Non-adrenergic Non-cholinergic Research Articles

Pharmacological evidence that GABA-induced relaxation of rat proximal duodenum longitudinal muscle depends on NKCC cotransporter activity and Ca2+ influx.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adult central nervous system (CNS) synapses, but it excites immature CNS neurons as well as neurons in the myenteric plexus. The present work aimed to determine whether GABA-induced nonadrenergic, noncholinergic (NANC) neuronal-mediated relaxation of the rat duodenum is dependent on the activity of Na+ K+ Cl- cotransporters (NKCC) and requires calcium influx. In the presence of guanethidine (3µmol/L), atropine (3µmol/L), and indomethacin (1µmol/L), relaxations induced by GABA (100µmol/L), KCl (5-10mmol/L) and electrical field stimulation (1-8Hz, 2ms, 60V), but not those induced by bradykinin (10-100nmol/L) were abolished by lidocaine (300µmol/L). However, only GABA-induced relaxations were reduced in a concentration-dependent manner by the NKCC1/2 inhibitors bumetanide (0.1-1µmol/L) and furosemide (1-10µmol/L). GABA-induced NANC neuronal relaxation was abolished by bicuculline (30µmol/L) and inhibited by N-nitroarginine methyl ester (l-NAME, 300µmol/L). The ω-conotoxin GVIA (1µmol/L), which acts exclusively on neuronal CaV2 channels, but not on smooth muscle voltage-gated Ca2+ CaV1 channels, and nonselective blockers of these channels (verapamil 100nmol/L and ruthenium red 10µmol/L), reduced GABA-induced relaxations. These results showed that the activation of GABAA receptors induces NANC nitrergic neuronal relaxations in the rat duodenum, which depend on NKCC activity and CaV2 channel activation, suggesting that this phenomenon results from neuronal depolarization promoted by Cl- efflux through GABAA receptors, with subsequent Ca2+ influx and nitric oxide release.

Neurochemical profile of the myenteric plexus in the rat colorectal region.

The enteric nervous system, a major subdivision of the autonomic nervous system, is known for its neurochemical heterogeneity and complexity. The myenteric plexus, one of its two principal components, primarily controls peristalsis and its dysfunction may lead to a number of gastrointestinal motility disorders. The myenteric neurons have been described to use a wide variety of neurotransmitters although no evidence has been reported for the existence of adrenergic neurons in the hindgut. This study aims at elucidating the chemical coding of neurons in the myenteric plexus of the rat colon and anorectal region with particular emphasis on cholinergic and the so-called nonadrenergic, noncholinergic (NANC) transmitter systems. The immunostaining for choline acetyltransferase revealed an intense staining of the myenteric ganglia with clear delineation of their neuronal cell bodies and without local distributional differences in the colonic region. The myenteric ATPergic structures were mostly limited to fiber bundles surrounding unstained myenteric neurons and penetrating the two muscle layers. We also observed an abundance of intensely stained varicose substance P-immunopositive fibers, ensheathing the immunonegative myenteric neuronal cell bodies in a basket-like manner. Applying NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry, we were able to demonstrate numerous nitrergic somata of myenteric neurons with Dogiel Type I morphology. Apart from the observed nitrergic distributional patterns, no distinct variations were found in the staining intensity or distribution of myenteric structures in the colon and anorectal area. Our results suggest that myenteric neurons in the distal intestinal portion utilize a broad spectrum of enteric transmitters, including classical and NANC transmitters.

