Graphical abstract Abstract Renin–angiotensin system plays a critical role in blood pressure control, and the abnormal activation of the AT1 receptor contributes to the development of renovascular hypertension. This study aimed to evaluate the underlying cellular signaling for AT1 receptor activation by Ang II and to compare this mechanism between aortas from 2K-1C and 2K rats. Effects of antagonists and inhibitors were investigated on Ang II-induced contractions in denuded or intact-endothelium aortas. The AT1 receptor antagonist abolished Ang II-induced contraction in 2K-1C and 2K rat aortas, while AT2 and Mas receptors antagonists had no effect. Endothelial nitric oxide synthase inhibition increased the maximal effect (Emax) of Ang II in 2K, which was not changed in 2K-1C aortas. It was associated with lower eNOS mRNA levels in 2K-1C. Endothelium removal increased the Emax of Ang II in 2K-1C and mainly in 2K rat aortas. Nox and COX inhibition did not alter Ang II-induced contraction in 2K and 2K-1C rat aortas. However, AT1 expression was higher in 2K-1C compared to 2K rat aortic rings, whereas expression of phosphorylated (active) IP3 receptors was lower in 2K-1C than in 2K rats. These results demonstrate that endothelium removal impairs Ang II-stimulated contraction in the aorta of 2K-1C rats, which is associated with the reduction of IP3 receptor phosphorylation and activation. In addition, eNOS plays a critical role in Ang II-induced contraction in 2K rat aortas. It is possible that the high Ang II plasma levels could desensitize AT1 receptor in 2K-1C rats, leading to impaired IP3 receptors activation.

We have synthesized cis-[Ru(bpy)2(NO2-κN)Ln-](n-1) and cis-[Ru(bpy)2(NO2-κO)L n-](n-1) (bpy = 2,2'-bipyridine; k = indication of the coordinated center to Ruthenium; L = pyridine type ligand) by reacting cis-[Ru(bpy)2(H2O)Ln-](n-2) with sodium nitrite or conducting basic cis-[Ru(bpy)2NO(Ln-)](n-3) hydrolysis. Photolysis at the metal-ligand charge transfer band (MLCT) of the isomers yielded nitric oxide (NO) as determined by NO measurement. The NO photorelease rates obtained upon 447 nm laser irradiation of the ruthenium complexes showed that cis-[Ru(bpy)2(NO2-κO)Ln-](n-1) released NO three times faster than cis-[Ru(bpy)2(NO2-κN)Ln-](n-1). We investigated endothelium-dependent vasodilation induced by cis-[Ru(bpy)2(4-pic)(NO2-κN)]+ and cis-[Ru(bpy)2(4-pic)(NO2-κO)]+ (4-pic = 4-picoline) in isolated 3 mm aortic rings precontracted with L-phenylephrine. Maximum vasodilation was achieved under 447 nm laser irradiation of 0.5 μMol.L-1 ruthenium complexes for 100 s.

Heart failure (HF) is a complex disease with multifactorial etiology. The degradation of the glycocalyx is a process thought to contribute to the progression of HF. The relative individual contribution of glycocalyx components to HF progression is unclear. Previous studies have shown that the expression of glypican 1 (Gpc1), a glycocalyx component, is reduced after aortic banding. Herein, we sought to determine if Gpc1 participates in the progression of HF induced by pressure overload and the associated mechanisms. Methods: Male, 5-8 weeks old, WT and Gpc1 knockout mice (Gpc1KO) were submitted to transaortic constriction (TAC) or sham procedure. Heart function was assessed by echocardiography; heart remodeling was assessed by histological analysis; heparan sulfate and glypican 1 content was assessed by immunofluorescence; the expression of ANP and myosin was assessed by immunoblotting. Results: Gpc1KO showed preserved heparan sulfate content in heart sections. Gpc1KO showed cardiac hypertrophy as evidenced by increased left ventricle mass (173.75mg vs. 123.96 mg in WT; p=0.004; n=10), left ventricle wall thickness (1.43mm vs . 1.13mm in WT; p=0.033; n=10), intraventricular septal end-diastolic thickness (1.83mm vs. 1.62mm in WT; p=0.001; n=11) and decreased cardiomyocyte cross-sectional area (203.86μm2 vs. 248.44μm2; p<0.0001; n=5). This was associated with increased stroke volume (52.89±2.1μL vs 46.42±2.0 μL; p=0.038, n=10), cardiac output (26.39 mL/min vs. 20.32 mL/min in WT), calcium deposits, ANP (0.86±0.02 vs. 0.57± 0.03; p<0.0001; n=5) and myosin (0.71±0.1 vs. 0.50±0.01 in WT; p=0.03; n=5) content in the heart. Gpc1KO were prone to TAC-induced HF as shown by decreased ejection fraction (34.37% vs . 58.41% in WT; p=0.047; n=3) and stroke volume (35.24μL vs. 44.60 μL in WT; p=0.05, n=3) 3 weeks after TAC. Moreover, Gpc1KO showed increased mortality rate, not surviving 5 weeks post-TAC. Conclusions: Gpc1KO shows basal cardiac remodeling associated with changes in calcium dynamics and increased contractility. The increased susceptibility of Gpc1KO to TAC-induced HF seems to be independent of glycocalyx degradation. Further studies are required to understand the mechanisms whereby Gpc1 regulates the progression of TAC-induced HF.

