Chronic mercury exposure at different concentrations produces opposed vascular responses in rat aorta.

Mercury chloride exposure for 30 days decreases NO bioavailability and increases oxidative stress. However, the mechanisms underlying the effects of mercury on the cardiovascular system are not completely understood, and it is not known if they are dose-dependent or if some concentrations have no harmful effects. Thus, we investigated the effects of chronic exposure to doses low (half) and high (2.5-fold higher) than that needed to obtain 29 nmol/L of HgCl2 on the vascular function. Three-month-old male Wistar rats received intramuscular (i.m.) HgCl2 for 30 days and were divided in three groups: lower (Low Hg); higher (High Hg); and saline was used as the control. High Hg exposure increased the contractile response to phenylephrine (PHE) in aortic rings, but Low Hg reduced it. The hyporesponsiveness in the Low Hg rats was blunted by endothelial denudation and NOS inhibition with l-NAME (100 µmol/L). The phosphorylated-eNOS/eNOS protein ratio increased in the aortas of Low Hg rats. In the High Hg group, endothelial denudation increased the PHE-induced contractions, while l-NAME had no effects and indomethacin (10 µmol/L), losartan (10 µmol/L) and apocynin (30 µmol/L) reduced this response. In the High Hg group, protein levels of the NADPH oxidase subunit gp91phox and cyclooxygenase-2 increased. Our results support previous suggestions that High Hg increases oxidative stress that might activate an inflammatory cascade and the renin-angiotensin system. However, very low Hg concentrations below the level considered safe still reduced vascular reactivity, suggesting the need for special attention to continuous exposure as a putative cause of increased cardiovascular risk.

The potential role of leptin in the vascular remodeling associated with obesity.

BACKGROUND/OBJECTIVES: Extracellular matrix (ECM) participates in the vascular remodeling associated with obesity. We investigated the effects of leptin on the production of ECM components in primary cultured vascular smooth muscle cells (VSMCs) and whether leptin could be a mediator of obesity-induced vascular remodeling. METHODS: T he effects of leptin (100 ng ml(-1)) on ECM components and superoxide anion production (O(2)(.-)) were evaluated in presence or absence of the antioxidant melatonin (10(-)(3) mmol l(-1)) or the inhibitor of phosphatidylinositol 3'-kinase (PI3K), LY294002 (2 × 10(-)(4) mmol l(-1)) in VSMCs from adult rats in order to explore the role of both oxidative stress and the participation of PI3K/Akt pathway in the effects of leptin. ECM components and O(2)(.-) were quantified in the aortic media of male Wistar rats fed a high-fat diet (HFD; 33.5% fat), or a standard diet (CT; 3.5% fat) for 6 weeks. RESULTS: In VSMCs, leptin enhanced gene and protein levels of collagen I, fibronectin, transforming growth factor (TGF)-ß and connective tissue growth factor (CTGF) but did not change those of collagen III and galectin-3. Leptin also increased O(2)(.-) and Akt phosphorylation in VSMCs. These effects were prevented by the presence of either melatonin or LY294002, except O(2)(.-) production in the case of PI3K inhibition. The increase in body weight in HFD rats was accompanied by aorta thickening due to an increase in media area. The aortic fibrosis observed in HFD rats was associated with high levels of leptin, collagen type I, fibronectin, TGF-ß, CTGF, phosphorylated Akt and O(2)(.-). Aortic leptin levels were positively correlated with total collagen, collagen I, TGF-ß and CTGF levels. No differences were observed in the levels of collagen III, elastin or galectin-3 between both the groups. CONCLUSIONS: Leptin could participate in the vascular remodeling and stiffness associated with obesity by ECM production in VSMCs through the activation of oxidative stress-PI3K/Akt pathway and the production of the profibrotic factors TGF-ß and CTGF.

Levetiracetam efficacy in patients with Lennox-Gastaut syndrome. Presentation of a case.

