L-nitroarginine Methyl Ester Research Articles

Role of Endothelial Nrf2 in the Regulation of Vasomotor Capacity and Arterial Blood Pressure

Introduction: Hypertension is a serious global public health issue. It is a complex disease with multiple factors contributing to its pathophysiology. One of these factors is oxidative stress, which results from an imbalance between the production and elimination of reactive oxygen species (ROS). Studies have shown that an increased ROS in endothelial cells (EC) impairs the vasodilatory capacity of blood vessels that contributes to blood pressure elevation. However, the modulation of endothelial ROS in the setting of hypertension is still not completely understood. To maintain cellular homeostasis, antioxidant enzymes continually adjust ROS levels under the orchestrating of transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of endogenous antioxidant responses. Modulation of endothelial Nrf2 expression may be a potential therapeutic option in hypertension. Therefore, the major objective of this study was to evaluate the role of Nrf2 in the modulation of endothelium associated vascular function and how it regulates vascular tone and blood pressure (BP). Methods: BP was measured in the conscious state using radiotelemetry transducers implanted into EC-specific Nrf2-knockout mice (Tie2-Cre-Nrf2 Floxed), EC specific Keap1 knockout mice (Tie2-Cre-Keap1 Floxed) and Wildtype (WT), all on the C57B6J background. After baseline BP recordings, mice were implanted with subcutaneous osmotic minipumps (7 days) for Angiotensin II (Ang II) (600ng/Kg/min) infusion or in a separate group L-nitroarginine methyl ester (L-NAME) (100 mg/kg/d in drinking water) for 2 weeks. For each intervention, recordings were done daily for 2 hours and one 24-hour measurement was done once per week. Endothelial specific downregulation or upregulation of Nrf2 was confirmed by immunohistochemistry on mouse aortic tissue and protein expression from mouse EC. For in-vitro studies: femoral-to-popliteal artery segments were harvested from mice of each genotype. Vasodilation was assessed in response to flow (30 uL·min−1) and acetylcholine (ACh, 10−7-10−3 M) with and without L-NAME, using video microscopy. Results: Ang II infusion significantly increased the mean arterial pressure (MAP) in each group when compared to their baseline values (Baseline: Nrf2KO = 92.6 ± 3.1, Keap1KO = 93.8 ± 4.1 and WT = 92.8 ± 1.7 mmHg; after Ang II Nrf2KO = 141.7 ± 3.8, Keap1KO = 121.9±5.4 and WT = 120.4±5.4 mmHg). The response to ANG II was significantly (p<0.05) augmented in Nrf2KO mice and stayed elevated for a longer period after emptying of the pump. The response to L-NAME was similar in both Nrf2KO and WT mice. Protein expression of Nrf2 was decreased in Nrf2KO mice in comparison to WT mice. Nrf2KO mice exhibited reduced endothelium-dependent vasodilation induced by flow (WT: 35.8± 1.3%, Nrf2KO: 24.1 ±1.7%; p<0.0001) and ACh (10−3M) (WT: 60.4±4.1%, Nrf2KO: 44.7± 2.1%; p<0.0001) compared to WT. In contrast, endothelial dependent vasodilatory responses to flow and Ach were markedly elevated in Keap1KO mice (51.3± 6.8%, p<0.0001; 71.6±11.6%, p<0.0087, respectively) compared to both WT and Nrf2KO mice. Conclusion: These results suggest that antioxidant defense mediated by Nrf2 in the endothelium plays a significant role in the modulation of blood pressure and vascular tone. Based on the L-NAME data, these effects are most likely due to an increase in the nitric oxide bioavailability and reduced ROS. University of Nebraska Theodore Hubbard foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Vasodilator Effect and Mechanism of 18β-glycyrrhetinic Acid on Isolated Pulmonary Artery Rings in Rats

Background: The purpose of this study was to evaluate the effect of 18β-glycyrrhetinic acid (18β-GA) on isolated rat pulmonary artery vascular rings as well as the mechanism that lies behind the diastolic effects of this compound. Methods: To examine the effects of various doses of 18β-GA on resting normal vascular rings, we used isolated rat pulmonary artery vascular rings. The isolated rat pulmonary artery vascular ring was precontracted with phenylephrine (PE) and potassium chloride solution (KCl), which allowed for the observation of the diastolic effects of 18β-GA, as well as the effects of various concentrations of 18β-GA on blocking the four potassium channels of glibenclamide, barium chloride (BaCl2), tetraethylamine (TEA), or 4-aminopyridine; the impact of various 18β-GA concentrations on the pulmonary artery vasodilation effect pre-use of the nitric oxide synthase inhibitors l-nitroarginine methyl ester and indomethacin. Results: It was shown that 18β-GA has concentration-dependent diastolic effects on isolated rat pulmonary vascular rings that have been precontracted with PE and KCl but it has no effect on resting isolated thoracic aortic vascular rings. Conclusion: The two Kir and Kv channels may be connected to the endothelium-dependent vasodilation mechanism of 18β-GA.

