Nitric Oxide Synthase Blockade Research Articles

Coronary vascular growth matches IGF-1-stimulated cardiac growth in fetal sheep.

As loss of contractile function in heart disease could often be mitigated by increased cardiomyocyte number, expansion of cardiomyocyte endowment paired with increased vascular supply is a desirable therapeutic goal. Insulin-like growth factor 1 (IGF-1) administration increases fetal cardiomyocyte proliferation and heart mass, but how fetal IGF-1 treatment affects coronary growth and function is unknown. Near-term fetal sheep underwent surgical instrumentation and were studied from 127 to 134 d gestation (term=147 d), receiving either IGF-1 LR3 or vehicle. Coronary growth and function were interrogated using pressure-flow relationships, an episode of acute hypoxia with progressive blockade of adenosine receptors and nitric oxide synthase, and by modeling the determinants of coronary flow. The main findings were that coronary conductance was preserved on a per-gram basis following IGF-1 treatment, adenosine and nitric oxide contributed to hypoxia-mediated coronary vasodilation similarly in IGF-1-treated and Control fetuses, and the relationships between coronary flow and blood oxygen contents were similar between groups. We conclude that IGF-1-stimulated fetal myocardial growth is accompanied by appropriate expansion and function of the coronary vasculature. These findings support IGF-1 as a potential strategy to increase cardiac myocyte and coronary vascular endowment at birth.

P0978THE EFFECTS OF CHRONIC DAPAGLIFLOZIN TREATMENT ON THE RENAL MICROVASCULATURE IN A RAT MODEL OF TYPE 2 DIABETES

Abstract Background and Aims Chronic kidney disease (CKD) is an ever-growing concern and CKD associated with diabetes accounted for ∼ 35% of new cases of end-stage renal failure. Clinical trials with insulin-independent sodium-glucose co-transporter 2 (SGLT2) inhibitors not only showed significant improvements in hyperglycemia and thus renal damage, but also reduced the risk of adverse cardiovascular events. Microvessels supply local tissue oxygen demands and are important controllers of regional perfusion. Endothelial dysfunction has been posited to be an important factor driving the pathogenesis of diabetic nephropathy, largely through impaired eNOS activity and thus reduced nitric oxide bioavailability. Thus, we aim to determine the effects of SGLT2 inhibition on vasodilator function in the renal vasculature. We hypothesize that rats chronically treated with dapagliflozin, a SGLT2 inhibitor, improves renal endothelial function and the microvessels are better able to maintain perfusion in response to the inhibition of nitric oxide synthase and prostaglandin blockade. Method Male Zucker fatty diabetic rats (8 wk old), a model of type 2 diabetes, were given daily oral gavage of 1 mg/kg dapagliflozin or its vehicle for up to 22 wks of age (n=6/group). Renal excretory function, glomerular filtration rate (GFR) and indices of renal injury was assessed before treatment and after every 4 wks up until 22 wks of age. GFR was assessed via transcutaneous clearance of FITC-sinistrin. Vasodilator function of the renal microvasculature was assessed via X-ray microangiography. We conducted successive imaging of the microvessels before and during infusion of acetylcholine (ACh) and sodium nitroprusside (SNP) which facilitated the assessment of endothelium dependent and independent dilation respectively. After which, rats were given successive boluses of L-NAME and indomethacin to assess the vasodilatory function of the microvasculature independent of nitric oxide and prostaglandins respectively. These inhibitors were given under the conditions of SNP clamp which allowed for us to titrate the blood pressure to baseline levels. Lastly, we assessed the ability of endothelium hyperpolarizing factors (EDHFs) to maintain microvascular perfusion when SNP infusion ceased and an infusion of ACh commenced while still under the effects of L-NAME and indomethacin. Results As expected, dapagliflozin alleviated hyperglycemia across the treatment period. There was a tendency for dapagliflozin to ameliorate the decline of GFR, although this apparent effect was not statistically significant. Dapagliflozin did not appear to improve indices of renal injury. Treatment with dapagliflozin alleviated polyuria but did not appear to have an impact on urine osmolarity or sodium excretion. The responses (vessel diameter) of renal microvessels to ACh and SNP was greater in dapagliflozin than in vehicle fed rats. The microvessels of vehicle fed rats appeared to undergo relative constriction in response to L-NAME and indomethacin even under the effects of SNP clamp. In contrast, microvessels of dapagliflozin fed rats appeared to be relatively well-perfused after NOS and COX blockade. This suggests that dapagliflozin may improve endothelial dysfunction commonly associated with diabetic nephropathy. Following NOS and COX blockade, ACh was infused in rats to determine the status of vasodilatory function mediated by EDHFs. The microvessels in diabetic rats did not appear to be dilated after infusion of ACh, suggesting that vasodilatory effects of EDHFs on the vasculature is diminished in diabetic rats. Dapagliflozin appeared to improve this effect in that the renal microvessels were dilated even when NOS and COX production was blocked/inhibited. Conclusion Chronic treatment of dapagliflozin may improve endothelial dysfunction and thus retard the progression of diabetic nephropathy in a rat model of type 2 diabetes.