INTRODUCTION: The Lennox-Gastaut syndrome (LGS) is one of the most severe epileptic encephalopathies of childhood, characterized by electro-clinical triad of generalized spike-wave activity, slow (POL) in the electroencephalogram (EEG), multiple types of seizures and development delay. This paper intends to describe the syndrome in a patient with a history of hypoxic-ischaemic encephalopathy and Lennox-Gastaut syndrome, and a good response to treatment with levetiracetam (LEV). METHOD: Descriptive study on the development of a 3 year old child with intrauterine asphyxia, multiorgan failure, metabolic acidosis, hypovolemic shock, and seizures with cerebral oedema, who developed a West syndrome, resistant to drug treatment. The semiology of seizures progressively changed to generalized episodes of hypertonia and myoclonus, with slow spike-wave electroencephalographic activity. RESULTS: With the diagnosis of Lennox-Gastaut syndrome the patient was treated with levetiracetam, showing a substantial improvement in the cognitive sphere, in the control of seizures, and electroencephalographic findings. CONCLUSIONS: Lennox-Gastaut syndrome is one of the most severe epileptic syndromes in paediatric patients. Levetiracetam can help cognitive improvement, and contribute to seizure control in these patients.

Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats.

BACKGROUND AND PURPOSE: To analyse the influence of hypertension in the modulation induced by inducible NOS (iNOS)-derived NO and superoxide anion (O(2) (*-)) of vasoconstrictor responses and the sources of O(2) (*-) implicated. EXPERIMENTAL APPROACH: Vascular reactivity experiments were performed in segments of aorta from normotensive, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR); protein and mRNA expressions were respectively measured by western blot and quantitative reverse transcription-polymerase chain reaction and O(2) (*-) production was evaluated by ethidium fluorescence. KEY RESULTS: The contractile responses to phenylephrine (1 nM-30 microM) and 5-hydroxytryptamine (0.1-100 microM) were greater in aortic segments from SHR than WKY. The selective iNOS inhibitor, 1400W (10 microM), increased the phenylephrine contraction only in WKY segments; however, iNOS protein and mRNA expressions were greater in aorta from SHR than WKY. Superoxide dismutase (SOD, 150 U ml(-1)) reduced phenylephrine and 5-hydroxytryptamine responses only in aorta from SHR; the NAD(P)H oxidase inhibitor apocynin (0.3 mM) decreased phenylephrine and 5-hydroxytryptamine responses more in vessels from SHR than WKY. Co-incubation with SOD plus 1400W potentiated the phenylephrine and 5-hydroxytryptamine responses more in segments from SHR than WKY. O(2) (*-) production was greater in aorta from SHR than WKY; apocynin abolished this difference. CONCLUSIONS AND IMPLICATIONS: Increased O(2) (*-) formation from NADP(H) oxidase in vessels from hypertensive rats contributes to the vasoconstrictor responses and counteract the increase of NO from iNOS and the consequent modulation of these responses.

Aldosterone induces endothelial dysfunction in resistance arteries from normotensive and hypertensive rats by increasing thromboxane A2 and prostacyclin.

BACKGROUND AND PURPOSE: The present study was designed to assess whether cyclooxygenase-2 (COX-2) activation is involved in the effects of chronic aldosterone treatment on endothelial function of mesenteric resistance arteries (MRA) from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). EXPERIMENTAL APPROACH: Relaxation to acetylcholine was measured in MRA from both untreated and aldosterone-treated strains. Vasomotor responses to prostacyclin and U46619 were also analysed. Release of 6-oxo-prostaglandin (PG)F1alpha and thromboxane B2 (TxB2) was determined by enzyme immunoassay. COX-2 protein expression was measured by western blot. KEY RESULTS: Aldosterone reduced acetylcholine relaxation in MRA from both strains. In MRA from both aldosterone-treated strains the COX-1/2 or COX-2 inhibitor (indomethacin and NS-398, respectively), TxA2 synthesis inhibitor (furegrelate), prostacyclin synthesis inhibitor (tranylcypromine) or TxA2/ PGH2 receptor antagonist (SQ 29 548), but not COX-1 inhibitor SC-560, increased acetylcholine relaxation. In untreated rats this response was increased only in SHR. Prostacyclin elicited a biphasic vasomotor response: lower concentrations elicited relaxation, whereas higher concentrations elicited contraction that was reduced by SQ 29 548. Aldosterone increased the acetylcholine-stimulated production of 6-oxo-PGF(1alpha) and TxB2 in MRA from both strains. COX-2 expression was higher in both strains of rats treated with aldosterone. CONCLUSIONS AND IMPLICATIONS: Chronic treatment with aldosterone impaired endothelial function in MRA under normotensive and hypertensive conditions by increasing COX-2-derived prostacyclin and thromboxane A2. As endothelial dysfunction participates in the pathogenesis of many cardiovascular disorders we hypothesize that anti-inflammatory drugs, specifically COX-2 inhibitors, could ameliorate vascular damage in patients with elevated aldosterone production.

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation.

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca2+-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.

