Nitrergic Nerves Research Articles

Nitric Oxide Is Essential for Generating the Minute Rhythm Contraction Pattern in the Small Intestine, Likely via ICC-DMP.

Nitrergic nerves have been proposed to play a critical role in the orchestration of peristaltic activities throughout the gastrointestinal tract. In the present study, we investigated the role of nitric oxide, using spatiotemporal mapping, in peristaltic activity of the whole ex vivo mouse intestine. We identified a propulsive motor pattern in the form of propagating myogenic contractions, that are clustered by the enteric nervous system into a minute rhythm that is dependent on nitric oxide. The cluster formation was abolished by TTX, lidocaine and nitric oxide synthesis inhibition, whereas the myogenic contractions, occurring at the ICC-MP initiated slow wave frequency, remained undisturbed. Cluster formation, inhibited by block of nitric oxide synthesis, was fully restored in a highly regular rhythmic fashion by a constant level of nitric oxide generated by sodium nitroprusside; but the action of sodium nitroprusside was inhibited by lidocaine indicating that it was relying on neural activity, but not rhythmic nitrergic nerve activity. Hence, distention-induced activity of cholinergic nerves and/or a co-factor within nitrergic nerves such as ATP is also a requirement for the minute rhythm. Cluster formation was dependent on distention but was not evoked by a distention reflex. Block of gap junction conductance by carbenoxolone, dose dependently inhibited, and eventually abolished clusters and contraction waves, likely associated, not with inhibition of nitrergic innervation, but by abolishing ICC network synchronization. An intriguing feature of the clusters was the presence of bands of rhythmic inhibitions at 4–8 cycles/min; these inhibitory patches occurred in the presence of tetrodotoxin or lidocaine and hence were not dependent on nitrergic nerves. We propose that the minute rhythm is generated by nitric oxide-induced rhythmic depolarization of the musculature via ICC-DMP.

The male external urethral sphincter is autonomically innervated.

The aim of the present study was to describe autonomic urethral sphincter (US) innervation using specific muscular and neuronal antibody markers and 3D reconstruction. We performed en-bloc removal of the entire pelvis of three male human fetuses between 18 and 40 weeks. Serial whole mount sections (5 μm intervals) were stained and investigated. The sections were stained with Masson's trichrome and Eosin Hematoxylin, and immunostained with: anti-SMA antibody for smooth muscle; anti-S100 antibody for all nerves; and anti-PMP22 antibody, anti-TH antibody, anti-CGRP antibody, anti-NOS antibody for somatic, adrenergic, sensory and nitrergic nerve fibers, respectively. The slides were digitized for 3D reconstruction to improve topographical understanding. An animated reconstruction of the autonomic innervation of the US was generated. The external and internal US are innervated by autonomic nerves of the inferior hypogastric plexus (IHP). These nerves are sympathetic (positive anti-TH antibody), sensory (positive anti-CGRP antibody), and nitrergic (positive anti-NOS antibody). Some autonomic fibers run within the neurovascular bundles, posterolaterally. Others run from the IHP to the posteromedial aspect of the prostate apex, above an through the rectourethral muscle. The external US is also innervated by somatic nerves (positive anti-PMP22 antibody) arising from the pudendal nerve, joining the midline but remaining below the rectourethral. This study provides anatomical evidence of an autonomic component in the innervation of the external US that travels in the neurovascular bundle. During radical prostatectomy, the rectourethral muscle and the neurovascular bundles are to be preserved, particularly during apical dissection.

Anatomical evidence of non-parasympathetic cardiac nitrergic nerve fibres in rat.

