NO Blockade Research Articles

Anti-inflammatory effects and related mechanisms in vitro and in vivo of Hedychium coccineum rhizome essential oil

Ethnopharmacological relevanceHedychium coccineum rhizome is an anti-inflammatory ethnomedicine used to remedy inflammation-related swelling and bronchial asthma. Aim of the studyThe study aimed to analyze the phytochemical constituents of H. coccineum rhizome essential oil (EO) and evaluate its in vitro and in vivo anti-inflammatory effects and underlying mechanisms. Materials and methodsPhytochemical constituents of H. coccineum rhizome EO were analyzed using GC-FID/MS. In RAW264.7 macrophages induced by LPS, blockade of PGE2, NO, IL-1β, IL-6, and TNF-α secretion by H. coccineum rhizome EO was measured, and then Western blot, qRT-PCR, and immunofluorescent staining were used to evaluate its underlying mechanisms. Moreover, we used the xylene-induced ear edema model for testing anti-inflammatory potential in vivo and examined auricular swelling as well as tissue and serum contents of IL-1β, IL-6, and TNF-α. ResultsEO's main components were E-nerolidol (40.5%), borneol acetate (24.8%), spathulenol (4.5%), linalool (3.8%), elemol (3.5%), and borneol (3.4%). In RAW264.7 cells stimulated by LPS, EO downregulated the expression of pro-inflammatory enzyme (iNOS and COX-2) genes and proteins, thereby suppressing pro-inflammatory mediators (NO and PGE2) secretion. Simultaneously, it reduced TNF-α, IL-1β, and IL-6 release by downregulating their mRNA expression. Besides, H. coccineum EO attenuated LPS-stimulated activation of NF-κB (by reducing IκBα phosphorylation and degradation to inhibit NF-κB nuclear translocation) and MAPK (by downregulating JNK, p38, and ERK phosphorylation). In xylene-induced mouse ear edema, EO relieved auricular swelling and lowered serum and tissue levels of TNF-α, IL-1β, and IL-6. ConclusionsH. coccineum EO had powerful in vivo and in vitro anti-inflammatory effects by inhibiting MAPK and NF-κB activation. Hence, H. coccineum EO should have great potential for application in the pharmaceutical field as a novel anti-inflammatory agent.

AT1R blocker prevents mental stress induced retrograde blood flow in overweight/obese men.

The main goal was to determine the impact of mental stress (MS) on blood flow regulation in overweight/obese men. Fourteen overweight/obese men (27 ± 7 years; 29.8 ± 2.6kg/m2 ) participated in two randomized experimental sessions with oral administration of the AT1R blocker Olmesartan (40 mg; AT1RB) or placebo (PL). After 2 h, a 5-min acute MS session (Stroop Color Word Test) was administered. Blood flow was assessed at baseline and during the first 3 min of MS by vascular ultrasound in the brachial artery. Blood was collected before (baseline) and during mental stress (MS) for measurement of nitrite (chemiluminescence) and endothelin-1 (ELISA kit). The AT1R blocker was able to reverse the MS responses observed in the placebo session for retrograde flow (p < 0.01), retrograde SR (p < 0.01) and oscillatory shear index (p=0.01). Regarding vasoactive substances, no differences were observed in ET-1 (p > 0.05) responses to MS between experimental sessions. However, for nitrite responses, the administration of the AT1R blocker was able to increase circulating levels of NO (p=0.03) Blockade of AT1R appears to prevent the decrease in endothelial function by reducing low shear stress and maintaining the vasoactive substances balance after MS in overweight/obese men.

