Nitric Oxide-mediated Relaxation Research Articles

β3 Receptor Signaling in Pregnant Human Myometrium Suggests a Role for β3 Agonists as Tocolytics.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the β2 adrenergic receptor relax airway smooth muscle and are effective in the treatment of asthma led to the notion that β2 mimetics would prevent preterm birth by relaxing uterine smooth muscle. The activation of cAMP-dependent protein kinase by β2 receptors is unable to provide meaningful tocolysis. The failure of β2 agonists such as ritodrine and terbutaline to prevent preterm birth suggests that the regulation of uterine smooth muscle is disparate from that of airway. Other smooth muscle quiescent-mediating molecules, such as nitric oxide, relax vascular smooth muscle in a cGMP-protein kinase G-dependent manner; however, nitric oxide activation of protein kinase G fails to explain the relaxation of the myometrium to nitric oxide. Moreover, nitric oxide-mediated relaxation is blunted in preterm labor, and thus, for this reason and because of the fall in maternal blood pressure, nitric oxide cannot be employed as a tocolytic. The β3 adrenergic receptor-mediated relaxation of the human myometrium is claimed to be cAMP-dependent protein kinase-dependent. This is scientifically displeasing given the failure of β2 agonists as tocolytics and suggests a non-canonical signaling role for β3AR in myometrium. The addition of the β3 agonist mirabegron to pregnant human myometrial strips in the tissue bath relaxes oxytocin-induced contractions. Mirabegron stimulates nitric oxide production in myometrial microvascular endothelial cells, and the relaxation of uterine tissue in vitro is partially blocked by the addition of the endothelial nitric oxide synthase blocker Nω-Nitro-L-arginine. Recent data suggest that both endothelial and smooth muscle cells respond to β3 stimulation and contribute to relaxation through disparate signaling pathways. The repurposing of approved medications such as mirabegron (Mybetriq™) tested in human myometrium as uterine tocolytics can advance the prevention of preterm birth.

Influence of lauric acid on the relaxation of corpus cavernosum in streptozotocin-induced diabetic male Wistar rats

BackgroundDiabetes mellitus is a global health challenge and has been recognised as a risk factor for erectile dysfunction. Dissatisfaction with standard medications has been reported by some patients, hence therapeutic plants are being considered as a viable alternative therapy, with their active components being investigated to create a standard regimen. Lauric acid is the most abundant constituent of coconut oil and is proposed to be responsible for its therapeutic properties. The corpus cavernosum plays an important role in erectile function with its relaxation favouring erection. This study thus sought to investigate the possible relaxant action and mechanism of lauric acid on the corpus cavernosum of diabetic male Wistar rats. Diabetes was induced by intraperitoneal injection of streptozotocin after which graded doses of lauric acid were administered orally to three groups of diabetic rats, once daily for 4 weeks. At the end of the experiment, the corpus cavernosal tissues of the rat penis were extracted. Following phenylephrine or potassium chloride (KCl)—induced contraction, relaxation response to acetylcholine and sodium nitroprusside was used to evaluate endothelium-dependent and nitric oxide-mediated relaxation, respectively.ResultsRelaxation response to acetylcholine, following pre-contraction with phenylephrine, was significantly decreased in the cavernosal tissues of diabetic untreated rats and was not significantly improved in lauric acid treated diabetic groups. Relaxation response to acetylcholine, following pre-contraction with KCl, was significantly decreased in the diabetic untreated group but was significantly improved in lauric acid treated diabetic groups at the lowest dose. Decreased relaxation response to sodium nitroprusside, following pre-contraction with phenylephrine in tissues of diabetic untreated rats, was significantly improved in lauric acid-treated diabetic groups at lower doses. Decreased relaxation response to sodium nitroprusside, following pre-contraction with KCl, was significantly improved in lauric acid-treated diabetic groups at all doses.ConclusionLauric acid improved relaxation of corpus cavernosum muscle in diabetic male rats by enhancing nitric oxide-mediated relaxing action of sodium nitroprusside and possibly inhibiting KCl-induced contraction.

Carbonic anhydrase inhibition improves pulmonary artery reactivity and nitric oxide-mediated relaxation in sugen-hypoxia model of pulmonary hypertension.

