Nitric Oxide Synthase Antagonist Research Articles

Solution-processed graphene films for electrochemical monitoring of extracellular nitric oxide released by breast cancer cells

Nitric oxide (NO) plays an important role in cardiovascular function, immune response, and intercellular signaling. However, due to its short lifetime, real-time detection of NO is challenging. Herein, an electrochemical sensor based on fibronectin-modified, solution-processed graphene ink for NO detection is developed using a facile fabrication method involving spin-coating and hot-plate annealing. The sensor is first electrochemically characterized with a NO donor, spermine NONOate, exhibiting a dynamic range of 10–1000 μM. The fibronectin-functionalized graphene supports the attachment and growth of MDA-MB-231 breast cancer cells, as confirmed by optical microscopy. Extracellular NO production is stimulated using the amino acid L-arginine. NO production results in morphological changes to the adhered cells, which are reversible upon the addition of the NO synthase antagonist Nω-nitro-L-arginine methyl ester. The production of NO is also confirmed using real-time amperometric measurements with the fibronectin-functionalized graphene sensors. While this work focuses on NO detection, this potentially scalable platform could be extended to other cell types with envisioned applications including the high-throughput evaluation of therapeutics and biocompatible coatings.

Perinatal N(G)-Nitro-L-arginine methyl ester administration decreases anxiety- and depression-like behaviors in adult mice.

We hypothesized that perinatal manipulations of the nitrergic system would affect adult animal behaviors. We tested this hypothesis by perinatally administering N(G)-Nitro-L-arginine methyl ester (L-NAME), a non-specific antagonist of nitric oxide synthase for 15 days and assessed anxiety- and depression-like behaviors in adult mice. At 70 days of age, the mice were subjected to a battery of tests consisting of the open-field, light/dark box, forced swim, and tail-flick tests. The tests were performed at two-day intervals, and the order of the tests within the battery was determined according to the progressive invasiveness degree. L-NAME-treated animals exhibited decreased anxiety-like behavior in the light/dark box and open field tests, with no change in locomotor activity. Additionally, they demonstrated decreased depression-like behavior in the forced swim test and no change in pain perception in the tail-flick test. The nitrergic system is possibly involved in neural circuitry development that regulates behaviors since blocking perinatal nitric oxide production decreases anxiety- and depression-like behaviors in adult mice.

Influence of formaldehyde exposure on the molecules of the NO/cGMP-cAMP signaling pathway in different brain regions of Balb/c mice.

This toxicology study was conducted to assess the impact of formaldehyde, a common air pollutant found in Chinese gymnasiums, on the brain function of athletes. In this research, a total of 24 Balb/c male mice of SPF-grade were divided into four groups, each consisting of six mice. The mice were exposed to formaldehyde at different concentrations, including 0 mg/m3, 0.5mg/m3, 3.0mg/m3, and 3.0mg/m3 in combination with an injection of L-NMMA (NG-monomethyl-L-arginine), which is a nitric oxide synthase antagonist. Following a one-week test period (8h per day, over 7days), measurements of biomarkers related to the nitric oxide (NO)/cGMP-cAMP signaling pathway were carried out on the experimental animals post-treatment. The study found that: (1) Exposure to formaldehyde can lead to brain cell apoptosis and neurotoxicity; (2) Additionally, formaldehyde exposure was found to alter the biomarkers of the NO/cGMP-cAMP signaling pathway, with some changes being statistically significant (p < 0.05 or p < 0.01); (3) The use of L-NMMA, an antagonist of the NO/cGMP-cAMP signaling pathway, was found to prevent these biomarker changes and had a protective effect on brain cells. The study suggests that the negative impact of formaldehyde on the brain function of mice is linked to the regulation of the NO/cGMP-cAMP signaling pathway.

Sulfasalazine for the treatment of preeclampsia in a nitric oxide synthase antagonist mouse model

