Nitro L-arginine Research Articles

Contractile properties of periosteal arterioles in the guinea-pig tibia.

The periosteal arterioles of the compact bone may play a critical role in bone growth. To explore the contractile properties of tibial arterioles, spontaneous and nerve-evoked constrictions were compared in preparations from 3-week-old and 1-year-old guinea-pigs. Changes in arteriole diameters were measured using video microscopy. Their innervation was investigated using fluorescence immunohistochemistry. Fifty per cent and 40% of tibial arterioles from 3-week-old and 1-year-old guinea-pigs, respectively, exhibited spontaneous phasic constrictions that were inhibited by 1μM nifedipine, 10μM cyclopiazonic acid or 100μM 2-APB. Nerve-evoked phasic constrictions in both age groups were largely suppressed by phentolamine (1μM), an α-adrenoceptor antagonist, or sympathetic neurotransmitter depletion using guanethidine (10μM) but were enhanced by spanttide (1μM), a substance P receptor antagonist, or L-nitro arginine (L-NA; 100μM), an inhibitor of nitric oxide synthase (NOS). Nerve-evoked constrictions in 1-year-old animals were smaller than those in younger animals but greatly enhanced by L-NA. Immunohistochemistry revealed sympathetic and substance P-positive primary afferent nerves running along the arterioles as well as endothelial NOS expression in both age groups. Spontaneous arteriolar constrictions appear to rely on both Ca2+ release from the sarcoplasmic reticulum and Ca2+ influx through L-type Ca2+ channels. Noradrenaline released from sympathetic nerves triggers arteriolar constriction, while substance P released from primary afferent nerves dilates the arterioles by releasing nitric oxide (NO), presumably from the endothelium. Thus, the enhanced endothelial NO release in adult guinea-pigs may be important to increase the blood supply to meet the increased metabolic demands during bone growth.

The Role of Calcium-Sensing Receptors in Endothelin-1-Dependent Effects on Adult Rat Ventricular Cardiomyocytes: Possible Contribution to Adaptive Myocardial Hypertrophy.

Nitric oxide (NO)-deficiency as it occurs during endothelial dysfunction activates the endothelin-1 (ET-1) system and increases the expression of receptor activity modifying protein (RAMP)-1 that acts as a chaperon for calcium-sensing receptors (CaR) that have recently been identified to improve cardiac function. Here, we hypothesized that ET-1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP-1, endothelin receptors, and CaR were analyzed by RT-PCR in left ventricular tissues of L-NAME-treated rats. Effects of ET-1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP-1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1 , ETB2 , and ETA receptors. Non-specific NO synthase inhibition with L-Nitro arginine methyl ester (L-NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of ET-1 on cardiomyocytes. Indeed, ET-1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions, cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Inhibition of ETA and ETB1 receptors led to ET-1-dependent reduction in cell shortening and attenuated up-regulation of CaR. Down-regulation of RAMP-1 reduced CaR responsiveness. In conclusion, ET-1 causes an adaptive type of hypertrophy by up-regulation of CaR in cardiomyocytes via ETA and/or ETB1 receptors. J. Cell. Physiol. 232: 2508-2518, 2017. © 2016 Wiley Periodicals, Inc.

R-Type Ca2+ channels couple to inhibitory neurotransmission to the longitudinal muscle in the guinea-pig ileum.

What is the central question of this study? Subtypes of enteric neurons are coded by the neurotransmitters they synthesize, but it is not known whether enteric neuron subtypes might also be coded by other proteins, including calcium channel subtypes controlling neurotransmitter release. What is the main finding and its importance? Our data indicate that guinea-pig ileum myenteric neuron subtypes may be coded by calcium channel subtypes. We found that R-type calcium channels are expressed by inhibitory but not excitatory longitudinal muscle motoneurons. R-Type calcium channels are also not expressed by circular muscle inhibitory motoneurons. Calcium channel subtype-selective antagonists could be used to target subtypes of neurons to treat gastrointestinal motility disorders. There is evidence that R-type Ca2+ channels contribute to synaptic transmission in the myenteric plexus. It is unknown whether R-type Ca2+ channels contribute to neuromuscular transmission. We measured the effects of the nitric oxide synthase inhibitor nitro-l-arginine (NLA), Ca2+ channel blockers and apamin (SK channel blocker) on neurogenic relaxations and contractions of the guinea-pig ileum longitudinal muscle-myenteric plexus (LMMP) in vitro. We used intracellular recordings to measure inhibitory junction potentials. Immunohistochemical techniques localized R-type Ca2+ channel protein in the LMMP and circular muscle. Cadmium chloride (pan-Ca2+ channel blocker) blocked and NLA and NiCl2 (R-type Ca2+ channel blocker) reduced neurogenic relaxations in a non-additive manner. Nickel chloride did not alter neurogenic cholinergic contractions, but it potentiated neurogenic non-cholinergic contractions. Relaxations were inhibited by apamin, NiCl2 and NLA and were blocked by combined application of these drugs. Relaxations were reduced by NiCl2 or ω-conotoxin (N-type Ca2+ channel blocker) and were blocked by combined application of these drugs. Longitudinal muscle inhibitory junction potentials were inhibited by NiCl2 but not MRS2179 (P2Y1 receptor antagonist). Circular muscle inhibitory junction potentials were blocked by apamin, MRS2179, ω-conotoxin and CdCl2 but not NiCl2 . We conclude that neuronal R-type Ca2+ channels contribute to inhibitory neurotransmission to longitudinal muscle but less so or not all in the circular muscle of the guinea-pig ileum.