Pulmonary hypertension (PH) is a serious disease with pulmonary arterial fibrotic remodeling and limited responsiveness to vasodilators. Our data suggest that mild acidosis induced by carbonic anhydrase inhibition could ameliorate PH, but the vascular mechanisms are unclear. We tested the hypothesis that carbonic anhydrase inhibition ameliorates PH by improving pulmonary vascular reactivity and relaxation mechanisms. Male Sprague-Dawley rats were either control normoxic (Nx), or injected with Sugen 5416 (20 mg/kg, sc) and subjected to hypoxia (9% O2) (Su+Hx), or Su+Hx treated with acetazolamide (ACTZ, 100 mg/kg/day, in drinking water). After measuring the hemodynamics, right ventricular hypertrophy was assessed by Fulton's Index; vascular function was measured in pulmonary artery, aorta, and mesenteric arteries; and pulmonary arteriolar remodeling was assessed in lung sections. Right ventricular systolic pressure and Fulton's Index were increased in Su+Hx and reduced in Su+Hx + ACTZ rats. Pulmonary artery contraction to KCl and phenylephrine were reduced in Su+Hx and improved in Su+Hx + ACTZ. Acetylcholine (ACh)-induced relaxation and nitrate/nitrite production were reduced in pulmonary artery of Su+Hx and improved in Su+Hx + ACTZ. ACh relaxation was blocked by nitric oxide (NO) synthase and guanylate cyclase inhibitors, supporting a role of NO-cGMP. Sodium nitroprusside (SNP)-induced relaxation was reduced in pulmonary artery of Su+Hx, and ACTZ enhanced relaxation to SNP. Contraction/relaxation were not different in aorta or mesenteric arteries of all groups. Pulmonary arterioles showed wall thickening in Su+Hx that was ameliorated in Su+Hx + ACTZ. Thus, amelioration of pulmonary hemodynamics during carbonic anhydrase inhibition involves improved pulmonary artery reactivity and NO-mediated relaxation and may enhance responsiveness to vasodilator therapies in PH.

Experimental Periodontal Disease Triggers Coronary Endothelial Dysfunction in Middle-Aged Rats: Preventive Effect of a Prebiotic β-Glucan.

This study was aimed to verify the hypothesis that periodontal disease contributes to endothelial dysfunction in the coronary arteries of middle-aged rats. Besides we evaluated the effects of a prebiotic (β-glucan isolated from Saccharomyces cerevisiae) in preventing vascular dysfunction. The sample comprised young (sham and induced to periodontal disease) and middle-aged rats (sham, periodontal disease, sham-treated and periodontal disease-treated), at 12 and 57 weeks, respectively. The treated-groups received daily doses of β-glucan (50 mg/kg) orally (gavage) for 4 weeks, and periodontal disease was induced in the last 2 weeks by ligature. A myograph system assessed vascular reactivity. The expression of endothelial nitric oxide synthase (eNOS), cyclooxygenase 1 (COX-1), COX-2, p47phox, gp91phox, NF-KB p65, p53, p21, and p16 was quantified by western blotting. Serum hydroperoxide production was measured by the ferrous oxidation-xylenol orange (FOX-2) assay method. Interleukin-1 beta (IL-1β), IL-10, and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. Periodontal disease in middle-aged rats was associated with reduced acetylcholine-induced relaxations of coronary artery rings affecting the endothelium-dependent hyperpolarization- and the nitric oxide-mediated relaxations. The endothelial dysfunction was related to eNOS downregulation, pronounced impairment of the EDH-mediated relaxation, increased IL-1β and TNF-α proinflammatory cytokines, and also upregulation of NADPH oxidase and COXs, starting accumulate aging markers such as p53/p21 and the p16. Treatment with β-glucan effectively reduced bone loss in periodontal disease and delayed endothelial dysfunction in the coronary artery. Our data show that yeast β-glucan ingestion prevented oxidative stress and synthesis of proinflammatory marker and prevented eNOS reduction induced by periodontal disease in middle-aged rats. These results suggest that β-glucan has a beneficial effect on the coronary vascular bed.

Novel Drug Discovery Pathways in the Myometrium for the Treatment of Preterm Labor

IntroductionApproximately 10% of all US births occur preterm (<37 weeks). Half of preterm births are the result of spontaneous preterm labor (sPTL), which is idiopathic in nature. We are developing novel therapeutics that build upon our previous findings that nitric oxide‐mediated relaxation of the myometrium is independent of cGMP accumulation and occurs via protein S‐nitrosations (S‐NO). An important mediator of protein S‐nitrosation is the availability of S‐nitrosoglutathione (GSNO), an endogenous NO donor. Nitric oxide synthase (NOS) and GSNO reductase (GSNOR) alter GSNO availability in smooth muscle. We have previously determined that GSNOR is upregulated in preterm laboring myometrium, and we hypothesize that increasing endogenous NO availability will restore the quiescent phenotype in sPTL tissue. Here we describe that GSNOR inhibition (SPL‐334, N6022) and β3AR activation (mirabegron, nebivolol), both of which increase endogenous NO levels in the cell, exhibit tocolytic effects on human myometrium. These findings are significant because currently available tocolytics are ineffective at delaying birth beyond 48‐hours, and they are innovative as they constitute a novel approach to preventing preterm birth.MethodsStrips of (~0.5x15mM) were attached to a force transducer and isometrically stretched in an organ bath containing Krebs buffer. Tissues were maintained at 37°C and gently bubbled with balanced oxygen. Tissues were then challenged with KCl (60mM replacing NaCl) and oxytocin (8nM), followed by washout, then treated with either mirabegron (100μM), nebivolol (100μM), N6022 (100μm) or SPL‐334 (10μM). Data were analyzed with LabChart (version 8.1.13). Western blots were run on a 10–15% polyacrylamide gel with 30μg of total protein and using a student’s t‐test (sig. p<0.05).ResultsBoth GSNOR inhibition (SPL‐334 or N6022), and β3AR activation (mirabegron or nebivolol), exhibit negative inotropic effect on human myometrium. SPL334 decreased ‘area under the curve’ by 21.6% (p<0.001) and reduced the number of ‘contractions per unit time’ by 20.3%. Similarly, mirabegron decreased peak force by 18.2% (p=0.0113). The combined administration of N6022 and nebivolol had a synergistic effect that all but abolished contractile force (92% decrease in peak force), as compared to their individual effects (N6022 40.8%, nebivolol 86.7% decrease in peak force). Of note, β3ARs are expressed in both endothelium and uterine smooth muscle, and we found that both β3AR and eNOS are downregulated in PTL (n=6 p=0.0317, p=0.0012) as compared to other pregnancy states (n=12 each: TL, TNL, PTNL), indicating a potential role for therapies that target these pathways.ConclusionThe importance of protein S‐nitrosation in the myometrium, combined with the known dysregulation of NO metabolism in sPTL tissue, have led us to seek novel therapeutics (tocolytics) that will mitigate preterm labor by enhancing endogenous NO availability (and SNOs) in the myometrium. SPL‐334 and N6022 increase NO through inhibition of GSNOR, and mirabegron and nebivolol activate β3ARs on both uterine smooth muscle and endothelium, further bolstering relaxation pathway activity.Support or Funding InformationR01 HD091114 (Buxton) 9/15/18‐9/14/22