Systemic NOS inhibition reduces contracting muscle oxygenation more in intact female than male rats

Females respond to baroreceptor stimulation with enhanced modulation of heart rate (HR) to regulate blood pressure and also express greater reliance on nitric oxide (NO) for vascular control compared to males. Sex differences in muscle oxygenation consequent to central hemodynamic challenge induced by systemic NO synthase (NOS) inhibition are unknown. We tested the hypotheses that systemic NOS inhibition would induce lower contracting skeletal muscle oxygenation in females compared to males. The spinotrapezius of Sprague-Dawley rats (females (♀) = 9, males (♂) = 9) was surgically exposed and contracted by electrical stimulation (180s, 1 Hz, ~6 V) under pentobarbital sodium anesthesia. Oxyphor G4 was injected into the muscle and phosphorescence quenching was used to measure the interstitial PO2 (PO2is, determined by O2 delivery-to-utilization matching) under control (Krebs-Henseleit solution) and after intra-arterial infusion of nitro-l-arginine methyl ester (l-NAME; NOS blockade; 10 mg kg−1). At rest, females showed a greater PO2is increase (ΔPO2is/ΔMAP) and HR (ΔHR/ΔMAP) reduction than males in response to the elevated MAP induced by systemic NOS inhibition (both p < 0.05). Following l-NAME, during the contracting steady-state, females exhibited lower PO2is than males (♂: 17.1 ± 1.4 vs ♀: 10.8 ± 1.4 mmHg, p < 0.05). The rate pressure product was lower in females than males (♂: 482 ± 14 vs ♀: 392 ± 29, p < 0.05) and correlated with the steady-state PO2is (r = 0.66, p < 0.05). These results support that females express greater reductions in HR than males in response to l-NAME-induced elevation of MAP via the baroreceptor reflex and provide new insights on how central hemodynamics affect skeletal muscle oxygenation in a sex-specific manner.

The Cyclitol L-(+)-Bornesitol as an Active Marker for the Cardiovascular Activity of the Brazilian Medicinal Plant Hancornia speciosa.

The cyclitol bornesitol is the main constituent of the leaves from the antihypertensive medicinal plant Hancornia speciosa. This study aimed to investigate the ability of bornesitol to reduce blood pressure and its mechanism of action. Normotensive Wistar rats were divided into control group and bornesitol groups treated intravenously with bornesitol (0.1, 1.0 and 3.0 mg/kg). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded in non-anesthetized awake animals. Nitric oxide (NO) and angiotensin-converting enzyme (ACE) were measured in plasma by using colorimetric methods. Vascular reactivity study was performed in rat aorta rings and the involvement of nitric oxide synthase (NOS), calcium-calmodulin complex and phosphatidylinositol-3-kinase (PI3K)/Akt pathway in the vasodilator effect was investigated. Administration of bornesitol significantly reduced the SBP, increased the plasmatic level of nitrite, and decreased ACE activity in normotensive rats. In the rat aorta, bornesitol induced endothelium-dependent vasodilatation, which was abolished by NOS blockade. While calcium-calmodulin complex inhibition decreased the vasodilator effect of bornesitol, the inhibition of PI3K/Akt pathway did not alter it. Bornesitol reduced the blood pressure by a mechanism involving an increased production or bioavailability of NO, inhibition of ACE, and by an endothelium- and NO-dependent vasodilator effect. The present results support the use of bornesitol as an active marker for the cardiovascular activity of Hancornia speciosa.

ASYMMETRIC DIMETHYLARGININE IS A POSITITVE REGULATOR OF ENDOTHELIAL NITRIC OXIDE PRODUCTION BY MODULATING CALCIUM SENSING RECEPTOR SIGNALLING