UV-B filter octylmethoxycinnamate-induced vascular endothelial disruption on rat aorta: In silico and in vitro approach

Throughout human life, an extensive and varied range of emerging environmental contaminants, called endocrine disruptors (EDCs), cause adverse health effects, including in the cardiovascular (CV) system. Cardiovascular diseases (CVD) are worryingly one of the leading causes of all mortality and mobility worldwide. The UV-B filter octylmethoxycinnamate (also designated octinoxate, or ethylhexyl methoxycinnamate (CAS number: 5466-77-3)) is an EDC widely present in all personal care products. However, to date, there are no studies evaluating the OMC-induced effects on vasculature using animal models to improve human cardiovascular health. This work analysed the effects of OMC on rat aorta vasculature and explored the modes of action implicated in these effects. Our results indicated that OMC relaxes the rat aorta by endothelium-dependent mechanisms through the signaling pathways of cyclic nucleotides and by endothelium-independent mechanisms involving inhibition of L-Type voltage-operated Ca2+ channels (L-Type VOCC). Overall, OMC toxicity on rat aorta may produce hypotension via vasodilation due to excessive NO release and blockade of L-Type VOCC. Moreover, the OMC-induced endothelial dysfunction may also occur by promoting the endothelial release of endothelin-1. Therefore, our findings demonstrate that exposure to OMC alters the reactivity of the rat aorta and highlight that long-term OMC exposure may increase the risk of human CV diseases.

Cardioprotective Effect of Rumex vesicarius Linn. Leaf Extract against Catecholamine-Induced Cardiotoxicity.

Rumex vesicarius (L.) is a folklore medicinal herb that has been used for centuries to cure cardiovascular diseases. The present work was carefully designed to ascertain the pharmacological basis for R. vesicarius’s therapeutic efficacy in cardiovascular diseases, as well as the underlying mechanism. In the ex vivo investigation, the aqueous-methanolic leaf extract of R. vesicarius was shown to have endothelium-dependent vasorelaxant effects in rabbit aorta tissue preparations, and its hypotensive responses were quantified by pressure and force transducers coupled to the Power Lab Data Acquisition System. Furthermore, when rabbits were subjected to adrenaline-induced myocardial infarction, R. vesicarius demonstrated cardioprotective characteristics. In contrast to the intoxicated group, the myocardial infarction model showed lower ALP, CK-MB, CRP, LDH, ALT, troponin, and AST levels (p > 0.005–0.000), as well as edema, necrosis, apoptosis, inflammatory cell enrolment, and necrosis. R. vesicarius exhibited significant antioxidant activity and delayed noradrenaline-induced platelet aggregation. Its cardioprotective, anticoagulant, and vasorelaxant properties in both investigations (in vivo and ex vivo) are mediated through partial endothelium-dependent, NO and calcium channel blockade mediated vasorelaxation. The minimizing of adrenaline, oxidative stress, and tissue damage demonstrate its therapeutic efficacy in cardiovascular diseases.

In Vitro Irradiation of Lymphatic Vessels Enhances Lymphatic Pump Function

Active lymphatic pumping is critical for propulsion of lymph against a pressure gradient as well as transport of lipids and immune cells to the central venous circulation. When lymphatic pumping is impaired, diminished lymph flow leads to interstitial edema, immune cell accumulation and sustained inflammation. However, a majority of radiation biology research has primarily focused on the effects of ionizing radiation (IR) on blood vessels. These studies reported that IR induces imbalance between vaso‐dilatory and ‐constrictive pathways and impairs vascular tone. Only a handful of the studies have investigated the acute effects of IR on lymphatic vessels. Furthermore, the studies evaluating IR‐induced changes in lymphatic tone and pumping in vivo are limited because, in addition to the intrinsic contractility of the lymphatic muscle, lymphatic tone and pumping in vivo is modulated by a variety of substances present in the luminal lymph and the surrounding interstitial fluid. Therefore, the goal of the present study was to characterize the effects of IR on lymphatic tone and pumping independent of the confounding effects of IR on the other tissues.Mesenteric lymphatic vessels (MLVs), isolated from male Sprague Dawley rats, were randomly divided into SHAM and IR groups and were placed in chilled APSS. MLVs from the IR group were then irradiated with X‐rays at 10 Gray Dose. Immediately after irradiation, MLVs from both groups were placed in chilled APSS until further use. MLVs were then cannulated and perfused with warm APSS. Lymphatic pumping was assessed in spontaneously contracting vessels. Role of nitric oxide (NO) and cyclooxygenase (COX) pathways was evaluated by assessing pump function in separate MLVs before and after incubation in N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME; 100 µM) and Indomethacin (INDO; 10 µM).Evaluation of the lymphatic pumping in response to changes in transmural pressure revealed significantly increased contraction frequency and calculated active lymph flow in IR MLVs compared to those in SHAM MLVs. However, stroke volume normalized to passive volume and diastolic tone were not significantly different between the IR and SHAM MLVs. The increase in contraction frequency in IR MLVs was partially restored by the blockade of COX pathways with INDO. Blockade of NOS with L‐NAME, however, led to further increase in contraction frequency of IR MLVs, which was significantly higher than that in SHAM MLVs.Early studies investigating the IR effects on lymphatic endothelial cells reported that lymphatic endothelium is resistant to IR. Conflicting findings of the in vivo lymphatic studies have further confounded the insights into the lymphatic responses to IR. It has been widely believed that lymphatic vessels are unaffected by IR, and therefore, the role of the lymphatic system‐ the other half of the circulatory system, in radiation injury has been mostly ignored. However, it is evident from the findings of the present study that lymphatic vessels are indeed affected by IR. Furthermore, IR‐induced increase in contraction frequency observed in the present study suggests that lymphatic acute responses to IR differ from the blood vessel responses.