[Cryptogenic stroke in a young patient with heart disease and kidney failure]. / Ictus criptogenico en un paciente joven con cardiopatia y fallo renal.

INTRODUCTION: Fabry's disease is an infrequent metabolic pathology linked to the X chromosome which causes a wide variety of signs and symptoms. CASE REPORT: A 39-year-old male who was admitted to our stroke unit with right-side hemiparesis (1 + 0) and dysarthria (1). The score on the National Institute of Health Stroke Scale was 2. The patient presented angiokeratomas in both thighs. A computerised axial tomography scan of the head showed left thalamic acute infarction. The duplex scan of the supra-aortic trunks was normal, and the transcranial Doppler reflected a generalised increase in the pulsatility indices. Transthoracic echocardiography showed left ventricular hypertrophy and left atrial dilatation. He was discharged five days later, with antiaggregating medication but asymptomatic. The prolonged Holter-electrocardiogram recording showed paroxysmal atrial fibrillation. One notable value in the urine analysis was microalbuminuria of 281 mg/L. In view of the multi-organic involvement and the family history, a study for Fabry's disease was performed. Activity of the enzyme alpha-galactosidase A was diminished, and the presence of a mutation in the GLA gene was found. The patient's brother, who suffered from kidney failure and atrial fibrillation, was positive for this mutation. The patient is on treatment with agalsidase beta. CONCLUSIONS: Fabry's disease must be suspected in young males with heart disease, stroke or peripheral neuropathy, skin lesions, kidney failure and a history of cases in the family. Hormone replacement therapy must be established at an early stage, as it can improve the prognosis.

TITLE: Ictus criptogenico en un paciente joven con cardiopatia y fallo renal.Introduccion. La enfermedad de Fabry es una patologia metabolica infrecuente ligada al cromosoma X, que provoca una amplia variedad de signos y sintomas. Caso clinico. Varon de 39 antilde;os que ingreso en nuestra unidad de ictus con hemiparesia derecha (1 + 0) y disartria (1). La puntuacion en la National Institute of Health Stroke Scale era de 2. Presentaba angioqueratomas en ambos muslos. La tomografia axial computarizada craneal mostraba un infarto agudo talamico izquierdo. El duplex de los troncos supraaorticos era normal, y el Doppler transcraneal reflejaba un aumento generalizado de los indices de pulsatilidad. El ecocardiograma transtoracico mostraba hipertrofia ventricular izquierda y dilatacion de la auricula izquierda. Recibio el alta cinco dias despues, asintomatico, con antiagregacion. El registro Holter-electrocardiografico prolongado mostraba fibrilacion auricular paroxistica. En la analitica de orina destacaba microalbuminuria de 281 mg/L. En vista de la afectacion multiorganica y la historia familiar, se curso estudio de enfermedad de Fabry. La actividad de la enzima alfa-galactosidasa-A se encontro disminuida, y se demostro la presencia de una mutacion en el gen GLA. Su hermano, que padecia insuficiencia renal y fibrilacion auricular, fue positivo para dicha mutacion. El paciente se encuentra en tratamiento con agalsidasa beta. Conclusiones. La enfermedad de Fabry debe sospecharse en varones jovenes con cardiopatia, ictus o neuropatia periferica, lesiones cutaneas, fallo renal e historia de familiares afectos. El tratamiento hormonal sustitutivo debe comenzarse precozmente, ya que puede mejorar el pronostico.

Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension.

BACKGROUND AND PURPOSE: Prostanoids derived from COX-2 and EP receptors are involved in vascular remodelling in different cardiovascular pathologies. This study evaluates the contribution of COX-2 and EP1 receptors to vascular remodelling and function in hypertension. EXPERIMENTAL APPROACH: Spontaneously hypertensive rats (SHR) and angiotensin II (AngII)-infused (1.44 mg · kg(-1) · day(-1), 2 weeks) mice were treated with the COX-2 inhibitor celecoxib (25 mg · kg(-1) · day(-1) i.p) or with the EP1 receptor antagonist SC19220 (10 mg · kg(-1) · day(-1) i.p.). COX-2(-/-) mice with or without AngII infusion were also used. KEY RESULTS: Celecoxib and SC19220 treatment did not modify the altered lumen diameter and wall : lumen ratio in mesenteric resistance arteries from SHR-infused and/or AngII-infused animals. However, both treatments and COX-2 deficiency decreased the augmented vascular stiffness in vessels from hypertensive animals. This was accompanied by diminished vascular collagen deposition, normalization of altered elastin structure and decreased connective tissue growth factor and plasminogen activator inhibitor-1 gene expression. COX-2 deficiency and SC19220 treatment diminished the increased vasoconstrictor responses and endothelial dysfunction induced by AngII infusion. Hypertensive animals showed increased mPGES-1 expression and PGE2 production in vascular tissue, normalized by celecoxib. Celecoxib treatment also decreased AngII-induced macrophage infiltration and TNF-α expression. Macrophage conditioned media (MCM) increased COX-2 and collagen type I expression in vascular smooth muscle cells; the latter was reduced by celecoxib treatment. CONCLUSIONS AND IMPLICATIONS: COX-2 and EP1 receptors participate in the increased extracellular matrix deposition and vascular stiffness, the impaired vascular function and inflammation in hypertension. Targeting PGE2 receptors might have benefits in hypertension-associated vascular damage.