Effect of asiaticoside on systolic blood pressure and relaxation of isolated thoracic aorta of rats

To investigate the effect of asiaticoside on blood pressure and relaxation of thoracic aorta in rats and explore the underlying mechanism. SD rats treated with 50 and 100 mg/kg asiaticoside by daily gavage for 2 weeks were monitored for systolic blood pressure changes, and histological changes of the thoracic aorta were evaluated using HE staining. In isolated rat endothelium-intact and endothelium-denuded thoracic aorta rings, the effects of asiaticoside on relaxation of the aortic rings were tested at baseline and following norepinephrine (NE)- and KCl-induced constriction. The vascular relaxation effect of asiaticoside was further observed in NE-stimulated endothelium-intact rat aortic rings pretreated with L-nitroarginine methyl ester, indomethacin, zinc protoporphyrin Ⅸ, tetraethyl ammonium chloride, glibenclamide, barium chloride, Iberiotoxin, 4-aminopyridine, or TASK-1-IN-1. The aortic rings were treated with KCl and NE followed by increasing concentrations of CaCl2 to investigate the effect of asiaticoside on vasoconstriction induced by external calcium influx and internal calcium release. Asiaticoside at 50 and 100 mg/kg significantly lowered systolic blood pressure in rats without affecting the thoracic aorta histomorphology. While not obviously affecting resting aortic rings with intact endothelium, asiaticoside at 100 mg/kg induced significant relaxation of the rings constricted by KCl and NE, but its effects differed between endothelium-intact and endothelium-denuded rings. In endothelium-intact aortic rings pretreated with indomethacin, ZnPP Ⅸ, barium chloride, glyburide, TASK-1-IN-1 and 4-aminopyridine, asiaticoside did not produce significant effect on NE-induced vasoconstriction, and tetraethylammonium, Iberiotoxin and L-nitroarginine methyl ester all inhibited the relaxation effect of asiaticoside. In KCland NE-treated rings, asiaticoside obviously inhibited CaCl2-induced vascular contraction. Asiaticoside induces thoracic aorta relaxation by mediating high-conductance calcium-activated potassium channel opening, promoting nitric oxide release from endothelial cells and regulating Ca2+ influx and outflow, thereby reducing systolic blood pressure in rats.

Endothelial Nrf2 modulates blood pressure and vasoconstrictor responses in conscious mice

Introduction: Excessive oxidant stress has been shown to mediate a variety of cardiovascular pathological conditions, including hypertension. The level of reactive oxygen species (ROS) in tissues is regulated by the balance between the production of ROS and the ability to scavenge superoxide and hydrogen peroxide by a myriad of antioxidant enzymes and small molecules. One of the most important regulators of antioxidant activity is the ubiquitous transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 binds to antioxidant response elements of multiple genes following its release from its inhibitory protein Keap1 and part of the E3 ubiquitin ligase complex. In this study we hypothesized that endothelial Nrf2 is a critical factor in blood pressure regulation following vasoconstrictor stimuli. Methods: Transgenic mice were created using the endothelial Tie2 promoter expressing Cre recombinase (Tie2-Cre) crossed with either Nrf2 floxed or Keap1 floxed mice so that Nrf2 would be deleted or upregulated respectively, and selectively in the endothelium. Cre negative mice were used as wild type (WT) controls. Mice were instrumented with radiotelemetry transducers to chronically record arterial blood pressure and heart rate in the conscious state. Blood pressure and heart rate were recorded for 24 hours once per week. After baseline recordings, animals were challenged with an osmotic minipump infusion of Angiotensin II (Ang II) (600ng/Kg/min) for 7 days or L-nitroarginine methyl ester (L-NAME) (100 mg/kg/d in drinking water). Results: Baseline mean arterial pressure (MAP) was 101.6 ± 2.5 mm Hg (mean±SEM; n=7) in WT mice (daytime =95.4±1.5; nighttime =106.1±1.0). In mice with endothelial Nrf2 deletion baseline MAP was significantly higher(p<0.01) during nighttime (112.1±1.1). In addition, the response to Ang II was significantly augmented (p < 0.001) in Nrf2 KO mice compared to WT mice (148.9±5.3 vs 116.8±6.7 mm Hg). Both day and nighttime values were higher in Nrf2 KO mice. Furthermore, Nrf2 KO mice exhibited a delayed return to baseline upon pump emptying. In contrast, in one Keap1 KO mouse there was a response to Ang II that was similar to WT (92.1 mm Hg baseline to 116.3 mmHg post Ang II). In Nrf2 deleted mice, the hypertensive response to L-NAME was no different than that of WT mice. Conclusion: In summary, these preliminary data suggest that endothelial Nrf2 plays an important role in modulation of blood pressure at rest and during oxidative stress induced by Ang II. The L-NAME data suggests that upregulation of Nrf2 increases the bioavailability of nitric oxide to blunt Ang II mediated vasoconstriction. Supported by NHLBI P01-62222 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of the Non-steroidal Anti-inflammatory Drug Diclofenac on Ischemia-Reperfusion Injury in Rat Liver: A Nitric Oxide-Dependent Mechanism.

