Endothelinergic Contractile Hyperreactivity in Rat Contralateral Carotid to Balloon Injury: Integrated Role for ETB Receptors and Superoxide Anion.

Temporal consequences of neurocompensation to balloon injury on endothelinergic functionality in rat contralateral carotid were evaluated. Rats underwent balloon injury in left carotid and were treated with CP-96345 (NK1 antagonist). Concentration-response curves for endothelin-1 were obtained in contralateral (right) carotid at 2, 8, 16, 30, or 45 days after surgery in the absence or presence of BQ-123 (ETA antagonist), BQ-788 (ETB antagonist), or Tempol (superoxide-dismutase mimic). Endothelin-1-induced calcium mobilization was evaluated in functional assays carried out with BQ-123, BQ-788, or Tempol. Endothelin-1-induced NADPH oxidase-driven superoxide generation was measured by lucigenin chemiluminescence assays performed with BQ-123 or BQ-788. Endothelin-1-induced contraction was increased in contralateral carotid from the sixteenth day after surgery. This response was restored in CP-96345-treated rats. Endothelium removal or BQ-123 did not change endothelin-1-induced contraction in contralateral carotid. This response was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced calcium mobilization, which was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced lucigenin chemiluminescence, which was restored by BQ-788. We conclude that the NK1-mediated neurocompensatory response to balloon injury elicits a contractile hyperreactivity to endothelin-1 in rat contralateral carotid by enhancing the muscular ETB-mediated NADPH oxidase-driven generation of superoxide, which activates calcium channels.

Chronic restraint stress increases angiotensin II potency in the rat carotid: role of cyclooxygenases and reactive oxygen species.

OBJECTIVES: To investigate the mechanisms underlying the effects of chronic restraint stress on the vascular contractile response induced by angiotensin (Ang) II in rat carotid. METHODS: Concentration-response curves for AngII were obtained in endothelium-intact or endothelium-denuded carotid rings, in the absence or presence of SC-560 (COX-1 inhibitor), SC-236 (COX-2 inhibitor), wortmannin (PI3 K-Akt inhibitor), ML171 (NOX-1 inhibitor), VAS2870 (NOX-4 inhibitor), tiron (O2- scavenger) or PEG-catalase (H2 O2 scavenger). 6-ketoPGF1α , TXB2 , O2- or H2 O2 levels and superoxide dismutase and catalase activity or expression were also measured in rat carotid. KEY FINDINGS: Stress increased AngII potency in rat carotid. Muscular COX-1 or COX-2-derived metabolites negatively modulated AngII-induced contraction in control rat carotid. Endothelial COX-1 or COX-2-derived metabolites positively modulated AngII-induced contraction in stressed rat carotid. PI3 K-Akt, NOX-1, NOX-4, O2- and H2 O2 positively modulated AngII-induced contraction in stressed rat carotid. Stress increased 6-ketoPGF1α or H2 O2 generation and reduced catalase activity in rat carotid. Protein expression of COX-1, NOX-4 or p-Akt was increased in stressed rat carotid. CONCLUSIONS: Stress increases AngII potency in rat carotid by a mechanism that involves the increased generation of PGI2 and H2 O2 and the activation of Akt pathway. Such mechanism could play a pathophysiological role in cardiovascular diseases correlated with stress.

Enhanced nitric oxide generation from nitric oxide synthases as the cause of increased peroxynitrite formation during acute restraint stress: Effects on carotid responsiveness to angiotensinergic stimuli in type-1 diabetic rats.

Diabetes mellitus is associated with reactive oxygen and nitrogen species accumulation. Behavioral stress increases nitric oxide production, which may trigger a massive impact on vascular cells and accelerate cardiovascular complications under oxidative stress conditions such as Diabetes. For this study, type-1 Diabetes mellitus was induced in Wistar rats by intraperitoneal injection of streptozotocin. After 28 days, cumulative concentration-response curves for angiotensin II were obtained in endothelium-intact carotid rings from diabetic rats that underwent to acute restraint stress for 3h. The contractile response evoked by angiotensin II was increased in carotid arteries from diabetic rats. Acute restraint stress did not alter angiotensin II-induced contraction in carotid arteries from normoglycaemic rats. However acute stress combined with Diabetes increased angiotensin II-induced contraction in carotid rings. Western blot experiments and the inhibition of nitric oxide synthases in functional assays showed that neuronal, endothelial and inducible nitric oxide synthase isoforms contribute to the increased formation of peroxynitrite and contractile hyperreactivity to angiotensin II in carotid rings from stressed diabetic rats. In summary, these findings suggest that the increased superoxide anion generation in carotid arteries from diabetic rats associated to the increased local nitric oxide synthases expression and activity induced by acute restrain stress were responsible for exacerbating the local formation of peroxynitrite and the contraction induced by angiotensin II.