Возрастные изменения механизмов эндотелий-зависимой дилатации пиальных артериальных сосудов у крыс SHR

The study investigated the role of age-related changes in the contribution of NO and IKCa-channels to acetylcholine-induced dilation of pial arteries in spontaneously hypertensive rats (SHR). We used intravital microphotography (×470) to evaluate the responses of pial arteries to acetylcholine chloride (ACh, 10−7 M, 5 min) with and without blockade of NO (L-NAME, 10−3 M) and IKCa-channels (klotrimazole,10−5 M) in SHR rats aged 4 and 18 months. The number and degree of vascular dilatation was measured from the erythrocyte flow width in three individual groups of arteries: small (less than 20 μm in diameter), medium (20–40 μm) and large (more than 40 μm). It was found that in young SHR rats, NO plays a significant role in the ACh-mediated dilation of small and large-diameter vessels. The role of IKCa-channels in endothelium-dependent dilatation mainly manifests itself in the group of small vessels and decreases with an increase in artery diameter. Aging, accompanied by hypertension, reduces the role of IKCa-channels and increases the contribution of NO to the dilatation of pial arterial vessels of all sizes.

Clopidogrel-Induced Gastric Injury in Rats is Attenuated by Stable Gastric Pentadecapeptide BPC 157.

AimAlthough Clopidogrel is safe in healthy volunteers, it can induce recurrence of gastric ulcers in high-risk patients. Here, we investigated the protective effect of the natural product, stable gastric pentadecapeptide 157 (BPC 157) on Clopidogrel-induced gastric injury.MethodsWe used acetic acid to induce gastric ulcer in Sprague Dawley rats. Clopidogrel alone or in combination with BPC 157 or L-NAME (nitric oxide system blockade) were administered after healing of acetic acid-induced ulcer. One percent methylcellulose solution was used as control. Ulcer recurrence rate and the ulcer index were compared between these groups. Gastric mucosal apoptosis rate, microscopic inflammation activity and angiogenesis markers vascular endothelial growth factor A (VEGF-A) and CD34 were examined by TUNEL, histological evaluations (HE) and immunohistochemistry (IHC). Pathways involved, expressions of endoplasmic reticulum (ER) stress apoptosis marker CHOP, angiogenic markers VEGF-A and its receptor VEGFR1, and endothelial NO synthase (eNOS) were all analyzed by Western blot.ResultsThis study indicated that Clopidogrel significantly induced the gastric ulcers recurrence, severe inflammation and ER stress related apoptosis of the gastric mucosa, suppressed the synthesis of angiogenic markers and eNOS. Furthermore, Clopidrogel intervention resulted in the activation of protein kinase B (AKT) and p38 mitogen-activated protein kinase (p38/MAPK). BPC 157 attenuated the gastric mucosal damage caused by Clopidogrel and reversed these molecular effects. However, NO blockade L-NAME weakened the protective effect and thus the molecular effects of BPC 157 on gastric mucosa.ConclusionIn conclusion, these results suggest that BPC 157 inhibited Clopidogrel-induced gastric mucosa injury partially by inhibition of gastric mucosa cell ER stress-mediated apoptosis and inflammation, and promoting gastric mucosa angiogenesis via VEGF-A/VEGFR1 mediated-AKT/p38/MAPK signaling pathways.