IntroductionDevelopment of a therapeutic that targets the pathophysiological elements of preeclampsia would be a major advance for obstetrics, with potential to save the lives of countless mothers and babies. We recently identified anti-inflammatory drug sulfasalazine as a prospective candidate therapeutic for treatment of preeclampsia. In primary human cells and tissues in vitro, sulfasalazine potently decreased secretion of anti-angiogenic sFlt-1 and sENG, increased production of pro-angiogenic PlGF, mitigated endothelial dysfunction, and promoted whole vessel vasodilation. MethodsUsing nitric oxide synthase antagonist Nω-Nitro-l-arginine methyl ester hydrochloride, a preeclampsia-like phenotype was induced in pregnant mice, including high blood pressure, fetal growth restriction, and elevated circulating sFlt-1. Mice were treated with sulfasalazine or vehicle from gestational day (D)13.5, with blood pressure measurements across gestation, fetal measurements at D17.5, and wire myograph assessment of vasoactivity. ResultsSulfasalazine had a modest effect on blood pressure, decreasing diastolic and mean blood pressure on D13.5, but not later in gestation, or systolic blood pressure. Sulfasalazine was not able to rescue fetal growth, in male or female fetuses. There was a suggestion of improved vasoactivity with sulfasalazine, but further clarification is required. DiscussionIn this mouse model of preeclampsia, sulfasalazine did not sustain reductions in blood pressure nor affect fetal parameters of size and weight, both desirable attributes of a viable preeclampsia therapeutic. While these data suggest sulfasalazine might improve vasoactivity, murine toxicity considerations limited the dose range of sulfasalazine that could be tested in the current study.

Calcium transients in intramuscular interstitial cells of Cajal of the murine gastric fundus and their regulation by neuroeffector transmission.

Enteric neurotransmission is critical for coordinating motility throughout the gastrointestinal (GI) tract. However, there is considerable controversy regarding the cells that are responsible for the transduction of these neural inputs. In the present study, utilization of a cell-specific calcium biosensor GCaMP6f, the spontaneous activity and neuroeffector responses of intramuscular ICC (ICC-IM) to motor neural inputs was examined. Simultaneous intracellular microelectrode recordings and high-speed video-imaging during nerve stimulation was used to reveal the temporal relationship between changes in intracellular Ca2+ and post-junctional electrical responses to neural stimulation. ICC-IM were highly active, generating intracellular Ca2+ -transients that occurred stochastically, from multiple independent sites in single ICC-IM. Ca2+ -transients were not entrained in single ICC-IM or between neighbouring ICC-IM. Activation of enteric motor neurons produced a dominant inhibitory response that abolished Ca2+ -transients in ICC-IM. This inhibitory response was often preceded by a summation of Ca2+ -transients that led to a global rise in Ca2+ . Individual ICC-IM responded to nerve stimulation by a global rise in Ca2+ followed by inhibition of Ca2+ -transients. The inhibition of Ca2+ -transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in intracellular Ca2+ was inhibited by the muscarinic antagonist, atropine. Simultaneous intracellular microelectrode recordings with video-imaging revealed that the rise in Ca2+ was temporally associated with rapid excitatory junction potentials and the inhibition of Ca2+ -transients with inhibitory junction potentials. These data support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach. KEY POINTS: The cells responsible for mediating enteric neuroeffector transmission remain controversial. In the stomach intramuscular interstitial cells of Cajal (ICC-IM) were the first ICC reported to receive cholinergic and nitrergic neural inputs. Utilization of a cell specific calcium biosensor, GCaMP6f, the activity, and neuroeffector responses of ICC-IM were examined. ICC-IM were highly active, generating stochastic intracellular Ca2+ -transients. Stimulation of enteric motor nerves abolished Ca2+ -transients in ICC-IM. This inhibitory response was preceded by a global rise in intracellular Ca2+ . Individual ICC-IM responded to nerve stimulation with a rise in Ca2+ followed by inhibition of Ca2+ -transients. Inhibition of Ca2+ -transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in Ca2+ was inhibited by the muscarinic antagonist atropine. Simultaneous intracellular recordings with video imaging revealed that the global rise in intracellular Ca2+ and inhibition of Ca2+ -transients was temporally associated with rapid excitatory junction potentials followed by more sustained inhibitory junction potentials. The data presented support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach.

Acute systemic inhibition of inflammation augments endothelium-dependent dilation in women with a history of preeclamptic pregnancy.