Ischemia/reperfusion injury (IRI) is an inevitable complication of liver surgery and transplantation. The purpose of this study was to examine the beneficial effects of diclofenac on hepatic IRI and the mechanism behind it. Wistar rats' livers were subjected to warm ischemia for 60min followed by 24h of reperfusion. Diclofenac was administered intravenously 15min before ischemia at 10, 20, and 40mg/kg body weight. To determine the mechanism of diclofenac protection, the NOS inhibitor L-Nitro-arginine methyl ester (L-NAME) was administered intravenously 10min after diclofenac injection (40mg/kg). Liver injury was evaluated by aminotransferases (ALT and AST) activities and histopathological analysis. Oxidative stress parameters (SOD, GPX, MPO, GSH, MDA, and PSH) were also determined. Then, eNOS gene transcription and p-eNOS and iNOS protein expressions were evaluated. The transcription factors PPAR-γ and NF-κB in addition to the regulatory protein IκBα were also investigated. Finally, the gene expression levels of inflammatory (COX-2, IL-6, IL-1β, IL-18, TNF-α, HMGB-1, and TLR-4) and apoptosis (Bcl-2 and Bax) markers were measured. Diclofenac, at the optimal dose of 40mg/kg, decreased liver injury and maintained histological integrity. It also reduced oxidative stress, inflammation, and apoptosis. Its mechanism of action essentially depended on eNOS activation rather than COX-2 inhibition, since pre-treatment with L-NAME abolished all the protective effects of diclofenac. To our knowledge, this is the first study demonstrating that diclofenac protects rat liver against warm IRI through the induction of NO-dependent pathway. Diclofenac reduced oxidative balance, attenuated the activation of the subsequent pro-inflammatory response and decreased cellular and tissue damage. Therefore, diclofenac could be a promising molecule for the prevention of liver IRI.

EXPERIMENTAL PARTICIPATION OF PHARMACOLOGICAL SUBSTANCES IN MECHANISMS OF LEAD ACETATE TOXICITY

The aim of the work is to study pharmacological substances that play a role of eNOS expression regulators in the modification of lead intoxication effects in the experiment.Materials and methods. In the experiment, linear male rats of the same age were used: intact and with lead intoxication (120 heads). The study design was the following: group 1 – control; group 2 – intoxication with a lead acetate solution; group 3 – intact + L-nitroarginine methyl ester; group 4 – lead acetate + L-nitroarginine methyl ester; group 5 – intact + L-arginine; group 6 – lead acetate + L-arginine. The research carried out the study state of the redox reactions, the content of nitric oxide (NOx) stable metabolites, a lipid profile, the level of NO-synthase (eNOS) expression in the vascular endothelium, the main processes of urination and the activity of Na+/K+-ATPase in the renal tissue layers, as well as in the liver. The results were subjected to statistical processing.Results. Saturnism caused the oxidative stress development, a decrease in the NOx content in blood plasma, a violation of the L-arginine for eNOS bioavailability, and an endothelial dysfunction. Indicators of the impaired renal function were a decrease in the glomerular filtration rate (GFR), the tubular reabsorption of water, sodium, and the Na+/K+-ATPase activity. The damage to hepatocytes was evidenced by changes in the activity of organ-specific enzymes in the blood and Na+/K+-ATPase. L-arginine exhibited antioxidant properties, increased the NOx content and the level of eNOS expression. The eNOS L-nitroarginine methyl ester inhibitor showed the effects opposite to L-arginine.Conclusion. Biochemical markers of damage to kidney and liver cells during saturnism are indicators of the oxidative stress, NOx deficiency and hemodynamic disturbances in them. These mechanisms involved the following pharmacological substances: an eNOS inhibitor, L-nitroarginine methyl ester, which caused a decrease in the expression level of the enzyme, and an eNOS inducer, L-arginine, which increased this indicator severity. The lead toxicity mechanisms have been implicated in the impaired cholesterol metabolism, contributing to the L-arginine reduced availability for eNOS and the NOx production. Therefore, the use of L-arginine can be recommended as a regulator of the oxidative stress and an NO-producing endothelial function in other pathologies.

Exploring the possible mechanism involved in the anti-nociceptive effect of β-sitosterol: modulation of oxidative stress, nitric oxide and IL-6.

Β-sitosterol is a phytosterol, documented to possess various activities including protection againstinflammation, diabetes and Alzheimer's disease. The current investigation was designed to explore the analgesic potential of β-sitosterol and the possible molecular mechanism involved in the observed effect. β-sitosterol was administered at varying doses of 10, 20, and 40mg/kg before subjecting the mice to acetic acid and formalin challenges. The number of writhings in acetic acid and the number of flinchings and foot tappings were quantified in the formalin test. For mechanistic studies, substance P (cyclooxygenase-2 (COX-2) stimulator) and L-Nitro arginine methyl ester (L-NAME) (nitric oxide synthetases (NOS) inhibitor) and L-arginine (nitric oxide precursor) were administered before β-sitosterol treatment. β-sitosterol (10, 20, 40mg/kg) treatment significantly reduced acetic acid-induced writhings and ameliorated the formalin-induced inflammatory phase dose-dependently. Whereas, 40mg/kg dose of β-sitosterol abrogated the formalin-induced neurogenic phase. Substance-P abrogated the effect of β-sitosterol in both neurogenic and inflammatory phases. Whereas, L-arginine only abrogated the inflammatory phase. In biochemical analysis, β-sitosterol treatment reduced the level of interleukin-6 (IL-6), thiobarbituric acid reactive substances (TBARS) and increased the level of reduced glutathione (GSH). Furthermore, L-arginine and substance-P abrogated the GSH increasing and TBARS lowering effect of β-sitosterol (40mg/kg). Overall, the current study delineated that β-sitosterol may induce an anti-nociceptive effect via inhibiting the IL-6, oxidative stress, cyclo-oxygenase and nitric oxide.