Toll-like receptor 4 contributes to vascular remodelling and endothelial dysfunction in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: Toll-like receptor 4 (TLR4) signalling contributes to inflammatory cardiovascular diseases, but its role in hypertension and the associated vascular damage is not known. We investigated whether TLR4 activation contributed to angiotensin II (AngII)-induced hypertension and the associated vascular structural, mechanical and functional alterations. EXPERIMENTAL APPROACH: AngII was infused (1.44 mg · kg(-1) · day(-1), s.c.) for 2 weeks in C57BL6 mice, treated with a neutralizing anti-TLR4 antibody or IgG (1 µg · day(-1); systolic BP (SBP) and aortic cytokine levels were measured. Structural, mechanical and contractile properties of aortic and mesenteric arterial segments were measured with myography and histology. RT-PCR and Western blotting were used to analyse these tissues and cultured vascular smooth muscle cells (VSMC) from hypertensive rats (SHR). KEY RESULTS: Aortic TLR4 mRNA levels were raised by AngII infusion. Anti-TLR4 antibody treatment of AngII-treated mice normalised: (i) increased SBP and TNF-α, IL-6 and CCL2 levels; (ii) vascular structural and mechanical changes; (iii) altered aortic phenylephrine- and ACh-induced responses; (iv) increased NOX-1 mRNA levels, superoxide anion production and NAD(P)H oxidase activity and effects of catalase, apocynin, ML-171 and Mito-TEMPO on vascular responses; and (v) reduced NO release and effects of L-NAME on phenylephrine-induced contraction. In VSMC, the MyD88 inhibitor ST-2825 reduced AngII-induced NAD(P)H oxidase activity. The TLR4 inhibitor CLI-095 reduced AngII-induced increased phospho-JNK1/2 and p65 NF-κB subunit nuclear protein expression. CONCLUSIONS AND IMPLICATIONS: TLR4 up-regulation by AngII contributed to the inflammation, endothelial dysfunction, vascular remodelling and stiffness associated with hypertension by mechanisms involving oxidative stress. MyD88-dependent activation and JNK/NF-κB signalling pathways participated in these alterations.

HuR mediates the synergistic effects of angiotensin II and IL-1ß on vascular COX-2 expression and cell migration.

BACKGROUND AND PURPOSE: Angiotensin II (AngII) and IL-1ß are involved in cardiovascular diseases through the induction of inflammatory pathways. HuR is an adenylate- and uridylate-rich element (ARE)-binding protein involved in the mRNA stabilization of many genes. This study investigated the contribution of HuR to the increased expression of COX-2 induced by AngII and IL-1ß and its consequences on VSMC migration and remodelling. EXPERIMENTAL APPROACH: Rat and human VSMCs were stimulated with AngII (0.1 µM) and/or IL-1ß (10 ng · mL(-1)). Mice were infused with AngII or subjected to carotid artery ligation. mRNA and protein levels were assayed by quantitative PCR, Western blot, immunohistochemistry and immunofluorescence. Cell migration was measured by wound healing and transwell assays. KEY RESULTS: In VSMCs, AngII potentiated COX-2 and tenascin-C expressions and cell migration induced by IL-1ß. This effect of AngII on IL-1ß-induced COX-2 expression was accompanied by increased COX-2 3' untranslated region reporter activity and mRNA stability, mediated through cytoplasmic HuR translocation and COX-2 mRNA binding. These effects were blocked by ERK1/2 and HuR inhibitors. VSMC migration was reduced by blockade of ERK1/2, HuR, COX-2, TXAS, TP and EP receptors. HuR, COX-2, mPGES-1 and TXAS expressions were increased in AngII-infused mouse aortas and in carotid-ligated arteries. AngII-induced tenascin-C expression and vascular remodelling were abolished by celecoxib and by mPGES-1 deletion. CONCLUSIONS AND IMPLICATIONS: The synergistic induction of COX-2 by AngII and IL-1ß in VSMCs involves HuR through an ERK1/2-dependent mechanism. The HuR/COX-2 axis participates in cell migration and vascular damage. HuR might be a novel target to modulate vascular remodelling.

