Non-adrenergic Non-cholinergic Relaxation Research Articles

BDNF rescues aging-associated internal anal sphincter dysfunction.

Aging can lead to rectoanal incontinence due to internal anal sphincter (IAS) dysfunction, which is characterized by a decrease in IAS tone and contractility and an increase in nonadrenergic noncholinergic (NANC) relaxation. We aimed to determine whether brain-derived neurotropic factor (BDNF) rescues this aging-associated IAS dysfunction (AAID). To do so, we studied the effects of BDNF on the basal and G protein-coupled receptors (GPCR)-stimulated IAS smooth muscle tone and on NANC relaxation in Fischer 344 rats representing different age groups [26-mo-old (aging) vs. 6-mo-old (young)], before and after tyrosine kinase receptor B (TrkB) antagonist K252a. We also used isolated smooth muscle cells (SMCs) to determine the effects of BDNF before and after different agonists. For some studies, we monitored NO release using smooth muscle perfusates. BDNF reversed AAID by rescuing the basal IAS tone and agonists [thromboxane A2 analog (U46619) and angiotensin II (ANG II)]-induced contractility, and NANC relaxation. These rescue effects of BDNF were selective as K252a attenuated the changes in the IAS without modifying the effects of K+depolarization. Because of the direct association between the basal and GPCR-stimulated IAS tone and RhoA/ROCK activation, we speculate that this pathway in the rescue effects of BDNF. Conversely, our data suggest that aging-associated increased NANC relaxation is reversed by decreased release of NO and decrease in the sensitivity of the released inhibitory neurotransmitter. In summary, BDNF rescue of AAID involves RhoA/ROCK and inhibitory neurotransmission. These data have direct implications for the role of BDNF in the pathophysiology and therapeutic targeting of aging-associated rectoanal motility disorders.NEW & NOTEWORTHY These studies demonstrate that brain-derived neurotropic factor (BDNF) rescues the aging-associated internal anal sphincter (IAS) dysfunction, characterized by a decrease in IAS tone, and increase in non-adrenergic noncholinergic relaxation. We determined the effects of BDNF on the basal and GPCR (TXA2 and ANG II)-stimulated IAS tone, and on NANC relaxation, before and after TrkB inhibitor K252a. BDNF may have an important role in the pathophysiology and therapeutic targeting of certain rectoanal motility disorders.

Involvement of Enteric Glia in Small Intestine Neuromuscular Dysfunction of Toll-Like Receptor 4-Deficient Mice.

Enteric glial cells (EGCs) influence nitric oxide (NO)− and adenosine diphosphate (ADP)− mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4−/− mice. Ileal segments from male TLR4−/− and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4−/− ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO− and ADP− mediated relaxation in the TLR4−/− mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4−/− myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4−/− mice.

BDNF augments rat internal anal sphincter smooth muscle tone via RhoA/ROCK signaling and nonadrenergic noncholinergic relaxation via increased NO release.

Presently, there are no studies examining the neuromodulatory effects of brain-derived neurotropic factor (BDNF) on the basal internal anal sphincter (IAS) tone and nonadrenergic noncholinergic (NANC) relaxation. To examine this, we determined the neuromuscular effects of BDNF on basal IAS smooth muscle tone and the smooth muscle cells (SMCs) and the effects of NANC nerve stimulation before and after high-affinity receptor tyrosine kinase receptor B (TrkB) antagonist K252a. We also investigated the mechanisms underlying BDNF-augmented increase in the IAS tone and NANC relaxation. We found that BDNF-increased IAS tone and SMC contractility were TTX resistant and attenuated by K252a. TrkB-specific agonist 7,8-dihydroxyflavone, similar to BDNF, also produced a concentration-dependent increase in the basal tone, whereas TrkB inhibitors K252a and ANA-12 produced a decrease in the tone. In addition, BDNF produced leftward shifts in the concentration-response curves with U46619 and ANG II (but not with bethanechol and K+ depolarization), and these shifts were reversed by K252a. Effects of Y27632 and Western blot data indicated that the BDNF-induced increase in IAS tone was mediated via RhoA/ROCK. BDNF-augmented NANC relaxation by electrical field stimulation was found to be mediated via the nitric oxide (NO)/soluble guanylate cyclase (sGC) pathway rather than via increased sensitivity to NO. In conclusion, the net effect of BDNF was that it caused an increase in the basal IAS tone via RhoA/ROCK signaling. BDNF also augmented NANC relaxation via NO/sGC. These findings may have relevance to the role of BDNF in the pathophysiology and therapeutic targeting of the IAS-associated rectoanal motility disorders.NEW & NOTEWORTHY These studies for the first time to our knowledge demonstrate that increased levels of brain-derived neurotrophic factor (BDNF; conceivably released from smooth muscle cells and/or the enteric neurons) has two major effects. First, BDNF augments the internal anal sphincter (IAS) tone via tyrosine kinase receptor B/thromboxane A2-receptor, angiotensin II receptor type 1/RhoA/ROCK signaling; and second, it increases nonadrenergic noncholinergic relaxation via nitric oxide/soluble guanylate cyclase. These studies may have relevance in therapeutic targeting in the anorectal motility disorders associated with the IAS.