Interplay of the adenosine system and NO in control of renal haemodynamics and excretion: Comparison of normoglycaemic and streptozotocin diabetic rats

The adenosine (Ado) system may participate in regulation of kidney function in diabetes mellitus (DM), therefore we explored its role and interrelation with NO in the control of renal circulation and excretion in normoglycemic (NG) and streptozotocin-diabetic (DM) rats. Effects of theophylline (Theo), a non-selective Ado receptor antagonist, were examined in anaesthetized NG or in streptozotocin induced diabetic (DM) rats, untreated or after blockade of NO synthesis with l-NAME. We measured arterial blood pressure (MABP), whole kidney blood flow and renal regional flows: cortical and outer- and inner-medullary (IMBF), determined as laser-Doppler fluxes. Renal excretion of water, total solutes and sodium and in situ renal tissue NO signal (selective electrodes) were also determined. Theo experiments disclosed minor baseline vasoconstrictor and vasodilator tone in the kidney of NG and DM rats, respectively. NO blockade increased baseline MABP and decreased renal haemodynamics, similarly in NG and DM rats, indicating comparable vasodilator influence of NO in the two groups. Unexpectedly, in all rats with intact NO synthesis, Ado receptor blockade increased kidney tissue NO. In NO-deficient NG and DM rats, Ado receptor blockade induced comparable renal vasodilatation, suggesting similar vasoconstrictor influence of the Ado system. However, DM rats showed an unexplained association of decreased MABP and IMBF and increased NO signal. Higher baseline renal excretion in DM rats indicated inhibition of renal tubular reabsorption due to the prevalence of natriuretic A2 over antinatriuretic A1 receptors. In conclusion, the experiments provided new insights in functional interrelation of adenosine and NO in normoglycaemia and streptozotocin-diabetes.

Hypertension Programmed in Adult Hens by Isolated Effects of Developmental Hypoxia In Ovo.

In mammals, pregnancy complicated by chronic hypoxia can program hypertension in the adult offspring. However, mechanisms remain uncertain because the partial contributions of the challenge on the placenta, mother, and fetus are difficult to disentangle. Here, we used chronic hypoxia in the chicken embryo-an established model system that permits isolation of the direct effects of developmental hypoxia on the cardiovascular system of the offspring, independent of additional effects on the mother or the placenta. Fertilized chicken eggs were exposed to normoxia (N; 21% O2) or hypoxia (H; 13.5%-14% O2) from the start of incubation (day 0) until day 19 (hatching, ≈day 21). Following hatching, all birds were maintained under normoxic conditions until ≈6 months of adulthood. Hypoxic incubation increased hematocrit (+27%) in the chicken embryo and induced asymmetrical growth restriction (body weight, -8.6%; biparietal diameter/body weight ratio, +7.5%) in the hatchlings (all P<0.05). At adulthood (181±4 days), chickens from hypoxic incubations remained smaller (body weight, -7.5%) and showed reduced basal and stimulated in vivo NO bioavailability (pressor response to NG-nitro-L-arginine methyl ester, -43%; phenylephrine pressor response during NO blockade, -61%) with significant hypertension (mean arterial blood pressure, +18%), increased cardiac work (ejection fraction, +12%; fractional shortening, +25%; enhanced baroreflex gain, +456%), and left ventricular wall thickening (left ventricular wall volume, +36%; all P<0.05). Therefore, we show that chronic hypoxia can act directly on a developing embryo to program hypertension, cardiovascular dysfunction, and cardiac wall remodeling in adulthood in the absence of any maternal or placental effects.