Effects of baicalin on mitochondria apoptotic pathway of trophoblast cells in a preeclampsia rat model

Objective To explore the effects of baicalin in the treatment of a preeclampsia (PE) rat model by detecting the expression of X-linked inhibitor of apoptosis protein (XIAP) and cysteine containing aspartate-9 (Caspase-9) and observing the ultrastructure of mitochondria in trophoblast cells. Methods Forty-eight pregnant Wistar rats were randomly divided into two groups: 12 in the control group and 36 in the PE model group. The PE model was established with subcutaneous injection of l-nitro arginine methyl ester with 100 mg/kg per day from the 13th day of pregnancy. Beginning from the 16th day of pregnancy, the PE rats were injected with different doses of baicalin till cesarean section, and divided into three groups: non-intervention PE model group treated with saline (NIP group), low-dose baicalin intervention group (IDB group) at 50 mg/kg per day, and high-dose baicalin intervention group (HDB group) at 100 mg/kg per day. The rat tail artery blood pressure and 24-h urine protein level were measured at pregnant day 10, 16 and 20. The levels of XIAP and Caspase-9 in placenta were measured by immunohistochemistry. The ultrastructure of mitochondria of trophoblast cells of the rat placenta was observed under electron microscope. T test, F test and LSD-t test were applied for statistical analysis. Results (1) On pregnant day 10, no significant differences were observed in rat tail artery systolic blood pressure, diastolic blood pressure and 24-h urine protein level between the control group and PE model group (all P>0.05). On pregnant day 16 and 20, the systolic blood pressure, diastolic blood pressure and 24-h urine protein level of NIP, IDB and HDB groups were significantly higher than those of control group [pregnant day 16: systolic blood pressure: (137.74±5.21),(136.15±4.86),(138.28±4.79) and (110.57±3.79) mmHg (1 mmHg=0.133 kPa), diastolic blood pressure:(89.58±5.50), (88.45±8.59), (89.42±6.29) and (80.28±7.36) mmHg, 24-h urine protein: (7.78±0.45), (7.53±0.54), (7.86±0.57) and (6.45±0.56) mg; pregnant day 20: systolic blood pressure: (145.26±4.67), (131.28±4.34), (130.93±5.33) and (110.40±6.92) mmHg, diastolic blood pressure: (89.87±6.55), (85.34±7.33), (84.64±7.36) and (80.19±7.34) mmHg, 24-h urine protein: (11.18±0.42), (9.65±0.54), (9.06±0.56) and (6.31±0.45) mg] (all P 0.05). (2)Compared with NIP group, the expression of XIAP in control group, IDB and HDB groups were significantly increased(210.39±0.78,180.56±0.82,195.36±0.96 and 192.84±1.06, all P<0.01). There was no significant difference in the expression of XIAP between IDB and HDB groups (P=0.66). The expression of Caspase-9 in control group, IDB and HDB groups were significantly decreased compared with NIP group (210.36±0.55, 195.53±0.96, 198.42±1.01 and 185.25±0.64, all P<0.01). There was no significant difference in the expression of Caspase-9 between IDB and HDB groups (P=0.65). Ultrastructure of mitochondria in NIP group showed different degrees of damage, matrix swelling, and mitochondrial cristae bresk or disappearance. In IDB group, mitochondrial matrix swelling was not obvious, and mitochondrial cristae were visible. In HDB group, mitochondrial cristae were neat and clear. Conclusions Baicalin may play an important role in the treatment of preeclampsia by reversing the trophoblast apoptosis and improving the ultrastructure of mitochondria through its regulation of XIAP expression and downregulation of Caspase-9 expression. Key words: Pre-eclampsia; Baicalin; Trophoblasts; X-Linked inhibitor of apoptosis protein; Caspase-9; Apoptosis; Rats

High-fat diet-induced obesity alters nitric oxide-mediated neuromuscular transmission and smooth muscle excitability in the mouse distal colon.