Effects of epoxygenases on the nonadrenergic noncholinergic relaxant responses induced by electrical field stimulation in rabbit corpus cavernosum.

We aimed to investigate the effects of epoxygenases on electrical field stimulation (EFS)-mediated nitric oxide (NO)-dependent and NO-independent nonadrenergic noncholinergic (NANC) relaxations in isolated rabbit corpus cavernosum. The tissues of 20 male adult albino rabbits (2.5-3kg) were suspended in organ baths containing aerated Krebs solution, and isometric contractions were recorded. EFS-mediated NANC relaxations were obtained on phenylephrin (3×10-5 M)-contracted tissues in the presence of guanethidine (10-6 M) and atropine (10-6 M). Miconazole (10-9 -10-4 M), 17-octadecynoic acid (ODYA) (10-10 -10-5 M), 14,15-epoxyeicosatrienoic acid (EET) (10-11 -10-8 M), 11,12-EET (10-12 -3×10-8 M) and 20-hydroxyeicosatetraenoic acid (HETE) (10-11 -3×10-8 M) were added cumulatively (n=5-7 for each set of experiments). For NO-independent relaxations, Nω -nitro-l-arginine methyl ester (l-NAME) (10-4 M) was added before a group of experiments. Depending on the concentration, miconazole, 17-ODYA, 14,15-EET, 11,12-EET, and 20-HETE significantly enhanced both NO-dependent and NO-independent EFS-mediated relaxations (p<0.05). Epoxygenases showed similar effect on NO-dependent and NO-independent relaxant responses except 20-HETE which caused significantly more enhanced relaxation on NO-dependent responses (p<0.05). No drug caused a significant relaxation response on tissues contracted with phenylephrine. Epoxygenases contribute to EFS-mediated NO-dependent and NO-independent NANC relaxations by presynaptic mechanisms, offering a new treatment alternative for erectile dysfunction which needs to be explored in further in vivo, molecular and clinical studies.

Testosterone Replacement Enhances Internal Pudendal Artery Relaxation to Reverse Erectile Dysfunction in a Rat Model of Androgen Deprivation Therapy