The inflammasome NLRP3 plays a dual role on mouse corpora cavernosa relaxation

NLRP3 plays a role in vascular diseases. Corpora cavernosa (CC) is an extension of the vasculature. We hypothesize that NLRP3 plays a deleterious role in CC relaxation. Male C57BL/6 (WT) and NLRP3 deficient (NLRP3−/−) mice were used. Intracavernosal pressure (ICP/MAP) measurement was performed. Functional responses were obtained from CC strips of WT and NLRP3−/− mice before and after MCC950 (NLRP3 inhibitor) or LPS + ATP (NLRP3 stimulation). NLRP3, caspase-1, IL-1β, eNOS, nNOS, guanylyl cyclase-β1 (GCβ1) and PKG1 protein expressions were determined. ICP/MAP and sodium nitroprusside (SNP)-induced relaxation in CC were decreased in NLRP3−/− mice. Caspase-1, IL-1β and eNOS activity were increased, but PKG1 was reduced in CC of NLRP3−/−. MCC950 decreased non-adrenergic non-cholinergic (NANC), acetylcholine (ACh), and SNP-induced relaxation in WT mice. MCC950 did not alter NLRP3, caspase-1 and IL-1β, but reduced GCβ1 expression. Although LPS + ATP decreased ACh- and SNP-, it increased NANC-induced relaxation in CC from WT, but not from NLRP3−/− mice. LPS + ATP increased NLRP3, caspase-1 and interleukin-1β (IL-1β). Conversely, it reduced eNOS activity and GCβ1 expression. NLRP3 plays a dual role in CC relaxation, with its inhibition leading to impairment of nitric oxide-mediated relaxation, while its activation by LPS + ATP causes decreased CC sensitivity to NO and endothelium-dependent relaxation.

Downregulation of endothelial transient receptor potential vanilloid type 4 channel underlines impaired endothelial nitric oxide-mediated relaxation in the mesenteric arteries of hypertensive rats.

The endothelium contributes to the maintenance of vasodilator tone by releasing endothelium-derived relaxing factors, including nitric oxide (NO). In hypertension, endothelial nitric oxide synthase (eNOS) produces less NO and could be one of the contributing factors to the increased peripheral vascular resistance. Agonist-induced Ca(2+) entry is essential for the activation of eNOS. The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca(2+)-permeant cation channel, is expressed in the endothelial cells and involved in the regulation of vascular tone. The present study aimed to investigate the role of TRPV4 channel in endothelium-dependent NO-mediated relaxation of the resistance artery in hypertensive rats. Using a wire myograph, relaxation response to the TRPV4 activator, 4alpha-phorbol-12,13-didecanoate (4alphaPDD) was assessed in mesenteric arteries obtained from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). Compared to WKY, SHR demonstrated a significantly attenuated 4alphaPDD-induced endothelium-dependent NO-mediated relaxation. Immunohistochemical analysis revealed positive staining for TRPV4 in the endothelium of mesenteric artery sections in both WKY and SHR. Furthermore, TRPV4 mRNA and protein expressions in SHR were significantly lower than their expression levels in WKY rats. We conclude that 4alphaPDD-induced endothelium-dependent NO-mediated vasorelaxation is reduced in SHR and downregulation of TRPV4 could be one of the contributing mechanisms.

Embryogenesis of the peristaltic reflex.