We tested the hypothesis that colonic enteric neurotransmission and smooth muscle cell (SMC) function are altered in mice fed a high-fat diet (HFD). We used wild-type (WT) mice and mice lacking the β1-subunit of the BK channel (BKβ1 (-/-)). WT mice fed a HFD had increased myenteric plexus oxidative stress, a 28% decrease in nitrergic neurons, and a 20% decrease in basal nitric oxide (NO) levels. Circular muscle inhibitory junction potentials (IJPs) were reduced in HFD WT mice. The NO synthase inhibitor nitro-l-arginine (NLA) was less effective at inhibiting relaxations in HFD compared with control diet (CD) WT mice (11 vs. 37%, P < 0.05). SMCs from HFD WT mice had depolarized membrane potentials (-47 ± 2 mV) and continuous action potential firing compared with CD WT mice (-53 ± 2 mV, P < 0.05), which showed rhythmic firing. SMCs from HFD or CD fed BKβ1 (-/-) mice fired action potentials continuously. NLA depolarized membrane potential and caused continuous firing only in SMCs from CD WT mice. Sodium nitroprusside (NO donor) hyperpolarized membrane potential and changed continuous to rhythmic action potential firing in SMCs from HFD WT and BKβ1 (-/-) mice. Migrating motor complexes were disrupted in colons from BKβ1 (-/-) mice and HFD WT mice. BK channel α-subunit protein and β1-subunit mRNA expression were similar in CD and HFD WT mice. We conclude that HFD-induced obesity disrupts inhibitory neuromuscular transmission, SMC excitability, and colonic motility by promoting oxidative stress, loss of nitrergic neurons, and SMC BK channel dysfunction.

Role of the ER/NO/cGMP Signaling Pathway in the Promotion of Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells by Actaea racemosa Extract.

Purpose/Objective. To investigate the effect of Actaea racemosa (AR) extract on in vitro osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) via the ER/NO/cGMP signaling pathway. Methods/Materials. Rat BMSCs were treated with osteogenic differentiation-inducing medium containing AR; estrogen receptor antagonist, ICI 182,780 (10−6 mol/L); and nitric oxide synthase inhibitor, L-nitro arginine methyl ester (L-NAME, 6 × 10−3 mol/L). Markers of osteogenic differentiation (alkaline phosphatase [ALP] activity, osteocalcin secretion, and calcium ion deposit levels) and the levels of key signaling molecules (nitric oxide synthase [NOS], nitric oxide [NO], and cyclic guanosine monophosphate [cGMP]) were assessed. Results. AR (10−1–10−6 g/L) increased ALP activity in a dose-dependent manner, and the highest ALP, osteocalcin, and osteoprotegerin activities were achieved at an AR concentration of 10−4 g/L. Therefore, the concentration of 10−4 g/L was used for promoting osteogenic differentiation of BMSCs in subsequent analyses. At this concentration, AR increased the levels of NO and cGMP, and such effects could be blocked by the estrogen receptor antagonist (ICI 182,780) and nitric oxide synthase inhibitor (L-NAME). Conclusion. AR induced osteogenic differentiation of rat BMSCs through the ER/NO/cGMP signaling pathway. This finding provides the theoretical foundation for the mechanism of AR in the treatment of postmenopausal osteoporosis.

Skeletal Muscle Vascular Control During Exercise: Impact of Nitrite Infusion During Nitric Oxide Synthase Inhibition in Healthy Rats.

The nitric oxide synthase (NOS)-independent pathway of nitric oxide (NO) production in which nitrite (NO2 (-)) is reduced to NO may have therapeutic applications for those with cardiovascular diseases in which the NOS pathway is downregulated. We tested the hypothesis that NO2 (-) infusion would reduce mean arterial pressure (MAP) and increase skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise in the face of NOS blockade via L-NAME. Following infusion of L-NAME (10 mg kg(-1), L-NAME), male Sprague-Dawley rats (3-6 months, n = 8) exercised without N(G)-nitro-L arginine methyl ester (L-NAME) and after infusion of sodium NO2 (-) (7 mg kg(-1); L-NAME + NO2 (-)). MAP and hindlimb skeletal muscle BF (radiolabeled microsphere infusions) were measured during submaximal treadmill running (20 m min(-1), 5% grade). Across group comparisons were made with a published control data set (n = 11). Relative to L-NAME, NO2 (-) infusion significantly reduced MAP (P < 0.03). The lower MAP in L-NAME+NO2 (-) was not different from healthy control animals (control: 137 ± 3 L-NAME: 157 ± 7, L-NAME + NO2 (-): 136 ± 5 mm Hg). Also, NO2 (-) infusion significantly increased VC when compared to L-NAME (P < 0.03), ultimately negating any significant differences from control animals (control: 0.78 ± 0.05, L-NAME: 0.57 ± 0.03, L-NAME + NO2 (-); 0.69 ± 0.04 mL min(-1) 100 g(-1) mm Hg(-1)) with no apparent fiber-type preferential effect. Overall, hindlimb BF was decreased significantly by L-NAME; however, in L-NAME + NO2 (-), BF improved to a level not significantly different from healthy controls (control: 108 ± 8, L-NAME: 88 ± 3, L-NAME + NO2 (-): 94 ± 6 mL min(-1) 100 g(-1), P = 0.38 L-NAME vs L-NAME + NO2 (-)). Individuals with diseases that impair NOS activity, and thus vascular function, may benefit from a NO2 (-)-based therapy in which NO bioavailability is elevated in an NOS-independent manner.