cis-[Ru(bpy)2(py)NO2](PF6) (RuBPY) is a ruthenium complex nitric oxide (NO) donor that presents a nitrite in its moiety and has been shown to induce vasodilation in various arteries, as well as arterial pressure reduction with no changes in heart rate. Because vascular tone is highly dependent on the cytosolic calcium concentration ([Ca2+ ]c), the current study aimed to investigate the effects of RuBPY on the intracellular mobilization of calcium stores of rat aortic vascular smooth muscle cells. Vascular reactivity experiments were performed in isolated aortic rings that were contracted with a high concentration of KCl or phenylephrine (Phe). Moreover, primary cultured vascular smooth muscle cells were used to measure [Ca2+ ]c by confocal microscopy. The NO donor RuBPY decreased the [Ca2+ ]c and reduced KCl and Phe-induced contractile responses. The selective inhibitor of sarco-endoplasmic Ca-ATPase (SERCA) with thapsigargin impaired the effect of RuBPY on Phe-induced contractile response. RuBPY also reduced caffeine-induced contraction, and the contraction dependent on the capacitive Ca2+ influx. Therefore, our results suggest that NO released from RuBPY decreased [Ca2+ ]c by calcium influx blockade and activation of guanylyl-cyclase-cGMP-GK pathway. These results indicate that RuBPY increases Ca2+ storage in the sarcoplasmic reticulum by SERCA activation and also by capacitive Ca2+ influx inhibition, which is dependent on the intracellular release of nitric oxide from this compound.

K+ channel activation is one of the major mechanisms involved in vasodilation. Vasoconstrictor agonists such as angiotensin II promote ATP-dependent potassium channels (KATP ) dysfunction. This study evaluates whether thromboxane-prostanoid (TP receptor) activation by the agonist U46619 increases reactive oxygen species (ROS) production in rat aortas, which could contribute to KATP channel dysfunction and impaired NO-dependent vasodilation. TP receptor activation with the selective agonist U46619 increased ROS in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), but the TP receptor antagonist SQ29548 abolished this effect. ECs and VSMCs incubation with ROS scavengers like Tiron or PEG-Catalase impaired U46619-induced ROS production. U46619 at the concentrations of 0.1 and 1 µmol/L induced contractions with similar amplitude. KATP channel activation with pinacidil-induced relaxation was lower for the contractions induced with 0.1 or 1 µmol/L U46619 than with 10 nmol/L U46619. Acetylcholine-induced relaxation provided similar results. In aortas pre-contracted with 10 nmol/L U46619, neither Tiron (100 µmol/L) nor catalase (300 U/mL) affected pinacidil-induced relaxation. However, in aortas pre-contracted with 0.1 µmol/L U46619, catalase potentiated pinacidil-induced relaxation. Pinacidil potentiated acetylcholine-induced relaxation in aortas pre-contracted with 0.1 and 1 µmol/L U46619. Incubation with 10 nmol/L U46619 increased NO concentration in ECs. Taken together, these results show that high concentrations of the TP receptor agonist U46619 impair KATP channels, which is probably due to ROS production. It is likely that hydrogen peroxide is the ROS.