Neurogenic gut movements start after longitudinal smooth muscle differentiation in three species (mouse, zebrafish, chicken), and at E16 in the chicken embryo. The first activity of the chicken enteric nervous system is dominated by inhibitory neurons. The embryonic enteric nervous system electromechanically couples circular and longitudinal spontaneous myogenic contractions, thereby producing a new, rostro-caudally directed bolus transport pattern: the migrating motor complex. The response of the embryonic gut to mechanical stimulation evolves from a symmetric, myogenic response at E12, to a neurally mediated, polarized, descending inhibitory, 'law of the intestine'-like response at E16. High resolution, whole-mount 3D reconstructions are presented of the enteric nervous system of the chicken embryo at the neural-control stage E16 with the iDISCO+ tissue clarification technique. Gut motility is a complex transport phenomenon involving smooth muscle, enteric neurons, glia and interstitial cells of Cajal. Because these different cells differentiate and become active at different times during embryo development, studying the ontogenesis of motility offers a unique opportunity to 'time-reverse-engineer' the peristaltic reflex. Working on chicken embryo intestinal explants in vitro, we found by spatio-temporal mapping and signal processing of diameter and position changes that motility follows a characteristic sequence of increasing complexity: (1) myogenic circular smooth muscle contractions from E6 to E12 that propagate as waves along the intestine, (2) overlapping and independent, myogenic, low-frequency, bulk longitudinal smooth muscle contractions around E14, and (3) tetrodotoxin-sensitive coupling of longitudinal and circular contractions by the enteric nervous system as from E16. Inhibition of nitric oxide synthase neurons shows that the coupling consists in nitric oxide-mediated relaxation of circular smooth muscle when the longitudinal muscle layer is contracted. This mechanosensitive coupling gives rise to a directional, cyclical, propagating bolus transport pattern: the migrating motor complex. We further reveal a transition to a polarized, descending, inhibitory reflex response to mechanical stimulation after neuronal activity sets in at E16. This asymmetric response is the elementary mechanism responsible for peristaltic transport. We finally present unique high-resolution 3D reconstructions of the chicken enteric nervous system at the neural-control stage based on confocal imaging of iDISCO+ clarified tissues. Our study shows that the enteric nervous system gives rise to new peristaltic transport patterns during development by coupling spontaneous circular and longitudinal smooth muscle contraction waves.

Chronic Intermittent Hypoxia Alters Rat Ophthalmic Artery Reactivity Through Oxidative Stress, Endothelin and Endothelium-Derived Hyperpolarizing Pathways.

Obstructive sleep apnea recently has been associated with a higher frequency of ischemic optic neuropathies. Intermittent hypoxia (IH) has been proposed as a major component of obstructive sleep apnea cardiovascular consequences. However, there currently are no pathophysiologic data regarding the effect of IH on the ocular vascular system. Thus, we assessed the impact of chronic IH exposure on the morphology and vascular reactivity of the rat ophthalmic artery (OA). Rats were exposed to 14 days of IH or normoxia (NX). Ophthalmic artery reactivity was studied using wire myography in rats treated or not with tempol (1 mM/day). Expression of endothelin-1 (ET-1) and its receptors, and of the three nitric oxide synthase (NOS) isoform genes was quantified using quantitative polymerase chain reaction (qPCR) in the retina and optic nerve. Structural alterations (optical and electron microscopy) and superoxide anion production were studied in OA sections. Superoxide ion expression in the OA wall was increased by 23% after IH exposure. Ophthalmic artery contractile response to 3.10-8 M ET-1 was increased by 18.6% and nitric oxide-mediated relaxation was significantly delayed in IH compared to NX rats. In the absence of nitric oxide, cytochrome P450 blockade increased relaxation to acetylcholine in IH rats and delayed it in NX rats. Tempol treatment abolished the IH-induced changes in OA reactivity. These results strongly suggest that chronic IH induces oxidative stress in the rat OA, associated with endothelial dysfunction through alterations of nitric oxide and endothelium-derived hyperpolarising factors (EDHF) pathways.

β-adrenergic Receptor Blocker ICI 118,551 Selectively Increases Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa )-Mediated Relaxations in Rat Main Mesenteric Artery.

Endothelial IKCa and/or SKCa channels play an important role in the control of vascular tone by participating in endothelium-dependent relaxation. Whether β-AR antagonists, mainly used in hypertension, affect endothelial KCa channel function is unknown. In this study, we examined the effect of the β2-AR antagonist and inverse agonist ICI 118,551 on the IKCa /SKCa channel activity by assessing functional relaxation responses to several agonists that stimulate these channels. Mesenteric arterial rings isolated from male Sprague Dawley mounted to organ baths. Acetylcholine elicited IKCa - and SKCa -mediated relaxations that were abolished by TRAM-34 and apamin, respectively. ICI 118,551, which did not dilate the arteries per se, increased the IKCa -mediated relaxations, whereas SKCa -mediated relaxations remained unaltered. Same potentiating effect was also detected on the IKCa -mediated relaxations to carbachol and A23187, but not to NS309. Neither acetylcholine-induced nitric oxide-mediated relaxations nor SNP relaxations changed with ICI 118,551. The PKA inhibitor KT-5720, the selective β2-AR agonist salbutamol, the selective β2-AR antagonist butoxamine, the non-selective β-AR antagonist propranolol, and the inverse agonists carvedilol or nadolol failed to affect the IKCa -mediated relaxations. ICI 118,551-induced increase was not reversed by salbutamol or propranolol as well. Besides, low potassium-induced relaxations in endothelium-removed arteries remained the same in the presence of ICI 118,551. These data demonstrate a previously unrecognized action of ICI 118,551, the ability to potentiate endothelial IKCa channel-mediated vasodilation, through a mechanism independent of β2-AR antagonistic or inverse agonistic action. Instead, the enhancement of acetylcholine relaxation seems likely to occur by a mechanism secondary to endothelial calcium increase.