Consequence of hyperhomocysteinaemia on α1-adrenoceptor-mediated contraction in the rat corpus cavernosum: the role of reactive oxygen species.

OBJECTIVES: Our main objective was to investigate the mechanisms underlying the effects of hyperhomocysteinaemia (HHcy) on contractile response mediated by α1-adrenoceptors in the rat corpus cavernosum. METHODS: Concentration-response curves for phenylephrine (PE) were obtained in strips of corpus cavernosum, in absence or after incubation with tiron, tempol or polyethylene glycol (PEG)-catalase combined or not with tempol. We also measured the superoxide anion (O2(-)) and hydrogen peroxide (H2O2) generation, superoxide dismutase (SOD) and catalase activity and α-actin expression in rat corpus cavernosum from both groups. KEY FINDINGS: HHcy increased PE-induced contraction in cavernosal strips. Tiron, PEG-catalase or tempol increased PE-induced contraction in strips from control rats, but it was not altered by tiron or PEG-catalase in HHcy rats, whereas tempol reduced this response. The combination of PEG-catalase and tempol did not alter the contractile response to PE in both groups. HHcy increased O2(-) generation and SOD activity, whereas H2O2 concentration was reduced. Finally, HHcy did not alter catalase activity or expression of α-actin. CONCLUSIONS: The major new finding from this study is that HHcy induced a marked increase in PE-induced contraction in rat corpus cavernosum by a mechanism that involves increased O2(-) generation and it could play a role in the pathogenesis of erectile dysfunction associated with HHcy.

Acute restraint stress increases carotid reactivity in type-I diabetic rats by enhancing Nox4/NADPH oxidase functionality.

Hyperglycemia increases the generation of reactive oxygen species and affects systems that regulate the vascular tone including renin-angiotensin system. Stress could exacerbate intracellular oxidative stress during Diabetes upon the activation of angiotensin AT1/NADPH oxidase pathway, which contributes to the development of diabetic cardiovascular complications. For this study, type-I Diabetes was induced in Wistar rats by intraperitoneal injection of streptozotocin. 28 days after streptozotocin injection, the animals underwent to acute restraint stress for 3 h. Cumulative concentration-response curves for angiotensin II were obtained in carotid rings pre-treated or not with Nox or cyclooxygenase inhibitors. Nox1 or Nox4 expression and activity were assessed by Western blotting and lucigenin chemiluminescence, respectively. The role of Nox1 and Nox4 on reactive oxygen species generation was evaluated by flow cytometry and Amplex Red assays. Cyclooxygenases expression was assessed by real-time polymerase chain reaction. The contractile response evoked by angiotensin II was increased in diabetic rat carotid. Acute restraint stress increased this response in this vessel by mechanisms mediated by Nox4, whose local expression and activity in generating hydrogen peroxide are increased. The contractile hyperreactivity to angiotensin II in stressed diabetic rat carotid is also mediated by metabolites derived from cyclooxygenase-2, whose local expression is increased. Taken together, our findings suggest that acute restraint stress exacerbates the contractile hyperreactivity to angiotensin II in diabetic rat carotid by enhancing Nox4-driven generation of hydrogen peroxide, which evokes contractile tone by cyclooxygenases-dependent mechanisms. Finally, these findings highlight the harmful role played by acute stress in modulating diabetic vascular complications.

MAS receptors mediate vasoprotective and atheroprotective effects of candesartan upon the recovery of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality.