Objective: Asymmetric dimethylarginine (ADMA) is generated through the post-translational modification of arginine residues and released into the cytosol following proteolysis. Elevated plasma ADMA concentration is a known risk factor for cardiovascular disease due, in part, to ADMA's role as a competitive inhibitor of the nitric oxide (NO) synthase enzymes. We have recently discovered that physiological concentrations of ADMA signal via the Calcium Sensing Receptor (CaSR). CaSR is a member of family C GPCR modulated by the binding of both Ca2+ and amino acids. Endothelial cells express CaSR where it positively regulates NO production. We hypothesized that low concentrations of ADMA increase NO signalling through modulation of CaSR while high concentration of ADMA inhibit NO synthesis. Design and method: ADMA modulation of CaSR signalling was assessed using human lung microvascular endothelial cells (HuLMVEC). CaSR is a GPCR which can signal through Gq intracellular Ca2+ mobilization; therefore, HuLMVEC were incubated with the Ca2+ sensitive dye CAL520 and stimulated with increasing concentrations of Ca2+ in the presence of ADMA. Nitric oxide production was assessed using the fluorescent sensor DAF-AM and stimulated with Ca2+ in the presence of ADMA. NPS-2143 was used as a CaSR selective antagonist. Results: Calcium stimulates HuLMVEC intracellular Ca2+ mobilization in a dose-dependent manner. Incubation of HuLMVEC with 10 μM ADMA shifts the dose-response curve left sensitizing CaSR to fluctuations in Ca2+ concentration an effect that is blocked by the CaSR antagonist NSP-2143. CaSR activation by Ca2+ increased DAF-AM fluorescence demonstrating increased NO synthesis. Low concentrations (10 μM) of ADMA again sensitized the HuLMVEC to Ca2+ increasing NO production; in contrast high concentrations of ADMA (100 μM) reduced NO through blockade of nitric oxide synthase. Conclusions: The Calcium sensing receptor is expressed in human endothelial cells and links fluctuations in extracellular Ca2+ concentrations to intracellular Ca2+ mobilization, resulting in increased endothelial NO production. ADMA positively modulated the CaSR in HuLMVEC increasing downstream intracellular Ca2+ release. These data suggest that low doses of ADMA may have a positive effect on vasodilation, while high concentrations ADMA reduce vascular reactivity through NO blockade.

ANGIOTENSIN II TYPE 1 RECEPTOR MEDIATED INHIBITORY MODULATION OF THE BAROREFLEX BY ANGIOTENSIN II INVOLVES NITRIC OXIDE DEPENDENT GABA RELEASE IN THE NUCLEUS TRACTUS SOLITARII

Objective: It is well established that angiotensin II exerts a dampening effect on the baroreflex within the nucleus tractus solitarii, the principal brainstem site for termination of baroreceptor afferents and which is densely populated with gamma-aminobutyric acid-ergic neurons and nerve terminals. The present study was designed to investigate whether local release of gamma-aminobutyric acid is involved in the effects mediated by local angiotensin II within the nucleus tractus solitarii. Design and method: In vivo microdialysis was used for measurement of extracellular glutamate and gamma-aminobutyric acid levels and for infusion of angiotensin II and other compounds within the nucleus tractus solitarii of normotensive Wistar rats. The mean arterial pressure and heart rate responses were monitored with a pressure transducer. Angiotensin II type 1 receptor selectivity was assessed using the angiotensin II type 1 receptor antagonist candesartan; the nitric oxide synthase inhibitor was used to assess the involvement of nitric oxide in the evoked responses by infusion of angiotensin II. Results: Local infusion of angiotensin II into the nucleus tractus solitarii significantly increased extracellular gamma-aminobutyric acid levels as well as mean arterial pressure. These responses were both abolished by co-infusion of either the angiotensin II type 1 receptor antagonist candesartan, or the nitric oxide synthase inhibitor, N (ω)-nitro-L-arginine methyl ester. The pressor response to angiotensin II was reversed by co-infusion with the gamma-aminobutyric acid A receptor antagonist, bicuculline. Local blockade of nitric oxide synthase decreased both, gamma-aminobutyric acid and glutamate concentrations. Conclusions: Our results suggest that the inhibitory modulation of the baroreflex by angiotensin II involves angiotensin II type 1 receptor mediated nitric oxide dependent local gamma-aminobutyric acid release within the nucleus tractus solitarii in normotensive rats. Nitric oxide produced within the nucleus tractus solitarii tonically potentiates local gamma-aminobutyric acid and glutamate release.

Pattern of sympathetic vasomotor activity in a model of hypertension induced by nitric oxide synthase blockade.

We aimed to investigate the effects of nitric oxide (NO) synthesis inhibition by NO synthase inhibitor N‐nitro‐L‐arginine‐methyl ester (L‐NAME) treatment on the sympathetic vasomotor nerve activity (SNA) on two sympathetic vasomotor nerves, the renal and splanchnic. NO plasma level and systemic oxidative stress were assessed. Hypertension was induced by L‐NAME (20 mg/kg per day, by gavage, for seven consecutive days) in male Wistar rats. At the end of the treatment, blood pressure, heart rate, arterial baroreflex sensitivity, renal SNA (rSNA), and splanchnic SNA (sSNA) were assessed in urethane anesthetized rats. L‐NAME‐treated rats presented increased blood pressure (152 ± 2 mmHg, n = 17) compared to the control group (101 ± 2 mmHg, n = 15). Both rSNA (147 ± 10, n = 15 vs. 114 ± 5 Spikes/s, n = 9) and sSNA (137 ± 13, n = 14 vs. 74 ± 13 spikes/s, n = 9) were significantly increased in the L‐NAME‐treated compared to the control group. A differential response on baroreflex sensitivity was found, with a significant reduction for rSNA but not for sSNA arterial baroreceptor sensitivity in L‐NAME‐treated rats. The adjusted regression model revealed that the reduction of systemic NO levels partially explains the variation in sSNA and blood pressure, but not rSNA. Taken together, our data show that hypertension induced by NO synthase blockade is characterized by increased SNA to the rSNA and sSNA. In addition, we found that the rats that had the greatest reduction in NO levels in plasma by L‐NAME were those that developed higher blood pressure levels. The reduction in the NO level partially explains the variations in sSNA but not in rSNA.