Analysis of oxidative modification of rat epididymal mitochondrial proteins under normobaric chronic hypoxia and modulation of nitric oxide (II) synthesis

Background. The molecular mechanism of NO effects in adaptation to hypoxia is of interest as a potential point of application in the therapy of fertility disorders.
 Aim. To assess the degree of oxidative modification of epididymal mitochondrial proteins during hypoxia under conditions of experimentally modified NO synthesis.
 Material and methods. Sexually mature rats were divided into four groups of 8 individuals: (1) control (animals without hypoxia); (2) chronic normobaric hypoxia, the animals were kept in a sealed chamber with oxygen reduced to 10% once a day for 14 days; (3) animals with hypoxia were injected with an inhibitor of the synthesis of nitric oxide (II) L-nitroarginine methyl ester at a dose of 25 mg/kg intraperitoneally once a day for 7 days; (4) animals with hypoxia were injected with the substrate for NO synthesis L-arginine at a dose of 500 mg/kg intraperitoneally once a day for 10 days. In the mitochondrial fraction of the head and tail of the epididymis, the activity of superoxide dismutase, the amount of NO metabolites, and the degree of oxidative modification of proteins were evaluated. Statistical analysis was performed using the ShapiroWilk, MannWhitney, and Spearman tests; differences were considered significant at p 0.05.
 Results. Hypoxia led to an increase in the oxidative modification of proteins in the mitochondria of the head of the epididymis 319.12 [240.98; 363.63] c.u./mg protein relative to control 17.89 [15.31; 27.62] c.u./mg protein, p=0.0009. In the mitochondria of the tail of the epididymis, the oxidation of proteins under the studied conditions was less pronounced. The use of L-nitroarginine methyl ester, as well as L-arginine, led to a decrease in the level of oxidative modification of proteins in the head of the epididymis relative to the hypoxia model 39.89 [29.25; 43.17] and 37.25 [34.91; 40.96] c.u./mg of protein, respectively, p=0.0009.
 Conclusion. Mitochondrial proteins in the head of the epididymis are more susceptible to oxidative damage during hypoxia; a decrease in the level of NO metabolites under conditions of oxygen deficiency is associated with a decrease in the oxidative modification of mitochondrial proteins.

Malate reduced blood pressure and exerted differential effects on renal hemodynamics; role of the nitric oxide system and renal epithelial sodium channels (ENaC)

Malate regulates blood pressure via nitric oxide production in salt-sensitive rats, a genetic model of hypertension. This study investigated the possible contributions of malate to blood pressure regulation and renal haemodynamics in normotensive rats. Malate (0.1, 0.3 and 1 μg/kg, iv) was injected into rats or L-nitro-arginine methyl ester (L-NAME)-treated rats and mean arterial blood pressure (MABP), cortical blood flow (CBF), and medullary blood flow (MBF), was measured. The clearance study involved infusion of malate at 0.1 μg/kg/h into rats, and MABP, CBF, MBF, glomerular filtration rate (GFR), urine volume (UV) and sodium output (UNaV) were determined. Mechanistic studies to evaluate the role of renal sodium channels involved the treatment with malate (600 mg/kg, po), amiloride (2.5 mg/kg, po) or hydrochlorothiazide (HCTZ) (10 mg/kg, po), and UV and UNaV were determined. Malate elicited significant peak reductions in MABP (124 ± 6.5 vs 105 ± 3.1 mmHg) at 0.1 μg/kg), CBF (231 ± 18.5 vs 205 ± 10.9 PU). L-NAME did not reverse the effect of malate on MABP but tended to blunt the effect on CBF (40%) and MBF (87%) at 0.3 μg/kg. Infusion of malate reduced MABP, CBF, and MBF in a time-dependent manner (p<0.05). Malate exerted a three-fold decrease in GFR in a time-related fashion (p<0.05) as well as increased UV. UNaV increased by 86% in malate-treated-amiloride rats (p<0.05). These data indicate that malate modulates blood pressure and exerts vascular and tubular effects on renal function that may involve epithelial sodium channels (ENaC).

Prophylactic Treatment with Hydrogen Sulphide Can Prevent Renal Ischemia-Reperfusion Injury in L-NAME Induced Hypertensive Rats with Cisplatin-Induced Acute Renal Failure.