Women who have had preeclampsia demonstrate microvascular endothelial-dysfunction, mediated in part by reduced nitric oxide (NO)-dependent dilation. Preeclamptic pregnancies are associated with elevated inflammation, and inhibition of inflammation attenuates endothelial damage in animal models of preeclampsia. However, it is unclear if inhibition of vascular inflammation improves endothelial function in women after a preeclamptic pregnancy. Using the cutaneous microcirculation as a model, we hypothesized that acute systemic inhibition of vascular inflammation (oral salsalate; 1500mg/twice daily, 4days) would improve endothelium- and NO-dependent vasodilation in women with a history of preeclampsia (PE) but not in women with a history of uncomplicated pregnancy (HC). Twelve HC (30±1yrs, 10±2months postpartum) and 10 PE (30±2yrs, 8±2months postpartum) participated in a double-blind placebo-controlled study. Following each treatment, 2 intradermal microdialysis fibers were placed in the skin of the ventral forearm for graded infusion of acetylcholine (Ach, 10-7-102mM) or Ach+15mM L-NAME (NO synthase antagonist). Red blood cell flux was measured over each site by laser-Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP+local heat 43°C). ACh-induced (77±3 vs. 92±3%CVCmax; p=0.01) and NO-dependent (20±6 vs. 33±4%; p=0.02) vasodilation were attenuated in PE compared to HC. Salsalate augmented ACh-induced (95±2%CVCmax; p=0.002) and NO-dependent (39±3%; p=0.009) dilation in PE compared to placebo but had no effect in HC (all p>0.05). Salsalate treatment augmented endothelium-dependent vasodilation via NO-mediated pathways in women who have had preeclampsia, suggesting that inflammatory signaling mediates persistent endothelial dysfunction following preeclampsia.

L–arginine alleviates postmenopausal complications in female rats by stimulating ovarian dopamine beta hydroxylase

Objective: To evaluate the levels of estrogen, albumin and gonadotropins (luteinizing hormone, follicle-stimulating hormone) as well as the activity of dopamine beta hydroxylase (DAß H) in aged female rats treated with nitric oxide precursor L-arginine and neuronal nitric oxide synthase antagonist L-NAME.Methods: A total of 224 Wistar rats (36 weeks old, weighing 250 g) based on a random sampling were divided into the control and experimental groups after Pap smear test. The control group received only saline (1 mL/kg) intraperitoneally (i.p.). The experiential groups were treated with L-arginine (5, 25 and 50 mg/kg, i.p.) and L-NAME (5 and 25 mg/kg, i.p.) for 3 to 21 days, once a day. Blood samples were taken from the rats and the levels of estrogen and albumin and gonadotropins in the serum were monitored by enzyme-linked immunosorbent assay kit, and the ovaries were examined immunohistopathologically for DAßH activity.Results: L-arginine (5 mg/kg) significantly increased estrogen level (P<0.05), which was associated with DAßH activation in the ovaries. L-NAME reduced this effect when administered prior to L-arginine dose. L-arginine caused no significant change in the levels of luteinizing hormone and follicle-stimulating hormone. Except for the lowest dose of L-arginine in the shortest period, albumin levels significantly decreased in other treatments compared to the control group (P<0.05).Conclusions: L-arginine is likely to reduce postmenopausal problems due to an increased nitric oxide level.

Vasorelaxant effects of benzodiazepines, non-benzodiazepine sedative-hypnotics, and tandospirone on isolated rat arteries

Benzodiazepines (BDZs) and non-BDZ sedative-hypnotics are effective for the management of chronic insomnia; however, they are associated with adverse effects such as headache, dizziness, and palpitations. Furthermore, long-term use of these medications is associated with decreased blood pressure (BP) or depressed baroreflex function. Therefore, here, we assessed whether BDZs and non-BDZs cause vasorelaxation directly. Vasorelaxation in response to 22 BDZs, 2 non-BDZs, and tandospirone was determined by myograph methods using isolated Wistar rat thoracic aortas. All the drugs relaxed phenylephrine-contracted rat aortas in a concentration-dependent manner. Zolpidem and tandospirone caused over 80% relaxation at a concentration of 10 μM; diazepam, estazolam, etizolam, and tofisopam caused 60–70% relaxation; whereas 18 other BDZs (alprazolam, bromazepam, brotizolam, chlordiazepoxide, clobazam, clonazepam, clorazepate, ethyl loflazepate, flunitrazepam, flurazepam, lorazepam, lormetazepam, midazolam, nimetazepam, nitrazepam, oxazepam, temazepam, and triazolam) and zaleplon caused less than 50% relaxation. The relaxation was partially but significantly inhibited to the same extent by a nitric oxide (NO) synthase antagonist and after endothelium removal. Binding assay of gamma-aminobutyric acid type A receptors was performed using [3H]flunitrazepam. No correlation was observed between vasorelaxation at a concentration of 10 μM and the binding affinities for 23 drugs. The study demonstrated that zaleplon, zolpidem, tandospirone, and many BDZs cause vasorelaxation to different extents via endothelial NO-dependent and endothelium-independent pathways. In conclusion, the direct vasodilatory effects of these drugs may be involved in the mechanisms underlying their adverse effects. Additionally, the decreased BP observed in persons who take BDZs or non-BDZs may be partly due to direct vasodilation.