Age-Related Changes in the Role of Calcium-Activated Potassium Channels in Acetylcholine Mediated Dilatation of Pial Arterial Vessels in Rats

Age-related changes in the contribution of endothelial hyperpolarization evoked by activation of intermediate-conductance Ca2+-activated K+ (IKca) channels and NO to acetylcholine-mediated dilatation of pial arterial vessels in rats were studied. Using intravital microphotography (×470), a comparative evaluation of pial arterial responses to acetylcholine chloride (ACh, 10–7 M, 5 min) with and without blockade of IKCa channels (clotrimazole, 10–5 M) and NO (L-NAME, 10–3 M) was carried out in Wistar rats aged 4 and 26 months. The contribution of endothelial hyperpolarization and NO to vascular dilatation was evaluated by changes in the number of dilated arteries and the degree of their response to ACh after application of the blockers. For the latter purpose, the erythrocyte flow width was measured in three individual groups of arteries: small- (< 20 µm), medium- (20–40 µm) and large- (> 40 µm) caliber. The obtained data indicate an age-related decrease in the contribution of endothelial hyperpolarization evoked by activation of IKca channels to ACh-induced dilatation of small-caliber pial arteries in rats. At the same time, the role of this mechanism in medium-caliber arteries increases. Apparently, changes in the vasodilatation mechanisms that occur during aging are due to an increasing role of NO-mediated regulation of endothelium-dependent dilatation responses.

Cerebral oxygen sensing and the integrated regulation of hypoxic vasodilatation.

Cerebral oxygen sensing and the integrated regulation of hypoxic vasodilatation.

Chronic ethanol consumption enhances inducible endothelium-dependent hyperpolarizing factor-mediated relaxation in the rat artery

The inducible endothelium-dependent hyperpolarizing factor (iEDHF) pathway is activated as a compensatory response to adverse changes in the body. It causes vasorelaxation and maintains circulatory homeostasis in the organs. Small to moderate quantities of ethanol enhance vascular relaxation. However, its mechanism and the involvement of the iEDHF pathway in this process are unknown. Therefore, we studied iEDHF-mediated, acetylcholine-induced, endothelium-dependent relaxation in the superior mesenteric arteries (SMAs) of rats chronically fed ethanol. Rats were administered a standard diet (S-Control group), Lieber's control diet (L-Control group), or Lieber's ethanol diet (EtOH group). SMA relaxation was assessed by isometric tension measurements. Arachidonate 15-lipoxygenase (ALOX15) and soluble epoxide hydrolase (sEH) were determined by immunoblot. Acetylcholine-induced, endothelium-dependent relaxation was significantly greater in the EtOH than the control groups. These differences persisted after PGI2 and NO blockade. Thus, the increase in acetylcholine-induced relaxation was EDHF-mediated. In the EtOH group, however, it was prevented by iEDHF inhibitors. ALOX15 and sEH protein expression levels were higher in the EtOH than the L-Control group. The increase in acetylcholine-induced relaxation by chronic ethanol consumption was mediated by the iEDHF pathway. This mechanism may compensate for the blood pressure elevation induced by ethanol. This study suggests that iEDHF is induced during proper drinking and may help prevent the onset of cardiovascular conditions.

The flavonoid compound apigenin prevents colonic inflammation and motor dysfunctions associated with high fat diet-induced obesity.