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) plays a major role in the neural control of circulation and in many cardiovascular diseases. However, the exact mechanism of how NO regulates these processes is still not fully understood. This study was designed to determine the possible sources of nitrergic nerve fibres supplying the heart attempting to imply their role in the cardiac neural control. Sections of medulla oblongata, vagal nerve, its rootlets and nodose ganglia, vagal cardiac branches, Th1 -Th5 spinal cord segments, dorsal root ganglia of C8 -Th5 spinal nerves, and stellate ganglia from 28 Wistar rats were examined applying double immunohistochemical staining for nNOS combined with choline acetyltransferase (ChAT), peripherin, substance P, calcitonin gene-related peptide, tyrosine hydroxylase or myelin basic protein. Our findings show that the most abundant population of purely nNOS-immunoreactive (IR) neuronal somata (NS) was observed in the nodose ganglia (37.4±1.3%). A high number of nitrergic NFs spread along the vagal nerve and entered its cardiac branches. All nitrergic neuronal somata (NS) in the nucleus ambiguus were simultaneously immunoreactive (IR) to ChAT and composed only a small subset of neurons (6%). In the dorsal nucleus of vagal nerve, biphenotypic nNOS-IR/ChAT-IR neurons composed 7.0±1.0%, while small purely nNOS-IR neurons were scarce. Nitrergic NS were plentifully distributed within the nuclei of solitary tract. In the examined dorsal root and stellate ganglia, a few nitrergic NS were sporadically present. The majority of sympathetic NS in the intermediolateral nucleus were simultaneously immunoreactive for nNOS and ChAT. In conclusion, an abundant population of nitrergic NS in the nodose ganglion implies that neuronal NO is involved in afferent cardiac innervation. Nevertheless, nNOS-IR neurons identified within vagal nuclei may play a role in the transmission of preganglionic parasympathetic nerve impulses.

The Influence of Bisphenol a on the Nitrergic Nervous Structures in the Domestic Porcine Uterus.

Bisphenol A (BPA) is one of the most common environmental pollutants among endocrine disruptors. Due to its similarity to estrogen, BPA may affect estrogen receptors and show adverse effects on many internal organs. The reproductive system is particularly vulnerable to the impact of BPA, but knowledge about BPA-induced changes in the innervation of the uterus is relatively scarce. Therefore, this study aimed to investigate the influence of various doses of BPA on nitrergic nerves supplying the uterus with the double immunofluorescence method. It has been shown that even low doses of BPA caused an increase in the number of nitrergic nerves in the uterine wall and changed their neurochemical characterization. During the present study, changes in the number of nitrergic nerves simultaneously immunoreactive to substance P, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating peptide, and/or cocaine- and amphetamine-regulated transcript were found under the influence of BPA. The obtained results strongly suggest that nitrergic nerves in the uterine wall participate in adaptive and/or protective processes aimed at homeostasis maintenance in the uterine activity under the impact of BPA.

Nitrergic perivascular innervation in health and diseases: Focus on vascular tone regulation.

For a long time, the vascular tone was considered to be regulated exclusively by tonic innervation of vasoconstrictor adrenergic nerves. However, accumulating experimental evidence has revealed the existence of nerves mediating vasodilatation, including perivascular nitrergic nerves (PNN), in a wide variety of mammalian species. Functioning of nitrergic vasodilator nerves is evidenced in several territories, including cerebral, mesenteric, pulmonary, renal, penile, uterine and cutaneous arteries. Nitric oxide (NO) is the main neurogenic vasodilator in cerebral arteries and acts as a counter-regulatory mechanism for adrenergic vasoconstriction in other vascular territories. In the penis, NO relaxes the vascular and cavernous smooth muscles leading to penile erection. Furthermore, when interacting with other perivascular nerves, NO can act as a neuromodulator. PNN dysfunction is involved in the genesis and maintenance of vascular disorders associated with arterial and portal hypertension, diabetes, ageing, obesity, cirrhosis and hormonal changes. For example defective nitrergic function contributes to enhanced sympathetic neurotransmission, vasoconstriction and blood pressure in some animal models of hypertension. In diabetic animals and humans, dysfunctional nitrergic neurotransmission in the corpus cavernosum is associated with erectile dysfunction. However, in some vascular beds of hypertensive and diabetic animals, an increased PNN function has been described as a compensatory mechanism to the increased vascular resistance. The present review summarizes current understanding on the role of PNN in control of vascular tone, its alterations under different conditions and the associated mechanisms. The knowledge of these changes can serve to better understand the mechanisms involved in these disorders and help in planning new treatments.