Effects of Simulated Long Duration Spaceflight on Erectile Function

The goal of NASA along with other space agencies in the world is to send manned missions to the moon in the coming years, and eventually to Mars. The long‐term health risks of spaceflight have been attributed primarily to cosmic radiation and microgravity and the adverse effects as seen in the astronauts and model animals include DNA damage, mitochondrial dysregulation, oxidative damage, epigenetic changes, telomere length changes and microbiome shifts all of which affect the cardiovascular system. However, little is known about the effect of spaceflight on erectile function which is an important quality of life aspect for men. The aim of our study was to investigate the effect of simulated long duration space flight on erectile function. We hypothesized that both hindlimb unloading (HLU) and radiation will adversely affect erectile function. 86 adult male Sprague‐Dawley rats were used for these studies. Half the animals underwent 4 weeks of HLU, and all animals were divided into 3 groups: those that received sham radiation, 0.75 Gy, and 1.5 Gy galactic cosmic radiation, respectively at the Ground‐based GCR simulator at the NASA Space Radiation laboratory. The animals were then monitored for 6‐9 months before they were sacrificed, following which Corpus cavernosum (CC) and the distal internal pudendal arteries (dIPA) were harvested and used for ex vivo functional assessment. Tissue and wire myography studies were performed to assess adrenergic and endothelium‐dependent vasoconstrictions using phenylephrine (PE), norepinephrine, and endothelin. This was done by measuring the cumulative dose‐responses to the agonists in both the CC and dIPA. Endothelium‐dependent and ‐independent relaxations were also assessed using acetylcholine and diethylamine nononate. Non‐adrenergic non‐cholinergic (NANC) transmission was assessed using electrical stimulation (Estim); 30V electric pulses were delivered to the tissues with frequency ranging from 1‐32Hz following the administration of atropine and guanethidine and pre‐constriction with PE. Finally, Estim was done without atropine and guanethidine to assess the parasympathetic mediated relaxation in the tissues. The effects of HLU and radiation on the contractile and relaxation responses were assessed by two‐way repeated measures ANOVA. HLU and radiation significantly reduced both endothelium‐dependent and ‐independent vasodilatation in the dIPA and CC. Also, HLU and radiation significantly reduced the adrenergic constriction of CC in response to PE and norepinephrine. However, neither HLU nor radiation influenced the endothelium‐dependent vasoconstriction in the dIPA. Finally, radiation reduced the relaxation in both CC and dIPA while HLU increased the dIPA relaxation following Estim for both the parasympathetic mediated and NANC mediated responses. These results therefore showed the effects of HLU and radiation on erectile function, with exposure to cosmic radiation being primarily responsible for the detrimental effects. Further research needs to be done on this subject to show more mechanistically how spaceflight can affect erectile function and ways to combat the problem to ensure that astronauts can return to their normal sexual lives after long trips to space.

Pleasurable ways to spontaneously expulse distal ureteral stones: A systematic review and meta-analysis of randomized controlled trials

ABSTRACT Introduction Numerous non-invasive ways of managing distal ureteral stones have been recommended, such as administering treatments to inhibit receptors responsible for ureteral tone and peristaltic movement frequency. Nitric oxide, which is released during penile erection, causes smooth muscle relaxation by inhibiting the non-adrenergic non-cholinergic (NANC) nerves of the distal ureter and ureterovesical junction. Bladder neck contraction followed by relaxation, occurring during orgasm and ejaculation could also facilitate the passage of stones. However, the exact role of sexual activities involving these mechanisms has yet to be established. Objective This review aimed to evaluate the efficacy of sexual acts, including sexual intercourse and masturbation, to promote distal ureteral stones expulsion based on the findings of relevant randomized controlled trials (RCT)s. Methods A systematic search adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist was performed in the Embase, Scopus, and Medline databases in July 2021. The initial search generated 50 articles. The articles were screened based on the eligibility criteria. Their quality was assessed using the Cochrane Risk of Bias (RoB) tool 2 for randomized trials. Measured outcomes included stone expulsion rate, expulsion time, analgesic injections frequency, and the need for a ureteroscopic (URS) lithotripsy. A fixed effects model was used for analyzing parameters with homogeneity among studies, whereas a random effects model was used for heterogenous studies. The odds ratio (OR) and mean difference (MD) among studies were displayed in forest plots using the Review Manager software version 5.4.1. Results A total of five RCTs with an overall low risk of bias, consisting of 406 subjects, were included in this review. Four studies evaluated sexual intercourse and one study evaluated masturbation. Both interventions were instructed to be performed for three to four times a week. The stone expulsion rate was higher in the sexual activity group compared to the control group at the two-week follow-up (OR 5.77 95%CI 3.70 - 9.01, p<0.00001) and four-week follow-up (OR 4.44 95%CI 2.73 - 7.23, p<0.00001) with a significantly lower expulsion time of the sexual activity group (MD -3.79 95% CI -6.38 - -1.20, p=0.004). The number of subjects requiring additional analgesic injections (MD -0.62 95% CI -1.03 - -0.21, p=0.003) and URS lithotripsy (OR 0.22 95% CI 0.13 – 0.37, p<0.00001) was also lower in the sexual activity group compared to the control group. Conclusions Performing sexual intercourse or masturbation for three to four times a week is beneficial for promoting spontaneous expulsion of distal ureteral stones. It could also reduce the use of analgesic injections and the need for a URS lithotripsy.