Introduction/ObjectivesAndrogen deprivation therapy (ADT) is frequently used to manage prostate cancer; however, erectile dysfunction (ED) and cardiovascular disease are common side effects. After cessation of ADT, testosterone (T) levels may take years to recover leaving men prone to cardiovascular disease and long‐term ED. The internal pudendal arteries (IPA) supply blood to the penis and vascular injury to these vessels can lead to ED. This study will determine if castration impairs vascular function in systemic (aorta and mesenteric arteries) and penile vasculature (IPA), and if T can restore erectile and vascular physiology. We hypothesize the IPA will develop impaired relaxation prior to systemic arteries following castration, and T therapy will recover both vascular and erectile function.MethodsMale Sprague Dawley rats (12 wks) were divided into 3 groups (n=8/grp): control (CON), CAST (6 wks castration), and CAST+T (6 wks castration+T (3mg/kg) in last 2 wks). Erections were assessed via cavernous nerve stimulation and measurement of intracavernosal to mean arterial pressures (ICP/MAP). Aortas, mesenteric arteries, and IPA were excised, cut (2 mm) and mounted into tissue baths. Contractility to high concentration potassium solution (KCl), electrical field stimulation (EFS), and phenylephrine (PE) were measured. Endothelial dependent acetylcholine (ACh) relaxation and endothelial independent relaxation to DEA NONOate were assessed. Non‐adrenergic non‐cholinergic (NANC) relaxation was evaluated by EFS in the presence of guanethidine and atropine. Serum testosterone levels were measured via ELISA. Androgen receptor protein expression was measured in IPA.ResultsCAST markedly impaired erectile function (ICP/MAP; CON: 0.83±0.03, CAST: 0.31±0.02; p&lt;0.05) and decreased IPA ACh relaxation (CON: 50.4±3.5%, CAST: 31.2±2.3%; p&lt;0.05). DEA NONOate relaxation was unchanged with CAST. CAST diminished IPA NANC relaxation (CON: 40.8±5.1%, CAST: 22.1±3.9%; p&lt;0.05). However, CAST did not impact IPA contraction. Interestingly, CAST did not change vascular contraction or relaxation in aortas or mesenteric arteries. IPA androgen receptor expression decreased after CAST (CON: 1±0.17, CAST: 0.39±0.03; p&lt;0.05). T supplementation restored serum T (T (ng/ml); CON: 4.2±0.8, CAST: 0.1±0.02, CAST+T: 32.9±3.2: p&lt;0.05), erectile function (ICP/MAP; CAST+T 0.74±0.01 p&lt;0.05) and improved ACh relaxation in IPA to values greater than CON (CAST+T: 63.9±2.9%; p&lt;0.05). Similarly, CAST+T enhanced IPA NANC relaxation to levels greater than CON (CAST+T: 69.2±1.4%; p&lt;0.05). T supplementation did not impact IPA contractions and had no effect on aortic or mesenteric vasoreactivity.ConclusionsCastration induces severe IPA vascular damage, yet systemic vasculature is unaffected. T therapy dramatically enhances IPA endothelial dependent and NANC‐mediated relaxation. Though T therapy safety in cancer survivors remains controversial, a growing body of literature is finding no correlation between T therapy and cancer progression. In certain populations of prostate cancer survivors, T replacement is warranted.Support or Funding InformationBSOM start‐up grant, SMSNA, AUAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

The expression, localization and function of α7 nicotinic acetylcholine receptor in rat corpus cavernosum.

Alpha7 nicotinic acetylcholine receptor (α7-nAChR), an emerging pharmacological target for a variety of medical conditions, is expressed in the most mammalian tissues with different effects. So, this study was designed to investigate the expression, localization and effect of α7-nAChR in rat corpus cavernosum (CC). Reverse transcription polymerase chain reaction (RT-PCR) revealed that α7-nAChR was expressed in rat CC and double immunofluorescence studies demonstrated the presence of α7-nAChR in corporal neurons. The rat CC segments were mounted in organ bath chambers and contracted with phenylephrine (0.1μm -300μm) to investigate the relaxation effect of electrical field stimulation (EFS,10Hz) assessed in the presence of guanethidine (adrenergic blocker, 5μm) and atropine (muscarinic cholinergic blocker, 1μm) to obtain non-adrenergic non-cholinergic (NANC) response. Cumulative administration of nicotine significantly potentiated the EFS-induced NANC relaxation (-log EC50=7.5±0.057). Whereas, the potentiated NANC relaxation of nicotine was significantly inhibited with different concentrations of methyllycaconitine citrate (α7-nAChR antagonist, P<0.05) in preincubated strips. L-NAME (non-specific nitric oxide synthase inhibitor, 1μm) completely blocked the neurogenic relaxation induced by EFS plus nicotine. To conclude α7-nAChR is expressed in rat CC and modulates the neurogenic relaxation response to nicotine.

Toll-Like Receptor 4 Modulates Small Intestine Neuromuscular Function through Nitrergic and Purinergic Pathways.