AT1 antagonists effectively prevent atherosclerosis since AT1 upregulation and angiotensin II-induced proinflammatory actions are critical to atherogenesis. Despite the classic mechanisms underlying the vasoprotective and atheroprotective actions of AT1 antagonists, the cross-talk between angiotensin-converting enzyme-angiotensin II-AT1 and angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axes suggests other mechanisms beyond AT1 blockage in such effects. For instance, angiotensin-converting enzyme 2 activity is inhibited by reactive oxygen species derived from AT1-mediated proinflammatory signaling. Since angiotensin-(1-7) promotes antiatherogenic effects, we hypothesized that the vasoprotective and atheroprotective effects of AT1 antagonists could result from their inhibitory effects on the AT1-mediated negative modulation of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality. Interestingly, our results showed that early atherosclerosis triggered in thoracic aorta from high cholesterol fed-Apolipoprotein E-deficient mice impairs angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality by a proinflammatory-redox AT1-mediated pathway. In such mechanism, AT1 activation leads to the aortic release of tumor necrosis factor-α, which stimulates NAD(P)H oxidase/Nox1-driven generation of superoxide and hydrogen peroxide. While hydrogen peroxide inhibits angiotensin-converting enzyme 2 activity, superoxide impairs MAS functionality. Candesartan treatment restored the functionality of angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis by inhibiting the proinflammatory-redox AT1-mediated mechanism. Candesartan also promoted vasoprotective and atheroprotective effects that were mediated by MAS since A779 (MAS antagonist) co-treatment inhibited them. The role of MAS receptors as the final mediators of the vasoprotective and atheroprotective effects of candesartan was supported by the vascular actions of angiotensin-(1-7) upon the recovery of the functionality of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis.

Vasoprotective effects of neurocompensatory response to balloon injury during diabetes involve the improvement of Mas signaling by TGFß1 activation.

Balloon injury in diabetic rats triggers a sensory neurocompensatory response that restores the blood flow in contralateral carotid. These vasoprotective effects result from H2O2-mediated relaxation that counteracts AT1-dependent contractile hyperreactivity. The most important mechanism from the renin-angiotensin-system in counteracting AT1-mediated effects is that one is mediated by Mas receptors. Thus, we hypothesized that the vasoprotective effects of balloon neurocompensation in diabetic rats could result from the improvement of Mas signaling by H2O2-mediated sensory mechanisms. NK1 receptors are sensory components whose activation could lead to H2O2 generation upon TGFß1 release and ALK5-mediated Nox4 upregulation. Based on this, we aimed to investigate: (1) the role of the TGFß1/ALK5-Nox4-H2O2 pathway on modulating Mas signaling in diabetic rat contralateral carotid; and (2) the contribution of Mas signaling in the control of local blood flow. Our results showed that balloon neurocompensation restored diabetic rat contralateral carotid flow by improving Mas signaling through NK1-mediated TGFß1 release. TGFß1/ALK5 activation enhanced Nox4 expression and Nox4-driven generation of H2O2. In turn, H2O2 enhanced the local Mas-mediated relaxation. Since restenosis impairs diabetic rat ipsilateral carotid flow, the restoration of diabetic rat contralateral carotid flow may prevent further damages in cerebral irrigation by carotid pathways after angioplasty during diabetes.

Impairment of α1-adrenoceptor-mediated calcium influx in contralateral carotids following balloon injury: beneficial effect of superoxide anions.

There are many evidences indicating a compensatory mechanism in contralateral carotids following balloon injury. Previously it was observed α1-adrenoceptor-mediated hyper-reactivity and impairment of calcium influx in contralateral carotids 4 days after injury. At a later stage, α1-adrenoceptor-mediated contraction is similar to the control and we hypothesized that downstream signaling was normal. In the present study, we aimed to evaluate α1-adrenoceptor-mediated calcium influx in contralateral carotids 15 days after balloon injury. Concentration-response curves for CaCl2 in presence of the α1-adrenoceptor agonist (phenylephrine), measurement of the intracellular calcium transient and the levels of reactive oxygen species using fluorescent dyes were performed in control and contralateral carotids. Phenylephrine-induced intracellular calcium mobilization in contralateral carotids was not altered, while phenylephrine-induced calcium influx was reduced in the contralateral artery. Nitric oxide synthase inhibitors, L-NAME or L-NNA, restored this response, but nitrite and nitrate levels were decreased in contralateral carotids. Additionally, a rise in oxygen free radicals was observed in contralateral carotids. Furthermore, Tiron, a superoxide anion scavenger, restored α1-adrenoceptor-mediated calcium influx in contralateral carotids to the control level. Similar results were observed with the selective potassium channels blockers 4-aminopyridine and charybdotoxin. In conclusion, data showed that balloon catheter injury resulted in increased superoxide anions levels, activation of potassium channels (Kv and BKCa), inhibition of calcium channels (Cav) and preservation of α1-adrenoceptor-mediated contraction at a later stage after injury.

Ethanol consumption increases endothelin-1 expression and reactivity in the rat cavernosal smooth muscle.