Localisation analysis of nerves in the mouse pancreas reveals the sites of highest nerve density and nociceptive innervation.

Neuropathy and neuro-inflammation drive the severe pain and disease progression in human chronic pancreatitis and pancreatic cancer. Mice, especially genetically induced-mouse models, have been increasingly utilized in mechanistic research on pancreatic neuropathy, but the normal "peripheral neurobiology" of the mouse pancreas has not yet been critically compared to human pancreas. We introduced a standardized tissue-harvesting technique that preserves the anatomic orientation of the mouse pancreas and allows complete sectioning in an anterior to posterior fashion. We applied immunohistochemistry and quantitative colorimetry of all nerves from the whole organ for studying pancreatic neuro-anatomy. Nerves in the mouse pancreas appeared as "clusters" of nerve trunks in contrast to singly distributed nerve trunks in the human pancreas. Nerve trunks in the mouse pancreas were exclusively found around intrapancreatic blood vessels, and around lymphoid structures. The majority of nerve trunks were located in the pancreatic head (0.15±0.08% of tissue area) and the anterior/front surface of the corpus/body (0.17±0.27%), thus significantly more than in the tail (0.02±0.02%, P=.006). Nerves in the tail included a higher proportion of nociceptive fibers, but the absolute majority, ie, ca. 70%, of all nociceptive fibers, were localized in the head. Mice heterozygous for Bdnf knockout allele (Bdnf+/- ) exhibited enrichment of nitrergic nerve fibers specifically in the head and corpus. Neuro-anatomy of the "mesenteric type" mouse pancreas is highly different from the "compact" human pancreas. Studies that aim at reproducing human pancreatic neuro-phenomena in mouse models should pay diligent attention to these anatomic differences.

Effects of the Astrocyte Endozepine Octadecaneuropeptide (ODN) on Hypoglycemia Patterns of Ventromedial Hypothalamic Nucleus (VMN) Metabolic‐Sensory Neuron Transmitter Signaling and Energy Sensor Activation in Male and Female Rats

The astrocyte gliotransmitter octadecaneuropeptide (ODN) is a cleavage product of diazepam‐binding inhibitor (DBI) and an endogenous central benzodiazepine receptor ligand. ODN regulation of feeding responses to excess or diminished glucose availability in the brain infers ability to modulate neural metabolic‐sensory function [Lanfray et al., 2013]. The ventromedial hypothalamic nucleus (VMN), a principal component of the neural glucostatic network, integrates nutrient and other cues to shape glucose counter‐regulation. Our studies show that VMN gluco‐stimulatory nitric oxide (NO) and gluco‐inhibitory g‐aminobutyric acid (GABA) neurons express the ultra‐sensitive energy sensor 5′‐AMP‐activated protein kinase (AMPK). This research examined the premise that ODN exerts sex‐specific effects on hypoglycemic patterns of VMN nitrergic and/or GABAergic nerve cell sensor activation and neurotransmitter signaling. Testes‐intact male and ovariectomized, estradiol‐replaced female rats were pretreated by intracerebroventricular administration of the ODN octapeptide (OP) fragment or vehicle prior to insulin injection. VMN NO and GABA cells were identified by immunocytochemistry prior to combinatory laser‐catapult dissection/high‐sensitivity Western blotting. Results identified nitrergic cells exhibiting OP‐reversible effects of hypoglycemia on expression profiles of the NO marker protein neuronal nitric oxide synthase (nNOS), AMPK, and phospho (activated)‐AMPK in distinctive regions of the male and female VMN. In each sex, OP effects on GABA neuron AMPK protein content and activation were also observed at specific levels of the VMN. Outcomes provide novel evidence that ODN may critically impact VMN substrate fuel screening. Further studies are needed to characterize neuroanatomical connectivity of OP‐sensitive nitrergic and GABAergic neurons within central metabolic pathways, and to examine whether metabolic cues that regulate VMN astrocyte ODN release, astrocyte reactivity to ODN‐controlling signals, and metabolic‐sensory nerve cell receptivity to ODN are sex‐dimorphic.Support or Funding InformationNIH DK 109382