Objective: Toll-like receptors (TLRs) play a pivotal role in the homeostatic microflora-host crosstalk. TLR4-mediated modulation of both motility and enteric neuronal survival has been reported mainly for colon with limited information on the role of TLR4 in tuning structural and functional integrity of enteric nervous system (ENS) and in controlling small bowel motility.Methods: Male TLR4 knockout (TLR4-/-, 9 ± 1 weeks old) and sex- and age-matched wild-type (WT) C57BL/6J mice were used for the experiments. Alterations in ENS morphology and neurochemical code were assessed by immunohistochemistry whereas neuromuscular function was evaluated by isometric mechanical activity of ileal preparations following receptor and non-receptor-mediated stimuli and by gastrointestinal transit.Results: The absence of TLR4 induced gliosis and reduced the total number of neurons, mainly nNOS+ neurons, in ileal myenteric plexus. Furthermore, a lower cholinergic excitatory response with an increased inhibitory neurotransmission was found together with a delayed gastrointestinal transit. These changes were dependent on increased ileal non-adrenergic non-cholinergic (NANC) relaxations mediated by a complex neuronal-glia signaling constituted by P2X7 and P2Y1 receptors, and NO produced by nNOS and iNOS.Conclusion: We provide novel evidence that TLR4 signaling is involved in the fine-tuning of P2 receptors controlling ileal contractility, ENS cell distribution, and inhibitory NANC neurotransmission via the combined action of NO and adenosine-5′-triphosphate (ATP). For the first time, this study implicates TLR4 at regulating the crosstalk between glia and neurons in small intestine and helps to define its role in gastrointestinal motor abnormalities during dysbiosis.

AB278. SPR-05 Purinergic regulation of nitric oxide mediated vasodilation in rat internal pudendal arteries

ObjectiveInternal pudendal arteries (IPA) provide 70% of total blood blow resistance to penile arteries and have been shown to undergo significant vascular remodeling due to aging, diabetes, chronic kidney disease, and cardiovascular disease. Balloon angioplasty and stent placement is a common treatment option to restore blood flow within IPAs; however, stent failure is disproportionately higher than coronary artery stents despite similar stents and placement techniques suggesting a unique IPA physiology. Non-adrenergic non-cholinergic (NANC) mediated vasodilation is well characterized in the penis, but IPA neurotransmitter release has yet to be characterized. The objective of this study is to characterize electric field stimulated (EFS) mediated neurotransmitter release in rat IPA and address the novel role of ATP as a neurotransmitter.MethodsProximal (internal iliac to gluteal artery) and distal (gluteal artery to penis) IPA segments excised from male Sprague-Dawley rats (350–475 g) were mounted into myographs. NANC vasodilation was assessed in precontracted IPAs following incubation with inhibitors of norepinephrine release (guanethidine; 10-6 M) and cholinergic receptors (atropine, 10-5 M) to assess neuronal nitric oxide (NO) release. This procedure was repeated with additional incubation of a NO synthase (NOS) inhibitor (L-NAME; 10-6 M), selective P2X1 inhibitor (NF-449; 10-5 M), or selective P2Y1 inhibitor (MRS 2500; 10-6 M). EFS relaxation parameters used were 5 ms delay, 0.3 ms duration at 20V and 0.5–32 Hz. Concentration response curves to assess ADP, a P2Y1 receptor agonist, relaxation and α,β-MetATP, a stable P2X1 receptor agonist, contraction were also performed.ResultsNANC stimulation lead to greater relaxation in distal IPA (30% vs. 15%, P<0.05). Additional inhibition of NOS completely eliminated NANC relaxation in distal IPA; however, did not reduce proximal IPA relaxation. P2X1 or P2Y1 inhibition caused further distal IPA relaxation, but combined P2X1 and P2Y1 inhibition increased distal IPA relaxation from 30% to 53% without affecting proximal IPA relaxation. No significant differences were noted to α,β-MetATP mediated contractions via the P2X1 receptor. ADP mediated relaxation was markedly increased in proximal IPA (16%) compared to distal IPA (6%).ConclusionsFunctional physiological differences exist within the proximal and distal segments of the IPA. NANC-mediated relaxation in the distal IPA is greater than the proximal IPA and is mediated by NO-dependent mechanisms. Furthermore, the distal IPA undergoes additional purinergic regulation which is NO-independent. Understanding the physiology of this critical feeder blood vessel to the penis is crucial when developing novel treatments that target the vasculature to recover erectile function.Funding Source(s)None

Role of the nitrergic pathway in motor effects of oxytocin in rat proximal colon.