Effect and mechanism of prophylactic use of tadalafil during pregnancy on l-NAME-induced preeclampsia-like rats

IntroductionPreeclampsia (PE) is a serious maternal inflammatory disease with endothelial cell dysfunction, and there is a lack of effective treatment and prevention. Tadalafil is considered to be a promising drug for PE. This study aimed to determine whether and how tadalafil use during early pregnancy alleviates PE induced by N-nitro-l-arginine-methyl-ester (l-NAME), an antagonist of nitric oxide synthase, in rats. MethodsTwenty-eight Sprague-Dawley (SD) rats were randomly divided into 4 equal groups on gestational day 0 (GD0): a pregnant control group, an l-NAME-treated PE group and two prophylactic low-dose and high-dose tadalafil groups. Blood pressure was measured on GD0, 5, 10, 15 and 20. Proteinuria was assessed on GD0 and 18. Femoral artery ultrasound was performed on GD19. Tissue sampling was performed on GD20. The perinatal outcomes, placenta and kidney tissue morphology, and endothelial and inflammatory markers were examined. ResultsProphylactic administration of low and high doses of tadalafil improved l-NAME induced hypertension, proteinuria, maternal weight loss during pregnancy, fetal growth restriction and flow-mediated dilatation, balanced endothelial-relative factors, and alleviated inflammation activation in placenta and kidney tissue. What's more, in some results, the HT group performed better than the LT group. DiscussionOur results indicate that prophylactic use of tadalafil in l-NAME-induced PE-like rat models alleviates PE symptoms, promotes fetal growth, protects endothelial function and reduces inflammation, suggesting that tadalafil may be a potential drug for the prevention of PE.

The Hypoxia Tolerance of the Goldfish (Carassius auratus) Heart: The NOS/NO System and Beyond.

The extraordinary capacity of the goldfish (Carassius auratus) to increase its cardiac performance under acute hypoxia is crucial in ensuring adequate oxygen supply to tissues and organs. However, the underlying physiological mechanisms are not yet completely elucidated. By employing an ex vivo working heart preparation, we observed that the time-dependent enhancement of contractility, distinctive of the hypoxic goldfish heart, is abolished by the Nitric Oxide Synthase (NOS) antagonist L-NMMA, the Nitric Oxide (NO) scavenger PTIO, as well as by the PI3-kinase (PI3-K) and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) pumps’ inhibition by Wortmannin and Thapsigargin, respectively. In goldfish hearts exposed to hypoxia, an ELISA test revealed no changes in cGMP levels, while Western Blotting analysis showed an enhanced expression of the phosphorylated protein kinase B (pAkt) and of the NADPH oxidase catalytic subunit Nox2 (gp91phox). A significant decrease of protein S-nitrosylation was observed by Biotin Switch assay in hypoxic hearts. Results suggest a role for a PI3-K/Akt-mediated activation of the NOS-dependent NO production, and SERCA2a pumps in the mechanisms conferring benefits to the goldfish heart under hypoxia. They also propose protein denitrosylation, and the possibility of nitration, as parallel intracellular events.

Acute Systemic Inhibition of Inflammation Augments Endothelium‐dependent Dilation in Women with a History of Preeclamptic Pregnancy

Women who have had preeclampsia demonstrate microvascular endothelial‐dysfunction, mediated in part by reduced nitric oxide (NO)‐dependent dilation. Preeclamptic pregnancies are associated with elevated inflammation, and inhibition of inflammation attenuates endothelial damage in animal models of preeclampsia. However, it is unclear if systemic inhibition of inflammation improves endothelial function in women after a preeclamptic pregnancy. Using the cutaneous microcirculation as a model, we hypothesized that acute systemic inhibition of inflammation (oral salsalate; 1500mg/twice daily/4 days) would improve endothelium‐ and NO‐dependent vasodilation in women with a history of preeclampsia (PrEC) but not in women with a history of uncomplicated pregnancy (HC). Eleven HC (30±1yrs, 9±2 months post‐partum) and 8 PrEC (29±3yrs, 6±1 months post‐partum) participated in a double‐blind placebo‐controlled study. Following each treatment, 2 intradermal microdialysis fibers were placed in the skin of the ventral forearm for graded infusion of acetylcholine (ACh, 10−7–102 mM) or ACh+15mM L‐NAME (NO synthase antagonist). Red cell flux was measured over each site by laser‐Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP + local heat 43°C). ACh‐induced (75±3 vs. 92±4%CVCmax; p=0.002) and NO‐dependent (18±8 vs. 32±4%; p=0.04) vasodilation were attenuated in PrEC compared to HC following placebo. Salsalate augmented ACh‐induced (92±3%CVCmax; p=0.001) and NO‐dependent (38±4%; p=0.04) dilation in PrEC compared to placebo but had no effect in HC (all p&gt;0.05). Women who have had preeclampsia demonstrate attenuated microvascular endothelial function compared to women with a history of uncomplicated pregnancy. Salsalate treatment augmented endothelium‐dependent vasodilation via NO‐mediated pathways in these women, suggesting that inflammatory signaling plays a role in mediating persistent endothelial‐dysfunction in women with a history of preeclampsia. ClinicalTrials.gov registration # NCT03482440Support or Funding InformationNIH K99 HL138133 (Stanhewicz); NIH R01 HL093238 (Alexander); NIH T32 AG049676 (Serviente)

