase (COX) inhibitor indomethacin and the selective tromboxane (TXA(2)) receptor antagonist SQ29548 reduce the effect of the endothelium on TERPY relaxation (pD(2) INDO: 6.80+/-0.17 and SQ29548: 6.85+/-0.15, respectively). However, a selective prostaglandin F(2alpha) receptor antagonist (AH6809) does not change the endothelium effect. Moreover, TERPY enhances the concentration of TXA(2) stable metabolite (TXB(2)), but this effect is blocked by L-NAME and TIRON. The present findings indicate that TERPY induces uncoupling of eNOS, enhancing O(2)(-) concentration. This enhancement in O(2)(-) concentration induces COX activation, producing TXA(2), which negatively modulates the rat aorta relaxation induced by the NO donor TERPY.

Pimarane diterpene from Viguiera arenaria (Asteraceae) inhibit rat carotid contraction

ent-Pimara-8(14),15-dien-19-oic acid (PA) isolated from Viguiera arenaria (Asteraceae; Heliantheae) inhibited rat carotid rings contraction induced by phenylephrine (10(-8) mol/l) or potassium chloride (45 mmol/l) at concentration ranging from 5 to 20 microg/ml. This inhibitory effect was not reversible after the removal of this compound from the medium bath.

Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3β-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-3beta-ol (PA-3beta-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3beta-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-3beta-ol (10, 50 and 100 micromol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3beta-ol also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 micromol/l). PA-3beta-ol (1-300 micromol/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-3beta-ol (100 micromol/l) and verapamil (1 micromol/l) inhibited the increase in Ca(2+)-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 micromol/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 micromol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 micromol/l) produced a rightward displacement of the PA-3beta-ol concentration-response curves. On the other hand, 7-nitroindazole (100 micromol/l), 1400 W (1 micromol/l), indomethacin (10 micromol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3beta-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3beta-ol involve extracellular Ca(2+) influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway.

Role of the Aryl Hydrocarbon Receptor in the Immune Response Profile and Development of Pathology during Plasmodium berghei Anka Infection

ABSTRACT Infection with Plasmodium falciparum may result in severe disease affecting various organs, including liver, spleen, and brain, resulting in high morbidity and mortality. Plasmodium berghei Anka infection of mice recapitulates many features of severe human malaria. The aryl hydrocarbon receptor (AhR) is an intracellular receptor activated by ligands important in the modulation of the inflammatory response. We found that AhR-knockout (KO) mice infected with P. berghei Anka displayed increased parasitemia, earlier mortality, enhanced leukocyte-endothelial cell interactions in the brain microvasculature, and increased inflammation in brain (interleukin-17 [IL-17] and IL-6) and liver (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) compared to infected wild-type (WT) mice. Infected AhR-KO mice also displayed a reduction in cytokines required for host resistance, including TNF-α, IL-1β, and IFN-γ, in the brain and spleen. Infection of AhR-KO mice resulted in an increase in T regulatory cells and transforming growth factor β, IL-6, and IL-17 in the brain. AhR modulated the basal expression of SOCS3 in spleen and brain, and P. berghei Anka infection resulted in enhanced expression of SOCS3 in brain, which was absent in infected AhR-KO mice. These data suggest that AhR-mediated control of SOCS3 expression is probably involved in the phenotype seen in infected AhR-KO mice. This is, to our knowledge, the first demonstration of a role for AhR in the pathogenesis of malaria.

NO donors-relaxation is impaired in aorta from hypertensive rats due to a reduced involvement of K+ channels and sarcoplasmic reticulum Ca2+-ATPase