Endothelial role in ouabain-induced contractions in guinea pig carotid arteries.

The influence of endothelium on the direct contractile effects of ouabain in vascular smooth muscle was analyzed in isolated perfused guinea pig carotid arteries. After blocking the neurogenic component of the glycoside contraction with alpha-adrenergic receptor blocking drugs or treating the animals with reserpine, ouabain-induced contractions were markedly reduced in vessels with intact endothelium. However, removal of the vascular endothelium from reserpinized carotid arteries resulted in ouabain-induced contractions similar to those observed in control arteries. These effects were not mimicked by the inhibitor of nitric oxide NG-monomethyl L-arginine or by the cyclooxygenase blocker indomethacin. Bioassay experiments suggested that these endothelial effects are mediated by diffusible factors. Uptake of 86Rb to measure sodium pump activity was significantly reduced by removal of the endothelium. These results suggest the existence of an inhibitory modulation by the endothelium of contractions induced by ouabain, likely mediated by a diffusible factor (or factors) released from these cells. The nature of this substance is unknown, but it is neither related to prostaglandins nor a nitric oxide-related compound. Its mechanism of action could be the stimulation of vascular sodium pump activity, the antagonism of the pump's inhibition by ouabain, or both.

Endothelial stimulation of sodium pump in cultured vascular smooth muscle.

We studied vascular sodium pump activity and its regulation by vasoactive agents and endothelium in cultured aortic vascular smooth muscle cells from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Baseline sodium pump activity (ouabain-inhibitable 86Rb+ uptake) was similar in cells from both rat strains. Angiotensin II and endothelin-1 increased ouabain-inhibitable 86Rb+ uptake more in SHR than WKY cells, whereas no effects were obtained with sodium nitroprusside, 8-bromo-cGMP, or iloprost. We examined the influence of endothelium on vascular sodium pump activity either by coculturing smooth muscle and endothelial cells or by using conditioned medium. Both coculture for 24 hours with endothelial cells and treatment with conditioned medium increased smooth muscle cell sodium pump activity, this effect being higher in SHR cells. These results suggest that the endothelium may modulate sodium pump activity in the underlying smooth muscle by releasing a diffusible compound, which is more active on SHR smooth muscle. The conditioned medium obtained in the presence of inhibitors of angiotensin-converting enzyme, endothelin-1-converting enzyme, cyclooxygenase, lipoxygenase, and nitric oxide synthase had no effect on the ability of conditioned medium to increase sodium pump activity, suggesting that angiotensin II, endothelin-1, eicosanoids, and nitric oxide are not involved in this stimulatory effect. The nature of the possible endothelial factor involved is still unknown, but it possesses a molecular weight between 25 and 50 kD, is heat stable, and is sensitive to trypsin treatment. We propose it could be a growth factor.

Neurogenic component of ouabain-evoked contractions is modulated by the endothelium.

The influence of endothelium on the neurogenic component of ouabain-induced contractions in isolated perfused guinea pig carotid arteries was analyzed. Ouabain (0.1 mumol/L to 0.1 mmol/L) evoked concentration-dependent increases of perfusion pressure. Phentolamine (0.3 to 10 mumol/L) and prazosin (30 nmol/L to 10 mumol/L) (nonselective antagonist of alpha-adrenergic receptors and selective antagonist of alpha 1-adrenergic receptors, respectively) induced a concentration-dependent relaxation in segments precontracted with ouabain (0.1 mmol/L). When the arteries were preincubated with those blockers (both at 3 mumol/L) or the animals were pretreated with reserpine, the contractions to the glycoside were diminished, indicating that they are partially mediated by norepinephrine release from adrenergic nerve endings. De-endothelialization abolished the effect of adrenergic blockade on ouabain-induced contractions. On the other hand, de-endothelialization did not modify significantly the effect of the adrenergic blockade on norepinephrine-induced contractions. The nitric oxide blocker oxyhemoglobin, at concentrations (10 mumol/L) that abolished endothelium-dependent relaxations induced by 3 mumol/L acetylcholine; or the cyclooxygenase blocker indomethacin (10 mumol/L) did not modify the relaxation caused by phentolamine. In bioassay experiments, 30 mumol/L phentolamine induced a relaxation on the ouabain-elicited contraction only when the glycoside was added through a donor segment with endothelium. Ouabain-induced tritiated norepinephrine release was significantly reduced by the removal of endothelium but not by 1 mumol/L oxyhemoglobin or 1 mumol/L indomethacin. These results suggest that the endothelium modulates the neurogenic component involved in contractions evoked by the glycoside by a diffusible factor (or factors) whose nature is unknown; however, the factor is neither nitric oxide nor a cyclooxygenase-related compound.