Acanthopanax senticosus Induces Vasorelaxation via Endothelial Nitric Oxide-Dependent and -Independent Pathways.

Although Acanthopanax senticosus root extract (ASRE), a functional food used in Japan, improves peripheral blood circulation and exerts vasorelaxant effects in rats under healthy conditions, the underlying mechanisms currently remain unclear. Therefore, we investigated the mechanisms responsible for ASRE-induced relaxation in isolated thoracic aortas using organ bath techniques and examined whether ASRE affects systemic and peripheral circulation using a photoplethysmographic tail-cuff system and noncontact laser tissue blood flow meter in Wistar rats. Similar to acetylcholine (ACh), ASRE induced dose-dependent relaxation in aortas pre-contracted with phenylephrine; however, in contrast to ACh, ASRE-induced relaxation was partially inhibited by treatments with antagonists of nitric oxide (NO) synthase and soluble guanylyl cyclase as well as by endothelium removal. Contractile responses to phenylephrine or potassium chloride were observed in the presence of ASRE. The oral administration of ASRE (900 mg/kg/d for 1 wk) decreased systolic blood pressure in rats 3 h after the treatment and did not affect heart rate, tail blood flow, mass, or velocity; this decreasing effect was not observed on day 2. A 1-wk treatment with ASRE did not affect vasorelaxation in response to ASRE. These results demonstrate that ASRE induces vasorelaxation via endothelial NO production and an NO-independent pathway in rats. Based on these findings, positive impacts of ASRE on blood pressure and peripheral blood circulation cannot be expected under healthy conditions as the systemic effects of ASRE are temporary. Instead, caution is needed to prevent the occurrence of side effects (i.e., orthostatic dizziness) at the beginning of ASRE dosing.

L-NAME releases nitric oxide and potentiates subsequent nitroglycerin-mediated vasodilation

L-NG-Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS). Herein, we show that L-NAME slowly releases NO from its guanidino nitro group. Daily pretreatment of rats with L-NAME potentiated mesenteric vasodilation induced by nitrodilators such as nitroglycerin, but not by NO. Release of NO also occurred with the NOS-inactive enantiomer D-NAME, but not with L-arginine or another NOS inhibitor L-NMMA, consistent with the presence or absence of a nitro group in their structure and their nitrodilator-potentiating effects. Metabolic conversion of the nitro group to NO-related breakdown products was confirmed using isotopically-labeled L-NAME. Consistent with Fenton chemistry, transition metals and reactive oxygen species accelerated the release of NO from L-NAME. Both NO production from L-NAME and its nitrodilator-potentiating effects were augmented under inflammation. NO release by L-NAME can confound its intended NOS-inhibiting effects, possibly by contributing to a putative intracellular NO store in the vasculature.

Effect of Electroacupuncture of "Weizhong" (BL 40) and "Huantiao" (GB 30) on cAMP-PKA-CREB Signaling of Spinal Cord in Rats with Neuropathic Pain