Erectile dysfunction in diabetes mellitus: A review

The International Diabetes Federation estimates that globally there are 425 million people with diabetes. Estimates of the prevalence of erectile dysfunction (ED) in men with diabetes range from 20% to 85% in different studies. Among men with ED, those with diabetes are likely to experience the problem 10–15 years earlier than men without diabetes. This review aims to provide an update of the epidemiology, pathophysiology and management of ED in diabetes patients in India. The proposed mechanisms of ED in diabetic patients include elevated advanced glycation end-products and increased levels of oxygen-free radicals, impaired nitric oxide (NO) synthesis, increased endothelin B receptor binding sites and ultrastructural changes, upregulated RhoA/Rho-kinase pathway, NO-dependent selective nitrergic nerve degeneration and impaired cyclic guanosine monophosphate-dependent kinase-1. Modifiable risk factors for ED include smoking, lack of physical activity, wrong diets, overweight or obesity, metabolic syndrome and excessive alcohol consumption. Therefore, the promotion of healthful lifestyles would yield great benefits in reducing the burden of ED. The treatment of diabetic ED is multimodal. The treatment of the underlying hyperglycaemia and comorbidities is of utmost importance to prevent the progression of the disease. The peripherally acting oral phosphodiesterase type 5 inhibitors are the mainstay of oral medical treatment of ED in diabetics. Vacuum erection devices are an additional a non-invasive treatment option. The local administration of vasoactive medication through urethral suppository or intracorporal injection can be effective with minimal side-effects.

Smooth muscle of the male pelvic floor: An anatomic study.

Knowledge of the anatomy of the male pelvic floor is important to avoid damaging the pelvic floor muscles during surgery. We set out to explore the structure and innervation of the smooth muscle (SM) of the whole pelvic floor using male fetuses. We removed en-bloc the entire pelvis of three male fetuses. The specimens were serially sectioned before being stained with Masson's trichrome and hematoxylin and eosin, and immunostained for SMs, and somatic, adrenergic, sensory and nitrergic nerve fibers. Slides were digitized for three-dimensional reconstruction. We individualized a middle compartment that contains SM cells. This compartment is in close relation with the levator ani muscle (LAM), rectum, and urethra. We describe a posterior part of the middle compartment posterior to the rectal wall and an anterior part anterior to the rectal wall. The anterior part is split into (1) a centro-levator area of SM cells localized between the right and left LAM, (2) an endo-levator area that upholsters the internal aspect of the LAM, and (3) an infra-levator area below the LAM. All these areas are innervated by autonomic nerves coming from the inferior hypogastric plexus. The core and the infra-levator area receive the cavernous nerve and nerves supplying the urethra. We thus demonstrate that these muscular structures are smooth and under autonomic influence. These findings are relevant for the pelvic surgeon, and especially the urologist, during radical prostatectomy, abdominoperineal resection and intersphincteric resection. Clin. Anat., 2019. © 2019 Wiley Periodicals, Inc.

Berberine restored nitrergic and adrenergic function in mesenteric and iliac arteries from streptozotocin-induced diabetic rats