Endothelium stimulates the vascular smooth muscle cell Na/K pump of normotensive and hypertensive rats by a protein kinase C pathway.

OBJECTIVE: To investigate the mechanisms involved in the endothelial stimulation of the vascular smooth muscle cell Na/K pump and their possible alteration by hypertension. METHODS: The Na/K pump activity of vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) was studied using the radioactive analogue of K+ 86Rb+. Conditioned medium of bovine endothelial aortic cells was used to investigate the endothelial modulation of VSMC Na/K pump activity. RESULTS: Conditioned medium enhanced VSMC Na/K pump activity (ouabain-sensitive 86Rb+ uptake), this effect being higher in SHR cells. This stimulatory effect was neither modified in Na(+)-loaded cells from both rat strains nor inhibited by the Na/H exchange blocker amiloride. Permeable analogues of cyclic adenosine and guanosine monophosphates did not modify the baseline VSMC Na/K pump activity of WKY rats and SHR, and subsequently the guanylate cyclase inhibitor methylene blue did not alter the conditioned medium-induced stimulation of the pump. However, the Ca(2+)-channel inhibitor nifedipine reduced the Na/K pump stimulation by conditioned medium, this decrease being higher in WKY rat than in SHR VSMC. Moreover, treatment with phorbol 12,13-dibutyrate for 24 h or with the protein kinase C inhibitor staurosporine for 15 min reduced the conditioned medium-induced Na/K pump activation in both VSMC cultures. CONCLUSIONS: Na/K pump stimulation by conditioned medium of endothelial cells is mediated mainly via activation of protein kinase C in VSMC from either WKY or SHR VSMC. However, SHR VSMC show some alterations in their intracellular signalling pathways.

Nitric oxide synthase induction by ouabain in vascular smooth muscle cells from normotensive and hypertensive rats.

OBJECTIVE: To investigate the effect of ouabain on inducible nitric oxide synthase (iNOS) activity and expression in cytokine-stimulated vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: VSMC were treated for 24 h and afterwards, nitric oxide (NO) release was determined by the production of nitrite, a stable metabolite of NO. Activity of iNOS was measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline and iNOS protein expression by Western blotting. RESULTS: Ouabain (0.01-1 mmol/l) further enhanced interleukin-1beta (II-1beta)-induced nitrite production by WKY and SHR VSMC, although a more pronounced effect was observed in SHR cells (maximum response 52.1 +/- 5.2 and 71.2 +/- 6.4% of 11-1beta effect in WKY and SHR cells, respectively). Such response on NO release was mimicked by the calcium ionophore A 23187 (0.01-1 micromol/l) and abolished by the voltage-operated calcium channels (VOCC) nifedipine (0.1 micromol/l). Expression of iNOS showed that ouabain increased the synthesis of the enzyme in WKY and SHR VSMC stimulated with II-1beta, and this effect was higher in SHR cells. The increased iNOS expression was significantly reduced by nifedipine. CONCLUSIONS: Ouabain stimulation of iNOS expression and activity in II-1beta-stimulated VSMCs from WKY rats and SHR seems to be related to increased intracellular calcium influx through VOCC. The more pronounced effect observed in SHR VSMC could be explained by an altered calcium entry in the hypertensive strain.

Effects of ouabain on isolated cerebral and femoral arteries of the cat: a functional and biochemical study.