AIMS: We investigated the effects of chronic ethanol consumption on the cavernosal smooth muscle (CSM) reactivity to endothelin-1 (ET-1) and the expression of ET system components in this tissue. METHODS: Male Wistar rats were treated with heavy dose of ethanol (20% v/v) for 6 weeks. Reactivity experiments were performed in the isolated rat CSM. Plasma and CSM nitrate generation and also superoxide anion generation in rat CSM were measured by chemiluminescence. Protein and mRNA levels of pre-pro-ET-1, endothelin-converting enzyme-1 (ECE-1), ETA and ETB receptors, eNOS, nNOS and iNOS were assessed by western immunoblotting and quantitative real-time polymerase chain reaction, respectively. RESULTS: Chronic ethanol consumption increased plasma ET-1 levels and the contractile response induced by this peptide in the isolated CSM. The relaxation induced by acetylcholine, but not IRL1620, a selective ETB receptor agonist, was reduced in CSM from ethanol-treated rats. BQ123, a selective ETA receptor antagonist, produced a rightward displacement of the ET-1 concentration-response curves in CSM from control, but not ethanol-treated rats. Reduced levels of nitrate were found in the plasma and CSM from ethanol-treated rats. Ethanol consumption increased superoxide anion generation in the rat CSM. The mRNA levels of pre-pro-ET-1, ECE-1, ETA and ETB receptors, eNOS, nNOS and iNOS were not altered by ethanol consumption. Protein levels of ET-1, ETA receptor and iNOS were higher in the CSM from rats chronically treated with ethanol. CONCLUSION: The major findings of the present study are that heavy ethanol consumption increases plasma ET-1 levels and the contraction induced by the peptide in the CSM. Increased CSM reactivity to ET-1 and altered protein levels of ET-1 and ETA receptors could play a role in the pathogenesis of erectile dysfunction associated with chronic ethanol consumption.

Diabetes confers a vasoprotective role to the neurocompensatory response elicited by carotid balloon injury: consequences on contralateral carotid tone and blood flow.

The purpose from this study was to investigate the consequences of sensory neurocompensation to carotid balloon injury in diabetic rats on angiotensin II-induced contraction and basal blood flow in contralateral carotid. Concentration-response curves for angiotensin II and blood flow were obtained in contralateral carotid from non-treated or capsaicin-treated streptozotocin-induced diabetic rats that underwent carotid balloon injury. Diabetes increased angiotensin II-induced contraction and impaired the blood flow in non-operated rat carotid. In diabetic rats, balloon injury led to neointima formation, which reduced the blood flow in ipsilateral carotid. Carotid balloon injury in diabetic rats reduced angiotensin II-induced contraction and restored the blood flow in contralateral carotid when compared to diabetic non-operated rat carotid. Capsaicin inhibited the effects evoked by carotid balloon injury on diabetic rat contralateral carotid. Endothelium removal, PEG-catalase (hydrogen peroxide scavenger) or l-NPA (neuronal nitric oxide synthase, nNOS, inhibitor) increased angiotensin II-induced contraction in contralateral carotid from diabetic operated rats to the levels observed in diabetic non-operated rat carotid. Our findings suggest that carotid balloon injury in diabetic rats elicits a neurocompensation that attenuates the diabetic hyperreactivity to angiotensin II in contralateral carotid by a sensory nerves-dependent mechanism mediated by hydrogen peroxide derived from endothelial nNOS. This sensory mechanism also restored the blood flow in this vessel, compensating the impaired blood flow in diabetic rat ipsilateral carotid. Thus, our major conclusions are that Diabetes confers a vasoprotective significance to the neurocompensation to carotid balloon injury in preventing further damage at carotid cerebral irrigation after angioplasty in diabetic subjects.

Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE-/- mice.

AIMS: Wall shear stress differentially regulates the arginase pathway in carotid arteries perfused ex vivo. Specific patterns of wall shear stress can locally determine atherosclerotic plaque size and composition in vivo. The present work investigates the effects of arginase inhibition on shear stress induced plaque composition. METHODS AND RESULTS: Carotid arteries of apolipoprotein E deficient mice were exposed to high (HSS), low (LSS) and oscillatory (OSS) shear stress conditions by the placement of a local shear stress modifier device for 9 weeks with or without the administration of the arginase inhibitor N-ω-Hydroxy-nor-L-arginine (nor-Noha) (10 mg/kg, i.p., 5 days/week). Carotid arginase activity was measured by colorimetric determination of urea. Atherosclerotic plaque size and composition, arginase expression and cellular localization were assessed by immunohistochemistry. Arginase activity was significantly increased in both LSS and OSS regions as compared to HSS. In the lesions, arginase II isoform co-localized preferentially with EC. Inhibition of arginase by nor-Noha decreased arginase activity and reduced plaque size in both LSS and OSS regions. Arginase inhibition affected mainly the composition of plaques developed in LSS regions by decreasing the total vascular ROS, the number of macrophages, apoptosis rate, lipid and collagen contents. CONCLUSIONS: Arginase activity is modulated by patterns of wall shear stress in vivo. Chronic inhibition of vascular arginase decreased the size of atherosclerotic lesions in both OSS and LSS regions, whereas changes on plaque composition were more pronounced in plaques induced by LSS. We identified wall shear stress as a key biomechanical regulator of arginase during plaque formation and stability.