(Background and Objectives): Renal ischemia perfusion injury is one of the major issues in kidney transplant. The aim of the study was to investigate the hypothesis that prophylactic treatment-with a hydrogen sulphide donor to an acute renal failure case of hypertensive rats-can minimize the ischemia reperfusion injury of the kidney which is beneficial for kidney transplant. To check this hypothesis, the present study was designed to investigate the effect of chronic administration of a hydrogen sulphide (H2S) donor and sodium hydrosulfide (NaHS) on nuclear factor kappa B (NF-kB) and inter cellular adhesion molecule-1 (ICAM-1) concentration in non-renal failure (NRF) and acute renal failure (ARF) rats in the ischemia-reperfusion injury (IRI) model of the kidney in both normotensive WKY and hypertensive rats (L-nitro arginine methyl ester (L-NAME-induced); (Materials and Methods): A total number of 48 Sprague-Dawley rats were recruited into eight groups each consisting of six animals. Each of these eight groups was used to measure systemic and renal parameters, H2S, antioxidant parameters in plasma, plasma concentration of NF-kB and ICAM-1 and renal cortical blood pressure. ARF was induced by single intraperitoneal (i.p.) cisplatin injection (5 mg/kg). Hypertension was induced by oral administration of L-NAME in drinking water for four weeks at 40 mg/kg/day. NaHS was administered (i.p) at 56 µmol/kg for five weeks while dL-propargylglycine (PAG), a H2S generation inhibitor, was administered as a single intra-peritoneal injection (50 mg/kg). An acute surgical experiment was performed for the induction of renal ischemia for 30 min by renal artery clamping followed by reperfusion for three hours; (Results): Chronic administration of NaHS attenuated the severity of ARF in both normotensive and hypertensive animals (L-NAME) along with lowering the blood pressure in hypertensive groups. NaHS improved the oxidative stress parameters such as total superoxide dismutase (T-SOD), glutathione (GSH) and reduced the malondialdehyde (MDA) concentration along with reduction of NF-kB and ICAM-1 following renal IRI; Conclusions: These findings demonstrate that H2S not only reduced the severity of cisplatin induced ARF but also reduced the severity of renal IRI by upregulating antioxidants along with decreased concentrations of NF-kB and ICAM-1 in normotensive and L-NAME induced hypertensive rats.

Ajuga bracteosa Exerts Antihypertensive Activity in l-NAME-Induced Hypertension Possibly through Modulation of Oxidative Stress, Proinflammatory Cytokines, and the Nitric Oxide/Cyclic Guanosine Monophosphate Pathway

Ajuga bracteosa has been used in traditional medicine to treat hypertension and other ailments. The present study has been designed to investigate the beneficial effects of A. bracteosa in l-nitro arginine methyl ester (l-NAME)-induced hypertensive rats. Hypertension was induced by intraperitoneal injection of l-NAME (185 μmol kg–1 i.p.). The aqueous methanol extract of A. bracteosa (AMEAB, 250 and 500 mg kg–1) and coumarin (30 and 70 mg kg–1) were administered orally from day 8 to day 35 of the study. In vivo antihypertensive activity was assessed by measuring the blood pressure using a PowerLab data system. The effects of the AMEAB and coumarin on nitric oxide (NO), cyclic guanosine monophosphate (cGMP), interleukin-6 (IL-6), the tumor necrosis factor (TNF-α), and oxidative stress markers were also assessed using kit methods. Phytochemical profiling of the AMEAB was carried out through high-performance liquid chromatography (HPLC) where quercetin, gallic acid, caffeic acid, vanillic acid, benzoic acid, syringic acid, p-coumaric acid, and ferulic acid were labeled as plant constituents including coumarin. The AMEAB and coumarin significantly reduced blood pressure at the tested doses of 500 and 70 mg kg–1, respectively. Serum levels of NO and cGMP were found to be significantly increased in AMEAB- and coumarin-treated groups when compared with only l-NAME-challenged rats. In addition, a marked decrease was noticed in the serum concentrations of proinflammatory cytokines (IL-6 and TNF-α) in AMEAB- and coumarin-treated rats. Moreover, in AMEAB- and coumarin-treated animals, a noticeable improvement was observed in the levels of antioxidant enzymes including catalase, superoxide dismutase, and malonaldehyde, and the total oxidant status when compared with those of only l-NAME-challenged rats. The data of real-time polymerase chain reaction (RT-PCR) experiments supported that the antihypertensive and anti-inflammatory activities of the AMEAB and coumarin are possibly mediated through modulation of endothelial nitric oxide synthase (eNOS), angiotensin-converting enzyme (ACE), nuclear factor (NF)-kB, and COX-2 gene expressions. This study concludes that A. bracteosa possesses an antihypertensive effect mediated through the modulation of the antioxidant, anti-inflammatory, and NO/cGMP pathways, thus providing a rationale to the antihypertensive use of A. bracteosa in traditional medicine.

Leonurine Reduces Oxidative Stress and Provides Neuroprotection against Ischemic Injury via Modulating Oxidative and NO/NOS Pathway.