We have demonstrated that electroacupuncture (EA) of "Weizhong" (BL 40) and "Huantiao" (GB 30) could evidently relieve mechanical hyperalgesia in spared nerve injury (SNI) rats. The present study was designed to observe the effect of EA on levels of cAMP, and protein kinase A (PKA) and cAMP response element binding protein (CREB) in the lumbar spinal cord of the same pain model rats, so as to explore its mechanisms underlying improvement of neuropathic pain. One hundred male SD rats were randomly divided into 5 groups: sham operation, model, EA, AP-5 and L-NAME groups (n＝20 in each group). The sham operation group underwent a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right BL 40 and GB 30 for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg•kg－1•d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase, NOS, 60 mg•kg－1•d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, the cAMP content of spinal cord (L 4－L 6) was determined by radioimmunoassay, and the expression of PKA, p-PKA and CREB proteins of spinal cord (L 4－L 6) was detected by Western blot. The content of cAMP and the expression levels of PKA, p-PKA and CREB were significantly higher in the model group than in the sham operation group (P<0.01), and considerably lower in the EA group than in the model group (P<0.01, P<0.05). Compared to the model group, the expression levels of PKA and p-PKA were significantly decreased in the AP-5 and L-NAME groups (P<0.01, P<0.05). Compared to the EA group, the content of cAMP and the expression levels of CREB were significantly higher (P<0.05), and the expression of p-PKA was significantly lower in the AP-5 and L-NAME groups (P<0.05)．. EA intervention-induced down-regulation of cAMP, PKA and CREB levels in the lumbar spinal cord may contribute to its analgesic effect in neuropathic pain rats, suggesting an involvement of reduction of cAMP/PKA/CREB signaling of spinal cord in EA analgesia.

Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton flower extract on isolated rat ileum contractions and the mechanisms of action

Azadirachta indica A. Juss var. siamensis Valeton or commonly known as Siamese neem is one of the most well-known plant in traditional Ayurvedic medicine. The aim of the present study was to investigate the relaxant effects of A. indica on isolated rat ileum contractions and its potential underlying mechanisms involved. The isometric contractions of ileum segments were investigated in organ baths for spontaneous activity and response to aqueous extract of Siamese neem flower (SNF). The spasmolytic action of the extract was also assessed on contraction induced by acetylcholine and high potassium. Our findings indicate that cumulative concentrations of SNF aqueous extract induced relaxant effect on spontaneous rat ileum contractions. The extract has also suppressed the cumulative concentration response curve for acetylcholine and pottasium ions-induced contraction. The presence and absence of propranol (antagonist of β-adrenergic receptor) and l-Name (antagonist of nitric oxide synthase) in SNF aqeous extract co-treatment demonstrated no significant different in term of contraction activity when compared to SNF extract treatment alone. The treatment of SNF extract caused a significant inhibition in tissue contraction stimulated by accumulation of calcium ions. Our results showed the relaxant effect of SNF aqueous extract on the isolated rat ileum. In short, the SNF aqueous extract exhibited an inhibitory effect on the spontaneous ileum contactions particularly on the contraction stimulated by acetylcholine and high potassium. The observed effect might acted through the modulation of calcium channels. This findings provide a pharmacological basis for the traditional use of SNF for the treatment of gastrointestinal spasms.

Local Angiotensin 1–7 Administration Augments Microvascular Endothelial Function in Women Who Have Had Preeclampsia

Women with a history of preeclampsia are at increased risk of developing cardiovascular disease. Despite remission of the clinical symptoms of preeclampsia postpartum, women who have had preeclampsia demonstrate microvascular endothelial dysfunction, mediated in part by increased sensitivity to angiotensin II. Angiotensin 1–7 (ang 1–7) is an endogenous inhibitor of the actions of angiotensin II and therefore a biologically plausible target for improving endothelial function in postpartum women who have had preeclampsia. Using the cutaneous microcirculation as a model, we hypothesized that women who have had preeclampsia (PreEC) would have attenuated endothelium‐dependent‐ and reduced nitric oxide (NO)‐dependent vasodilation compared to control women who had a normal pregnancy (HC). We further hypothesized that localized ang 1–7 administration would increase endothelium‐dependent vasodilation and NO‐dependent vasodilation in PreEC, but not HC. The subject sample included 8 PreEC (26±2 yrs of age; 6±2 months post‐partum) and 7 HC (32±2 yrs; 6±1 months post‐partum). Four intradermal microdialysis fibers were placed in the skin of the ventral forearm for the graded infusion of acetylcholine (ACh, 10−7 – 102 mM), ACh + 15 mM NG Nitro‐L‐arginine methyl ester (L‐NAME; nitric oxide synthase antagonist), ACh + 100μM ang 1–7, or ACh + 15 mM L‐NAME + 100μM ang 1–7. Red cell flux was measured over each site by laser‐Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/MAP) and normalized to the respective site‐specific maximum (%CVCmax; 28 mM SNP + local heating to 43°C). ACh‐induced vasodilation was attenuated in PreEC compared to HC (logEC50: −1.0±0.2 vs. −1.8±0.1; p=0.04). There was no difference between PreEC and HC when ACh was co‐infused with L‐NAME (PreEC logEC50: −0.9±0.3 vs. HC: −1.2±0.2 logEC50). ACh + Ang 1–7 co‐infusion augmented the vasodilation response in PreEC (logEC50: −1.9±0.1) compared to ACh alone (logEC50: −1.0±0.2) (p&lt;0.001) but had no effect in HC. Co‐infusion of ang1–7 + L‐NAME + ACh did not differ from ACh + L‐NAME in either group. Women who have had preeclampsia demonstrate attenuated endothelium‐dependent and NO‐dependent vasodilation compared to women who have had a healthy pregnancy. Ang 1–7 increased endothelium‐dependent vasodilation via NOS‐mediated pathways in women who have had preeclampsia, suggesting that ang 1–7 may be a viable therapeutic target for improved microvascular endothelial function in women who have had a preeclamptic pregnancy.Support or Funding InformationNIH F32 HL129677‐03 (Stanhewicz), R01 HL093238‐07 (Alexander)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Electroacupuncture on Intracellular Calcium Ion Concentration and Expression of Calcium/Calmodulin Dependent Protein Kinase Ⅱ in Lumbar Spinal Cord in Rats with Neuropathic Pain