1. This study analyzes the mechanisms involved in the responses to ouabain in cat cerebral and femoral arteries and characterizes the electrogenic Na+ pump present in these vessels. The latter was accomplished by measurement of [3H]-ouabain binding to arterial membrane fractions, K+-elicited relaxation and ouabain-sensitive 86Rb+ uptake. 2. Ouabain induced transient contraction in cylindrical segments of cerebral arteries. This contraction was reduced by verapamil (3 X 10(-6) M) and Ca2+-removal from the medium but was not modified by phentolamine (3 X 10(-6) M) or pretreatment with reserpine. However, the contraction elicited by ouabain in femoral artery segments lasted longer, and was reduced by Ca2+-omission, phentolamine or reserpine, but remained unaffected by verapamil. 3. The immersion of the arteries in low-Na+ (25 mM) medium abolished the contraction caused by ouabain. 4. The exposure of the arteries to a K+-free medium induced a small transient increase in tension, and the subsequent application of K+ (7.5 mM) elicited a marked relaxation. This effect was greater in cerebral than in peripheral arteries, and was suppressed by ouabain (10(-4) M). 5. Scatchard analysis of the [3H]-ouabain binding to arterial membrane fractions suggested a single class of binding sites. The KD values for both kinds of arteries were of similar order, while the Bmax value was greater in cerebral than in femoral arteries. 6. Total and ouabain-sensitive 86Rb+ uptakes were greater in cerebral than in femoral vessels. 7. These results indicate that: (1) ouabain-induced contraction of cerebral arteries is due to a direct effect on vascular smooth muscle cells, while in femoral arteries it is due to noradrenaline release from adrenergic nerve terminals; and (2) the electrogenic Na+ pump activity is greater in cerebral than in peripheral arteries.

Effect of neuropeptide Y on adrenergic and non-adrenergic, non-cholinergic responses in the rat anococcygeus muscle.

1. The effects of neuropeptide Y (NPY) were examined on adrenergic and non-adrenergic, non-cholinergic (NANC) neurotransmission in the rat anococcygeus muscle. 2. NPY (0.1-0.3 microM) greatly potentiated the contractile responses induced by field stimulation. Prazosin (0.1 microM) completely abolished the stimulation-induced responses either in the absence or presence of NPY. 3. NPY (0.1-0.3 microM) enhanced only the contractile responses to low doses of noradrenaline (NA, 0.003-0.01 microM). Responses to tyramine were unaffected by the same concentrations of NPY. 4. In superfused anococcygeus, previously loaded with [3H]-NA, NPY (0.1-0.3 microM) failed to modify the basal, as well as the stimulation-evoked, release of tritium at 2 and 4 Hz. 5. NANC relaxations induced by electrical stimulation were significantly reduced, in a concentration-related manner, by 0.1-0.3 microM NPY. 6. L-NG-nitro-arginine (L-NOARG, 30 microM) enhanced the stimulation (0.25-1 Hz)-induced motor responses. In the presence of L-NOARG (30 microM), NPY (0.1 microM) did not modify the motor responses induced by field stimulation (0.25-0.5 Hz). L-Arginine did not reverse the NPY-induced potentiation of stimulation-induced motor responses. 7. The relaxations of anococcygeus muscle induced by sodium nitroprusside (SNP, 0.01-0.3 microM) were diminished by NPY (0.1-0.3 microM). 8. Our study suggests that NPY, at concentrations devoid of contractile effect, potentiates the motor responses of rat anococcygeus muscle as a consequence, at least in part, of the inhibition of NANC relaxing responses by a different mechanism from L-NOARG.

Role of iNOS in the vasodilator responses induced by L-arginine in the middle cerebral artery from normotensive and hypertensive rats.

1. The substrate of nitric oxide synthase (NOS), L-arginine (L-Arg, 0.01 microM - 1 mM), induced endothelium-independent relaxations in segments of middle cerebral arteries (MCAs) from normotensive Wistar-Kyoto (WKY) and hypertensive rats (SHR) precontracted with prostaglandin F2alpha (PGF2alpha). These relaxations were higher in SHR than WKY arteries. 2. L-N(G)-nitroarginine methyl ester (L-NAME) and 2-amine-5,6-dihydro-6-methyl-4H-1,3-tiazine (AMT), unspecific and inducible NOS (iNOS) inhibitors, respectively, reduced those relaxations, specially in SHR. 3. Four- and seven-hours incubation with dexamethasone reduced the relaxations in MCAs from WKY and SHR, respectively. 4. Polymyxin B and calphostin C, protein kinase C (PKC) inhibitors, reduced the L-Arg-induced relaxation. 5. Lipopolysaccharide (LPS, 7 h incubation) unaltered and inhibited these relaxations in WKY and SHR segments, respectively. LPS antagonized the effect polymyxin B in WKY and potentiated L-Arg-induced relaxations in SHR in the presence of polymyxin B. 6. The contraction induced by PGF2alpha was greater in SHR than WKY arteries. This contraction was potentiated by dexamethasone and polymyxin B although the effect of polymyxin B was higher in SHR segments. LPS reduced that contraction and antagonized dexamethasone- and polymyxin B-induced potentiation, these effects being greater in arteries from SHR. 7. These results suggest that in MCAs: (1) the induction of iNOS participates in the L-Arg relaxation and modulates the contraction to PGF2alpha; (2) that induction is partially mediated by a PKC-dependent mechanism; and (3) the involvement of iNOS in such responses is greater in the hypertensive strain.