Enhanced Survival and Accelerated Perfusion of Skin Flap to Recipient Site Following Administration of Human α1-Antitrypsin in Murine Models.

Objective: Skin flaps are routinely used in reconstructive surgery yet remain susceptible to ischemia and necrosis. Distant flaps require lengthy time to detach causing patient discomfort. Human α1-antitrypsin (hAAT) is a clinically available serum glycoprotein. hAAT was shown to support mature vessel formation and enhance tissue survival following ischemia-reperfusion injuries. The purpose of the presented study was to examine the effect of hAAT on skin flap survival and distant "tube" flap perfusion through its recipient site. Approach: Random-pattern skin flaps were performed on mice treated with clinical-grade hAAT using three unique routes of administration (transgenic, i.p. and s.c. infiltration); necrotic area and tissue perfusion were assessed. Blockade of vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) were used to explore aspects of mechanism of action. A distant tube flap model was performed to examine time to perfusion. Results: hAAT-treated mice displayed approximately two-fold smaller necrotic flap areas versus controls across all hAAT administration routes. Flaps displayed greater perfusion as early as 3 days postsurgery (64.6% ± 4.0% vs. 43.7% ± 1.7% in controls; p = 0.007). hAAT-mediated flap survival was prominently NOS dependent, but only partially VEGF dependent. Finally, distant flaps treated with hAAT displayed significantly earlier perfusion versus controls (mean 9.6 ± 1.6 vs. 13.1 ± 1.0 days; p = 0.0005). Innovation: The established safety record of hAAT renders it an attractive candidate toward improving skin flap surgery outcomes, particularly during VEGF blockade. Conclusions: hAAT treatment enhances survival and accelerates perfusion of skin flaps in animal models in a NOS-dependent manner, partially circumventing VEGF blockade. Further mechanistic studies are required.

AT1 Receptor Mediated Hypertensive Response to Ang II in the Nucleus Tractus Solitarii of Normotensive Rats Involves NO Dependent Local GABA Release.

AimIt is well-established that angiotensin II exerts a dampening effect on the baroreflex within the nucleus tractus solitarii (NTS), the principal brainstem site for termination of baroreceptor afferents and which is densely populated with gamma-aminobutyric acid (GABA)ergic neurons and nerve terminals. The present study was designed to investigate whether local release of GABA is involved in the effects mediated by local angiotensin II within the NTS.MethodsIn vivo microdialysis was used for measurement of extracellular glutamate and GABA levels and for infusion of angiotensin II within the NTS of conscious normotensive Wistar rats. The mean arterial pressure (MAP) and heart rate response to local infusion of angiotensin II were subsequently monitored with a pressure transducer under anesthesia. The angiotensin II type 1 receptor (AT1R) antagonist, candesartan, was used to assess whether responses were AT1R dependent and the nitric oxide (NO) synthase inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME), was used to assess the involvement of NO in the evoked responses by infusion of angiotensin II. The MAP and heart rate responses were monitored with a pressure transducer.ResultsLocal infusion into the NTS of angiotensin II induced a significant to ninefold significantly increase in extracellular GABA levels; as well as MAP was increased by 15 mmHg. These responses were both abolished by co-infusion of either, the angiotensin II type 1 receptor antagonist, candesartan, or the NO synthase inhibitor, L-NAME, demonstrating that the effect is not only AT1R dependent but also NO dependent. The pressor response to angiotensin II was reversed by co-infusion with the GABAA receptor antagonist, bicuculline. Local blockade of NO synthase decreased both, GABA and glutamate concentrations.ConclusionOur results suggest that the AT1R mediated hypertensive response to angiotensin II within the NTS in normotensive rats is GABA and NO dependent. Nitric oxide produced within the NTS tonically potentiates local GABA and glutamate release.

Mechanisms of sex differences in exercise capacity.

Sex differences are an important component of National Institutes of Health rigor. The goal of this investigation was to test the hypothesis that female mice have greater exercise capacity than male mice, and that it is due to estrogen, nitric oxide, and myosin heavy chain expression. Female C57BL6/J wild-type mice exhibited greater (P < 0.05) maximal exercise capacity for running distance (489 ± 15 m) than age-matched male counterparts (318 ± 15 m), as well as 20% greater work to exhaustion. When matched for weight or muscle mass, females still maintained greater exercise capacity than males. Increased type I and decreased type II myosin heavy chain fibers in the soleus muscle from females are consistent with fatigue resistance and better endurance in females compared with males. After ovariectomy, female mice no longer demonstrated enhanced exercise, and treatment of male mice with estrogen resulted in exercise capacity similar to that of intact females (485 ± 37 m). Nitric oxide synthase, a downstream target of estrogen, exhibited higher activity in female mice compared with male mice, P < 0.05, whereas ovariectomized females exhibited nitric oxide synthase levels similar to males. Nitric oxide synthase activity also increased in males treated with chronic estrogen to levels of intact females. Nitric oxide synthase blockade with Nω-nitro-l-arginine methyl ester eliminated the sex differences in exercise capacity. Thus estrogen, nitric oxide, and myosin heavy chain expression are important mechanisms mediating the enhanced exercise performance in females.