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).

Impaired calcium influx despite hyper-reactivity in contralateral carotid following balloon injury: eNOS involvement.

Balloon catheter injury results in hyper-reactivity to phenylephrine in contralateral carotids. Decreased nitric oxide (NO) modulation and/or increased intracellular calcium concentration triggers vascular smooth muscle contraction. Therefore, this study explores the participation of NO signaling pathway and calcium mobilization on hyper-reactivity to phenylephrine in contralateral carotids. Concentration-response curves for calcium (CaCl(2)) and phenylephrine were obtained in control and contralateral carotids four days after balloon injury, in the presence and absence of the inhibitors (L-NAME, L-NNA, 1400W, 7-NI, Oxyhemoglobin, ODQ or Tiron). Confocal microscopy using Fluo-3AM or DHE was performed to detect the intracellular levels of calcium and reactive oxygen species, respectively. The modulation of NO on phenylephrine-induced contraction was absent in the contralateral carotid. Phenylephrine-induced intracellular calcium mobilization was not altered in contralateral carotids. However, extracellular calcium mobilization by phenylephrine was reduced in the contralateral carotid compared to control arteries, and this result was confirmed by confocal microscopy. L-NAME increased phenylephrine-induced extracellular calcium mobilization in the contralateral carotid to the control levels. Results obtained with L-NNA, 1400W, 7-NI, OxyHb, ODQ or Tiron showed that this response was mediated by products from endothelial NOS (eNOS) different from NO and without soluble guanylate cyclase activation, but it involved superoxide anions. Furthermore, Tiron or L-NNA reduced the levels of reactive oxygen species in contralateral carotids. Data suggest that balloon catheter injury promoted eNOS uncoupling in contralateral carotids, which generates superoxide rather than NO, and reduces phenylephrine-induced extracellular calcium mobilization, despite the hyper-reactivity to phenylephrine in contralateral carotids.

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.

Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3beta-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.

Mechanisms underlying the biphasic effect of vitamin K1 (phylloquinone) on arterial blood pressure.

Phylloquinone (vitamin K(1), VK(1)) is widely used therapeutically and intravenous administration of this quinone can induce hypotension. We aimed to investigate the mechanisms underlying the effects induced by VK(1) on arterial blood pressure. With this purpose a catheter was inserted into the abdominal aorta of male Wistar rats for blood pressure and heart rate recording. Bolus intravenous injection of VK(1) (0.5-20 mgkg(-1)) produced a transient increase in blood pressure followed by a fall. Both the pressor and depressor response induced by VK(1) were dose-dependent. On the other hand, intravenous injection of VK(1) did not alter heart rate. The nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10 and 20 mgkg(-1)) reduced both the increase and decrease in blood pressure induced by VK(1) (5 mgkg(-1)). On the other hand, indometacin (10 mg kg(-1)), a non-selective cyclooxygenase inhibitor, did not alter the increase in mean arterial pressure (MAP) induced by VK(1). However, VK(1)-induced fall in MAP was significantly attenuated by indometacin. We concluded that VK(1) induces a dose-dependent effect on blood pressure that consists of an acute increase followed by a more sustained decrease in MAP. The hypotension induced by VK(1) involves the activation of the nitric oxide (NO) pathway and the release of vasodilator prostanoid(s).