The Impact of Long‐Duration Exposure to Microgravity and Galactic Cosmic Radiation on Corpus Cavernosum Function

During spaceflight missions, astronauts are exposed to an extreme environment with high levels of radiation and microgravity, which negatively influence the cardiovascular system. With an increased development in space exploration and growing interest in manned missions to mars, it is of high importance to evaluate the health risks associated with long‐duration spaceflights. The increased risk of oxidative stress and inflammatory damage post‐spaceflight has been associated with cardiovascular dysfunction, but little is known about the influence of space exposure on erectile function, which is a critical component for quality of life in men. The purpose of this investigation was to determine the influence of long‐duration spaceflight on corpus cavernosum (CC) function. 86 adult male Sprague‐Dawley rats were randomized into 6 different groups with half of them enduring 4‐weeks of hindlimb unloading (HLU), and exposed to sham, 0.75Gy or 1.5Gy of simulated galactic cosmic radiation at the Ground‐based GCR Simulator at the NASA Space Radiation Laboratory (NSRL). Following a 6–9‐month recovery the rats were sacrificed, and CC tissue segments were harvested and mounted into a muscle strip myograph system for ex vivo functional assessment. CC reactivity to six 10‐s,30V‐electric field stimulations with progressive frequencies in the range of 1‐32 Hz targeting the non‐adrenergic non‐cholinergic (NANC) stimulation was assessed. The effects of HLU and radiation were determined by two‐way repeated measures ANOVA. High levels of radiation significantly decreased NANC‐mediated relaxation of the CC. Treatment with nitric oxide synthase (NOS) inhibitor L‐NAME completely inhibited tissue response to electric stimulation, showing that the relaxation was mainly driven by nitric oxide. Incubation of the CC tissue with the xanthine oxidase inhibitor allopurinol, the mitochondria‐targeted antioxidant mito‐TEMPO, and the superoxide dismutase (SOD) mimetic TEMPOL increased the relaxation response of the erectile tissue for the groups exposed to radiation, with little or opposite effect on the groups under no radiation exposure. Upon treatment with the Arginase inhibitor S‐(2‐boronoethyl)‐l‐cysteine (BEC), CC tissue for the groups under radiation exposure showed a significant improvement in relaxation under NANC electric field stimulation. Comparably, western blot analysis showed increased levels of oxidative stress and lipid peroxidation in the corpus cavernosum on the groups under radiation exposure by assessment of 4‐hydroxynonenal (HNE), as well as higher levels of arginase 1 and 2. This information suggests that increased oxidative stress from radiation exposure in long spaceflight trips impairs corpus cavernosum vasoreactivity and exposes new factors to consider with space exploration to ensure that astronauts can return to normal life after long trips in space.