Ethnopharmacological relevancePerivascular neuropathy was reported to involve in the vascular disorders associated with diabetes. The dried rhizomes of Coptis chinensis Franch. (Latin name: Coptidis Rhizoma; common name: Huang Lian in China), used frequently in Traditional Chinese medicine to treat diabetes (Xiaoke), have been confirmed to possess beneficial effects on diabetic peripheral neuropathy by modern clinical and pharmacological studies. Berberine (BBR), the main effective component of Huang Lian in the treatment of diabetes, is reported to ameliorate diabetic central and peripheral neuropathy. However, the effects of BBR on nerve function of mesenteric and iliac arteries are unclear. Aim of the studyTo investigate the effects of BBR on the diabetes-induced changes in nitrergic and adrenergic function in mesenteric and iliac arteries. Materials and methodsIn this study, the animals were randomized into three groups: control rats, diabetic rats, and diabetic rats gavaged with BBR. We established diabetic rat model using intraperitoneal injection of streptozotocin (STZ, 55 mg kg−1). Two weeks after model establishment, those in the BBR-treated groups were gavaged with berberine chloride (Sichuan Xieli Fharmaceutical. Co., Ltd; 200 mg·kg−1·day−1) diluted in distilled water for another 2 weeks. The superior mesenteric artery and iliac artery were excised. Electric field stimulation (EFS) was used to induce arterial vasoconstriction and explore (1) the diabetes-induced changes in neurogenic function of the superior mesenteric artery and iliac artery; (2) the effects of BBR on neurovascular dysfunction in the early stage of STZ-induced diabetic rats. Nitric oxide (NO) and noradrenaline (NA) released from the nitrergic and adrenergic nerves were quantified using fluorescence assays and ELISA, respectively. ResultsEFS induced frequency-dependent vasoconstrictions in both superior mesenteric and iliac artery, and the contractile responses of arteries were abolished by 0.1 μmol·L−1 tetrodotoxin (TTX), or inhibited by 1 μmol·L−1 phentolamine or increased by 0.1 mmol·L−1 Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). In superior mesenteric artery, but not in iliac artery, the changes of contractile responses with L-NAME were significantly decreased in diabetic rats, and NO release was less also. In contrast, in iliac artery of diabetic rats, but not in superior mesenteric artery, the changes of contractile responses with phentolamine were increased, and NA release was increased significantly. All these changes in diabetic rats on both superior mesenteric artery and iliac artery were reversed by treated with BBR. ConclusionsIn the STZ-induced early diabetic rats, neural control of mesenteric and iliac vasomotor tone are altered differently. The diminished nitrergic nerve in superior mesenteric artery and enhanced adrenergic nerve in iliac artery both contributed to increased vasocontrictor responses. All these changes in diabetic rats were reversed by BBR, suggesting a novel mechanism of BBR in balance of neural regulation of vascular tone.

Sex-dimorphic estrogen receptor regulation of ventromedial hypothalamic nucleus glucoregulatory neuron adrenergic receptor expression in hypoglycemic male and female rats

The ventromedial hypothalamic nucleus (VMN) is a vital component of the neural circuitry that regulates glucostasis. Norepinephrine (NE) controls VMN gluco-inhibitory γ-aminobutyric acid (GABA) and gluco-stimulatory nitric oxide (NO) transmission. Sex-specific insulin-induced hypoglycemic (IIH) patterns of VMN GABA signaling are estrogen receptor-alpha (ERα)- and -beta (ERβ)-dependent. Current research utilized combinatory immunocytochemistry, laser-microdissection, and Western blot techniques in a pharmacological approach to address the hypothesis that ERα and/or -β mediate sex-dimorphic VMN GABAergic and/or nitrergic nerve cell receptivity to NE and estradiol during IIH. The impact of these ER on expression of the pyruvate recycling pathway marker proteins glutaminase (GLS) and malic enzyme-1 (ME-1) was also examined. Both VMN neuron populations express ERα, ERβ, and G protein-coupled estrogen receptor-1 (GPER), along with alpha1, alpha2, and beta1 adrenergic receptor (AR) proteins. NO neurons exhibited ERα/β-dependent (beta1 AR, GPER) and -independent (alpha1 AR) sex differences in receptor protein responses to hypoglycemia. Similarly, sex-dimorphic effects of IIH on alpha1 AR, alpha2 AR, and ERα profiles in GABA neurons involve ERα/β. These ERs also underlie divergent adjustments in gluco-regulatory nerve cell GLS and ME-1 protein expression in hypoglycemic males and females. Sex-specific nitrergic and GABAergic nerve cell sensitivity to NE and E, respectively, during IIH may contribute to sex-contingent patterns of neurotransmitter signaling.

Different vasodilator mechanisms in intermediate- and small-sized arteries from the hindlimb vasculature of the toad Rhinella marina.