Oxytocin (OT) reduces rat duodenal tone and mouse intestinal transit; however, the underlying mechanisms are not totally understood. Consequently, this study was designed to investigate the influence of OT on spontaneous mechanical activity and neurally evoked responses, to characterize the mechanisms of the action, and to determine the distribution of the OT receptor (OTR) in rat proximal colonic muscle strips. The organ bath technique with electrical field stimulation, western blotting, and immunofluorescence were used. In rat proximal colon, exogenous OT induced a concentration-dependent reduction of the spontaneous mechanical activity without affecting the resting basal tone, which was abolished by atosiban, an OTR antagonist, by tetrodotoxin (TTX), a neural blocker or by Nω-propyl-l-arginine hydrochloride, an inhibitor of neuronal nitric oxide synthase (nNOS). The inhibitory effects of OT were not affected by atropine or the vasoactive intestinal peptide (VIP) receptor antagonist [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP (VIPHyb). Proximal colon responses to electrical field stimulation were characterized by nonadrenergic, noncholinergic (NANC) relaxation, which was followed by an off-contraction. Oxytocin enhanced only NANC relaxation. Oxytocin stimulated spontaneous NO release from the longitudinal muscle myenteric plexus preparation of rat proximal colon. Western blot and immunohistochemistry experiments showing the presence of the OTR in proximal colon, and its co-localization with nNOS established that myenteric nitrergic neurons express OTR. The activation of OTR located on nitrergic neurons may negatively modulate colonic spontaneous contraction and enhance electrically evoked NANC relaxation through excitation of NO release.

Effects of chronic l-DOPA administration on neurogenic and endothelium-dependent relaxation responses in rabbit corpus cavernosum

Dopamine is a crucial central neurotransmitter that plays a fundamental role in the autonomic and somatic components of penile reflexes in animals and humans. Similar to the erectile responses of dopamine, systemic administration of l-DOPA induces yawning and penile erection in some species. The possible effects of l-DOPA on nitric oxide (NO)-dependent and -independent non-adrenergic non-cholinergic (NANC) relaxation responses mediated by electrical field stimulation (EFS) and endothelium-dependent relaxation were investigated in this study. Thirty-two adult albino male rabbits, in two- and four-week-treatment groups, were divided into three subgroups: control group (saline-injected) (n=4), 3mg/kg/day (low dose) l-DOPA-injected groups (n=6) and 12mg/kg/day (high dose) l-DOPA-injected groups (n=6). After the intraperitoneal injection treatments, the corpus cavernosum tissues were placed in organ bath chambers. The EFS-mediated responses, and the concentration-response curve to carbachol, sodium nitroprusside (SNP), sildenafil were assessed. The two-week treatment with high-dose l-DOPA decreased the NO-dependent NANC relaxation responses, while there was no change in the low-dose two- and four-week treatment groups. The NO-independent NANC relaxation responses in the two-week groups decreased, and the responses in the four-week groups were unchanged when compared to the controls. The relaxation responses to carbachol showed no differences among all groups except for the high-dose four-week l-DOPA group. The relaxation responses of SNP and sildenafil were increased in all of the treatment groups when compared to the controls. The observed increases in SNP- and sildenafil-induced responses, along with the decreased EFS-mediated responses, suggest increased sensitivity in the NO-signalling pathway following l-DOPA administration.

Bimodal effect of oxidative stress in internal anal sphincter smooth muscle.