Geoffrey Burnstock 1929–2020

Geoffrey Burnstock was a biomedical scientist who gained renown for his discovery that adenosine 5′-triphosphate (ATP) functions as an extracellular signalling molecule. Born in London and educated at King’s and University Colleges, he did postdoctoral work at Mill Hill and Oxford. He moved in 1959 to the Department of Zoology at the University of Melbourne because he sensed there a greater freedom to challenge established thinking in physiology. His group found that transmission from sympathetic and parasympathetic autonomic nerves to smooth muscle was in some places not mediated by the accepted chemical messengers (noradrenaline and acetylcholine). He amassed evidence that ATP was this non-adrenergic, non-cholinergic (NANC) transmitter, using biochemical, histological and electrophysiological approaches: heretically, he styled this ‘purinergic transmission’. Geoff further upset dogma in the 1970s by proposing ‘co-transmission’ in which some nerves released ATP in addition to either noradrenaline or acetylcholine. He distinguished pharmacologically P1 receptors (activated best by adenosine and blocked by xanthines) and P2 receptors (activated best by purine nucleotides such as ATP) and he proposed in 1985 that the latter embraced P2X (ion channel) and P2Y (G protein-coupled) subtypes: about ten years later these categories were substantiated by cDNA cloning. From 1975 until his retirement in 1997, Geoff was head of Anatomy and Embryology at University College London (UCL), which he developed energetically into a large and strong research department. Later, as head of the Autonomic Research Institute at the Royal Free (part of UCL), he continued to collaborate extensively, and founded several journals and international professional societies. He widely sought clinical benefit for his discoveries, and both P2X and P2Y receptors have been developed as the targets of useful therapeutics (gefapixant, clopidogrel). Geoff was proud of his modest, rather humble, background and eschewed formality. He may have smiled when his early discoveries were met with cynicism, even ridicule (‘pure-imagine’ transmission noted one amusing critic), but this just reinforced his resolve and encouraged his encyclopaedic oeuvre.

Nitric Oxide: From Gastric Motility to Gastric Dysmotility

It is known that nitric oxide (NO) plays a key physiological role in the control of gastrointestinal (GI) motor phenomena. In this respect, NO is considered as the main non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter responsible for smooth muscle relaxation. Moreover, many substances (including hormones) have been reported to modulate NO production leading to changes in motor responses, further underlying the importance of this molecule in the control of GI motility. An impaired NO production/release has indeed been reported to be implicated in some GI dysmotility. In this article we wanted to focus on the influence of NO on gastric motility by summarizing knowledge regarding its role in both physiological and pathological conditions. The main role of NO on regulating gastric smooth muscle motor responses, with particular reference to NO synthases expression and signaling pathways, is discussed. A deeper knowledge of nitrergic mechanisms is important for a better understanding of their involvement in gastric pathophysiological conditions of hypo- or hyper-motility states and for future therapeutic approaches. A possible role of substances which, by interfering with NO production, could prove useful in managing such motor disorders has been advanced.

Pathophysiological Changes in the Enteric Nervous System of Rotenone-Exposed Mice as Early Radiological Markers for Parkinson's Disease.

Parkinson's disease (PD) is known to involve the peripheral nervous system (PNS) and the enteric nervous system (ENS). Functional changes in PNS and ENS appear early in the course of the disease and are responsible for some of the non-motor symptoms observed in PD patients like constipation, that can precede the appearance of motor symptoms by years. Here we analyzed the effect of the pesticide rotenone, a mitochondrial Complex I inhibitor, on the function and neuronal composition of the ENS by measuring intestinal contractility in a tissue bath and by analyzing related protein expression. Our results show that rotenone changes the normal physiological response of the intestine to carbachol, dopamine and electric field stimulation (EFS). Changes in the reaction to EFS seem to be related to the reduction in the cholinergic input but also related to the noradrenergic input, as suggested by the non-adrenergic non-cholinergic (NANC) reaction to the EFS in rotenone-exposed mice. The magnitude and direction of these alterations varies between intestinal regions and exposure times and is associated with an early up-regulation of dopaminergic, cholinergic and adrenergic receptors and an irregular reduction in the amount of enteric neurons in rotenone-exposed mice. The early appearance of these alterations, that start occurring before the substantia nigra is affected in this mouse model, suggests that these alterations could be also observed in patients before the onset of motor symptoms and makes them ideal potential candidates to be used as radiological markers for the detection of Parkinson's disease in its early stages.

Signaling and function of cardiac autonomic nervous system receptors: Insights from the GPCR signalling universe.

