Carlos R. Tirapelli

Functional characterization and expression of endothelin receptors in rat carotid artery: involvement of nitric oxide, a vasodilator prostanoid and the opening of K+ channels in ETB-induced relaxation

We aimed to functionally characterize endothelin (ET) receptors in the rat carotid artery. mRNA and protein expressions of both ETA and ETB receptors, evaluated by reverse transcription–polymerase chain reaction (RT–PCR) and Western immunoblotting, were detected in carotid segments. Immunohistochemical assays showed that ETB receptors are expressed in the endothelium and smooth muscle cells, while ETA receptors are expressed only in the smooth muscle cells. In endothelium‐denuded vessels, levels of ETB receptor mRNA were reduced. Vascular reactivity experiments, using standard muscle bath procedures, showed that ET‐1 induces contraction in endothelium‐intact and ‐denuded carotid rings in a concentration‐dependent manner. Endothelial removal enhanced ET‐1‐induced contraction. BQ123 and BQ788, selective antagonists for ETA and ETB receptors, respectively, produced concentration‐dependent rightward displacements of the ET‐1 concentration–response curves. IRL1620, a selective agonist for ETB receptors, induced a slight vasoconstriction that was abolished by BQ788, but not affected by BQ123. IRL1620‐induced contraction was augmented after endothelium removal. ET‐1 concentration dependently relaxed phenylephrine‐precontracted rings with intact endothelium. The relaxation was augmented in the presence of BQ123, reduced in the presence of BQ788 and completely abolished after endothelium removal. IRL1620 induced vasorelaxation that was abolished by BQ788 and endothelium removal, but not affected by BQ123. Preincubation of intact rings with NG‐nitro‐L‐arginine methyl ester (L‐NAME), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), indomethacin or tetraethylammonium (TEA) reduced IRL1620‐induced relaxation. The combination of L‐NAME, indomethacin and TEA completely abolished IRL1620‐induced relaxation while sulfaphenazole did not affect this response. 4‐aminopyridine (4‐AP), but not apamin, glibenclamide or charybdotoxin, reduced IRL1620‐induced relaxation. The major finding of this work is that it firstly demonstrated functionally the existence of both ETA and ETB vasoconstrictor receptors located on the smooth muscle of rat carotid arteries and endothelial ETB receptors that mediated vasorelaxation via NO–cGMP pathway, vasodilator cyclooxygenase product(s) and the activation of voltage‐dependent K+ channels.

Hypotensive action of naturally occurring diterpenes: a therapeutic promise for the treatment of hypertension.

Plants have always been an exemplary source of drugs and many of the currently available medicines have been directly or indirectly derived from them. For this reason, the research, development and use of natural products as therapeutic agents, especially those derived from plants, have been increasing in recent years. A great deal of attention has focused on the naturally occurring antispasmodic phytochemicals as potential drugs for the treatment of cardiovascular diseases. Arterial hypertension is a common and progressive disorder that poses a major risk for cardiovascular and renal diseases. Recent data have revealed that the global burden of hypertension is an important and increasing public health problem worldwide and that the level of awareness, treatment and control of hypertension varies considerably among countries. The research on naturally occurring blood pressure-lowering agents is rapidly expanding due to the high potential of such molecules as new antihypertensive drugs. Recently, a great number of plant-derived substances, such as diterpenoids, have been evaluated as possible antihypertensive agents. Naturally occurring diterpenes such as forskolin and stevioside, exhibit vasorelaxant action and inhibit vascular contractility by different mechanisms of action. In this review we will discuss the mechanisms underlying the hypotensive action displayed by diterpenes and their potential use in human hypertension. We will also discuss the use of these compounds in the treatment of glaucoma, which is characterized by increased intraocular pressure (IOP).

Ethanol Consumption Enhances Endothelin-1-Induced Contraction in the Isolated Rat Carotid

We investigated the mechanisms involved in the enhancement of endothelin (ET)-1 vascular reactivity induced by ethanol consumption. Ethanol intake for 2, 6, and 10 weeks enhanced the ET-1-induced contractile response of endothelium-intact but not endothelium-denuded rat carotid rings independently of the treatment duration. Conversely, phenylephrine-induced contraction was not affected by ethanol intake. The contraction induced by IRL1620 [succinyl-(Glu9,Ala11,15)-ET-1-(8-21)], a selective ETB agonist, was increased after treatment with ethanol in endothelium-intact but not in endothelium-denuded carotid rings. Moreover, ET-1- and IRL1620-induced relaxation was reduced in endothelium-intact phenylephrine-precontracted rings from ethanol-treated rats. Acetylcholine-induced relaxation was not affected by ethanol treatment. NG-Nitro-l-arginine methyl ester, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one, indomethacin, and tetraethylammonium reduced the relaxation induced by IRL1620 in carotid glands from control but not ethanol-treated rats. The mRNA levels for ETA and ETB receptors were not altered by ethanol consumption. However, ethanol treatment reduced the protein expression of ETB receptors. Furthermore, immunohistochemical assays showed reduced immunostaining for endothelial ETB receptors after treatment with ethanol. We conclude that ethanol consumption enhances ET-1-induced contraction in the rat carotid and that this response is not different among the three periods of treatment used in this study. Finally, the potentiation of ET-1-induced vascular reactivity is probably caused by reduced expression of relaxing endothelial ETB receptors.