Changes in oxidative stress may affect basal tone and relaxation of the internal anal sphincter (IAS) smooth muscle in aging. We examined this issue by investigating the effects of the oxidative stress inducer 6-anilino-5,8-quinolinedione (LY-83583) in basal as well as U-46619-stimulated tone, and nonadrenergic, noncholinergic (NANC) relaxation in rat IAS. LY-83583, which works via generation of reactive oxygen species in living cells, produced a bimodal effect in IAS tone: lower concentrations (0.1 nM to 10 μM) produced a concentration-dependent increase, while higher concentrations (50-100 μM) produced a decrease in IAS tone. An increase in IAS tone by lower concentrations was associated with an increase in RhoA/Rho kinase (ROCK) activity. This was evident by the increase in RhoA/ROCK in the particulate fractions, in ROCK activity, and in the levels of phosphorylated (p) (Thr696)-myosin phosphatase target subunit 1 and p(Thr18/Ser19)-20-kDa myosin light chain. Conversely, higher concentrations of LY-83583 produced inhibitory effects on RhoA/ROCK. Interestingly, both the excitatory and inhibitory effects of LY-83583 in the IAS were reversed by superoxide dismutase. The excitatory effects of LY-83583 were found to resemble those with neuronal nitric oxide synthase (nNOS) inhibition by l-NNA, since it produced a significant increase in the IAS tone and attenuated NANC relaxation. These effects of LY-83583 and l-NNA were reversible by l-arginine. This suggests the role of nNOS inhibition and RhoA/ROCK activation in the increase in IAS tone by LY-83583. These data have important implications in the pathophysiology and therapeutic targeting of rectoanal disorders, especially associated with IAS dysfunction.

Role of PGE2/EP2 Receptor Signaling on The Effects of Neonatal Hyperoxia on Airway Smooth Muscle

It was shown that neonatal hyperoxic‐exposure associates with airway hyperresponsiveness and impaired nonadrenergic noncholinergic relaxation. It was demonstrated that reduced relaxant responses were associated with an impairment of prostaglandin E2 (PGE2) release from airway smooth muscle (ASM). In this study we tested the hypothesis that supplementation of PGE2 or EP2 agonists to ASM restore relaxant responses. Tracheal cylinders were obtained from 12 days rat pups exposed to hyperoxia or room air for seven days. These cylinders were used to study contractile and relaxant responses. In hyperoxic tracheal preparations the contractile responses to bethanechol were enhanced as compared to room air controls. These enhanced contractile responses were attenuated by pre‐incubation of tissues with PGE2 (1µM). Similar effect was showed by using an EP2 agonist. This effect of PGE2 and EP2 agonist was reversed by an EP2 antagonist. PGE2 and EP2 induced dose response relaxations of ASM, which were reduced by an EP2 antagonist.We conclude that PGE2 induces relaxation of airways via EP2 receptors, and the use of EP2 agonists may serve as a therapeutic approach to overcome the adverse effects of neonatal hyperoxia on relaxant responses of ASM.Support by MEST

Role of PGE2/EP4 Receptor Signaling on The Effects of Hyperoxia on Airway Smooth Muscle

It was shown that neonatal hyperoxic‐exposure associates with airway hyperresponsiveness and impaired nonadrenergic noncholinergic relaxation. It was demonstrated that reduced relaxant responses were associated with an impairment of prostaglandin E2 (PGE2) release from airway smooth muscle (ASM). In this study we tested the hypothesis that supplementation of PGE2 or EP4 agonists to ASM restore relaxant responses. Tracheal cylinders were obtained from 12 days rat pups exposed to hyperoxia or room air for seven days. These cylinders were used to study contractile and relaxant responses. In hyperoxic trachea the contractile responses to bethanechol were enhanced as compared to room air controls. These enhanced contractile responses were attenuated by pre‐incubation of tissues with PGE2 (1µM). Similar effect showed an EP4 agonist. This effect of PGE2 and EP4 agonist was reversed by an EP4 antagonist. PGE2 and EP4 induced dose response relaxations of ASM, which were reduced by an EP4 antagonist.We conclude that PGE2 induces relaxation of airways via EP4 receptors, and the use of EP4 agonists may serve as a therapeutic approach to overcome the adverse effects of neonatal hyperoxia on relaxant responses of ASM.Supported by MEST

Effect of vagal stimulation in acute asthma

American lung association report,there were 1.8 million emergency department visits and 3,816 deaths per year attributable to asthma [1]. according to (GINA2012) the role of complementary and alternative medicine in adult asthma treatment are limited because these approaches have been insufficiently researched and their effect are largely unproven [2]. the parasympathetic nervous system are the primary regulators of pulmonary smooth muscle tone. Postganglionic parasympathetic nerves mediate both cholinergic contractions and inhibitory non-adrenergic non-cholinergic (iNANC) relaxations. It is the close balance between these two that ultimately determines airway caliber. These relaxation and contraction responses are through anatomical and physiological distinct vagus nerve pathways [3].