AIMS To examine the vasodilatation induce by the NO donors, [Ru(terpy)(bdq)NO](3+) (TERPY) and sodium nitroprusside (SNP), and to compare their effects in aortic rings from hypertensive 2K-1C and normotensive 2K rats. MAIN METHODS Vascular reactivity was performed in aortic rings pre-contracted with phenylephrine (Phe 100nM). We have analyzed the maximal relaxation (Emax) and potency (pD(2)) of NO donors. KEY FINDINGS Potency of SNP was greater than TERPY in both arterial groups. The vasodilatation induced by TERPY was greater in 2K than in 2K-1C, and it was inhibited by sGC inhibitor ODQ in 2K and in 2K-1C aortic rings. ODQ did not alter the efficacy to SNP, but it reduced its potency in 2K and 2K-1C. The blockade of K(+) channels reduced the potency of TERPY only in aortic rings of 2K. On the other hand, the potency of SNP was reduced in both 2K and 2K-1C. The combination of ODQ and TEA reduced the relaxation induced by TERPY and SNP in 2K and reduced the efficacy to SNP in 2K-1C aortic rings but it had no additional effect on the TERPY relaxation in 2K-1C aortas. The production of cGMP induced by TERPY was greater than that produced by SNP, which was similarly increased in 2K and 2K-1C. Sarcoplasmic reticulum Ca-ATPase inhibition only impaired the relaxation induced by SNP in 2K aortic rings. SIGNIFICANCE Taken together, our results provide evidences that in this model of hypertension, impaired K(+) channels activation by TERPY and SERCA activation by SNP may contribute to decreased vasodilatation.

Mechanisms Underlying the Vascular Actions of Endothelin 1, Angiotensin II and Bradykinin in the Rat Carotid

The carotid artery has a pivotal role in the body since it supplies the head and neck with oxygenated blood. Alterations in the functional and structural integrity of these vessels can decrease blood flow to the brain. For this reason, it is important to understand how the carotid artery responds to various stimuli. The organ bath is a traditional experimental set-up that has been used extensively to investigate the (patho)physiology and pharmacology of in vitro tissue preparations including the rat carotid artery. Molecular biology developed from related fields such as biochemistry, genetics and biophysics is now considered an important tool for understanding physiological pathways in a variety of tissues. Several local and systemic factors regulate carotid reactivity, including vaso-active peptides, such as endothelin 1 (ET-1), angiotensin II (Ang II) and bradykinin (BK). These vaso-active peptides play a fundamental role in controlling the functional and structural integrity of the arterial wall and may be important in physiological processes and in pathological mechanisms underlying vascular diseases. In the rat carotid, these peptides induce vasoconstriction or relaxation by the release of endothelium-derived relaxing factors, such as nitric oxide and prostacyclin. Identification of such signal transduction processes is essential for understanding the mechanisms that regulate vascular smooth muscle cell function, both physiologically and pathophysiologically. The present review discusses the mechanisms of action, distribution of ET-1, Ang II and BK and their receptors in the rat carotid. With this purpose, data obtained in functional studies using classical pharmacological approaches as well as data obtained in molecular biology experiments are discussed.

The semi-synthetic kaurane ent-16α-methoxykauran-19-oic acid induces vascular relaxation and hypotension in rats.

The present work investigates the mechanisms involved in the vasorelaxant effect of ent-16α-methoxykauran-19-oic acid (KA-OCH3), a semi-synthetic derivative obtained from the kaurane-type diterpene ent-kaur-16-en-19-oic acid (kaurenoic acid). Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats using standard muscle bath procedures. The cytosolic calcium concentration ([Ca2+]c) was measured by confocal microscopy using the fluorescent probe Fluo-3 AM. Blood pressure measurements were performed in conscious rats. KA-OCH3 (10, 50 and 100 μmol/l) inhibited phenylephrine-induced contraction in either endothelium-intact or endothelium-denuded rat aortic rings. KA-OCH3 also reduced CaCl2-induced contraction in a Ca2+-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 μmol/l). KA-OCH3 (0.1-300 μmol/l) concentration-dependently relaxed endothelium-intact and endothelium-denuded aortas pre-contracted with either phenylephrine or KCl, to a greater extent than kaurenoic acid. Moreover, a Ca2+ mobilisation study showed that KA-OCH3 (100 μmol/l) inhibited the increase in Ca2+ concentration in smooth muscle and endothelial cells induced by phenylephrine or KCl. Pre-incubation of intact or denuded aortic rings with NG-nitro-L-arginine methyl ester (L-NAME, 100 μmol/l), 7-nitroindazole (100 μmol/l), wortmannin (0.5 μmol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 μmol/l) produced a rightward displacement of the KA-OCH3 concentration-response curve. Intravenous administration of KA-OCH3 (1-10 mg/kg) reduced mean arterial blood pressure in normotensive rats. Collectively, our results show that KA-OCH3 induces vascular relaxation and hypotension. The mechanisms underlying the cardiovascular actions of KA-OCH3 involve blockade of Ca2+ influx and activation of the NO-cGMP pathway.