The two branches of the autonomic nervous system (ANS), adrenergic and cholinergic, exert a multitude of effects on the human myocardium thanks to the activation of distinct G protein-coupled receptors (GPCRs) expressed on the plasma membranes of cardiac myocytes, cardiac fibroblasts, and coronary vascular endothelial cells. Norepinephrine (NE)/epinephrine (Epi) and acetylcholine (ACh) are released from cardiac ANS terminals and mediate the biological actions of the ANS on the heart via stimulation of cardiac adrenergic or muscarinic receptors, respectively. In addition, several other neurotransmitters/hormones act as facilitators of ANS neurotransmission in the heart, taking part in the so-called nonadrenergic noncholinergic (NANC) part of the ANS's control of cardiac function. These NANC mediators also use several different cell membrane-residing GPCRs to exert their effects in the myocardium. Cardiac ANS dysfunction and an imbalance between the activities of its two branches underlie a variety of cardiovascular diseases, from heart failure and hypertension to coronary artery disease, myocardial ischemia, and arrhythmias. In this review, we present the main well-established signaling modalities used by cardiac autonomic GPCRs, including receptors for salient NANC mediators, and we also highlight the latest developments pertaining to cardiac cell type-specific signal transduction, resulting in cell type-specific cardiac effects of each of these autonomic receptors.

Mechanistic role of antioxidants in rescuing delayed gastric emptying in high fat diet induced diabetic female mice

Diabetic gastroparesis (DG) exhibits delayed gastric emptying (GE) due to impaired gastric non-adrenergic, non-cholinergic (NANC) relaxation. These defects are due to loss or reduction of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) that causes reduced expression and/or dimerization of neuronal nitric oxide synthase alpha (nNOSα) gene expression and function. We investigated the effect of potent Nrf2 activators (cinnamaldehyde [CNM] & curcumin [CUR]) on GE in obesity-induced diabetic female mice. We fed adult female homozygous Nfe2l2-/- (Nrf2 KO) and wild-type (WT) female mice with either a high-fat diet (HFD) or a normal diet (ND) for a period of 16 weeks. Groups of HFD mice were fed with CUR or CNM either at 6th or 10th week respectively. Our results demonstrate that supplementation of CNM or CUR restored impaired nitrergic relaxation and attenuated delayed GE in HFD fed mice. Supplementation of CNM or CUR normalized altered gastric antrum protein expression of (1) p-ERK/p-JNK/MAPK/p-GSK-3β, (2) BH4 (Cofactor of nNOS) biosynthesis enzyme GCH-1 and the GSH/GSSG ratio, (3) nNOSα protein & dimerization and soluble guanylate cyclase (sGC), (4) AhR and ER expression, (5) inflammatory cytokines (TNF α, IL-1β, IL-6), (6)TLR-4, as well as (7) reduced oxidative stress markers in WT but not in Nrf2 KO obesity-induced chronic diabetic female mice. Immunoprecipitation experiments revealed an interaction between nNOS and Nrf2 proteins. Our results conclude that Nrf2 activation restores nitrergic-mediated gastric motility and GE by normalizing inflammation and oxidative stress induced by obesity-induced chronic diabetes.

Postprandial cardiorespiratory responses and the regulation of digestion-associated tachycardia in Nile tilapia (Oreochromis niloticus).

Cardiorespiratory adjustments that occur after feeding are essential to supply the demands of digestion in vertebrates. The well-documented postprandial tachycardia is triggered by an increase in adrenergic activity and by non-adrenergic non-cholinergic (NANC) factors in mammals and crocodilians, while it is linked to a withdrawal of vagal drive and NANC factors in non-crocodilian ectotherms-except for fish, in which the sole investigation available indicated no participation of NANC factors. On the other hand, postprandial ventilatory adjustments vary widely among air-breathing vertebrates, with different species exhibiting hyperventilation, hypoventilation, or even no changes at all. Regarding fish, which live in an environment with low oxygen capacitance that requires great ventilatory effort for oxygen uptake, data on the ventilatory consequences of feeding are also scarce. Thus, the present study sought to investigate the postprandial cardiorespiratory adjustments and the mediation of digestion-associated tachycardia in the unimodal water-breathing teleost Oreochromis niloticus. Heart rate (fH), cardiac autonomic tones, ventilation rate (fV), ventilation amplitude, total ventilation and fH/fV variability were assessed both in fasting and digesting animals under untreated condition, as well as after muscarinic cholinergic blockade with atropine and double autonomic blockade with atropine and propranolol. The results revealed that digestion was associated with marked tachycardia in O. niloticus, determined by a reduction in cardiac parasympathetic activity and by circulating NANC factors-the first time such positive chronotropes were detected in digesting fish. Unexpectedly, postprandial ventilatory alterations were not observed, although digestion triggered mechanisms that were presumed to increase oxygen uptake, such as cardiorespiratory synchrony.