The Role of NO Synthase in the Cardioprotective Effect of Substances of Humic Origin on the Model of Ischemia and Reperfusion of Isolated Rat Heart.

The cardioprotective and inotropic effects of standardized active natural substance based on high-molecular-weight compounds of humic origin were studied on the model of global ischemia (40 min) and reperfusion of isolated perfused rat heart. Preventive administration of the test substance (0.1 mg/ml) before ischemia/reperfusion modeling reduced reperfusion contracture and necrotic death of cardiomyocytes and promoted recovery of myocardial contractility. Blockade of NO synthase with L-NAME (100 μM) abolished the above effects of the test substance. It was hypothesized that NO synthase plays an important role in the development of the cardioprotective and inotropic effects of the test natural substance.

Retinal arteriole reactivity in mice lacking the endothelial nitric oxide synthase (eNOS) gene

Dysfunctional vascular endothelial nitric oxide synthase (eNOS) has been proposed to play a main pathophysiological role in various ocular diseases. The aim of the present study was to test the hypothesis that the chronic lack of eNOS impairs endothelium-dependent vasodilation in retinal arterioles. The relevance of eNOS for mediating vascular responses was studied in retinal vascular preparations from eNOS-deficient mice (eNOS−/−) and wild-type controls in vitro. Changes in luminal diameter in response to vasoactive agents were measured by videomicroscopy. The thromboxane mimetic, U46619, induced similar concentration-dependent constriction of retinal arterioles in eNOS−/− and wild-type mice. Responses to the endothelium-independent vasodilator, nitroprusside, did not differ between both mouse genotypes, either. In contrast, responses to the endothelium-dependent vasodilator, acetylcholine, were blunted in eNOS−/− mice. Non-isoform-selective blockade of either nitric oxide synthase (NOS) or cyclooxygenase (COX) alone did not affect responses to acetylcholine. However, combined blockade of both enzyme families markedly attenuated cholinergic vasodilation. Also, combined blockade of COX and neuronal NOS (nNOS) blunted acetylcholine-induced vasodilation, while combined COX and inducible NOS (iNOS) inhibition had no effect. Simultaneous NOS and COX-1 blockade did not affect cholinergic vasodilation, whereas combined NOS and COX-2 inhibition markedly reduced vasodilation to acetylcholine. These findings are the first to demonstrate that the chronic lack of eNOS is associated with moderate endothelial dysfunction in retinal arterioles. However, eNOS-deficiency is partially compensated by nNOS and COX-2 metabolites, which are reciprocally regulated.

α1G T-type calcium channel determines the angiogenic potential of pulmonary microvascular endothelial cells.

Pulmonary microvascular endothelial cells (PMVECs) display a rapid angioproliferative phenotype, essential for maintaining homeostasis in steady-state and promoting vascular repair after injury. Although it has long been established that endothelial cytosolic Ca2+ ([Ca2+]i) transients are required for proliferation and angiogenesis, mechanisms underlying such regulation and the transmembrane channels mediating the relevant [Ca2+]i transients remain incompletely understood. In the present study, the functional role of the microvascular endothelial site-specific α1G T-type Ca2+ channel in angiogenesis was examined. PMVECs intrinsically possess an in vitro angiogenic "network formation" capacity. Depleting extracellular Ca2+ abolishes network formation, whereas blockade of vascular endothelial growth factor receptor or nitric oxide synthase has little or no effect, suggesting that the network formation is a [Ca2+]i-dependent process. Blockade of the T-type Ca2+ channel or silencing of α1G, the only voltage-gated Ca2+ channel subtype expressed in PMVECs, disrupts network formation. In contrast, blockade of canonical transient receptor potential (TRP) isoform 4 or TRP vanilloid 4, two other Ca2+ permeable channels expressed in PMVECs, has no effect on network formation. T-type Ca2+ channel blockade also reduces proliferation, cell-matrix adhesion, and migration, three major components of angiogenesis in PMVECs. An in vivo study demonstrated that the mice lacking α1G exhibited a profoundly impaired postinjury cell proliferation in the lungs following lipopolysaccharide challenge. Mechanistically, T-type Ca2+ channel blockade reduces Akt phosphorylation in a dose-dependent manner. Blockade of Akt or its upstream activator, phosphatidylinositol-3-kinase (PI3K), also impairs network formation. Altogether, these findings suggest a novel functional role for the α1G T-type Ca2+ channel to promote the cell's angiogenic potential via a PI3K-Akt signaling pathway.