Impaired Pulmonary Arterial Vasoconstriction and Nitric Oxide-Mediated Relaxation Underlie Severe Pulmonary Hypertension in the Sugen-Hypoxia Rat Model.

Pulmonary vasoreactivity could determine the responsiveness to vasodilators and, in turn, the prognosis of pulmonary hypertension (PH). We hypothesized that pulmonary vasoreactivity is impaired, and we examined the underlying mechanisms in the Sugen-hypoxia rat model of severe PH. Male Sprague-Dawley rats were injected with Sugen (20 mg/kg s.c.) and exposed to hypoxia (9% O2) for 3 weeks, followed by 4 weeks in normoxia (Su/Hx), or treated with Sugen alone (Su) or hypoxia alone (Hx) or neither (Nx). After hemodynamic measurements, the heart was assessed for right ventricular hypertrophy (Fulton's index); the pulmonary artery, aorta, and mesenteric arteries were isolated for vascular function studies; and contractile markers were measured in pulmonary arteries using quantitative polymerase chain reaction (PCR). Other rats were used for morphometric analysis of pulmonary vascular remodeling. Right ventricular systolic pressure and Fulton's index were higher in Su/Hx versus Su, Hx, and Nx rats. Pulmonary vascular remodeling was more prominent in Su/Hx versus Nx rats. In pulmonary artery rings, contraction to high KCl (96 mM) was less in Su/Hx versus Nx and Su, and phenylephrine-induced contraction was reduced in Su/Hx versus Nx, Hx, and Su. Acetylcholine (ACh)-induced relaxation was less in Su/Hx versus Nx and Hx, suggesting reduced endothelium-dependent vasodilation. ACh relaxation was inhibited by nitric oxide synthase (NOS) and guanylate cyclase blockade in all groups, suggesting a role of the NO-cGMP pathway. Nitrate/nitrite production in response to ACh was less in Su/Hx versus Nx, supporting reduced endothelial NO production. Sodium nitroprusside (10-8 M) caused less relaxation in Su/Hx versus Nx, Hx, and Su, suggesting a decreased responsiveness of vascular smooth muscle (VSM) to vasodilators. Neither contraction nor relaxation differed in the aorta or mesenteric arteries of all groups. PCR analysis showed decreased expression of contractile markers in pulmonary artery of Su/Hx versus Nx. The reduced responsiveness to vasoconstrictors and NO-mediated vasodilation in the pulmonary, but not systemic, vessels may be an underlying mechanism of severe PH in Su/Hx rats and appears to involve attenuation of the NO relaxation pathway and a switch of pulmonary VSM cells to a synthetic less reactive phenotype.

Erectile Dysfunction in Wistar Audiogenic Rats Is Associated With Increased Cavernosal Contraction and Decreased Neuronal Nitric Oxide Synthase Protein Expression

To test the hypothesis that naive Wistar audiogenic rats (WARs) display erectile dysfunction (ED), which is associated with increased sympathetic-mediated contractile tone and decreased nitric oxide-mediated relaxation responses of the cavernous tissue. Changes in the ratio of the maximal intracavernosal pressure-mean arterial pressure after the electrical stimulation of the right major pelvic ganglion were determined in vivo. Cavernosal contractility was induced by electrical field stimulation and phenylephrine. In addition, nonadrenergic-noncholinergic (NANC)-induced relaxation was determined. Rho-kinase (ROCK) pathway proteins, neuronal nitric oxide synthase (nNOS) protein expression, and endothelial nitric oxide synthase (eNOS) and extracellular signal-regulated kinase 1/2 activities were determined by Western blot. WARs display a significant decrease in maximal intracavernosal pressure-mean arterial pressure responses suggesting ED in this strain. Sympathetic-mediated contractile responses were increased in WARs and contractile responses to phenylephrine were not changed. The increased sympathetic-mediated contractile responses were not associated with changes in the ROCK pathway. On the other hand, NANC-mediated relaxation responses were significantly reduced in WARs. This functional response was accompanied by decreased nNOS and total eNOS protein expressions, augmented phosphorylated eNOS, and decreased extracellular signal-regulated kinase 1/2 phosphorylation levels. Our data have demonstrated that naive WARs display ED in vivo that is associated with increased sympathetic-mediated contractile responses and decreased NANC-mediated relaxation responses. The increase in contractile responses is independent of the ROCK pathway, and the changes in relaxation responses are associated with a decrease in nNOS protein expression, which may activate compensatory mechanisms in the cavernous tissue.