Background and purposeApigenin can exert beneficial actions in the prevention of obesity. However, its putative action on obesity-associated bowel motor dysfunctions is unknown. This study examined the effects of apigenin on colonic inflammatory and motor abnormalities in a mouse model of diet-induced obesity.Experimental approachMale C57BL/6J mice were fed with standard diet (SD) or high-fat diet (HFD). SD or HFD mice were treated with apigenin (10 mg/Kg/day). After 8 weeks, body and epididymal fat weight, as well as cholesterol, triglycerides and glucose levels were evaluated. Malondialdehyde (MDA), IL-1β and IL-6 levels, and let-7f expression were also examined. Colonic infiltration by eosinophils, as well as substance P (SP) and inducible nitric oxide synthase (iNOS) expressions were evaluated. Motor responses elicited under blockade of NOS and tachykininergic contractions were recorded in vitro from colonic longitudinal muscle preparations.Key resultsWhen compared to SD mice, HFD animals displayed increased body weight, epididymal fat weight and metabolic indexes. HFD mice showed increments in colonic MDA, IL-1β and IL-6 levels, as well as a decrease in let-7f expression in both colonic and epididymal tissues. HFD mice displayed an increase in colonic eosinophil infiltration. Immunohistochemistry revealed an increase in SP and iNOS expression in myenteric ganglia of HFD mice. In preparations from HFD mice, electrically evoked contractions upon NOS blockade or mediated by tachykininergic stimulation were enhanced. In HFD mice, Apigenin counteracted the increase in body and epididymal fat weight, as well as the alterations of metabolic indexes. Apigenin reduced also MDA, IL-1β and IL-6 colonic levels as well as eosinophil infiltration, SP and iNOS expression, along with a normalization of electrically evoked tachykininergic and nitrergic contractions. In addition, apigenin normalized let-7f expression in epididymal fat tissues, but not in colonic specimens.Conclusions and implicationsApigenin prevents systemic metabolic alterations, counteracts enteric inflammation and normalizes colonic dysmotility associated with obesity.

Possible mechanisms of diverse spaceflight effects on endothelial function in different vascular beds

Possible mechanisms of diverse spaceflight effects on endothelial function in different vascular beds

No Sex Differences in Muscle O2 Delivery-to-Utilization Matching Before or During Contractions in Rats

Pre-menopausal women express a reduced arterial blood pressure and risk of cardiovascular disease relative to age-matched men. The mechanism for these outcomes purportedly relate to elevated estrogen levels increasing endothelial nitric oxide (NO) synthase activity and NO-mediated vasorelaxation. PURPOSE: Based on the role that NO plays in the relationship between O2 delivery and utilization, we tested the hypothesis that females would show a fundamentally higher O2 delivery/utilization ratio; especially during muscle contractions, compared to males. METHODS: To test this hypothesis, the spinotrapezius muscle of Sprague Dawley rats (14 total; female = 7, male = 7) was surgically exposed and electrically stimulated at 1 Hz. Oxyphor G4 was injected into the muscle and phosphorescence quenching employed to determine the temporal profile of muscle interstitial space O2 partial pressure (PintO2, determined by O2 delivery/utilization ratio). This was performed under three conditions: control (CON), 300 μM sodium nitroprusside (SNP; NO donor) superfusion, and 1.5 mM L-arginine methyl ester (L-NAME; NOS blockade) superfusion. RESULTS: No differences were found for baseline PintO2 (CON: 21 ± 1 vs 17 ± 2; SNP: 40 ± 3 vs 36 ± 3; L-NAME: 16 ± 2 vs 14 ± 2 mmHg (all p > 0.05)); nor ΔPintO2 during contractions (CON: 13 ± 1 vs 12 ± 2; SNP 20 ± 2 vs 18 ± 2; L-NAME: 11 ± 1 vs 9 ± 1 mmHg (all p > 0.05)) between males and females, respectively. The kinetics response (mean response time) to contractions did not differ in any condition (CON: 17 ± 2 vs 16 ± 3; SNP: 21 ± 2 vs 28 ± 5; L-NAME: 15 ± 2 vs 14 ± 1 seconds (all p > 0.05)) between males and females, respectively. DISCUSSION: In direct contrast to our hypothesis, no sex differences were evident at rest or during contractions under any condition. Therefore, at rest and during muscle contractions, the effect of estrogen on NO bioavailability and vascular control are either insignificant or redundant to other vasodilatory pathways.