Contribution of Thyrotropin-Releasing Hormone to Cerebellar Long-Term Depression and Motor Learning.

Thyrotropin-releasing hormone (TRH) regulates various physiological activities through activation of receptors expressed in a broad range of cells in the central nervous system. The cerebellum expresses TRH receptors in granule cells and molecular layer interneurons. However, the function of TRH in the cerebellum remains to be clarified. Here, using TRH knockout (KO) mice we studied the role of TRH in the cerebellum. Immunohistochemistry showed no gross morphological differences between KO mice and wild-type (WT) littermates in the cerebellum. In the rotarod test, the initial performance of KO mice was comparable to that of WT littermates, but the learning speed of KO mice was significantly lower than that of WT littermates, suggesting impaired motor learning. The motor learning deficit in KO mice was rescued by intraperitoneal injection of TRH. Electrophysiology revealed absence of long-term depression (LTD) at parallel fiber-Purkinje cell synapses in KO mice, which was rescued by bath-application of TRH. TRH was shown to increase cyclic guanosine monophosphate (cGMP) content in the cerebellum. Since nitric oxide (NO) stimulates cGMP synthesis in the cerebellum, we examined whether NO-cGMP pathway was involved in TRH-mediated LTD rescue in KO mice. Pharmacological blockade of NO synthase and subsequent cGMP production prevented TRH-induced LTD expression in KO mice, whereas increase in cGMP signal in Purkinje cells by 8-bromoguanosine cyclic 3’,5’-monophosphate, a membrane-permeable cGMP analog, restored LTD without TRH application. These results suggest that TRH is involved in cerebellar LTD presumably by upregulating the basal cGMP level in Purkinje cells, and, consequently, in motor learning.

Severe steatosis induces portal hypertension by systemic arterial hyporeactivity and hepatic vasoconstrictor hyperreactivity in rats

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease. The presence of portal hypertension has been demonstrated in NAFLD prior to development of inflammation or fibrosis, and is a result of extrahepatic and intrahepatic factors, principally driven by vascular dysfunction. An increased intrahepatic vascular resistance potentially contributes to progression of NAFLD via intralobular hypoxia. However, the exact mechanisms underlying vascular dysfunction in NAFLD remain unknown. This study investigates systemic hemodynamics and both aortic and intrahepatic vascular reactivity in a rat model of severe steatosis. Wistar rats were fed a methionine-choline-deficient diet, inducing steatosis, or control diet for 4 weeks. In vivo hemodynamic measurements, aortic contractility studies, and in situ liver perfusion experiments were performed. The mean arterial blood pressure was lower and portal blood pressure was higher in steatosis compared to controls. The maximal contraction force in aortic rings from steatotic rats was markedly reduced compared to controls. While blockade of nitric oxide (NO) production did not reveal any differences, cyclooxygenase (COX) blockade reduced aortic reactivity in both controls and steatosis, whereas effects were more pronounced in controls. Effects could be attributed to COX-2 iso-enzyme activity. In in situ liver perfusion experiments, exogenous NO donation or endogenous NO stimulation reduced the transhepatic pressure gradient (THPG), whereas NO synthase blockade increased the THPG only in steatosis, but not in controls. Alpha-1-adrenergic stimulation and endothelin-1 induced a significantly more pronounced increase in THPG in steatosis compared to controls. Our results demonstrate that severe steatosis, without inflammation or fibrosis, induces portal hypertension and signs of a hyperdynamic circulation, accompanied by extrahepatic arterial hyporeactivity and intrahepatic vascular hyperreactivity. The arterial hyporeactivity seems to be NO-independent, but appears to be mediated by specific COX-2-related mechanisms. Besides, the increased intrahepatic vascular resistance in steatosis appears not to be NO-related but rather to vasoconstrictor hyperreactivity.In this study, systemic hemodynamics and both aortic and intrahepatic vascular reactivity in a rat model of severe steatosis were investigated. Portal hypertension and signs of a hyperdynamic circulation were demonstrated. NO-independent, COX-2 mediated extrahepatic arterial hyporeactivity, as well as NO-independent alpha-1-adrenergic and endotheline-1 mediated intrahepatic vascular hyperreactivity are demonstrated, likely contributing to the observed portal hypertension in steatosis.

The Newly Synthesized Pyrazole Derivative 5-(1-(3 Fluorophenyl)-1H-Pyrazol-4-yl)-2H-Tetrazole Reduces Blood Pressure of Spontaneously Hypertensive Rats via NO/cGMO Pathway