To observe the changes of intracellular calcium ([Ca2＋]i) concentration and expression of calcium/calmodulin dependent protein kinaseⅡ (CaMKⅡ) in spinal dorsal horn neurons of spared nerve injury (SNI) rats, so as to explore its mechanisms underlying improvement of neuropathic pain. One hundred and ten SD rats were randomly divided into 5 groups: sham control, model, EA, AP-5 and L-NAME groups. The sham group underwent only a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right "Weizhong" (BL 40) and "Huantiao" (GB 30) for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg·kg－1·d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase [NOS], 60 mg·kg－1·d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, and the calcium fluorescence intensity (shown by Fluo-3/AM calcium fluorescence indicator) of the superficial layer of the lumbar spinal cord (L 4－L 6) was measured by immunohistochemical staining and the expression of spinal cord (L 4－L 6) CaMK Ⅱ protein was detected by Western blot (WB). After modeling, the mechanical pain threshold was significantly decreased on day 10 and 16 after operation in comparison with the sham operation group and baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA, AP-5 and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05), while the effect of EA was significantly superior to that of AP-5 and L-NAME groups (P<0.05), suggesting a reduction of EA analgesia after administration of AP-5 and L-NAME. The concentration of intracellular [Ca2＋]i was significantly higher in the model group than in the sham group, and considerably lower in the EA, AP-5 and L-NAME groups than in the model group (P<0.01, P<0.05). Moreover, the expression level of CaMKⅡ shown by WB and immunohistochemical staining was significantly higher in the model group than in the sham group (P<0.05) and obviously lower in the EA group (not the AP-5 and L-NAME groups) than in the model group on day 16 after the intervention (P<0.05). It suggests an involvement of glutamate NMDA receptor and NMDAR-NOS/NO signaling in the analgesic effect and CaMKⅡ expression down-regulation of EA. EA can ease pain in rats with neuropathic pain, which is closely related to its effect in reducing the calcium concentration and the expression of CaMKⅡ in the lumbar spinal cord, possibly mediated by glutamate NMDA receptor and NMDAR-NOS/NO signaling.

Enhanced Electrical Field Stimulated Nitrergic and Purinergic Vasoreactivity in Distal vs Proximal Internal Pudendal Arteries