Inhibition of Myogenic Tone in Rat Cremaster and Cerebral Arteries by SKA-31, an Activator of Endothelial KCa2.3 and KCa3.1 Channels.

Endothelial KCa2.3 and KCa3.1 channels contribute to the regulation of myogenic tone in resistance arteries by Ca(2+)-mobilizing vasodilatory hormones. To define further the functional role of these channels in distinct vascular beds, we have examined the vasodilatory actions of the KCa channel activator SKA-31 in myogenically active rat cremaster and middle cerebral arteries. Vessels pressurized to 70 mm Hg constricted by 80-100 μm (ie, 25%-45% of maximal diameter). SKA-31 (10 μM) inhibited myogenic tone by 80% in cremaster and ∼65% in middle cerebral arteries, with IC50 values of ∼2 μM in both vessels. These vasodilatory effects were largely prevented by the KCa2.3 blocker UCL1684 and the KCa3.1 blocker TRAM-34 and abolished by endothelial denudation. Preincubation with N(G) nitro L-arginine methyl ester (L-NAME, 0.1 mM) did not affect the inhibitory response to SKA-31, but attenuated the ACh-evoked dilation by ∼45%. Penitrem-A, a blocker of BK(Ca) channels, did not alter SKA-31 evoked vasodilation but did reduce the inhibition of myogenic tone by ACh, the BKCa channel activator NS1619, and sodium nitroprusside. Collectively, these data demonstrate that SKA-31 produces robust inhibition of myogenic tone in resistance arteries isolated from distinct vascular beds in an endothelium-dependent manner.

Excess Nitric Oxide (NO) Blunts Presynaptic Adrenergic Transduction in Orthostatic Intolerance (OI)

NO is a fundamental signaling molecule during postural blood redistribution. NO can centrally modulate sympathetic vasoconstriction by reducing sympathetic outflow, and can peripherally reduce sympathetic transduction at pre- and post-junctional sites of the adrenergic synapse. Prior data suggest blunted adrenergic vasoconstriction as a fundamental mechanism in orthostatic intolerance. We hypothesized that the production of NO in OI is enhanced resulting in reduced sympathetic noradrenergic neurotransmission which may be at pre- or post-junctional sites. To investigate this we predilated cutaneous arteries using adenosine as a [mostly] NO-independent vasodilator and local heat as a[mostly] NO-dependent vasodilator. We investigated the dose response to pre-synaptic vasoconstriction with tyramine and post-synaptic vasoconstriction with norepinephrine with or without NOS inhibition using nitro-L-arginine (NLA). The dose-responses of 8 OI (19 ± 3 yrs) patients were compared with 11 healthy controls (21 ±3 yrs). Data from OI and control subjects with and without NLA were compared by repeated measures ANOVA. OI subjects exhibited blunted vasoconstriction responses (P<0.001) to tyramine following adenosine pre-dilation which were similar to control with NLA. Tyramine dose-response following heat pre-dilation were blunted in both OI and controls. Norepinephrine dose-response was similar for OI and controls with and without NLA. The data confirm that NO modulates cutaneous sympathetic nerve transduction both pre- and post-synaptically in OI and controls. However, excessive endogenous NO blunts pre-synaptic adrenergic transduction in OI which may account, in part, for impaired sympathetic vasoconstriction.

Analgesic and anti-inflammatory activities of Citrus aurantium L. blossoms essential oil (neroli): involvement of the nitric oxide/cyclic-guanosine monophosphate pathway.