0169 : Effects of intermittent hypoxia on rat ophthalmic artery reactivity: role of endothelin 1, oxidative stress and endothelium derived hyperpolarizing factors

Obstructive sleep apnea (OSA) is a common disease, characterized by recurrent desaturation-reoxygenation sequences. Intermittent hypoxia (IH) has been proposed as major component of OSA. OSA has been recently associated with a higher frequency of optic neuropathies. There are currently no pathophysiological data regarding the effect of HI or OSA on ocular vascular system. This study aims to characterize IH impact on rat ophthalmic artery (OA) reactivity. Particularly, the role of endothelin 1 (ET-1), oxidative stress and endothelium derived hyperpolarizing factors (EDHF) were studied. Rats were exposed to 14 days IH (IH rats) or normoxia (NX rats). Ophthalmic artery reactivity was studied using wire myography. Endothelin A receptor (ETRA) expression and superoxide anions production in OA were studied by immunohistolabelling and DHE labelling respectively. After 14 days IH, ETRA immunolabeling and superoxide anions expression in OA were increased by 22% (p = 0.015) and 23% (p = 0.04) respectively in IH rats. Ophthalmic artery contraction to 3.10-8 M ET-1 was increased by 16% (p<0.05) in IH rats. ETRA blockage (BQ-123) almost completely abolished contractile response to ET-1 in both groups. Endothelin B receptor (ETRB) blockage (BQ-788) increased response to ET-1 in NX rats but not in IH rats. Relaxation to acetylcholine (Ach) was significantly delayed in IH rats (p<0.0001). After NOS blockade with L-NAME, difference in Ach induced relaxation between groups was abolished. Cytochrome P450 inhibition by fluconazole had an opposite effect on response to Ach in NX and IH rats, increasing relaxation to Ach in IH rats and delaying it in NX rats. 14-day IH-exposure induces endothelial dysfunction in rat ophtalmic artery, associated with increase in oxidative stress and change in EDHF contribution to endothelial function. The increased contractile response to ET-1 in IH rats was due to an increase in ETRA -vasoconstriction and a decrease in ETRB vasorelaxation. The author hereby declares no conflict of interest

Abstract 15229: Critical Protective Role of Endogenous Nitric Oxide in Established Severe Pulmonary Arterial Hypertension in Rats

Background: It is widely believed that impared bioavailability of endothelium-derived nitric oxide (NO) contributes to the development of pulmonary arterial hypertension (PAH). However, there are reports that show high pulmonary endothelial NO synthase (eNOS) expression in PAH patients, and the functional roles of endogenous NO in established PAH remain unclear. Hypothesis: We tested the hypothesis that pulmonary eNOS expression is increased, and that endogenous NO plays an important functional role in a rat model of PAH. Method and Results: Adult male Sprague Dawley rats (BW: 150 - 200g) were injected subcutaneously with Sugen5416 (20 mg/kg; VEGF receptor blocker), and exposed to hypoxia (10% O2) for 3 weeks followed by a return to normoxia for 5 weeks. Hemodynamic examination was performed at the 1-, 3-, 5- and 8-week (wk) time points after Sugen5416 injection. RV systolic pressure increased time-dependently (RVSP: 47 ± 6, 83 ± 14, 101 ± 22, and 122 ± 16 mmHg at 1-, 3-, 5- and 8-wk time points, n=4-6) compared with normal rats (31 ± 2 mmHg, n=4, p&lt;0.01, respectively). Blockade of NOS with Nω-nitro-L- arginine methyl ester (L-NAME, 30 mg/kg, iv) further increased RVSP acutely (ΔRVSP from each baseline: 7 ± 11, 25 ± 11, 52± 21, and 43 ± 13 at 1-, 3-, 5- and 8-wk time points) compared with normal rats (0.8 ± 1.1 mmHg). At the 8-wk time point, inhibition of NOS caused a marked RVSP elevation (165 ± 11 mmHg), leading to RV failure and death in all rats tested. In vivo pulmonary angiogram using the synchrotron radiation microangiogram system showed severe small pulmonary arterial narrowing in response to L-NAME. Western blot analysis showed increased eNOS protein levels in PAH rat lungs (n=4 each time point; p&lt;0.01 vs. normal) and immunohistochemisty revealed high expression of eNOS in remodeled pulmonary arteries at the 5- and 8-time points. Conclusion: Based on our findings that L-NAME caused marked pulmonary vasoconstriction and further elevation in RVSP and that there was high lung eNOS protein levels in advanced PAH rats, we conclude, contrary to the general assumption, that endogenous NO presumably via eNOS may play a critical protective role by counteracting severe pulmonary vasoconstriction in established PAH.