This study aimed to investigate the antiproteinuric and hyperkalemic mechanisms activated by dual renin-angiotensin system (RAS) blockade in renovascular hypertensive rats (2-kidney 1-clip model [2K-1C]). Six weeks after clipping the left renal artery or sham operation (2K), rats were treated with losartan, enalapril, or both drugs for two weeks. We found that 2K-1C rats displayed higher tail-cuff blood pressure (BP), increased non-clipped kidney Ang II concentration, and more pronounced urinary albumin excretion than 2K. BP was decreased by the treatment with either enalapril or losartan, and the combination of both drugs promoted an additional antihypertensive effect in 2K-1C rats. Renal Ang II content and albuminuria were reduced by either enalapril or losartan in monotherapy and restored to control levels by dual RAS blockade. Albuminuria in 2K-1C rats was accompanied by downregulation of the glomerular slit protein podocin, reduction of the endocytic receptors megalin and cubilin, and a marked decrease in the expression of the ClC-5 chloride channel, compared to 2K animals. Treatment with losartan and enalapril in monotherapy or combination increased the expression of podocin, cubilin, and ClC-5. However, only the combined therapy normalized podocin, cubilin, and ClC-5 protein abundance in the non-clipped kidney of 2K-1C rats. Renovascular hypertensive 2K-1C rats had a lower concentration of plasma potassium compared to 2K rats. Single RAS blockade normalized potassium plasma concentration, whereas 2K-1C rats treated with dual RAS blockade exhibited hyperkalemia. Hypokalemia in 2K-1C rats was accompanied by an increase in the cleaved activated forms of α-ENaC and γ-ENaC and the expression of β-ENaC. Combined RAS blockade but not monotherapy significantly reduced the expression of these ENaC subunits in 2K-1C rats. Indeed, double RAS blockade reduced the abundance of cleaved-α-ENaC to levels lower than those of 2K rats. Collectively, these results demonstrate that the antiproteinuric effect of dual RAS blockade in 2K-1C rats is associated with the restored abundance of podocin and cubilin, and ClC-5. Moreover, double RAS blockade-induced hyperkalemia may be due, at least partially, to an exaggerated downregulation of cleaved α-ENaC in the non-clipped kidney of renovascular hypertensive rats.

Aims: Hypertension underlies endothelial dysfunction, and activation of vasorelaxation signaling with low dependence on nitric oxide (NO) represents a good alternative for vascular modulation. C-type natriuretic peptide (CNP) causes relaxation by increasing cyclic guanosine 3',5'-monophosphate (cGMP) or Gi-protein activation through its natriuretic peptide receptor-B or -C, respectively. We have hypothesized that CNP could exerts its effects and could overcome endothelial dysfunction in two kidney-one clip (2K-1C) hypertensive rat aorta. Here, we investigate the intracellular signaling involved in CNP effects in hypertension. Materials and methods: The 2K-1C hypertension was induced in male Wistar rats (200 g). CNP-induced vascular relaxation and cGMP production were investigated in rat thoracic aortas. The natriuretic peptide receptor-B and -C localization was evaluated by immunofluorescence. Calcium mobilization was assessed in endothelial cells from rat aortas. Key findings: CNP induced similar relaxation in normotensive and 2K-1C hypertensive rat aortas, which increased after endothelium removal. CNP-induced relaxation involved natriuretic peptide receptor-B and -C activation in 2K-1C rats. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) counter-regulated CNP-particulate GC (pGC) activation in aortas. CNP reduced endothelial calcium and increased cGMP production, which was lower in 2K-1C. CNP-induced cGMP-dependent protein kinase (PKG) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activation was impaired in 2K-1C rat aorta. Significance: Our results indicated CNP triggered relaxation through its natriuretic peptide receptor-B and -C in 2K-1C rat aortas, and that CNP-induced relaxation overcomes endothelial dysfunction in hypertension. In addition, NOS and sGC activities counter-regulate CNP-pGC activation to induce vascular relaxation.