The analgesic and anti-inflammatory properties of Citrus aurantium L. blossoms essential oil (neroli) were investigated in mice and rats. The analgesic activity of neroli was assessed by acetic acid-induced writhing and Eddy's hot plate methods, while acute and chronic anti-inflammatory effects were investigated by inflammatory paw edema in rat and the cotton pellet-induced granuloma tissue model, respectively. Mechanistic studies were conducted using L-nitro arginine methyl ester (L-NAME), an inhibitor of NO synthase. Neroli significantly decreased the number of acetic acid-induced writhes in mice compared to animals that received vehicle only. Also, it exhibited a central analgesic effect, as evidenced by a significant increase in reaction time in the hot plate method. The oil also significantly reduced carrageenan-induced paw edema in rats. The inhibitory activity of neroli (especially at 40mg/kg) was found to be very close to the standard drug, diclofenac sodium (50mg/kg). In cotton pellet-induced granuloma, neroli was effective regarding the transudate and granuloma formation amount. Neroli was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) and twenty-three constituents, representing 91.0% of the oil, were identified. The major components of neroli were characterized as linalool (28.5%), linalyl acetate (19.6%), nerolidol (9.1%), E,E-farnesol (9.1%), α-terpineol (4.9%), and limonene (4.6%), which might be responsible for these observed activities. The results suggest that neroli possesses biologically active constituent(s) that have significant activity against acute and especially chronic inflammation, and have central and peripheral antinociceptive effects which support the ethnomedicinal claims of the use of the plant in the management of pain and inflammation.

Central Antinociceptive and Mechanism of Action of Pereskia bleo Kunth Leaves Crude Extract, Fractions, and Isolated Compounds.

Pereskia bleo (Kunth) DC. (Cactaceae) is a plant commonly used in popular medicine in Malaysia. In this work, we evaluate the antinociceptive effect of P. bleo leaf extracts and isolated compounds in central antinociceptive model. Ethanol extract (E), hexane (H), ethyl acetate (EA), or butanol (B) fractions (30, 50, or 100 mg/kg, p.o.), sitosterol (from hexane) and vitexin (from ethyl acetate), were administered to mice. Antinociceptive effect was evaluated in the hot plate and capsaicin- or glutamate-induced licking models. Morphine (1 mg/kg, p.o.) was used as reference drug. Naloxone (1 mg/kg, i.p.), atropine (1 mg/kg, i.p.), and L-nitro arginine methyl ester (L-NAME, 3 mg/kg, i.p.) were administered 30 min earlier (100 mg/kg, p.o.) in order to evaluate the mechanism of the antinociceptive action. Higher dose of B developed an effect significantly superior to morphine-treated group. Naloxone prevented the antinociceptive effect of all fractions. L-NAME demonstrated effect against E, EA, and B. In all fractions, sitosterol and vitexin reduced the licking time after capsaicin injection. Glutamate-induced licking response was blocked by H, EA, and B. Our results indicate that Pereskia bleo fractions, sitosterol and vitexin, possessed a central antinociceptive effect. Part of this effect is mediated by opioid receptors and nitrergic pathway.

Vasoactive Properties of an Extract of Allium Sativum in the Rat Aorta

The aim of this study was to determine whether an hydro-alcoolic extract of cloves of Allium sativum is able to induce a relaxant effect in the rat aorta and so the mechanism underlying this effect. Rat aortic rings were suspended in organ chambers for recording of changes in isometric forces. Rings with endothelium were incubated or not with L-Nitro Arginine Methyl Ester (L-NAME) to block NO synthase, before contraction with adrenalin and a concentration relaxation curve to the hydro-alcoolic extract. In some experiments, endothelium was removed before contraction with adrenalin and concentration relaxation to the extract. Prevent effect of the extract was determine with contraction curve to adrenaline in rings with and without endothelium incubated with the extract of Allium sativum. The hydro-alcoolic extract of Allium sativum induces a vasodilatatory effect in the rat aorta contracted with adrenalin. This effect is not endothelium-dependent and is not mediated by NO. Furthermore the extract incubated with aorta rings, is able to prevent the contraction induced by adrenalin. Allium sativum induces vascular relaxation which can explain the benefic effect of this plant in treatment of high blood pressure in Senegal.

Stability of murine bradykinin type 2 receptor despite treatment with NO, bradykinin, icatibant, or C1-INH.