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 AT₁ 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 AT₁-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage.

Doxycycline Dose-dependently Inhibits MMP-2-Mediated Vascular Changes in 2K1C Hypertension

Abstract:  Hypertension induces vascular alterations that are associated with up‐regulation of matrix metalloproteinases (MMPs). While these alterations may be blunted by doxycycline, a non‐selective MMPs inhibitor, no previous study has examined the effects of different doses of doxycycline on these alterations. This is important because doxycycline has been used at sub‐antimicrobial doses, and the use of lower doses may prevent the emergence of antibiotic‐resistant microorganisms. We studied the effects of doxycycline at 3, 10 and 30 mg/kg per day on the vascular alterations found in the rat two kidney‐one clip (2K1C) hypertension (n = 20 rats/group). Systolic blood pressure (SBP) was monitored during 4 weeks of treatment. We assessed endothelium‐dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall was studied, and aortic MMP‐2 levels/proteolytic activity were determined by gelatin and in situ zymography, respectively. All treatments attenuated the increases in SBP in hypertensive rats (195.4 ± 3.9 versus 177.2 ± 6.2, 176.3 ± 4.5, and 173 ± 5.1 mmHg in 2K1C hypertensive rats treated with vehicle, or doxycycline at 3, 10, 30 mg/kg per day, respectively (all p < 0.01). However, only the highest dose prevented 2K1C‐induced reduction in endothelium‐dependent vasorelaxation (p < 0.05), vascular hypertrophy and increases in MMP‐2 levels (all p < 0.05). In conclusion, our results suggest that relatively lower doses of doxycycline do not attenuate the vascular alterations found in the 2K1C hypertension model, and only the highest dose of doxycycline affects MMPs and vascular structure. Our results support the idea that the effects of doxycycline on MMP‐2 and vascular structure are pressure independent.

Hypertension and chronic ethanol consumption: What do we know after a century of study?

The influences of life habits on the cardiovascular system may have important implications for public health, as cardiovascular diseases are among the leading causes of shorter life expectancy worldwide. A link between excessive ethyl alcohol (ethanol) consumption and arterial hypertension was first suggested early last century. Since then, this proposition has received considerable attention. Support for the concept of ethanol as a cause of hypertension derives from several epidemiologic studies demonstrating that in the general population, increased blood pressure is significantly correlated with ethanol consumption. Although the link between ethanol consumption and hypertension is well established, the mechanism through which ethanol increases blood pressure remains elusive. Possible mechanisms underlying ethanol-induced hypertension were proposed based on clinical and experimental observations. These mechanisms include an increase in sympathetic nervous system activity, stimulation of the renin-angiotensin-aldosterone system, an increase of intracellular Ca(2+) in vascular smooth muscle, increased oxidative stress and endothelial dysfunction. The present report reviews the relationship between ethanol intake and hypertension and highlights some mechanisms underlying this response. These issues are of interest for the public health, as ethanol consumption contributes to blood pressure elevation in the population.

Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms

The present study investigated the role of ROS (reactive oxygen species) and COX (cyclo-oxygenase) in ethanol-induced contraction and elevation of [Ca2+]i (intracellular [Ca2+]). Vascular reactivity experiments, using standard muscle bath procedures, showed that ethanol (1-800 mmol/l) induced contraction in endothelium-intact (EC50: 306+/-34 mmol/l) and endothelium -denuded (EC50: 180+/-40 mmol/l) rat aortic rings. Endothelial removal enhanced ethanol-induced contraction. Preincubation of intact rings with L-NAME [NG-nitro-L-arginine methyl ester; non-selective NOS (NO synthase) inhibitor, 100 micromol/l], 7-nitroindazole [selective nNOS (neuronal NOS) inhibitor, 100 micromol/l], oxyhaemoglobin (NO scavenger, 10 micromol/l) and ODQ (selective inhibitor of guanylate cyclase enzyme, 1 micromol/l) increased ethanol-induced contraction. Tiron [O2- (superoxide anion) scavenger, 1 mmol/l] and catalase (H2O2 scavenger, 300 units/ml) reduced ethanol-induced contraction to a similar extent in both endothelium-intact and denuded rings. Similarly, indomethacin (non-selective COX inhibitor, 10 micromol/l), SC560 (selective COX-1 inhibitor, 1 micromol/l), AH6809 [PGF2alpha (prostaglandin F2alpha)] receptor antagonist, 10 micromol/l] or SQ29584 [PGH2(prostaglandin H2)/TXA2 (thromboxane A2) receptor antagonist, 3 micromol/l] inhibited ethanol-induced contraction in aortic rings with and without intact endothelium. In cultured aortic VSMCs (vascular smooth muscle cells), ethanol stimulated generation of O2- and H2O2. Ethanol induced a transient increase in [Ca2+]i, which was significantly inhibited in VSMCs pre-exposed to tiron or indomethacin. Our data suggest that ethanol induces vasoconstriction via redox-sensitive and COX-dependent pathways, probably through direct effects on ROS production and Ca2+ signalling. These findings identify putative molecular mechanisms whereby ethanol, at high concentrations, influences vascular reactivity. Whether similar phenomena occur in vivo at lower concentrations of ethanol remains unclear.