The search for new antihypertensive drugs has grown in recent years because of high rate of morbidity among hypertensive patients and several side effects that are associated with the first-line medications. The current study sought to investigate the antihypertensive effect of a newly synthesized pyrazole derivative known as 5-(1-(3 fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazole (LQFM-21). Spontaneously hypertensive rats (SHR) were used to evaluate the effect of LQFM-21 on mean arterial pressure (MAP), heart rate (HR), renal vascular conductance (RVC), arterial vascular conductance (AVC), baroreflex sensitivity (BRS) index, and vascular reactivity. Acute intravenous (iv) administration of LQFM-21 (0.05, 0.1, 0.2, and 0.4 mg kg-1) reduced MAP and HR, and increased RVC and AVC. Chronic oral administration of LQFM-21 (15 mg kg-1) for 15 days reduced MAP without altering BRS. The blockade of muscarinic receptors and nitric oxide synthase by intravenous infusion of atropine and L-NAME, respectively, attenuated cardiovascular effects of LQFM-21. In addition, ex vivo experiments showed that LQFM-21 induced an endothelium-dependent relaxation in isolated aortic rings from SHR. This effect was blocked by guanylyl cyclase inhibitor (ODQ) and L-NAME. These findings suggest the involvement of muscarinic receptor and NO/cGMP pathway in the antihypertensive and vasodilator effects of LQFM-21.

Branched‐chain amino acids promote endothelial dysfunction through increased reactive oxygen species generation and inflammation

Branched‐chain amino acids (BCAA: leucine, isoleucine and valine) are essential amino acids implicated in glucose metabolism and maintenance of correct brain function. Elevated BCAA levels can promote an inflammatory response in peripheral blood mononuclear cells. However, there are no studies analysing the direct effects of BCAA on endothelial cells (ECs) and its possible modulation of vascular function. In vitro and ex vivo studies were performed in human ECs and aorta from male C57BL/6J mice, respectively. In ECs, BCAA (6 mmol/L) increased eNOS expression, reactive oxygen species production by mitochondria and NADPH oxidases, peroxynitrite formation and nitrotyrosine expression. Moreover, BCAA induced pro‐inflammatory responses through the transcription factor NF‐κB that resulted in the release of intracellular adhesion molecule‐1 and E‐selectin conferring endothelial activation and adhesion capacity to inflammatory cells. Pharmacological inhibition of mTORC1 intracellular signalling pathway decreased BCAA‐induced pro‐oxidant and pro‐inflammatory effects in ECs. In isolated murine aorta, BCAA elicited vasoconstrictor responses, particularly in pre‐contracted vessels and after NO synthase blockade, and triggered endothelial dysfunction, effects that were inhibited by different antioxidants, further demonstrating the potential of BCAA to induce oxidative stress with functional impact. In summary, we demonstrate that elevated BCAA levels generate inflammation and oxidative stress in ECs, thereby facilitating inflammatory cells adhesion and endothelial dysfunction. This might contribute to the increased cardiovascular risk observed in patients with elevated BCAA blood levels.

Impact of cell‐free hemoglobin on contracting skeletal muscle oxygen pressure dynamics: Potential therapeutic role of haptoglobin

Free hemoglobin (Hb) associated with hemolytic diseases (i.e., sickle cell anemia) extravasates into vascular tissue and depletes nitric oxide (NO), which leads to impaired vascular function and could disrupt skeletal muscle metabolic control during exercise. We tested the hypothesis that: 1) free Hb would extravasate into skeletal muscle tissue impairing the contracting skeletal muscle O2 delivery/O2 utilization ratio (microvascular PO2, PO2mv) to a similar extent as that observed following NO synthase (NOS) blockade, and 2) that the Hb scavenging protein haptoglobin (Hp) would prevent Hb extravasation and inhibit these skeletal muscle tissue effects. PO2mv was measured in eight healthy rats (phosphorescence quenching) at rest and during 180s of electrically induced (1‐Hz) twitch spinotrapezius muscle contractions (experiment 1). A second subset of seven rats was also used to investigate the effects of Hb+Hp (experiment 2). For both experiments, measurements were made: 1) during control conditions, 2) following a bolus infusion of either Hb (50 mg/kg) or Hb+Hp (50 mg/kg, bound Hb), and 3) following local superfusion of NG‐nitro‐l‐arginine methyl ester (L‐NAME; 10 mg/kg). Additional experiments were completed to visualize Hb extravasation into the muscular tissue using Click chemistry techniques. There were no significant differences in the PO2mv observed at rest for any condition in either experiment (p&gt;0.05 for all). In experiment 1, both Hb and L‐NAME reduced the PO2mv significantly during the steady‐state of muscle contractions when compared to control conditions with no differences between Hb and L‐NAME conditions (control: 24 ± 1, Hb: 21 ± 1, L‐NAME: 20 ± 1 mmHg, p&lt;0.05). In experiment 2, only L‐NAME resulted in a significantly lower PO2mv during the steady‐state of muscle contractions (control: 25 ± 1, Hb+Hp: 22 ± 2, L‐NAME: 18 ± 1 mmHg, p&lt;0.05). Free Hb lowered the blood‐myocyte O2 driving force to a level not significantly different from L‐NAME. However, infusing Hb bound to Hp resulted in no significant differences in steady‐state PO2mv during muscle contractions when compared to control. Surprisingly, we did not observe Hb accumulation in skeletal muscle tissue. Taken together these data suggest that free Hb impairs O2 delivery/utilization via a NO scavenging effect which would presumably disrupt oxidative metabolism and shorten the onset of fatigue during exercise. Furthermore, the unchanged PO2mv steady‐state observed following Hb+Hp further suggests that therapeutic administration of the Hb binding protein haptoglobin may restore skeletal muscle metabolic control and potentially exercise tolerance in those afflicted with hemolytic diseases.Support or Funding InformationFunded by NIH (R01HL125642) to DCI and Colorado Nutrition and Obesity Research Center (P30DK048520) award to SKF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Early life stress (ELS) protects against LNAME hypertension‐induced renal tubular damage