Little is known about factors which trigger and/or contribute to hereditary angioedema or ACE-inhibitor-mediated angioedema including variations in bradykinin type 2 receptor (B2R) expression and activity. Protein and mRNA expression of B2R and the increase of intracellular calcium (iCa) in response to bradykinin were monitored in porcine and murine endothelial cells in response to NO donors or bradykinin. B2R protein expression was evaluated in skin, heart, and lung of (i) mice with endothelial-specific overexpression of eNOS (eNOS(tg) ), (ii) in eNOS(-/-) mice and (iii) in C57BL/6 mice treated with the NO donor pentaerythritol tetranitrate (PETN), the NOS inhibitor l-nitroarginine (L-NA), plasma pool C1-INH, and the B2R antagonist icatibant. Aortic reactivity to bradykinin was investigated including eNOS(-/-) mice. B2R protein and mRNA expression remained unchanged in cells subjected to L-NA, NO donors, and bradykinin in a time- and concentration-dependent manner. Likewise, increases of iCa in murine brain endothelial cells remained unchanged. B2R protein levels were similar in eNOS(tg) and eNOS(-/-) as compared to transgene-negative littermates. Likewise, treatment of C57BL/6 mice with PETN, L-NA, C1-INH or icatibant did not change B2R protein expression. In aortic rings of C57BL/6 mice, bradykinin induced B2R-dependent constrictions which were attenuated by endothelial NO and abolished by diclofenac indicating the functional importance of B2R-induced activation of endothelial NO synthase and cyclooxygenase. These data suggest that alterations of B2R protein expression induced by NO, bradykinin, C1-INH, or icatibant unlikely contribute to bradykinin-induced angioedema. This finding does not rule out a role for NO in bradykinin-induced extravasation and/or angioedema.

Effect of aliskiren, telmisartan and torsemide on cardiac dysfunction in l-nitro arginine methyl ester (l-NAME) induced hypertension in rats

Comparative study of cardio protective effect of aliskiren, telmisartan, and torsemide was carried out on l-nitro arginine methyl ester (l-NAME) induced hypertension in rats. The three drugs were given daily for 8weeks simultaneously with l-NAME, with a control group for each drug and l-NAME. The degree of protection was assessed by measurement of systolic blood pressure and heart rate of animals every two weeks. At the end of the experimental period blood sampling was carried out for estimation of the level of NO2−/NO3−. After which animals were sacrificed for heart dissection to detect collagen types I and III gene expression. Histopathological study was done to evaluate the extension of collagen deposits. The study revealed that the three drugs decreased blood pressure significantly compared to l-NAME. There was no significant difference between aliskiren and telmisartan in all measurements, but there was significant decrease in measurements of both aliskiren and telmisartan treated groups compared to torsemide starting from 4th week. There were insignificant changes in pulse rate values between the three l-NAME treated groups through the experiment. The three drugs significantly increased NO compared to l-NAME. Collagen I and III gene expression was significantly decreased by the three drugs but the highest percentage of inhibition was with telmisartan compared to l-NAME. Comparing the percentage inhibition of cardiac fibrosis, there was insignificant difference between telmisartan and torsemide treated groups while both were superior to aliskiren. In conclusion, further experimental studies are required to elucidate the potential cardioprotective mechanisms of aliskiren, telmisartan and torsemide, and assess their efficacy in treatment of heart failure.

Maternal nutrient restriction during pregnancy impairs an endothelium-derived hyperpolarizing factor-like pathway in sheep fetal coronary arteries.

The mechanisms underlying developmental programming are poorly understood but may be associated with adaptations by the fetus in response to changes in the maternal environment during pregnancy. We hypothesized that maternal nutrient restriction during pregnancy alters vasodilator responses in fetal coronary arteries. Pregnant ewes were fed a control [100% U.S. National Research Council (NRC)] or nutrient-restricted (60% NRC) diet from days 50 to 130 of gestation (term = 145 days); fetal tissues were collected at day 130. In coronary arteries isolated from control fetal lambs, relaxation to bradykinin was unaffected by nitro-l-arginine (NLA). Iberiotoxin or contraction with KCl abolished the NLA-resistant response to bradykinin. In fetal coronary arteries from nutrient-restricted ewes, relaxation to bradykinin was fully suppressed by NLA. Large-conductance, calcium-activated potassium channel (BKCa) currents did not differ in coronary smooth muscle cells from control and nutrient-restricted animals. The BKCa openers, BMS 191011 and NS1619, and 14,15-epoxyeicosatrienoic acid [a putative endothelium-derived hyperpolarizing factor (EDHF)] each caused fetal coronary artery relaxation and BKCa current activation that was unaffected by maternal nutrient restriction. Expression of BKCa-channel subunits did not differ in fetal coronary arteries from control or undernourished ewes. The results indicate that maternal undernutrition during pregnancy results in loss of the EDHF-like pathway in fetal coronary arteries in response to bradykinin, an effect that cannot be explained by a decreased number or activity of BKCa channels or by decreased sensitivity to mediators that activate BKCa channels in vascular smooth muscle cells. Under these conditions, bradykinin-induced relaxation is completely dependent on nitric oxide, which may represent an adaptive response to compensate for the absence of the EDHF-like pathway.