In this study, myography was used to determine the effect of arterial size on nitric oxide (NO) vasodilatory mechanisms in the hindlimb vasculature of the toad Rhinella marina. Immunohistochemical analysis showed NO synthase (NOS) 1 immunoreactivity in perivascular nitrergic nerves in the iliac and sciatic arteries. Furthermore, NOS3 immunoreactivity was observed in the vascular smooth muscle of the sciatic artery, but not the endothelium. Acetylcholine (ACh) was used to facilitate intracellular Ca2+ signaling to activate vasodilatory pathways in the arteries. In the iliac artery, ACh-mediated vasodilation was abolished by blockade of the soluble guanylate cyclase pathway with the soluble guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, 10-5 M) and blockade of the prostaglandin signaling pathway with indomethacin (10-5 M). Furthermore, disruption of the endothelium had no effect on the ACh-mediated vasodilation in the iliac artery, and generic inhibition of NOS with Nω-nitro-l-arginine (3 × 10-4 M) significantly inhibited the vasodilation, indicating NO signaling. In contrast to the iliac artery, ACh-mediated vasodilation of the sciatic artery had a significant endothelium-dependent component. Interestingly, the vasodilation was not significantly affected by Nω-nitro-l-arginine, but it was significantly inhibited by the specific NOS1 inhibitor N5-(1-imino-3-butenyl)-l-ornithine (vinyl-l-NIO, 10-4 M). ODQ mostly inhibited the ACh-mediated vasodilation. In addition, indomethacin also significantly inhibited the ACh-mediated vasodilation, indicating a role for prostaglandins in the sciatic artery. This study found that the mechanisms of vasodilation in the hindlimb vasculature of R. marina vary with vessel size and that the endothelium is involved in vasodilation in the smaller sciatic artery.

The Adaptation of Small Intestine Nitregic Myenteric Neurons on Rats (Rattus norvegicus) to High Fat Diet

High fat diet can result in the loss of nitrergic neurons in the myenteric plexus. The study aimed at finding out the effect of high fat diet on the adaptation of nitrergic nerve of rat intestine. It used 15 male rats (Rattus norvegicus) of a month of age with mean body weight of 53.73 gr. The rats were adapted for 7 days to individual cages with ad libitum feeding. After random adaptation, all of the rats were assigned to 3 groups of five rats, namely K-7, K-10, and K-13 groups. Feed and drinking water were given ad libitum. The treatment of the high fat diet lasted for 7 weeks. After the treatment, all of the rats were fasted for 12 hours and then killed. Subsequently, small intestine segments (duodenum, jejunum, and ileum) were taken. The intestinal segments were prepared by using NADPH-d histopathological technique to determine the morphometric changes of nitrergic myenteric neurons. During the treatment the rats were weighed every week and at the end of the study orbitalis vein blood measurement was carried out to see its glucose, cholesterol and cholecystokinin (CCK) plasma levels. The data of body weight, glucose, cholesterol, CCK levels, the total number of the nippergenic myenteric segments of the small intestine were statistically analyzed using Anova. The results of the study showed that the treatment of 7% to 13% fat diets for 7 weeks did not indicate any weight gain and any increase in cholescystokinin level, and any decrease in glucose level. However, it indicated significant increase in cholesterol level. The treatment of 10% and13% fat diets increased the total number of neurons in the jejunum and the ileum. Thus, it was concluded that the treatments of the high-fat diet of the rats (K-7, K-10, and K-13) for 7 weeks had significant effect on the adaptation of the neurons of the jejunum and the ileum.

031 Low intensity-shockwave therapy (Li-ESWT) delivered by Aries® improves erectile function and decreases cavernosal fibrosis of spontaneously hypertensive rats (SHR)

Li-ESWT has been reported to improve erectile function in patients with vasculogenic ED, and convert PDE5i non-responders into responders. Erectile function of SHR, a validated rat model of hypertension associated ED, is partially improved by PDE5is. Here, we investigated whether Li-ESWT could affect erectile function with and without acute sildenafil in SHR. A pronounced accumulation of collagen in erectile tissue of SHR has been described, thus we also investigated the histological features of SHR erectile tissue after ESWT. 2 groups of male SHR (CTRL and ESWT; n=14/group) were treated twice/week for 6 weeks. Only in the ESWT group, shockwaves were delivered to the penis at the mid-shaft and base (Aries®, Dornier MedTech, Germany). Each session comprised 1000 shocks per site (total: 2000 shocks, EFD: 0.06 mJ/mm2). 4 weeks after the final session, erectile function was assessed by recording intracavernosal pressure (ICP) and mean arterial pressure (MAP) under electrical stimulation of the cavernous nerve (ES CN), first in absence then in presence of sildenafil (0.3mg/kg iv). Then rats were sacrificed, the corpora cavernosa were harvested and prepared for histological analysis. Cavernosal smooth muscle (SM)/collagen ratio was assessed by trichrome staining. Cavernosal microvasculature and nitrergic nerves were assessed by immunohistology against CD31 and nNOS.