In humans, early life stress (ELS) is a newly recognized cardiovascular disease risk factor and associated with increases in inflammation and risk of hypertension. Utilizing a rat model of ELS, maternal separation (MatSep), we recently reported that MatSep induced immune activation in the kidney compared to normally‐reared control animals. Chronic nitric oxide synthase blockade with LNAME in the drinking water is a model of hypertension with increased immune cells and inflammation. Therefore, we hypothesized that exposure to MatSep during early life and LNAME during adulthood would induce increased levels of renal injury markers and immune cells when compared to normally‐reared rats with LNAME during adulthood. MatSep involves separating rat pups from the dam for 3hrs/day from postnatal day 2 (PD2) to PD14. Normally‐reared littermate rats were used as controls. Twelve weeks old adult (n=6–8rats/group) control and MatSep rats were given LNAME (100mg/kg/rat) in drinking water for 7 days. LNAME was adjusted daily based on the amount of water intake. We compared urinary renal damage markers and renal immune cell populations in control and MatSep rats after LNAME treatment. On day 7 after LNAME, urine was collected for assessment of glomerular (albumin and nephrin) and tubular (KIM1 and NGAL) injury markers by enzyme‐linked immunosorbent assay (ELISA). Our results showed that urinary KIM1 excretion was significantly lower in MatSep rats compared to control rats (control LNAME vs. MatSep LNAME: 6.78mg/day ± 0.32 vs. 5.53mg/day ± 0.36; p = 0.029). No statistical differences were observed in urinary NGAL, albumin and nephrin excretion. At day 7 after LNAME, blood and kidneys were used for determination of immune cell populations by flow cytometry. The proportion of renal total T cells, T helper cells, macrophages, neutrophils, natural killer, and dendritic cells were similar in control and MatSep rats. However, there was a trend towards an increase in the proportion of cytotoxic T cells in MatSep compared to control animals (%CD45+ cells; control LAME vs. MatSep LNAME: 14.04 ± 0.97 vs. 17.37 ± 1.35; p = 0.087). No differences were observed in the proportion of circulating immune T helper cells, cytotoxic T cells, macrophages, neutrophils, natural killer, and dendritic cells in control and MatSep rats. Our findings suggest that MatSep during early life may protect from tubular injury in response to inhibition of nitric oxide synthase. Future studies are warranted to determine whether the observed renal tubular protection is linked to LNAME induced blood pressure elevation, loss of tubular nitric oxide production, and T cell activation.Support or Funding InformationFunded by Cardiovascular Pathophysiology NIH/NHLBI T32HL007918‐19 to IEO, R01DK059600 to JFG, and P01HL69999 to JSP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduction in nitric oxide bioavailability shifts serum lipid content towards atherogenic lipoprotein in rats

Nitric oxide (NO) is major endothelial relaxing factor and reduction in its bioavailabilty has been linked to hypertension. Furthermore, high lipid content is a strong risk factor predisposing to cardiovascular diseases. The principal focus of this study was to investigate the effect of blockade of nitric oxide synthase (NOS) on serum lipid content in rats. Male Wistar rats (150–170 g, n = 15) were randomly divided into two groups designated control (n = 5), and L-Name group (n = 10) and were gavage with distilled water and 60 mg/kg of L-NAME respectively daily for three weeks. After 3 weeks, the L-NAME group was sub-divided into two sub-groups (n = 5 each): L-NAME (60 mg/kg of L-NAME), and L-NAME plus ramipril (LR) (60 mg/kg of L-NAME plus 20 mg/kg of ramipril) and were treated daily for another three weeks. The blood pressure (BP) of the conscious rats was measured by tail-cuff method at the onset, at the third and at the sixth weeks of the experiment; while serum lipid contents and NO were measured at the third and sixth weeks. At the end of the experiment blood sample was drawn by ocular puncture for evaluation of lipid profile and NO, and the animals were later euthanized by overdose of anaesthesia. Data were analyzed using ANOVA at p < 0.05. There was a significant increase in BP, triglyceride, total cholesterol, low density lipoprotein-cholesterol, and atherogenic indices in L-NAME group compared to the control and LR group (p < 0.05); NO and high density lipoprotein-cholesterol was significant lower in the L-NAME group compared to control and LR (p < 0.05). In conclusion, reduction in NO bioavailability alters lipid metabolism, which was rectified by ramipril.