BackgroundThe internal pudendal arteries (IPAs) supply blood to the penis and are highly susceptible to vascular remodeling in rodent models of diabetes, hypertension, aging, and chronic kidney disease, thus contributing to erectile dysfunction. Interestingly, vascular remodeling primarily occurs in the distal and not in the proximal IPA, suggesting distinct local physiologic signaling differences within the IPA. AimTo examine the role of purinergic signaling and neurotransmitter release by electrical field stimulation (EFS) in the regulation of proximal and distal IPA vascular tone. MethodsProximal and distal IPAs were mounted in wire myographs and vascular responses to phenylephrine, acetylcholine, and 2-(N,N-diethylamino)-diazenolate-2-oxide, diethyl-ammonium salt (DEA NONOate) were measured. EFS-mediated contraction and non-adrenergic non-cholinergic (NANC) relaxation were evaluated in the absence and presence of a nitric oxide synthase antagonist. Purinergic agonist and NANC relaxation responses were assessed in the presence and absence of P2X1 and P2Y1 antagonists. Protein expression of P2X1 and P2Y1 receptors was measured by western blot. Main Outcome MeasuresProximal and distal IPA contraction and relaxation were measured during increasing agonist administration and EFS in the presence and absence of antagonists. ResultsProximal and distal IPA concentration response curves to phenylephrine, acetylcholine, and DEA NONOate did no differ. Interestingly, distal IPA exhibited greater EFS-mediated contraction and NANC relaxation compared with proximal IPA. Nitric oxide synthase inhibition completely inhibited distal IPA NANC relaxation but did not affect proximal IPA relaxation. P2X1 or P2Y1 receptor antagonism during NANC relaxation increased distal IPA relaxation but decreased proximal IPA relaxation. Combined P2X1 and P2Y1 receptor antagonism had no effect on proximal IPA relaxation but significantly increased distal IPA NANC relaxation. Clinical TranslationUnderstanding neurovascular regulation of IPA vascular tone through nitrergic and purinergic mechanisms could yield new therapeutic targets to improve IPA blood flow and treat vasculogenic erectile dysfunction. Strengths and LimitationsThis study is the first to illustrate the differences in mechanisms responsible for regulating vascular tone in the proximal and distal IPAs. All presented findings are currently limited to ex vivo vascular function. ConclusionThe regulation of vascular tone differs regionally in the IPA. The distal IPA is controlled through neurotransmitter-mediated NO-dependent mechanisms and increased sensitivity to purinergic P2X1 and P2Y1 receptor inhibition.Odom MR, Pak ES, Brown DA, Hannan JL. Enhanced Electrical Field Stimulated Nitrergic and Purinergic Vasoreactivity in Distal vs Proximal Internal Pudendal Arteries. J Sex Med 2017;14:1285–1296.

Reactivation of Tert in the medial prefrontal cortex and hippocampus rescues aggression and depression of Tert(-/-) mice.

The role of telomerase reverse transcriptase (TERT) has been extensively investigated in the contexts of aging and cancer. Interestingly, Tert−/− mice exhibit additional but unexpected aggressive and depressive behaviors, implying the potential involvement of TERT function in mood control. Our conditional rescue experiments revealed that the depressive and aggressive behaviors of Tert−/− mice originate from Tert deficiency in two distinct brain structures. Reactivation of Tert in the hippocampus was sufficient to normalize the depressive but not the aggressive behaviors of Tert−/− mice. Conversely, re-expression of Tert in the medial prefrontal cortex (mPFC) reversed the aggressive but not the depressive behavior of Tert−/− mice. Mechanistically, decreased serotonergic signaling and increased nitric oxide (NO) transmission in the hippocampus transduced Tert deficiency into depression as evidenced by our observation that the infusion of a pharmacological agonist for serotonin receptor 1a (5-HTR1A) and a selective antagonist for neuronal NO synthase into the hippocampus successfully normalized the depressive behavior of Tert−/− mice. In addition, increased serotonergic transmission by the 5-HTR1A agonist in the mPFC was sufficient to rescue the aggressive behavior of Tert−/− mice. Thus, our studies revealed a novel function of TERT in the pathology of depression and aggression in a brain structure-specific manner, providing direct evidence for the contribution of TERT to emotional control.

Hemoglobin induced NO/cGMP suppression Deteriorate Microcirculation via Pericyte Phenotype Transformation after Subarachnoid Hemorrhage in Rats.

Subarachnoid hemorrhage (SAH) usually results from ruptured aneurysm, but how leaked hemoglobin regulates the microcirculation in the pathophysiology of early brain injury after SAH is still unclear. In the present study, we sought to investigate the role and possible mechanism of hemoglobin induced pericyte phenotype transformation in the regulation of microcirculation after SAH. Endovascular perforation SAH rat model, brain slices and cultured pericytes were used, and intervened with endothelial nitric oxide synthase (eNOS) antagonist L-NNA and its agonist scutellarin, hemoglobin, DETA/NO (nitric oxide(NO) donor), PITO (NO scavenger), 8-Br-cGMP (cGMP analog). We found modulating eNOS regulated pericyte α-SMA phenotype transformation, microcirculation, and neurological function in SAH rats. Modulating eNOS also affected eNOS expression, eNOS activity and NO availability after SAH. In addition, we showed hemoglobins penetrated into brain parenchyma after SAH. And hemoglobins significantly reduced the microvessel diameters at pericyte sites, due to the effects of hemoglobin inducing α-SMA expressions in cultured pericytes and brain slices via inhibiting NO/cGMP pathway. In conclusion, pericyte α-SMA phenotype mediates acute microvessel constriction after SAH possibly by hemoglobin suppressing NO/cGMP signaling pathway. Therefore, by targeting the eNOS and pericyte α-SMA phenotype, our present data may shed new light on the management of SAH patients.