Leonurine (Leo) has been found to have neuroprotective effects against cerebral ischemic injury. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. The aim of the present study was to investigate whether Leo could provide protection through the nitric oxide (NO)/nitric oxide synthase (NOS) pathway. We firstly explored the effects of NO/NOS signaling on oxidative stress and apoptosis in in vivo and in vitro models of cerebral ischemia. Further, we evaluated the protective effects of Leo against oxygen and glucose deprivation (OGD)-induced oxidative stress and apoptosis in PC12 cells. We found that the rats showed anxiety-like behavior, and the morphology and number of neurons were changed in a model of photochemically induced cerebral ischemia. Both in vivo and in vitro results show that the activity of superoxide dismutase (SOD) and glutathione (GSH) contents were decreased after ischemia, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were increased, indicating that cerebral ischemia induced oxidative stress and neuronal damage. Moreover, the contents of NO, total NOS, constitutive NOS (cNOS) and inducible NOS (iNOS) were increased after ischemia in rat and PC12 cells. Treatment with L-nitroarginine methyl ester (L-NAME), a nonselective NOS inhibitor, could reverse the change in NO/NOS expression and abolish these detrimental effects of ischemia. Leo treatment decreased ROS and MDA levels and increased the activity of SOD and GSH contents in PC12 cells exposed to OGD. Furthermore, Leo reduced NO/NOS production and cell apoptosis, decreased Bax expression and increased Bcl-2 levels in OGD-treated PC12 cells. All the data suggest that Leo protected against oxidative stress and neuronal apoptosis in cerebral ischemia by inhibiting the NO/NOS system. Our findings indicate that Leo could be a potential agent for the intervention of ischemic stroke and highlighted the NO/NOS-mediated oxidative stress signaling.

Anti-cancer effect of in vivo inhibition of nitric oxide synthase in a rat model of breast cancer.

Increased expression of nitric oxide synthase (NOS) is associated with different cancers such as cervical, breast, lung, brain, and spinal cord. Inhibition of NOS activity has been suggested as potential tool to prevent breast cancer. The anti-tumor therapeutic effect of L-nitro arginine methyl ester (L-NAME), NOS inhibitor, using in vivo models is currently under investigation. We hypothesized that L-NAME will show an anti-tumor effect by delaying a progression of breast cancer via a modulation of cell death and proliferation, and angiogenesis. We used a novel model of anti-cancer treatment by the administration of L-NAME (30mg/kg in a day, intraperitoneal) injected every third day for five weeks to rat model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumor. Concentrations of nitrite anions, polyamines, malondialdehyde, NH4+ levels, and arginase activity in the blood were decreased in DMBA + L-NAME-treated rats compared with DMBA rats. The mortality rates, tumor number, weight, and volume, as well as the histopathological grade of breast cancer were also significantly reduced. In addition, L-NAME treatment showed a delay in tumor formation, and in body weight compared with rats administrated only with DMBA. In conclusion, our data show that L-NAME is a promising anti-cancer agent to treat breast cancer, which can lead to development of anti-tumor therapeutic tools in future.

Nitric oxide synthase inhibition reveals differences in the nitric oxide pathway in previously laminitic ponies

Previously laminitic (PL) ponies are reported to have higher blood pressure than non laminitic (NL) ponies. This relative hypertension may be related to endothelial cell dysfunction, similar to humans with metabolic syndrome. To investigate the relationship between laminitis predisposition and endothelial dysfunction, the effect of nitric oxide synthase (NOS) inhibition on the change in circulating nitric oxide (NO) concentrations and systemic blood pressure (BP) was determined. An intravenous NO sensor was used to measure changes in blood NO concentration during and after intravenous infusion of the NOS inhibitor L-nitroarginine methyl ester (L-NAME; 4 mg/kg IV) in PL and NL ponies. NO concentrations decreased and BP increased in response to L-NAME infusion, with a significantly (p = 0.02) greater rate of increase in systolic BP in PL (14.62 ± 1.88 mmHg/h) compared to NL (8.54 ± 1.88 mmHg/h) ponies. This greater effect on BP in PL compared to NL ponies is consistent with higher basal NO production in PL ponies, suggesting that the relative hypertension previously seen in PL ponies results in increased basal NO production, rather than being caused by reduced NO production as hypothesised. Up-regulation of the NO system may be a compensatory mechanism stimulated by the higher resting BP in PL ponies. Further investigation of the mechanism underlying the relative hypertension seen in PL ponies is required.

Sex‐Dependent Changes in Vascular Function in Adult Rats Following Prenatal Exposure to Methamphetamine