Upregulation of bile acid receptor TGR5 and nNOS in gastric myenteric plexus is responsible for delayed gastric emptying after chronic high-fat feeding in rats.

Chronic high-fat feeding is associated with functional dyspepsia and delayed gastric emptying. We hypothesize that high-fat feeding upregulates gastric neuronal nitric oxide synthase (nNOS) expression, resulting in delayed gastric emptying. We propose this is mediated by increased bile acid action on bile acid receptor 1 (TGR5) located on nNOS gastric neurons. To test this hypothesis, rats were fed regular chow or a high-fat diet for 2 wk. Rats fed the high-fat diet were subjected to concurrent feeding with oral cholestyramine or terminal ileum resection. TGR5 and nNOS expression in gastric tissue was measured by immunohistochemistry, PCR, and Western blot. Gastric motility was assessed by organ bath and solid-phase gastric emptying studies. The 2-wk high-fat diet caused a significant increase in neurons coexpressing nNOS and TGR5 in the gastric myenteric plexus and an increase in nNOS and TGR5 gene expression, 67 and 111%, respectively. Enhanced nonadrenergic, noncholinergic (NANC) relaxation, deoxycholic acid (DCA)-induced inhibition in fundic tissue, and a 26% delay in gastric emptying accompanied these changes. A 24-h incubation of whole-mount gastric fundus with DCA resulted in increased nNOS and TGR5 protein expression, 41 and 37%, respectively. Oral cholestyramine and terminal ileum resection restored the enhanced gastric relaxation, as well as the elevated nNOS and TGR5 expression evoked by high-fat feeding. Cholestyramine also prevented the delay in gastric emptying. We conclude that increased levels of circulatory bile acids induced by high-fat feeding upregulate nNOS and TGR5 expression in the gastric myenteric plexus, resulting in enhanced NANC relaxation and delayed gastric emptying.

Hematin supplementation protects against hyperoxia‐induced airway hyperresponsiveness (870.8)

It was shown that neonatal hyperoxic‐exposure associates with airway hyperresponsiveness and impaired nonadrenergic noncholinergic relaxation. Previously we have demonstrated the ability of endogenous carbon monoxide (CO) to normalize the airway relaxation under hyperoxic conditions. The aim of this study was to investigate the effect of CO on the hyperoxia‐induced airway hyperresponsiveness. Tracheal cylinders were excised from rat pups 12 days old, exposed to hyperoxia or room air for seven days. Electrical field stimulation (EFS) was applied to induce contraction. The preparations were supplemented with CO precursor ‐ hematin in presence or absence of heme oxygenase (HO) inhibitor tin protoporphyrin‐IX (SnPP‐IX; 100 µM), and/or soluble guanylyl cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a] quinoxalin‐1‐one (ODQ; 10 µM). In TSM obtained from hyperoxia‐exposed animals the contractile responses were increased significantly as compared to room air‐exposed animals, but supplementation of tissues with hematin reversed the contractile responses in hyperoxic group to almost normal level. Inhibiton of HO by SnPP‐IX diminished this effect of hematin. Similar effect was shown also by ODQ alone or in combination with SnPP‐IX. We conclude that increase of CO production overcomes the effects of hyperoxia on airway contractile responses via cGMP signaling and this may serve as a therapeutic approach for treatment of increased airway reactivity in hyperoxia‐exposed neonates.

2, 3, 5, 4'-Tetrahydroxystilbene-2-O-beta-D-glucoside improves gastrointestinal motility disorders in STZ-induced diabetic mice.