Background: Vascular dysfunction is a checkpoint to the development of hypertension. Heparan sulfate proteoglycans (HSPG) participate in nitric oxide (NO) and calcium signaling, key regulators of vascular function. The relationship between HSPG-mediated NO and calcium signaling and vascular dysfunction has not been explored. Likewise, the role of HSPG on the control of systemic blood arterial pressure is unknown. Herein, we sought to determine if the HSPG syndecan 1 and glypican 1 control systemic blood pressure and the progression of hypertension. Purpose: To determine the mechanisms whereby glypican 1 and syndecan 1 regulate vascular tone and contribute to the development of noradrenergic hypertension. Experimental approach and key results: By assessing systemic arterial blood pressure we observed that syndecan 1 (Sdc1(-/-)) and glypican 1 (Gpc1(-/-)) knockout mice show a similar phenotype of decreased systolic blood pressure that is presented in a striking manner in the Gpc1(-/-) strain. Gpc1(-/-) mice are also uniquely protected from a norepinephrine hypertensive challenge failing to become hypertensive. This phenotype was associated with impaired calcium-dependent vasoconstriction and altered expression of calcium-sensitive proteins including SERCA and calmodulin. In addition, Gpc1(-/-) distinctively showed decreased IP3R activity and increased calcium storage in the endoplasmic reticulum. Conclusions and implications: Glypican 1 is a trigger for the development of noradrenergic hypertension that acts via IP3R- and calcium-dependent signaling pathways. Glypican 1 may be a potential target for the development of new therapies for resistant hypertension or conditions where norepinephrine levels are increased.

Angiotensin-(1-7) [Ang-(1-7)]/Mas receptor is a counter-regulatory axis that counteracts detrimental renin-angiotensin system (RAS) effects, especially regarding systemic inflammation, vasopressin (AVP) release, and hypothalamic-pituitary-adrenal (HPA) activation. However, it is not completely understood whether this system may control centrally or systemically the late phase of systemic inflammation. Thus, the aim of this study was to determine whether intracerebroventricular (i.c.v.) administration of Ang-(1-7) can modulate systemic inflammation through the activation of humoral pathways in late phase of endotoxemia. Endotoxemia was induced by systemic injection of lipopolysaccharide (LPS) (1.5 mg/kg, i.v.) in Wistar rats. Ang-(1-7) (0.3 nmol in 2 µL) promoted the release of AVP and attenuated interleukin-6 (IL-6) and nitric oxide (NO) levels but increased interleukin-10 (IL-10) in the serum of the endotoxemic rats. The central administration of Mas receptor antagonist A779 (3 nmol in 2 µL, i.c.v.) abolished these anti-inflammatory effects in endotoxemic rats. Furthermore, Ang-(1-7) applied centrally restored mean arterial blood pressure (MABP) without affecting heart rate (HR) and prevented vascular hyporesponsiveness to norepinephrine (NE) and AVP in animals that received LPS. Together, our results indicate that Ang-(1-7) applied centrally promotes a systemic anti-inflammatory effect through the central Mas receptor and activation of the humoral pathway mediated by AVP.

This review highlights recent findings about the role that endothelial glycocalyx and caveolae play in vascular homeostasis. We describe the structure, synthesis, and function of glycocalyx and caveolae in vascular cells under physiological and pathophysiological conditions. Special focus will be given in glycocalyx and caveolae that are associated with impaired production of nitric oxide (NO) and generation of reactive oxygen species (ROS). Such alterations could contribute to the development of cardiovascular diseases, such as atherosclerosis, and hypertension.

This review highlights recent findings about the role that endothelial glycocalyx and caveolae play in vascular homeostasis. We describe the structure, synthesis, and function of glycocalyx and caveolae in vascular cells under physiological and pathophysiological conditions. Special focus will be given in glycocalyx and caveolae that are associated with impaired production of nitric oxide (NO) and generation of reactive oxygen species (ROS). Such alterations could contribute to the development of cardiovascular diseases, such as atherosclerosis, and hypertension.

OBJECTIVES:Evaluate whether the RAS dual blockade would induce additional beneficial effects on cardiovascular remodelling when compared to monotherapy in renal hypertensive two kidneys-one clip (2K-1C) rats. METHODS:Hypertensive 2K-1C and normotensive (2K) rats were treated for 14 days with submaximal doses of losartan (LOS), enalapril (ENA), losartan plus enalapril (LOS + ENA) or vehicle (water). Blood pressure and some parameters of cardiovascular remodelling were evaluated. RESULTS:Systolic blood pressure (SBP) was higher in 2K-1C (209 ± 3 mm Hg, P < .05) than in 2K (113 ± 1 mm Hg) rats. There was an additional effect in 2K-1C treated with LOS + ENA (153 ± 9 mm Hg) on lowering SBP when compared to LOS (184 ± 12 mm Hg) or ENA (177 ± 9 mm Hg). None of the treatments had effect on SBP in 2K rats. In 2K-1C, cardiomyocyte hypertrophy was reduced by all treatments, although the cardiac hypertrophy indexes remained unchanged. 2K-1C aortas presented medial thickening that was partially reduced by the treatments. Intimal hyperplasia observed in 2K-1C (15.56 ± 0.89 µm vs 8.24 ± 0.80 µm) was reversed by ENA (9.52 ± 0.45 µm) or LOS + ENA (8.17 ± 0.53 µm). Collagen deposition was increased in 2K-1C hearts (1.77 ± 0.16 vs 1.28 ± 0.09) and aortas (8.1 ± 0.6 vs 5.2 ± 0.2). Treatment with LOS reduced (1.12 ± 0.14%) and ENA (0.81 ± 0.11%) or LOS + ENA (0.86 ± 0.11%) additionally diminished collagen only in 2K-1C hearts. CONCLUSIONS:Submaximal doses of ACEi and/or ARB have inhibitory actions on cardiac remodelling and vascular hypertrophy not entirely dependent on their effects on blood pressure normalization in renovascular hypertensive rats. Combined therapy produced additional reduction in blood pressure than monotherapy despite a similar inhibition on cardiovascular remodelling.