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.

312 Endogenous Vasoactive Peptides in the Control of Penile Erectile Tissue: Is there a Role of Arginine-Vasopressin (AVP)?

The role of the adrenergic system in mediating the constant tone of corpus cavernosum (CC) smooth musculature during penile flaccidity and in returning the erect penis into the flaccid state is well established. However, it is not yet known whether additional non-adrenergic, non-cholinergic (NANC) transmitters are involved in this process. Arginine-Vasopressin (AVP, ADH), a pituitary peptide hormone with vasoconstrictor properties, has been assumed to be an important mediator contributing to such a control. The present study was conducted in order to determine whether there is a role of AVP in controlling CC smooth muscle tension. Plasma levels of AVP (ng/L) were determined in the systemic and cavernous blood of 25 healthy adult male volunteers who were exposed to visual and tactile erotic stimuli in order to elicit penile tumescence and erection. Whole blood was aspirated from the CC and a cubital vein. Following the separation of plasma volumes, AVP was quantified by means of radioimmunometric methods (RIA). The effects of AVP (0.1 nM - 100 nM) on isolated human penile erectile tissue at basal tension were examined using the tissue bath technique.

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.

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.

Akt phosphorylation of neuronal nitric oxide synthase regulates gastrointestinal motility in mouse ileum

Nitric oxide (NO) is a major inhibitory neurotransmitter that mediates nonadrenergic noncholinergic (NANC) signaling. Neuronal NO synthase (nNOS) is activated by Ca2+/calmodulin to produce NO, which causes smooth muscle relaxation to regulate physiologic tone. nNOS serine1412 (S1412) phosphorylation may reduce the activating Ca2+ requirement and sustain NO production. We developed and characterized a nonphosphorylatable nNOSS1412A knock-in mouse and evaluated its enteric neurotransmission and gastrointestinal (GI) motility to understand the physiologic significance of nNOS S1412 phosphorylation. Electrical field stimulation (EFS) of wild-type (WT) mouse ileum induced nNOS S1412 phosphorylation that was blocked by tetrodotoxin and by inhibitors of the protein kinase Akt but not by PKA inhibitors. Low-frequency depolarization increased nNOS S1412 phosphorylation and relaxed WT ileum but only partially relaxed nNOSS1412A ileum. At higher frequencies, nNOS S1412 had no effect. nNOSS1412A ileum expressed less phosphodiesterase-5 and was more sensitive to relaxation by exogenous NO. Under non-NANC conditions, peristalsis and segmentation were faster in the nNOSS1412A ileum. Together these findings show that neuronal depolarization stimulates enteric nNOS phosphorylation by Akt to promote normal GI motility. Thus, phosphorylation of nNOS S1412 is a significant regulatory mechanism for nitrergic neurotransmission in the gut.

Ganglia in the Human Fetal Lung.

Although pulmonary ganglia were considered to be an analogue of the myenteric ganglia of intestines in embryos, there seemed to be no morphological evaluation in the later stage of development. We conducted immunostainings of intrapulmonary nerves using 17 human fetuses at 14-18 and 28-34 weeks. The ganglion cells were small (15-20 μm in diameter) in the earlier group, but they increased in size (20-30 μm) in the late group. One ganglion, containing 5-30 cell bodies, was usually located "outside" of the bronchial smooth muscle or cartilage. In addition, a few ganglion was found beneath the mucosa of the trachea and principal bronchi. The highest density of ganglia (5-15 ganglia per section with 50 μm interval) was found at the origin of the subsegmental bronchi, but ganglia were absent along more peripheral bronchi those are responsible for contraction and obstruction of the airway. Therefore, in topographical relation between smooth muscle and nerve, intrapulmonary intrinsic neurons were different from intestinal myenteric neurons. Consequently, a previous hypothesis of "embryonic intramuscular bronchial ganglia" seemed not to be based on observations of the peripheral bronchus but on the central bronchus than the sub-subsegmental level. An extrinsic migration and redistribution of ganglia might occur at midterm to provide the final location outside of airway smooth muscles. Finally, no ganglion cell bodies were positive either for neuronal nitric oxide synthase or tyrosine hydroxylase. Instead of the classical entity of autonomic nerves, nonadrenergic noncholinergic (NANC) innervation might be dominant even in fetuses. Anat Rec, 302:2233-2244, 2019. © 2019 American Association for Anatomy.