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 Ca(2+) influx. To this end, the effect of PA on phenylephrine- and KCl-induced increases in cytosolic calcium concentration ([Ca(2+)](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 micromol L(-1)) and KCl (30 or 90 mmol L(-1)) in endothelium-denuded rat aortic rings in a concentration dependent manner. Pre-treatment with PA (10, 100, 200 micromol L(-1)) attenuated the contraction induced by CaCl(2) (0.5 nmol L(-1) or 2.5 mmol L(-1)) in denuded rat aorta exposed to Ca(2+)-free medium containing phenylephrine (0.1 micro mol L(-1)) or KCl (30 mmol L(-1)). Interestingly, the inhibitory effect displayed by PA on CaCl(2)-induced contraction was more pronounced when KCl was used as the stimulant. Phenylephrine- and KCl-induced increases in [Ca(2+)](c) were inhibited by PA. Similarly, verapamil, a Ca(2+)-channel blocker, also inhibited the increase in [Ca(2+)](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 Ca(2+) influx through smooth muscle cellular membrane, a mechanism that could mediate the hypotensive response induced by this diterpene in normotensive rats.

Hyperhomocysteinaemia-induced cardiovascular changes in rats.

(1) Increased plasma homocysteine content and increased blood pressure are independently associated with higher cardiovascular risks. The present study was designed to determine the effects of hyperhomocysteinaemia (HHcys) on the activity of the cardiovascular system in rats. (2) Using male Wistar rats, the effect of moderate HHcys, induced by treating rats with dl-homocysteine thiolactone (DL-HT; 1 g/kg per day) for 15 days, on arterial blood pressure, heart rate, baroreflex and vascular reactivity was determined. (3) Hyperhomocysteinaemia was observed after 15 days of treatment. Baseline arterial blood pressure and heart rate values of HHcys animals were significantly increased after 15 days of treatment. Plasma homocysteine and cardiovascular parameters returned to control values after termination of treatment. Baroreflex gain was significantly enhanced in HHcys rats. The pressor effect of an i.v. infusion of phenylephrine (50 mg/kg per mL) was decreased in HHcys rats and returned to control values after washout of DL-HT. Hypotensive responses to i.v. infusions of sodium nitroprusside (70 mg/kg per mL) or acetylcholine (10 mg/kg per mL) were increased in HHcys animals and returned to control values after washout of DL-HT. The increase in resting arterial blood pressure associated with the moderate HHcys was reversed by treatment with the b1-adrenoceptor antagonist atenolol, suggesting that HHcys-related hypertension is related to increase in cardiac sympathetic activity. (4) The present study showed significantly increased arterial blood pressure, heart rate and baroreflex activity in the early phase of moderate HHcys. In addition, HHcys was associated with alterations of vascular responsiveness to pressor and depressor agents, as well as increased cardiac sympathetic activity. The fact that cardiovascular changes observed in HHcys were reversed after DL-HT washout indicate that moderate HHcys evokes cardiovascular changes.

Ethanol consumption increases blood pressure and alters the responsiveness of the mesenteric vasculature in rats.

Chronic ethanol consumption and hypertension are related. In the current study we investigated whether changes in reactivity of the mesenteric arterial bed could account for the increased blood pressure associated with chronic ethanol intake. Changes in reactivity to phenylephrine and acetylcholine were investigated in the perfused mesenteric bed from rats treated with ethanol for 2 or 6 weeks and their age-matched controls. Mild hypertension was observed in chronically ethanol-treated rats. Treatment of rats for 6 weeks induced an increase in the contractile response of endothelium-intact mesenteric bed to phenylephrine, but not denuded rat mesenteric bed. The phenylephrine-induced increase in perfusion pressure was not altered after 2 weeks' treatment with ethanol. Moreover, acetylcholine-induced endothelium-dependent relaxation was reduced by ethanol treatment for 6 weeks, but not 2 weeks. Pre-treatment with indometacin, a cyclooxygenase inhibitor, reduced the maximum effect induced by phenylephrine (Emax) in endothelium-intact mesenteric bed from both control and ethanol-treated rats. No differences in the Emax values for phenylephrine were observed between groups in the presence of indometacin. L-NNA, a nitric oxide (NO) synthase (NOS) inhibitor, increased the Emax for phenylephrine in endothelium-intact mesenteric bed from control rats but not from ethanol-treated rats. Levels of endothelial NOS (eNOS) mRNA were not altered by chronic ethanol consumption. However, chronic ethanol intake strongly reduced eNOS protein levels in the mesenteric bed. This study shows that chronic ethanol consumption increases blood pressure and alters the reactivity of the mesenteric bed. Moreover, the increased vascular response to phenylephrine observed in the mesenteric bed is maintained by two mechanisms: an increased release of endothelial-derived vasoconstrictor prostanoids and a reduced modulatory action of endothelial NO, which seems to be associated with reduced post-transcriptional expression of eNOS.

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.