BackgroundPrenatal exposure to central nervous system stimulants such as cocaine, nicotine, and caffeine alters cardiovascular function in adult offspring. Methamphetamine use during pregnancy is associated with negative consequences in the offspring. However, most studies of methamphetamine use during pregnancy have focused on behavioral and neurological outcomes. Relatively little is known regarding the impact of prenatal methamphetamine exposure on the adult cardiovascular system. This study examined the hypothesis that methamphetamine use during pregnancy alters contractile function of the vasculature in the adult offspring. We also investigated the role of perivascular adipose tissue (PVAT) in methamphetamine‐induced changes in vascular function.MethodsPregnant female rats received daily injections of saline or methamphetamine (5 mg/kg) throughout gestation. Agonist‐induced contraction and relaxation responses were measured in aortas of 5 month old offspring in the presence and absence of perivascular adipose tissue.ResultsPrenatal methamphetamine significantly attenuated acetylcholine‐induced relaxation in male (but not female) aortas when PVAT remained intact. However, prenatal methamphetamine had no impact on acetylcholine‐induced relaxation when PVAT was removed. Nitroprusside‐induced relaxation was unaffected by prenatal methamphetamine. Prenatal exposure to methamphetamine had no impact on acetylcholine‐induced relaxation in third order mesenteric arteries of male or female offspring regardless of the presence of PVAT. Angiotensin II‐induced contractile responses were significantly potentiated in male (but not female) aortas regardless of the presence of PVAT. This effect was abolished by L‐Nitro arginine methyl ester (L‐NAME). Contractile responses to phenylephrine and serotonin were unaffected in both male and female aortas. Basal blood pressure in adult male and female offspring was also unaffected by prenatal exposure to methamphetamine.ConclusionsPrenatal exposure to methamphetamine sex‐dependently alters PVAT function in the aorta. Methamphetamine also enhances angiotensin II‐induced contraction through a mechanism that involves suppression of nitric oxide signaling. These data provide evidence that methamphetamine use during pregnancy induces sex‐dependent changes in the vasculature that may increase the risk of cardiovascular disease in adult offspring.

Prenatal Exposure to Methamphetamine Causes Vascular Dysfunction in Adult Male Rat Offspring.

Methamphetamine use during pregnancy can have negative consequences on the offspring. However, most studies investigating the impact of prenatal exposure to methamphetamine have focused on behavioral and neurological outcomes. Relatively little is known regarding the impact of prenatal methamphetamine on the adult cardiovascular system. This study investigated the impact of chronic fetal exposure to methamphetamine on vascular function in adult offspring. Pregnant female rats received daily saline or methamphetamine (5 mg/kg) injections starting on gestational day 1 and continuing until the pups were born. Vascular function was assessed in 5 month old offspring. Prenatal methamphetamine significantly decreased both the efficacy and potency of acetylcholine-induced relaxation in isolated male (but not female) aortas when perivascular adipose tissue (PVAT) remained intact. However, prenatal methamphetamine had no impact on acetylcholine-induced relaxation when PVAT was removed. Nitroprusside-induced relaxation of the aorta was unaffected by prenatal methamphetamine. Angiotensin II-induced contractile responses were significantly potentiated in male (but not female) aortas regardless of the presence of PVAT. This effect was reversed by L-nitro arginine methyl ester (L-NAME). Serotonin- and phenylephrine-induced contraction were unaffected by prenatal methamphetamine. Prenatal methamphetamine had no impact on acetylcholine-induced relaxation of third order mesenteric arteries and no effect on basal blood pressure. These data provide evidence that prenatal exposure to methamphetamine sex-dependently alters vasomotor function in the vasculature and may increase the risk of developing vascular disorders later in adult life.

Abstract 13900: Ex vivo Evaluation of Vascular Toxicity of Tobacco Products

Introduction: Tobacco smoke is the single most significant modifiable risk factor of cardiovascular disease (CVD), yet the contribution of electronic cigarettes (e-cig) to CVD risk is uncertain. Objective: The purpose of this study was to test an ex vivo approach for evaluation of vascular toxicity of tobacco products including e-cig aerosols. Methods: The assay relies on 2 facts of aortic function: 1) it is a biomarker of endothelial dysfunction; and, 2) it is nearly 100% dependent on endothelial-derived nitric oxide synthase (eNOS). To assess real-time eNOS function, phenylephrine-precontracted (PE) murine aortas were incubated with L-nitroargininemethylester (L-NAME) that subsequently increased tension and blocked (&gt;99%) acetylcholine-induced relaxation (ACh %). The ‘L-NAME PE Contraction Ratio’ (PECR) was calculated as: ‘PE Tension post-L-NAME’ divided by ‘PE Tension pre-L-NAME’, and regressed vs ACh % relaxation. Results: Validation 1: PECR and ACh % values of naïve aortas were strongly and positively correlated (PECR vs ACh %, r 2 =0.91, n=7). Validation 2: Retrospective analyses of published PECR and ACh % data of aortic function of female mice exposed to air, propylene glycol:vegetable glycerin (PG:VG), formaldehyde (FA), or acetaldehyde (AA) for 4d, 6h/d showed that the PECR in air-exposed mice (PECR=1.42±0.04, n=16) correlated positively with the ACh % (r 2 =0.40) as in naïve aortas. Similarly, PECR values were significantly decreased in aortas with endothelial dysfunction yet retained positive regression coefficients (PG:VG r 2 =0.54; FA r 2 =0.55). Unlike other toxicants, AA exposure significantly increased both PECR and ACh % values yet diminished regression (r 2 =0.09). Validation 3: Naïve aortas were incubated (1h) with particles of either mainstream cigarette smoke (MCS) or PG:VG, and the PECR was significantly increased by MCS (1.97±0.17; p&lt;0.05) yet not by PG:VG (1.34±0.05) compared with controls (1.48±0.05) indicating MCS induced vascular toxicity. Conclusions: Because the PECR reflects eNOS function directly, it is a robust tool for evaluating tobacco toxicity and predicting endothelial dysfunction. Thus, it strongly complements and extends insights gained using ex vivo measures of vascular toxicity.