AB302. SPR-29 Sphingosine-1-phosphate in vitro and in vivo modulates corpus cavernosum smooth muscle tone

ObjectiveThe bioactive lipid sphingosine-1-phosphate (S1P) regulates smooth muscle (SM) contractility predominantly via three G protein-coupled receptors. The S1P1 receptor is associated with nitric oxide-mediated SM relaxation while S1P2 & S1P3 receptors are linked to SM contraction via activation of the Rho-kinase (ROK) pathway. The objective of this study was to determine the role of S1P in the modulation of corpus cavernosum (CC) SM (CCSM) tone.MethodsHuman and rat samples were used. Plasma S1P levels were detected by high-performance liquid chromatography. The expression of S1P1-3 receptors and sphingosine kinase-1 (SphK1) was determined by real-time RT-PCR and western blot. In vitro organ bath contractility and in vivo intracavernous pressure (ICP) measurements were performed. Results were expressed as mean ± SEM for n experiments. Statistical analysis was performed using either the Student’s t-test (when two sample treatments were being compared) or using ANOVA when multiple means were compared. P<0.05 was considered significant.ResultsPlasma S1P levels were determined to be ~200 nanomolar. Human and rat CC both express SphK1 and all S1P1-3 receptors. Exogenous S1P and the S1P receptor agonist FTY720-P contracted while antagonist JTE-013 relaxed CCSM in vitro. Meanwhile, force produced by S1P and agonists could be totally reversed by ROK inhibitor. Also, intracavernous injection of FTY720-P inhibited ICP rise induced by submaximal electrical stimulation of cavernous nerve, while JTE-013 alone induced ICP increase in vivo. Finally, SphK1 siRNA knocked down rat CC SphK1 by 80% and ICP were significantly potentiated.ConclusionsIn conclusion, we provide novel data that S1P, possibly coupling S1P2 and S1P3 receptors via RhoA/ROK pathway, mediates CCSM in vitro and in vivo. Antagonizing S1P or its receptors induces CCSM relaxation and proerectile effects. Thus, we provide the first clear evidences that the S1P system is another key contractile regulatory system in CC and thus this pathway is a potential therapeutic target for the treatment of priapism and erectile dysfunction.Funding Source(s)NIH R01 DK077116 to ME DiSanto

Adipocytokine, progranulin, augments acetylcholine-induced nitric oxide-mediated relaxation through the increases of cGMP production in rat isolated mesenteric artery.

Progranulin (PGRN) is a novel adipocytokine with anti-inflammatory effects in vascular cells. The aim of this study was to clarify the effects of PGRN on reactivity of isolated blood vessel. Isometric contraction of rat isolated superior mesenteric artery was measured. Pre-treatment with PGRN (10-100ngmL-1 , 30min) had no effect on noradrenaline- or 5-hydroxytriptamine-induced contraction. On the other hand, pre-treatment with PGRN (100ngmL-1 ) augmented acetylcholine (ACh; 30nm)-induced endothelium-dependent relaxation. Pre-treatment with PGRN (100ngmL-1 ) augmented ACh (10μm)-induced nitric oxide (NO)-mediated relaxation in the presence of indomethacin (10μm), a cyclooxygenase inhibitor, and tetraethyl ammonium (10mm), a non-selective potassium channel blocker. In contrast, pre-treatment with PGRN (100ngmL-1 ) had no effect on ACh-induced endothelium-derived hyperpolarizing factor-mediated relaxation. Pre-treatment with PGRN (100ngmL-1 ) had no effect on ACh (10μm, 1min)-induced endothelial NO synthase phosphorylation (at Ser1177) as determined by Western blotting. Pre-treatment with PGRN (100ngmL-1 ) augmented an NO donor, sodium nitroprusside (SNP; 30nm-1μm)- but not a membrane-permeable cGMP analogue, 8-bromo-cGMP-induced relaxation. In the presence of 3-isobutyl-1-methylxanthine (100μm), a phosphodiesterase inhibitor, pre-treatment with PGRN (100ngmL-1 ) increased SNP (30nm, 5min)-induced cGMP production as determined by enzyme immunoassay. We for the first time demonstrate that PGRN augments ACh-induced NO-mediated relaxation through the increases of cGMP production in smooth muscle. These results indicate PGRN as a possible pharmacotherapeutic target against cardiovascular diseases including obesity-related hypertension.

Reduced nitric oxide-mediated relaxation and endothelial nitric oxide synthase expression in the tail arteries of streptozotocin-induced diabetic rats