Maternal protein restriction during pregnancy alters vasodilator mechanisms in fetal placental arteries (910.9)

Maternal undernutrition during pregnancy reduces uterine blood flow. We hypothesized that reduced maternal protein intake during pregnancy alters vasodilator responses in placental arteries. Pregnant ewes were fed an isocaloric diet containing 100% (control) or 60% (protein‐restricted) of protein requirements from day 100‐130 of gestation (term= 145 d). Fetal placental arteries (FPA) were isolated on day 130. Sensitivity to bradykinin (BK)‐induced relaxation was increased 5‐fold in FPA from protein‐restricted ewes. In control FPA, BK‐induced relaxation was unaffected by indomethacin (Indo) but was inhibited by nitro‐l‐arginine (NLA). In FPA from protein‐restricted ewes, neither Indo nor NLA affected the response to BK. Sodium nitroprusside‐induced relaxation, phenylephrine (PE)‐induced contraction, and expression of eNOS, guanylyl cyclase (GC), and BK‐receptors, was similar in FPA from both dietary groups. In ewes fed a control diet, BK‐induced relaxation of FPA is mediated primarily by NO. In ewes fed a protein‐restricted diet, FPA are more sensitive than controls to BK‐induced relaxation and the mechanism is independent of NO and PGI2. The altered BK response is not due to changes in sensitivity to NO or PE, or to altered expression of eNOS, GC or BK receptors and may represent an adaptive mechanism to ensure adequate blood supply to the fetus during adverse maternal dietary conditions.Grant Funding Source: Supported by NIH

Valproic acid attenuates blood pressure, vascular remodeling and modulates ET-1 expression in L-NAME induced hypertensive rats

The present study was considered to assess the antihypertensive and antioxidant effect of valproic acid, against Nω- nitro-L arginine methyl ester hydrochloride (L-NAME) induced hypertension in male Wistar rats. Hypertension was prompted in adult male albino rats of the Wistar strain, weighing 180–220g, by oral administration of the L-NAME (40mg/kg body weight/day) in drinking water for 4 weeks. The L- NAME hypertensive rats revealed significant (P<0.05) rise in the systolic and diastolic blood pressure, heart rate, water intake and heart weight L-NAME hypertensive rats also revealed significant (P<0.05) increase in the levels of thiobarbituric acid reactive substances, lipid hydroperoxides in plasma and tissues (heart and aorta), and significant (P<0.05) drop in the body weight, nitrite and nitrate levels in plasma and aorta. Activities of enzymic antioxidants such as superoxide dismutase, catalase and glutathione peroxidase in erythrocyte and tissues and the levels of non-enzymic antioxidant such as reduced glutathione in plasma and tissues, ET-1 mRNA expression in aorta was significantly (P<0.05) increased in L-NAME rats. Valproic acid (VPA) supplementation (100mg/kg) daily for four weeks brought back all the above parameters to near normal level. The above outcomes were confirmed by the histopathological examination. No significant (P<0.05) effect was observed in control rats treated with valproic acid (100mg/kg). These results suggest that valproic acid performed as an antihypertensive and antioxidant agent against L-NAME induced hypertension.

Neurohumoral regulation of spontaneous constrictions in suburothelial venules of the rat urinary bladder

Venules of the bladder suburothelium develop spontaneous phasic constrictions that may play a critical role in maintaining venular drainage of tissue metabolites. We aimed to investigate neurohumoral regulation of the spontaneous venular constrictions (SVCs). Changes in venular diameter of the rat bladder suburothelium were monitored using a video tracking system, whilst the effects of electrical field stimulation (EFS) and bath-applied bioactive substances were investigated. The innervation of the suburothelial microvasculature was examined by immunohistochemistry. EFS (10Hz for 30s) induced an increase in the frequency of SVCs that was prevented by phentolamine (1μM). In phentolamine-pretreated venules, EFS suppressed SVCs with a venular dilatation in a manner attenuated by propranolol (1μM) or l-nitro arginine (LNA, 10μM). BRL37344 (1μM), a β3 adrenoceptor agonist, dilated venules and reduced the frequency of SVCs in an LNA-sensitive manner. ACh (1–10μM) increased the frequency of SVCs. ATP (1μM) transiently constricted venules and then caused LNA-sensitive cessation of SVCs associated with a dilatation. Substance P (100nM) caused a venular constriction, whilst calcitonin gene related peptide (CGRP, 100nM) caused a dilatation of venules and suppression of SVCs that were not inhibited by LNA. Immunohistochemical staining demonstrated sympathetic as well as substance P- and CGRP-containing nerves running along the venules. Spontaneous constrictions of suburothelial venules are accelerated by sympathetic α-adrenergic stimulation, but suppressed upon β-adrenergic stimulation. In addition, suburothelial venular constrictions appear to be modulated by several bioactive substances that could be released from urothelium or suburothelial sensory nerves.