Diterpenes: A Therapeutic Promise for Cardiovascular Diseases

The research, development and use of natural products as therapeutic agents, especially those derived from plants, have been increasing in recent years. There has been great deal of focus on the naturally occurring antispasmodic phytochemicals as potential therapy for cardiovascular diseases. Naturally occurring diterpenes exert several biological activities such as anti-inflammatory action, antimicrobial and antispasmodic activities. Several diterpenes have been shown to have pronounced cardiovascular effects, for example, grayanotoxin I produces positive inotropic responses, forskolin is a well-known activator of adenylate cyclase, eleganolone and 14-deoxyandrographolide exhibit vasorelaxant properties and marrubenol inhibits smooth muscle contraction by blocking L-type calcium channels. In the last few years, we have investigated the biological activity of kaurane and pimarane-type diterpenes, which are the main secondary metabolites isolated from the roots of Viguiera robusta and V. arenaria, respectively. These diterpenoids exhibit vasorelaxant action and inhibit the vascular contractility mainly by blocking extracellular Ca(2+) influx. Moreover, kaurane and pimarane-type diterpenes decreased mean arterial blood pressure in normotensive rats. Diterpenes likely fulfil the definition of a pharmacological preconditioning class of compounds and give hope for the therapeutic use in cardiovascular diseases. This article will review patents, structure-activity relationship, pharmacology, antihypertensive efficiency, and the vascular mechanisms underlying the effects of diterpenes. Careful examination of the cardiovascular effects exhibited by kaurane and pimarane-type diterpenes will be provided.

Inhibitory action of kaurenoic acid from Viguiera robusta (Asteraceae) on phenylephrine-induced rat carotid contraction

ent-kaurenoic acid (KA) isolated from Viguiera robusta was tested for activity on vascular smooth muscle contactility. Cumulative concentration-response curves were obtained for a stepwise increase (10(-10)-10(-05) mol/l) in the concentration of phenylephrine (Phe), a selective alpha(1)-adrenergic agonist. The effects in the presence of KA (0.2, 2.0 and 20.0 microg/ml), and 90 min after the removal of KA from the medium bath were compared to controls. At 20.0 microg/ml, KA inhibited the in vitro contractility of rat carotid artery elicited by Phe, but had no effect at lower concentrations (0.2 and 2.0 microg/ml). The effect elicited by KA was reversible after 90 minutes.

Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress

OBJECTIVES We hypothesized that chronic ethanol intake enhances vascular oxidative stress and induces hypertension through renin-angiotensin system (RAS) activation. METHODS AND RESULTS Male Wistar rats were treated with ethanol (20% v/v). The increase in blood pressure induced by ethanol was prevented by losartan (10mg/kg/day; p.o. gavage), a selective AT1 receptor antagonist. Chronic ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels and serum aldosterone levels. No differences on plasma osmolality and sodium or potassium levels were detected after treatment with ethanol. Ethanol consumption did not alter ACE activity, as well as the levels of ANG I and ANG II in the rat aorta or mesenteric arterial bed (MAB). Ethanol induced systemic and vascular oxidative stress (aorta and MAB) and these effects were prevented by losartan. The decrease on plasma and vascular nitrate/nitrite (NOx) levels induced by ethanol was prevented by losartan. Ethanol intake did not alter protein expression of ACE, AT1 or AT2 receptors in both aorta and MAB. Aortas from ethanol-treated rats displayed decreased ERK1/2 phosphorylation and increased protein expression of SAPK/JNK. These responses were prevented by losartan. MAB from ethanol-treated rats displayed reduced phosphorylation of p38MAPK and ERK1/2 and losartan did not prevent these responses. CONCLUSIONS Our study provides novel evidence that chronic ethanol intake increases blood pressure, induces vascular oxidative stress and decreases nitric oxide (NO) bioavailability through AT1-dependent mechanisms.