Maternal vitamin D deficiency reduces docosahexaenoic acid, placental growth factor and peroxisome proliferator activated receptor gamma levels in the pup brain in a rat model of preeclampsia

Background: Preeclampsia is a pregnancy disorder characterized with abnormal placental angiogenesis. Vitamin D and long chain polyunsaturated fatty acids (LCPUFA) play a crucial role in pregnancy and are required for normal placental and fetal growth and development. This study reports the effect of maternal vitamin D on LCPUFA levels in the mother and offspring brain fatty acid levels and angiogenic markers in a rat model of preeclampsia.Methods: Female rats were divided into four groups from pre-pregnancy to pregnancy, viz Control; Preeclampsia (PE); Vitamin D deficient with PE (VDD-PE) and Vitamin D supplemented with PE (VDS-PE). Preeclampsia was induced by administering l-nitroarginine methyl ester (L-NAME) at the dose of 50 mg/kg body weight/day from day 14 to day 19 of gestation. Dams were sacrificed at d20 of gestation to collect dam blood, placenta and pup brain. LCPUFA levels from dam plasma, erythrocytes and placenta and its transcription factor peroxisome proliferator activated receptor gamma (PPAR-g) from placenta were estimated. Pup brain LCPUFA levels, angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) and transcription factor hypoxia inducible factor (Hif-1α) and PPAR-g were also estimated.Results: Maternal vitamin D status influences fatty acid levels. Placental PPAR-g levels were lower in the VDD-PE group as compared to the VDS-PE groups (p < 0.01). In the offspring brain, both PE and VDD-PE group showed lower levels of DHA (p < 0.05 for both) while saturated fatty acids (SFA) levels in the VDD-PE group were higher as compared to the control group (p < 0.05). VDD-PE group also showed lower levels of PlGF and PPAR-g (p < 0.01 and p < 0.05, respectively) in the pup brain while vitamin D supplementation demonstrated levels similar to control.Conclusion: This study for the first time demonstrates that maternal vitamin D status influences LCPUFA metabolism and angiogenesis in the offspring brain.

Systemic administration of choline acetyltransferase decreases blood pressure in murine hypertension

Acetylcholine (ACh) decreases blood pressure by stimulating endothelium nitric oxide-dependent vasodilation in resistance arterioles. Normal plasma contains choline acetyltransferase (ChAT) and its biosynthetic product ACh at appreciable concentrations to potentially act upon the endothelium to affect blood pressure. Recently we discovered a T-cell subset expressing ChAT (TChAT), whereby genetic ablation of ChAT in these cells produces hypertension, indicating that production of ACh by TChAT regulates blood pressure. Accordingly, we reasoned that increasing systemic ChAT concentrations might induce vasodilation and reduce blood pressure. To evaluate this possibility, recombinant ChAT was administered intraperitoneally to mice having angiotensin II-induced hypertension. This intervention significantly and dose-dependently decreased mean arterial pressure. ChAT-mediated attenuation of blood pressure was reversed by administration of the nitric oxide synthesis blocker l-nitro arginine methyl ester, indicating ChAT administration decreases blood pressure by stimulating nitic oxide dependent vasodilation, consistent with an effect of ACh on the endothelium. To prolong the half life of circulating ChAT, the molecule was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG-ChAT. Administration of PEG-ChAT to hypertensive mice decreased mean arterial pressure with a longer response duration when compared to ChAT. Together these findings suggest further studies are warranted on the role of ChAT in hypertension.

Hydrogen Sulphide Treatment Prevents Renal Ischemia-Reperfusion Injury by Inhibiting the Expression of ICAM-1 and NF-kB Concentration in Normotensive and Hypertensive Rats.

Our main objective was to investigate the effect of chronic administration of hydrogen sulphide donor (sodium hydrosulphide) on the expression of intercellular adhesion molecule-1 (ICAM-1) and concentration of nuclear factor-kappa B (NF-kB) in a renal ischemia-reperfusion injury (IRI) model of WKY and L-nitro-arginine-methyl-ester (L-NAME)-induced hypertensive rats. Sodium hydrosulphide (NaHS) was administered intraperitoneally (i.p.) for 35 days while cystathionine gamma lyase (CSE) inhibitor dL-propargylglycine (PAG) was administered at a single dose of 50 mg/kg. Animals were anesthetised using sodium pentobarbitone (60 mg/kg) and then prepared to induce renal ischemia by clamping the left renal artery for 30 min followed by 3 h of reperfusion. Pre-treatment with NaHS improved the renal functional parameters in both WKY and L-NAME-induced hypertensive rats along with reduction of blood pressure in hypertensive groups. Oxidative stress markers like malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione (GSH) were also improved by NaHS treatment following renal IRI. Levels of ICAM-1 and NF-kB concentration were reduced by chronic treatment with NaHS and increased by PAG administration after renal IRI in plasma and kidney. Treatment with NaHS improved tubular morphology and glomerulus hypertrophy. Pre-treatment with NaHS reduced the degree of renal IRI by potentiating its antioxidant and anti-inflammatory mechanism, as evidenced by decreased NF-kB concentration and downregulation of ICAM-1 expression.