Diabetes is associated with endothelial dysfunction, which is characterized by impaired endothelium-dependent relaxations. The present study aimed to examine the role of nitric oxide (NO), prostacyclin and endothelium-dependent hyperpolarization (EDH), in the relaxation of ventral tail arteries of rats under diabetic conditions. Relaxations of tail arteries of control and diabetic rats were studied in wire myograph. Western blotting and immunostaining were used to determine the presence of proteins. Acetylcholine-induced relaxations were significantly smaller in arteries of diabetic compared to control rats (Rmax; 70.81±2.48% versus 85.05±3.15%). Incubation with the combination of non-selective cyclooxygenase (COX) inhibitor, indomethacin and potassium channel blockers, TRAM 34 and UCL 1684, demonstrated that NO-mediated relaxation was attenuated significantly in diabetic compared to control rats (Rmax; 48.47±5.84% versus 68.39±6.34%). EDH-type (in the presence of indomethacin and NO synthase inhibitor, LNAME) and prostacyclin-mediated (in the presence of LNAME plus TRAM 34 and UCL 1684) relaxations were not significantly reduced in arteries of diabetic compared to control rats [Rmax: (EDH; 17.81±6.74% versus 34.16±4.59%) (prostacyclin; 15.85±3.27% versus 17.23±3.75%)]. Endothelium-independent relaxations to sodium nitroprusside, salbutamol and prostacyclin were comparable in the two types of preparations. Western blotting and immunostaining indicated that diabetes diminished the expression of endothelial NO synthase (eNOS), while increasing those of COX-1 and COX-2. Thus, since acetylcholine-induced NO-mediated relaxation was impaired in diabetes because of reduced eNOS protein expression, pharmacological intervention improving NO bioavailability could be useful in the management of diabetic endothelial dysfunction.

The ability of asymmetric dimethylarginine (ADMA) or monomethylarginine (L-NMMA) to block endothelium-dependent, nitric oxide-mediated relaxation in rat aorta is inversely related to the efficacy of the relaxant stimulus

Previous work on rat aorta has shown that L-NMMA and ADMA each enhance vasoconstrictor-induced tone, consistent with blockade of basal nitric oxide activity, whereas they exert little inhibitory effect on acetylcholine-induced relaxation when tone is matched carefully to that of control tissues. The aim of this study was to determine if the ability of L-NMMA or ADMA to inhibit nitric oxide-mediated relaxation was critically determined by the efficacy of the relaxant stimulus. The effects of L-NMMA or ADMA were examined on relaxation to a range of agonists producing different maximal responses, namely, acetylcholine, the muscarinic partial agonist, butyrylcholine, and calcitonin gene-related peptide-1 (CGRP-1). The effects of L-NMMA or ADMA were also examined on relaxation to acetylcholine when its apparent efficacy at the M3 muscarinic receptor was reduced using the irreversible receptor blocking agent, phenoxybenzamine. Maximal relaxation induced by butyrylcholine or CGRP-1 was lower than to acetylcholine. While acetylcholine-induced relaxation was largely resistant to blockade by L-NMMA or ADMA (0.1 or 1mM), relaxation to butyrylcholine or CGRP-1 was powerfully suppressed. Phenoxybenzamine (0.1–10μM for 30min) concentration-dependently reduced maximal acetylcholine-induced relaxation. When the efficacy of acetylcholine was reduced by phenoxybenzamine, its residual relaxant effect was powerfully inhibited by L-NMMA or ADMA (0.1 or 1mM). Thus, in rat aorta, the ability of L-NMMA or ADMA to block agonist-induced nitric oxide activity is critically determined by the efficacy of the relaxant stimulus.

Endothelin A (ETA) Receptors Are Involved in Augmented Adrenergic Vasoconstriction and Blunted Nitric Oxide-Mediated Relaxation of Penile Arteries from Insulin-Resistant Obese Zucker Rats

Endothelin 1 (ET-1) levels and receptors are up-regulated in the erectile tissue of diabetic patients and animal models of erectile dysfunction (ED). The present study assessed the role of ET-1 receptors in the impaired adrenergic vasoconstriction and nitrergic relaxation of penile arteries from a rat model of insulin resistance. The effect of ET receptor antagonists was evaluated on the contractile responses to electrical field stimulation (EFS) of penile arteries from obese Zucker rats (OZRs) compared with lean Zucker rats (LZRs). ET receptor expression was determined by immunohistochemistry. Changes in neural nitrergic relaxation and adrenergic vasoconstriction and the expression of ET receptors in perivascular nerves were assessed. ET-1 (10(-10) M) enhanced EFS-induced vasoconstriction, and treatment with the adrenergic neurotoxin guanethidine reduced the contractions induced by ET-1 in penile arteries from both LZRs and OZRs, thus supporting the hypothesis that ET-1 releases noradrenaline from adrenergic nerves. ET-1 antagonized neural nitric oxide (NO)-mediated relaxant responses in LZR arteries, antagonizing relaxations induced by the NO donor S-nitroso-N-acetylpenicillamine to a larger extent in arteries from OZRs. ET(A) and ET(B) receptors were expressed in perivascular fibers colocalized with the neuronal marker protein gene product 9.5 in penile arteries from OZRs. The ET(A) receptor antagonist BQ-123 reversed the enhancing effect of ET-1 on the vasoconstriction elicited by EFS and the ET-1-induced inhibition of nitrergic relaxations in LZRs, restoring them to control levels in penile arteries of OZRs. ET-1 enhances adrenergic vasoconstriction through presynaptic ET(A) receptors and antagonizes neural NO-mediated relaxation through postsynaptic smooth muscle ET(A) receptors in penile arteries from OZRs, which likely contributes to the augmented vasoconstriction and blunted nitrergic relaxation of erectile tissue under conditions of insulin resistance.