Actions of vanadate on vascular tension and sodium pump activity in cat isolated cerebral and femoral arteries.

1. The mechanisms involved in the responses induced by sodium vanadate (Va3 VO4) on cat cerebral and femoral arteries were studied. The possibility that these responses were due to Na+, K+-ATPase inhibition was investigated by measuring the effect of vanadate on [3H]-ouabain binding to arterial membrane fractions, K+-induced vasodilatation and ouabain-sensitive 86Rb+ uptake. 2. The vanadium compounds (Na3VO4, VOSO4, VCl3 and O5V3) induced similar, concentration-dependent contractions in each kind of artery, the cerebral vessels being the most sensitive to these compounds. 3. Exposure of the arteries to a low-Na+ (25 mM) solution suppressed the contraction caused by vanadate in femoral but not in cerebral arteries. 4. Vanadate-induced contractions were reduced in Ca2+-free medium but remained unaffected by 3 x 10(-6) M phentolamine, reserpine pretreatment or 3 x 10(-6) M verapamil in both kinds of artery. 5. The addition of 7.5 mM K+ to the arteries immersed in a K+-free solution induced vasodilatation, which was not modified by 10(-3) M vanadate. 6. The consecutive administration of ouabain (10(-4) M) and vanadate (10(-3) M) (or vice versa), or the simultaneous administration of both agents (10(-8) to 10(-3) M) appeared to produce an additive contraction in both types of artery. 7. Vanadate (10(-7) to 10(-3) M) did not displace the [3H]-ouabain binding to arterial membrane fractions of these arteries, whereas 10(-4) M ouabain did. 8. In both kinds of artery, total 86Rb+ uptake was reduced by ouabain (10(-8) to 10(-3) M), in a concentration-dependent manner, whereas it was not modified by vanadate (10(-8)-10(-3) M). 9. These results suggest that vanadate induces contraction in both types of artery by a mechanism unrelated to Na+, K+-ATPase inhibition. Such a mechanism is likely to be related to inhibition of the Ca2-ATPase of the cell membrane and/or the sarcoplasmic reticulum.

Influence of hypertension on nitric oxide synthase expression and vascular effects of lipopolysaccharide in rat mesenteric arteries.

1. Experiments were designed to investigate the effects of the inducible nitric oxide synthase (iNOS) stimulator, lipopolysaccharide (LPS), on noradrenaline (NA) responses and on NOS activity and its expression in intact mesenteric resistance arteries (MRAs) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. In MRAs from WKY, LPS (10 microg ml(-1); 1-5 h) reduced the vasoconstrictor responses to NA (0.1 - 30 microM) in the presence, but not in the absence of L-arginine (L-Arg, 10 microM). However, in SHR arteries, LPS induced an incubation-time dependent reduction of NA responses in the absence, as well as the presence, of L-Arg. The LPS inhibitory effect was reduced by the non-specific NOS inhibitor L-N(G)-nitroarginine methyl ester (L-NAME, 100 microM) and the selective iNOS inhibitor, aminoguanidine (100 microM). 3. L-NAME alone similarly shifted the concentration-response curve to NA leftward in arteries from both strains, while aminoguanidine had no effect. L-Arg shifted the curve to NA rightward only in SHR MRAs. 4. Basal activity of both iNOS and constitutive NOS (conversion of [(3)H]-L-Arg to [(3)H]-L-citrulline) was similar in arteries from both strains. After 5 h incubation with LPS, only iNOS activity in arteries from SHR was increased. 5. Basal iNOS protein expression was undetectable; basal endothelial (eNOS) protein expression was similar in arteries from both strains, while neuronal (nNOS) was greater in arteries from SHR. LPS induced iNOS protein expression, that was higher in arteries from SHR than in those from WKY. 6. These results indicate that NO production, via iNOS induction, is greater than in those from MRAs from SHR to WKY.