Differential effect of morphine on gastrointestinal transit, colonic contractions and nerve-evoked relaxations in Toll-Like Receptor deficient mice

Toll-like receptors (TLRs) are expressed in enteric neurons, glia, gastrointestinal (GI) smooth muscle and mucosa, yet their functional roles in the GI tract are not fully understood. TLRs have been linked to many of the undesirable central effects of chronic opioid administration including hyperalgesia and dependence via activation of central microglia. Opioid-induced bowel dysfunction (OIBD) remains a primary reason for the reduction or withdrawal of opioid analgesics. Morphine-induced inhibition of colonic motility was assessed in vivo by GI transit studies and in vitro using isolated colons from wildtype (WT) and TLR deficient mice. Morphine slowed movement of ingested content in WT but this retardation effect was attenuated in TLR4−/− and TLR2/4−/−. In isolated colons, morphine reduced amplitude and frequency colonic migrating motor contractions in both WT and TLR2/4−/−. Electrical field stimulation elicited distal colon relaxation that was potentiated by morphine in WT but not in TLR2/4−/−. Inhibitory junction potentials were of similar amplitude and kinetics in WT and TLR2/4−/− distal colon and not altered by morphine. Enteric nerve density and proportion of nitrergic nerves were similar in WT and TLR2/4−/− distal colon. These data suggest an involvement of TLRs in opioid pharmacodynamics and thus a potential interventional target for OIBD.

Galanin Administration Partially Restores Erectile Function After Cavernous Nerve Injury and Mediates Endogenous Nitrergic Nerve Outgrowth In Vitro

BackgroundPreviously, we found that the neuropeptide galanin was strongly upregulated soon after bilateral cavernous nerve injury (BCNI) and that galanin and its receptors were expressed in nitrergic erectile innervation. Galanin has been observed to exert neuroregenerative effects in dorsal root ganglion neurons, but evidence for these effects in the major pelvic ganglion (MPG) after BCNI is lacking. AimTo evaluate the neurotropic effects of galanin receptor agonists and antagonists in vitro in nitrergic neurons and MPG and in vivo in rats after BCNI. MethodsMale Sprague-Dawley rats underwent BCNI and sham surgery. Organ culture and single-cell neuron culture of the MPG were performed. Osmotic pump treatment with the galanin agonist in vivo and measurement of erectile response to electrostimulation after BCNI, immunohistochemical localization of galanin and receptors in the human neurovascular bundle, and myographic analysis of rat corpus cavernosum smooth muscle relaxation to galanin receptor agonists were investigated. OutcomesNeurite outgrowth in vitro and erectile response to electrostimulation after BCNI in vivo, immunohistochemical localization of galanin and receptors, and penile muscle relaxation in vitro. ResultsGalanin showed neurotrophic action in vitro and inhibition of endogenous galanin significantly impaired neurite outgrowth in nitrergic but not in sympathetic MPG neurons. In vivo administration of a selective galanin receptor-2 agonist, M1145, resulted in partial recovery of erectile function (EF) after BCNI. Galanin did not act as a direct vasodilator on corpus cavernosum muscle strips. Clinical TranslationEndogenous neurotrophins such as galanin could be used as a strategy to improve EF for patients after BCNI from radical prostatectomy. Strengths and LimitationsWe evaluated the effect of galanin on nerve regeneration and EF recovery in vivo and in vitro. Limitations include the lack of washout period for the in vivo experiment and absence of differences in the expression of neuronal markers between treatment groups. ConclusionsWe identified galanin as a potential endogenous mechanism for nerve regeneration after BCNI, which could play a physiologic role in EF recovery after radical prostatectomy. In vivo treatment with exogenous galanin was beneficial in enhancing EF recovery after BCNI, but further research is necessary to understand the underlying mechanisms.Weyne E, Hannan JL, Gevaert T, et al. Galanin Administration Partially Restores Erectile Function After Cavernous Nerve Injury and Mediates Endogenous Nitrergic Nerve Outgrowth In Vitro. J Sex Med 2018;15:480–491.