Oxidative stress has recently been considered as a pivotal player in the pathogenesis of diabetic gastrointestinal dysfunction. We therefore investigated the role of 2, 3, 5, 4′-tetrahydroxystilbene-2-O-beta-D-glucoside (THSG) that has a strong anti-oxidant property, in diabetic gastrointestinal dysmotility as well as the underlying protective mechanisms. THSG restored the delayed gastric emptying and the increased intestinal transit in streptozotocin (STZ)-induced diabetic mice. Loss of neuronal nitric oxide synthase (nNOS) expression and impaired nonadrenergic, noncholinergic (NANC) relaxations in diabetic mice were relieved by long-term preventive treatment with THSG. Meanwhile, THSG (10−7∼10−4 mol/L) enhanced concentration-dependently NANC relaxations of isolated colons in diabetic mice. Diabetic mice displayed a significant increase in Malondialdehyde (MDA) level and decrease in the activity of glutathione peroxidase (GSH-Px), which were ameliorated by THSG. Inhibition of caspase-3 and activation of ERK phosphorylation related MAPK pathway were involved in prevention of enhanced apoptosis in diabetes afforded by THSG. Moreover, THSG prevented the significant decrease in PPAR-γ and SIRT1 expression in diabetic ileum. Our study indicates that THSG improves diabetic gastrointestinal disorders through activation of MAPK pathway and upregulation of PPAR-γ and SIRT1.

D-serine modulates non-adrenergic non-cholinergic contraction of lower esophageal sphincter in rats

Endogenous d-serine is known to modulate glutamatergic transmission via interaction with the glycine site of N-methyl-d-aspartate (NMDA) receptors. d-serine is synthesized by racemization of l-serine using an enzymatic reaction catalyzed by serine racemase. Although much attention has been focused on the role of d-serine within the central nervous system, the physiological role of d-serine in enteric nervous system has not been investigated. Lower esophageal sphincter (LES) function is known to be modulated by NMDA-dependent mechanisms. The present study was aimed to study the expression of enzymes involved in d-serine metabolism and the function of d-serine in lower esophageal sphincter in rats. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting showed the expression of serine racemase in isolated rat LES. Electrical field stimulation was used to induce non-adrenergic non-cholinergic (NANC) contraction/relaxation of isolated rat LES in an organ bath using an isometric force transducer. The organ bath studies on isolated rat LES showed that incubation with d-serine (100μM) is associated with a significant increase in the NANC contraction of isolated LES. This effect of exogenous d-serine was inhibited by NMDA receptor antagonists (MK-801), suggesting that NMDA receptors are involved in the effects of d-serine on NANC contraction of LES. Incubation with d-serine did not show a significant effect on NANC relaxation within our experimental setting. The results of this study suggest that serine racemase is expressed in LES and d-serine modulates contraction of the lower esophageal sphincter in rats.

Abstract 647: Toll-like Receptor 4 (TLR4) Activation Impairs the Nitrergic Relaxation Response in Rat Corpus Cavernosum

Cardiovascular disease etiology of hypertension and atherosclerosis has been shown to involve pattern-recognition receptor activation, including toll-like receptor 4 (TLR4). Indeed, our lab showed that TLR4 activation was implicated in blood pressure regulation, and its blockade lowered blood pressure in spontaneously hypertensive rats. However, whether TLR4 activation plays a role in erectile dysfunction is not known. Therefore, we hypothesized that TLR4 activation using its ligand, lipopolysaccharide (LPS), would increase reactive oxygen species (ROS) and impair corpus cavernosum (CC) relaxation. To test our hypothesis, rat CC smooth muscle cells were incubated for 15 min with LPS (100 ng/mL) and ROS detected with a fluorescent probe. Treatment with LPS resulted in an increase in ROS, compared to baseline (Fig.1). To investigate vascular reactivity to TLR4 activation, we performed myograph studies on CC isolated from 13-15 week old Sprague-Dawley rats, then incubated 3 hours in DMEM with LPS (+LPS) or vehicle (VEH). Concentration response curves to phenyleprine (PE, 10 -9 -10 -4 M) and to non-adrenergic, non-cholinergic (NANC) stimulation were used to investigate CC reactivity. Contractility to PE did not differ between groups, but +LPS-induced relaxation responses decreased significantly to 20-volt electrical field stimulation compared to VEH (8Hz: 16.3 vs 24.9, p&lt; 0.01 and 16 Hz: 21.2 vs 35.1, p&lt; 0.001). In conclusion, treatment with the TLR4 agonist, LPS, decreased NANC relaxation in the CC. Increased ROS generation may result in scavenging and reducing availability of nitric oxide that could impair relaxation in the CC.