Angiotensin-(1-7) [Ang-(1-7)] is an angiotensin-derived neuropeptide with potential anti-hypertensive and anti-inflammatory properties. However, a possible action of Ang-(1-7) in neuroimmune interactions to regulate inflammatory response has not been explored. Thus, the aim of this study was to determine whether the intracerebroventricular (i.c.v.) administration of Ang-(1-7) can modulate systemic inflammation via sympathetic efferent circuits. Wistar male rats received systemic administration of lipopolysaccharide (LPS) (1.5 mg/Kg). Ang-(1-7) (0.3 nmol in 2 mu L) promoted the release of splenic norepinephrine and attenuated tumor necrosis factor (TNF) and nitric oxide (NO), but increased interleukin-10 (IL-10), levels in the serum, spleen, and liver in endotoxemic rats. Furthermore, 6-hydroxydopamine-induced chemical sympathectomy (100 mg/Kg, intravenous) or i.c.v. administration of Mas receptor antagonist A779 (3 nmol in 2 mu L) abolished the anti-inflammatory effects of central Ang-(1-7) injection. Moreover, this treatment did not alter the plasmatic LPS-induced corticosterone and vasopressin. The administration of Ang-(1-7) reverted the low resistance in response to catecholamines of rings of thoracic aorta isolated from endotoxemic rats, treated or not, with this peptide by a mechanism dependent on the regulation of NO released from perivascular adipose tissue. Together, our results indicate that Ang-(1-7) regulates systemic inflammation and vascular hyporesponsiveness in endotoxemia via activation of a central Mas receptors/sympathetic circuits/norepinephrine axis and provide novel mechanistic insights into the anti-inflammatory Ang-(1-7) properties.

Physiological or supraphysiological levels of testosterone appear to be associated with the development of risk factors for cardiovascular diseases such as hypertension, as this hormone modulates the release of endothelial factors. However, its actions are still controversial, especially in the coronary circulation of hypertensive animals. This study was designed to assess the effects of testosterone treatment (T) on endothelium-dependent coronary vascular reactivity in orchiectomized SHR. The animals were divided into SHAM, orchiectomized (ORX), ORX+T and ORX+T+aromatase inhibitor (AI). All treatments lasted 15 days. Blood pressure (BP) was measured. Dose-response curves to bradykinin (BK) were constructed using the Langendorff technique, followed by inhibition of endothelium mediators (NO, prostanoids, EETs) and potassium channels. The intensity of eNOS, COX-1, COX-2, Akt, and gp91phox protein expression was quantified by Western blotting. BP was elevated in SHAM, ORX+T, and ORX+T+AI groups. However, we did not observe differences in the ORX group. Baseline coronary perfusion pressure (CPP) remained unaffected. Orchiectomy did not change the BK-induced relaxation compared to the SHAM group, whereas testosterone treatment increased it. This response was diminished in the absence of NO, prostanoids, and EETs in the SHAM and ORX groups, while in ORX+T group the relaxation was diminished only in the absence of NO and EETs. There was no difference in eNOS, COX-1, COX-2, and gp91phox protein expression, though Akt expression was increased in ORX and ORX+T groups. These results show that testosterone treatment can modulate endothelial function, especially in the coronary circulation under hypertension conditions, via NO and EETs pathways.