Effect of Ferulic Acid on Bladder Over Activity in Diabetic Rat

AbstractBackground: Diabetic bladder dysfunction (DBD) shows a change in the neurotransmission patterns which could be of great importance for drug development.Aim of Study: This study aims to explore whether cholin-ergic or non-adrenergic non-cholinergic (NANC) pathways are altered in their participation in mediating the contraction of diabetic urinary bladder and if ferulic acid could improve DBD.Material and Methods: Rats were divided into three groups (n=12): Streptozotocin induced diabetic group, diabetic with ferulic acid supplemented group, and age matched control group. At the end of 10 weeks, blood samples were collected, and the bladders were removed, weighed, used to measure TBARS reduced glutathione (GSH), and super oxide dismutase (SOD) in bladder homogenate and to record contractility of bladder strips.Results: Diabetic rats received ferulic acid expressed significant reduction in blood glucose, fluid intake, urine output and improvement in bladder wt/body wt. ratio and oxidative stress markers. Ferulic group showed significant improvement in spontaneous contractility and bladder sensi-tivity to different agonist and antagonists when compared to those of diabetic group. The purinergic as well as cholinergic component are significantly decreased in FA treated rats than in diabetic ones which could be explained by its antioxidant activity that ameliorates oxidative stress associated with diabetes. So, in Conclusion: FA could be considered as a beneficial adjuvant in lines of DBD treatment.

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<0.05) and decreased IPA ACh relaxation (CON: 50.4±3.5%, CAST: 31.2±2.3%; p<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<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<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<0.05), erectile function (ICP/MAP; CAST+T 0.74±0.01 p<0.05) and improved ACh relaxation in IPA to values greater than CON (CAST+T: 63.9±2.9%; p<0.05). Similarly, CAST+T enhanced IPA NANC relaxation to levels greater than CON (CAST+T: 69.2±1.4%; p<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.

188 - nNOS phosphorylation mediates gastrointestinal motility

Purpose One third of the world’s population suffers from gastrointestinal (GI) dysmotility, a common feature of which is disrupted nitric oxide (NO) signaling and spastic contractility. In the healthy gut, neuronal NO synthase (nNOS) induces cGMP-dependent GI smooth muscle relaxation, which facilitates digestion. However, regulation of nNOS activity is poorly understood. PKA or Akt phosphorylation of nNOS S1412 enhances hypothalamic and penile NO synthesis. We hypothesized that nNOS S1412 phosphorylation promotes GI motility. Methods We quantified the contractile force of ileal segments from C57Bl/6 wildtype and nNOSS1412A mutant mice in an organ bath under non-adrenergic, non-cholinergic (NANC) conditions. Relaxation stimuli—neuronal depolarization (EFS; 20V, 2msec pulsewidth) or adenylate cyclase activation (FSK)—were applied to ileal segments in the presence of kinase, nNOS, or cGMP pathway inhibitors. We measured nNOS S1412 phosphorylation by western blot. We determined non-NANC ileal contraction velocity with the gastrointestinal motility monitor. Results EFS (1Hz) and FSK (300nM) increased nNOS S1412 phosphorylation by 310% (p Conclusions nNOS S1412 phosphorylation promotes GI motility by decreasing spastic contractility in murine ileal segments. Neuronal depolarization elicits Akt-dependent phosphorylation, while adenylate cyclase activation elicits PKA-dependent phosphorylation of nNOS. Common GI motility disorders like irritable bowel syndrome may benefit from therapies that upregulate nNOS S1412 phosphorylation.