Familial hypercholesterolemia impairs exercise-induced systemic vasodilation due to reduced NO bioavailability

Hypercholesterolemia impairs endothelial function [e.g., the nitric oxide (NO)-cyclic GMP-phosphodiesterase 5 (PDE5) pathway], limits shear stress-induced vasodilation, and is therefore expected to reduce exercise-induced vasodilation. To assess the actual effects of hypercholesterolemia on endothelial function and exercise-induced vasodilation, we compared the effects of endothelial NO synthase (eNOS) and PDE5 inhibition in chronically instrumented Yucatan (Control) and Rapacz familial hypercholesterolemic (FH) swine, at rest and during treadmill exercise. The increases in systemic vascular conductance produced by ATP (relative to nitroprusside) and exercise were blunted in FH compared with Control swine. The vasoconstrictor response to eNOS inhibition, with nitro-l-arginine (NLA), was attenuated in FH compared with Control swine, both at rest and during exercise. Furthermore, whereas the vasodilator response to nitroprusside was enhanced slightly, the vasodilator response to PDE5 inhibition, with EMD360527, was reduced in FH compared with Control swine. Finally, in the pulmonary circulation, FH resulted in attenuated vasodilator responses to ATP, while maintaining the responses to both NLA and EMD360527. In conclusion, hypercholesterolemia reduces exercise-induced vasodilation in the systemic but not the pulmonary circulation. This reduction appears to be the principal result of a decrease in NO bioavailability, which is mitigated by a lower PDE5 activity.

Effects of NO on the expression and function of bradykinin type 2 receptors. Role for angioedema?

Purpose: The pathophysiology of angioedema induced by angiotensin-converting enzyme inhibitors (ACEi) is not well understood. While activation of bradykinin (BK) type 2 receptors (B2) has been established, the triggering events remain obscure. In this study we investigated effect of NO as one product of B2 stimulation. Methods: Brain (bEND3), human umbilical vein (HUVEC) and porcine aortic endothelial cells (PAEC) were incubated with the NO-donors DEA/NO (10μM), DETA/NO (100μM) and SNAP (1-100μM) for 3, 6 and 12 h. B2 expression was assessed by RT-PCR and westernblot using monoclonal B2 antibodies staining either glycosylated or unglycosylated B2. Function of B2 was determined by monitoring iCa2+ upon activation with BK. Likewise, B2 protein levels were evaluated in aorta, heart and lung of (1) two different eNOS-/- strains, (2) mice with endothelial-specific overexpression of eNOS (eNOS-tg), (3) in C57Bl/6 mice treated with NOS inhibitor L-nitroarginine (L-NA). Here, B2 function was evaluated in aortic rings. Results: After 3h incubation with DEA/NO of bEND3, B2-mRNA was unchanged (160±32%, n=6, P>0.05). Likewise, B2 protein levels remained stable (98±13.6%, n=8, P>0.05). Similar results were obtained in PAECs and HUVECs and after extended incubation time and increased concentration. There was also a similar rise of iCa2+ in response to 0.01-100 μM BK in bEND3 treated with DEA/NO or vehicle as demonstrated by almost identical flourescence signals. These results obtained in cell lines and primary cultured PAEC were confirmed using transgenic mice with different levels of vascular NO-bioavailability. In aorta, neither the complete lack of endothelial NO in eNOS-/- (93±26%, n=5, P>0.05), nor overexpression in eNOS-tg (112±13.7%, n=10, P>0.05) changed B2 protein expression and a similar observation was made in C56Bl/6 treated with L-NA (110±19.4%, n=5, P>0.05). Aortic constrictor responses to 10 μM BK (related to maximal constriction to KCl) were identical in C57Bl/6 (24.3±2.7%, n=7) and eNOS-tg (30±11.8%, n=4, P>0.05). However, B2 signaling involved generation of NO as constrictor responses were significantly increased following L-NA in both C57BL/6 (64±18.3%, n=4) and eNOS-tg (67±3.6%, n=4) and a similar constriction was observed in eNOS-/- without (61±10.9%, n=7) and with L-NA (61±14.6%, n=6). None of these responses differed significantly. Conclusion: These data demonstrate that NO does not regulate vascular B2 gene expression and function suggesting that changes of B2 expression by this product of B2 activation are unlikely involved in triggering ACEi-induced angioedema.