Endothelial dysfunction and consequent vasoconstriction are a common condition in patients with hypertension and other cardiovascular diseases. Endothelial cells produce and release vasodilator substances that play a pivotal role in normal vascular tone. The mechanisms underlying endothelial dysfunction are multifactorial. However, enhanced reactive oxygen species (ROS) production and consequent vasoconstriction instead of endothelium-derived relaxant generation and consequent vasodilatation contribute to this dysfunction considerably. The main targets of the drugs that are currently used to treat vascular diseases concerning enzyme activities and protein functions that are impaired by endothelial nitric oxide synthase (eNOS) uncoupling and ROS production. Nitric oxide (NO) bioavailability can decrease due to deficient NO production by eNOS and/or NO release to vascular smooth muscle cells, which impairs endothelial function. Considering the NO cellular mechanisms, tackling the issue of eNOS uncoupling could avoid endothelial dysfunction: provision of the enzyme cofactor tetrahydrobiopterin (BH4) should elicit NO release from NO donors, to activate soluble guanylyl cyclase. This should increase cyclic guanosine-monophosphate (cGMP) generation and inhibit phosphodiesterases (especially PDE5) that selectively degrade cGMP. Consequently, protein kinase-G should be activated, and K+ channels should be phosphorylated and activated, which is crucial for cell membrane hyperpolarization and vasodilation and/or inhibition of ROS production. The present review summarizes the current concepts about the vascular cellular mechanisms that underlie endothelial dysfunction and which could be the target of drugs for the treatment of patients with cardiovascular disease.

Aim: Although progesterone (P4) has a beneficial effect on the cardiovascular system, P4 actions on the coronary bed have not yet been fully elucidated. This study evaluated the effect of progesterone treatment on endothelium-dependent coronary vascular reactivity in Wistar rats. Main methods: Eight-week-old adult rats were divided into Sham, Ovariectomized (OVX), Ovariectomized and progesterone treated (OVX P4). The OVX P4 group received daily doses of progesterone (2 mg/kg/day). Vascular reactivity was assessed by a modified Langendorff technique. The intensity of eNOS, Akt, and gp9lphox protein expression was quantified by Western blotting. Superoxide anion (O-2(center dot-)) and hydrogen peroxide (H2O2) production was measured by dihydroethidium and 2',7'-dichlorofluorescein, respectively. Key findings: Treatment with P4 was able to prevent the reduction in baseline coronary perfusion pressure induced by ovariectomy. We observed that endothelium-dependent coronary vasodilation was reduced in the OVX group and potentiated in the OVX P4 group. Following the inhibition of the nitric oxide (NO) pathway, the bradykinin-induced relaxing response was potentiated in the OVX P4 group. With regard to the combined inhibition of NO and prostanoids pathways, the OVX P4 group showed a greater relaxing response, similar to what was found upon individual inhibition of NO. After the combined inhibition of NO, prostanoids and epoxyeicosatrienoic acids' pathways, the vasodilatory response induced by BK was abolished in all groups. Significance: Treatment with P4 prevented oxidative stress induced by ovariectomy. These results suggest that progesterone has a beneficial action on the coronary vascular bed.

PURPOSE:This study investigated the intracellular mechanisms involved in the vasodilatation induced by the classic NO donor SNP and the non-classic NO donor cis-[Ru(bpy)2(py)(NO2)](PF6) (or RuBPY) in mesenteric resistance arteries obtained from renal hypertensive (2K-1C) and normotensive (2K) rats. METHODS:On the basis of fluorimetric assays in cultured vascular smooth muscle cells (VSMCs) isolated from 2K-1C and 2K rats, we measured NO release from SNP and RuBPY, cytosolic Ca2+ concentration ([Ca2+]c), and reactive oxygen species (ROS) with the selective probes DAF-2DA, Fluo-3AM and the more selective probe for peroxynitrite (7-CBA), respectively. We determined isometric tension in mesenteric arteries to assess SNP- and RuBPY-induced relaxation. RESULTS:SNP and RuBPY released NO in comparable amounts in cultured aortic VSMCs from hypertensive 2K-1C and normotensive 2K rats. The NO0 scavenger hydroxocobalamin blunted NO release. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibition with thapsigargin reduced [Ca2+]c in normotensive 2K rat VSMCs only. ROS amounts were greater in hypertensive 2K-1C than in normotensive 2K rat VSMCs, but neither SNP nor RuBPY altered ROS concentrations in any of the groups. SNP and RuBPY induced similar relaxation in hypertensive 2K-1C and normotensive 2K rat mesenteric resistance arteries. The SNP and RuBPY-induced relaxation involves sGC and PKG activation. On the other hand, SNP but not RuBPY activates K+ channels. Interestingly, SERCA inhibition reduces SNP induced relaxation only in normotensive 2K rat mesenteric arteries whereas RuBPY-induced relaxation does not involve SERCA activation in both normotensive and hypertensive arteries. CONCLUSION:Our results indicate that SNP and RuBPY-induced mesenteric resistance artery relaxation involves NO/sGC/cGMP/PKG pathway activation. K+ channels and SERCA activation is required to SNP but not for RuBPY-induced relaxation. Moreover, SERCA seems to be impaired in hypertensive 2K-1C rat mesenteric resistance arteries although it does not impact SNP- or RuBPY-induced relaxation.