Effects of LPS Endotoxemia on Cardiovascular Parameters and Plasma Nitric Oxide of Ovariectomized Rats

Females of reproductive age have a more active immune system than males, but influence of sex hormones on cardiovascular system have been controversial. The cardiovascular responses of ovariectomized female rats compared with females in estrus phase after immunological challenge with LPS infusion and nitric oxide production have not yet been elucidated. Wistar rats, eight weeks after being subjected to ovariectomy (OVX) or false-OVX (Sham), had their femoral artery and vein catheterized. Cardiovascular parameters were recorded in conscious rats for 2 hours after LPS injection (5 mg / kg, IV), previously treated with saline or L-NAME (10mg / kg). Plasma samples were collected for measurement of nitrite (NO2) by Griess method. LPS promoted four distinct phases in mean arterial pressure (MAP) of the sham group, with hypotension within 10 minutes (DMAP = -27.5 ± 6 mmHg, n = 8). OVX rats did not show this initial drop (DMAP = -4.4 ± 4 mmHg, n = 7); and pretreatment with L-NAME did not alter the pattern of response to LPS. After 2 hours of LPS injection, NO2 plasma levels were higher in OVX (29±0,9 µM, n = 9 versus 21 ± 2µM, n = 9) and group treated with L-NAME prevented this rise (19 ± 1uM, n = 8 versus 14 ± 2 µM, n = 4). Female rats in estrus decrease more pressure to LPS in the first minutes compared to ovarietomized rats. Blockade of NOS did not alter the decrease in MAP, so it is possible that other mediators participate in the hypotension induced by endotoxemia in female rats.

N (G)-nitro-L-arginine Methyl Ester Protects Against Hormonal Imbalances Associated with Nicotine Administration in Male Rats.

Background:The administration of nicotine is associated with altered hormonal imbalances and increased serum and testicular nitric oxide (NO) level.Aim:This study sought to investigate the effects of NO inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on altered hormonal imbalance in adult male albinorats.Materials and Methods:Rats were administered with 0.5 mg/kg body weight (BW) and 1.0 mg/kg BW nicotine and were treated with L-NAME in the drinking water or drinking water alone for 30 days. Serum was analyzed for testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin using radioimmunoassay.Results:Nicotine administration significantly decreased (P < 0.05) testosterone in the low and high dose treated groups and FSH in the high dose treated group when compared with the control group. There was a significant increase (P < 0.05) in mean LH and prolactin level in the high dose treated group when compared with the control. Concomitant treatment with nicotine and L-NAME produced significant increases in testosterone and FSH, and a decrease in prolactin in 1.0 mg/kg BW. L-NAME alone did not lead to a significant increase in testosterone when compared with control.Conclusion:These data demonstrate that the suppressive effects of nicotine on testosterone level of the adult male rat can be prevented by NOS blockade with L-NAME. It appears that these beneficial effects are mediated primarily within the gonad; however, the involvement of the pituitary cannot be totally ruled out.

Involvement of histamine in endothelium-dependent relaxation of mesenteric lymphatic vessels.

The knowledge of the basic principles of lymphatic function, still remains, to a large degree, rudimentary and will require significant research efforts. Recent studies of the physiology of the MLVs suggested the presence of an EDRF other than NO. In this study, we tested the hypothesis that lymphatic endothelium-derived histamine relaxes MLVs. We measured and analyzed parameters of lymphatic contractility in isolated and pressurized rat MLVs under control conditions and after pharmacological blockade of NO by L-NAME (100 μM) or/and histamine production by α-MHD (10 μM). Effectiveness of α-MHD was confirmed immunohistochemically. We also used immunohistochemical labeling and Western blot analysis of the histamine-producing enzyme, HDC. In addition, we blocked HDC protein expression in MLVs by transient transfection with vivo-morpholino oligos. We found that only combined pharmacological blockade of NO and histamine production completely eliminates flow-dependent relaxation of lymphatic vessels, thus confirming a role for histamine as an EDRF in MLVs. We also confirmed the presence of HDC and histamine inside lymphatic endothelial cells. This study supports a role for histamine as an EDRF in MLVs.