Notoginsenoside Ft1 activates both glucocorticoid and estrogen receptors to induce endothelium-dependent, nitric oxide-mediated relaxations in rat mesenteric arteries

Panax notoginseng (Burk.) F.H. Chen has been used traditionally for the treatment of cardiovascular diseases. Notoginsenoside Ft1 (Ft1) is a bioactive saponin from the leaves of P. notoginseng. Experiments were designed to determine whether or not Ft1 is an endothelium-dependent vasodilator. Rat mesenteric arteries were suspended in organ chambers for the measurement of isometric tension during phenylephrine-induced contractions. The cyclic guanosine monophosphate (cGMP) level was assessed using enzyme immunoassay. The phosphorylation and protein expressions of endothelial nitric oxide synthase (eNOS), glucocorticoid receptors (GR), estrogen receptors beta (ERß), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) were determined by Western blotting. The localization of GR and ERß were determined by immunofluorescence staining. Ft1 caused endothelium-dependent relaxations, which were abolished by l-NAME (inhibitor of nitric oxide synthases) and ODQ (inhibitor of soluble guanylyl cyclase). Ft1 increased the cGMP level in rat mesenteric arteries. GR and ERß were present in the endothelial layer and their antagonism by RU486 and PHTPP, respectively, inhibited Ft1-induced endothelium-dependent relaxations and phosphorylations of eNOS, Akt and ERK1/2. Inhibition of phosphoinositide-3-kinase (PI3K) by wortmannin and ERK1/2 by U0126 reduced Ft1-evoked relaxations and eNOS phosphorylation. Taken in conjunction, the present findings suggest that Ft1 stimulates endothelial GRs and ERßs with subsequent activation of the PI3K/Akt and ERK1/2 pathways in rat mesenteric arteries. This results in phosphorylation of eNOS and the release of NO, which activates soluble guanylyl cyclase in the vascular smooth muscle cells leading to relaxations.

Pravastatin improves the impaired nitric oxide-mediated neurogenic and endothelium-dependent relaxation of corpus cavernosum in aged rats

Aim: The aim of this study was to investigate the effect of pravastatin treatment on diminished corpus cavernosum (CC) function associated with aging.Methods: Male rats were divided into three groups as adult rats (12–14 weeks old), aged rats (72–80 weeks old) and aged rats given 10 mg/kg/d pravastatin in drinking water for six weeks. Blood pressure was measured by tail-cuff method. Total cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, triglycerides and testosterone levels were estimated in blood. Changes in expression levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS) (Ser-1177), neuronal nitric oxide synthase (nNOS), NADPH oxidase subunit gp91phox, Rho A and Rho kinase (ROCK2) in CC were assessed by immunohistochemistry. Nitric oxide (NO)-mediated endothelium-dependent and neurogenic CC relaxation were evaluated by acetylcholine (ACh, 0.1 nM–100 µM) and electrical field stimulation (EFS; 30 V, 5 ms, 2–32 Hz), respectively.Results: In aged rats, NO-mediated, both endothelium-dependent and neurogenic CC relaxation, were significantly impaired as compared to adult rats. Besides, eNOS, p-eNOS and nNOS expressions decreased significantly in CC from aged rats, while gp91phox, RhoA and ROCK2 expressions increased significantly. The diminished relaxation in response to ACh or EFS as well as the changes in expression of these proteins in aged rats were significantly improved by pravastatin treatment.Conclusion: Pravastatin improves NO-mediated CC relaxations of aged rats probably by inhibiting NADPH oxidase/Rho kinase pathways, and this effect does not seem to be associated with lipid lowering effect of this drug.

The human uterine smooth muscle S-nitrosoproteome fingerprint in pregnancy, labor, and preterm labor

Molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor at term are incompletely known. More than 10% of babies born worldwide are premature and 1,000,000 die annually. Preterm labor results in preterm delivery in 50% of cases in the United States explaining 75% of fetal morbidity and mortality. There is no Food and Drug Administration-approved treatment to prevent preterm delivery. Nitric oxide-mediated relaxation of human uterine smooth muscle is independent of global elevation of cGMP following activation of soluble guanylyl cyclase. S-nitrosation is a likely mechanism to explain cGMP-independent relaxation to nitric oxide and may reveal S-nitrosated proteins as new therapeutic targets for the treatment of preterm labor. Employing S-nitrosoglutathione as an nitric oxide donor, we identified 110 proteins that are S-nitrosated in 1 or more states of human pregnancy. Using area under the curve of extracted ion chromatograms as well as normalized spectral counts to quantify relative expression levels for 62 of these proteins, we show that 26 proteins demonstrate statistically significant S-nitrosation differences in myometrium from spontaneously laboring preterm patients compared with nonlaboring patients. We identified proteins that were up-S-nitrosated as well as proteins that were down-S-nitrosated in preterm laboring tissues. Identification and relative quantification of the S-nitrosoproteome provide a fingerprint of proteins that can form the basis of hypothesis-directed efforts to understand the regulation of uterine contraction-relaxation and the development of new treatment for preterm labor.