Acetaminophen (APAP) is one of the most widely consumed drugs in the world. Studies have shown renal and hepatic damage as the direct result of high oxidative stress induced by APAP. Since the cardiovascular system is sensitive to oxidative stress and literature describes increased cardiovascular dysfunction in APAP consumers, this work aimed to evaluate harmful effects of APAP on the vascular system. Rats were exposed to APAP (400 mg/kg/day in drinking water) for 14 days. Plasma and aortas were collected and stored in − 80 °C and a selection of arteries was prepared for isometric tension recordings, morphological, immunohistochemical and protein expression analysis. The APAP-treated group presented increased transaminases (ALT/AST) and malondialdehyde levels in the plasma compared to controls. Lipid peroxidation, glutathione reductase and superoxide dismutase levels were increased in the plasma and arteries of the APAP group. Nevertheless, glutathione level was reduced as compared to control group. The vasodilation response to acetylcholine and sodium nitroprusside (0.1 nM to 10 µM) was also impaired after APAP treatment; however, the vascular relaxation was restored after treatment with vitamin C (100 µM). Arteries from the APAP group presented reduced wall thickness, collagen deposition, elastic fibers and increased immunoreactivity to nitrotyrosine. eNOS and sGC protein expression remained unchanged and were at similar levels as controls. These findings showed higher oxidative stress and impaired vasodilation in rats exposed to APAP. Furthermore, arteries presented reduced cell layers, collagen, elastin deposition and significantly increased immunoreactivity to nitrotyrosine after APAP treatment.

Chronic treatment with apocynin reduces blood pressure and prevents endothelial dysfunction development in spontaneously hypertensive rats (SHR). Mechanisms underlying apocynin effects on SHR remain unclear. Compared to diapocynin and other drugs, apocynin is a weak antioxidant, which suggests that its effects on SHR are associated with other mechanisms besides its antioxidant capacity. Angiotensin (Ang) II regulates NOX, the major reactive oxygen species (ROS) source in the cardiovascular system. We hypothesized that, by inhibiting NOX, apocynin could alter Ang II pressor and vasoconstrictor effects on SHR. We analyzed how Ang II affects blood pressure and vascular reactivity in aorta and mesenteric resistance arteries and evaluated plasma antioxidant capacity, NOX isoforms and subunits, NOS isoforms, AT1 and AT2 receptors expression, ROS production, and NOS activity in apocynin-treated SHR blood vessels (30 mg/Kg/day, p.o.). In SHR, apocynin reduced Ang II pressor effects, increased plasmatic antioxidant capacity, and blunted aortic and mesenteric NOX-dependent oxidants production and NOX2 and p47phox overexpression, which demonstrated that apocynin inhibits NOX in SHR blood vessels. Moreover, apocynin raised plasmatic and aortic nitrate/nitrite levels, maintained NOS activity and eNOS, p-eNOS, nNOS, iNOS, sGC-α, and sGC-β expression in mesenteric bed, diminished AT1 expression in aorta and mesenteric bed, and elevated AT2 expression in SHR aorta. Apocynin increased Ang II vasoconstriction endothelial modulation in SHR resistance arteries. All these results showed that in vivo treatment with apocynin alters several mechanisms that reduce Ang II pressor and vasoconstrictor effects on SHR. Such apocynin effects involve other mechanisms besides vascular ROS modulation, which improves NO availability in SHR vascular cells. These integrated data could help us to understand the promising apocynin activity as an antihypertensive drug that acts differently from the drugs that are currently being used in the clinical setting.