Effects of endogenous nitric oxide on adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide-containing nerve-mediated vasodilation in pithed rats

Vascular adrenergic nerves mainly regulate the tone of blood vessels. Calcitonin gene-related peptide-containing (CGRPergic) vasodilator nerves also participate in the regulation of vascular tone. Furthermore, there are nitric oxide (NO)-containing (nitrergic) nerves, which include NO in blood vessels as vasodilator nerves, but it remains unclear whether nitrergic nerves participate in vascular regulation. The present study investigated the role of nitrergic nerves in vascular responses to spinal cord stimulation (SCS) and vasoactive agents in pithed rats. Wistar rats were anesthetized and pithed, and vasopressor responses to SCS and injections of norepinephrine were observed. To evaluate vasorelaxant responses, the BP was increased by a continuous infusion of methoxamine with hexamethonium to block autonomic outflow. After the elevated BP stabilized, SCS and injections of acetylcholine (ACh), sodium nitroprusside (SNP), and CGRP were intravenously administered. We then evaluated the effects of the NO synthase (NOS) inhibitor, N-ω-nitro-L-arginine methylester hydrochloride (L-NAME), on these vascular responses. Pressor responses to SCS and norepinephrine in pithed rats were enhanced by L-NAME, while the combined infusion of L-NAME and L-arginine had no effect on these responses. L-NAME infusion significantly increased the release of norepinephrine evoked by SCS. In pithed rats with artificially increased BP and L-NAME infusion, depressor response to ACh (except for 0.05nmol/kg) was suppressed and SNP (only 2nmol/kg) was enhanced. However, depressor responses to SCS and CGRP were similar to control responses. The present results suggest endogenous NO regulates vascular tone through endothelium function and inhibition of adrenergic neurotransmission, but not through CGRPergic nerves.

Nitrergic cardiovascular regulation in the African lungfish, Protopterus aethiopicus

As a ubiquitous signaling molecule, nitric oxide (NO) exerts various important effects on the cardiovascular system and is involved in the regulation of vascular tone and myocardial metabolism in vertebrates. Lungfishes are closely related to tetrapods and provide an interesting possibility to understand the transition from water to land. Lungfishes are endowed with both systemic and pulmonary circulations, and their incompletely divided ventricle allows for blood to bypass either circuit. Lungfishes inhabit ephemeral waterbodies that may enforce prolonged aestivation during drought, throughout which nitric oxide synthase (NOS) expression is upregulated. To better understand the physiological relevance of NO on cardiovascular regulation in this transitory group, we measured vascular reactivity to muscarinic agonist acetylcholine, α- and β-adrenergic agonists (phenylephrine and isoproterenol, respectively), or the NO donor, sodium nitroprusside (SNP) on four vessel segments—efferent branchial arteries, gill artery, ductus arteriosus and pulmonary artery—from the African lungfish, Protopterus aethiopicus. In a simultaneous study, we measured oxygen consumption and twitch force in myocardial preparations in the presence and absence of an NOS inhibitor (asymmetric dimethylarginine; ADMA). Only the ductus arteriosus vasodilated in response to SNP. Isoproterenol caused vasodilation, whereas acetylcholine and phenylephrine vasoconstricted all vessel segments. NOS inhibition decreased myocardial force relative to oxygen consumption, indicating a lowered efficiency. We provide novel evidence that NO affects the vasculature of lungfish that may be derived from perivascular nitrergic nerves limited to the ductus arteriosus. Our data also suggests that NO exerts a tonic dampening of myocardial oxygen consumption which may be particularly important during aestivation.

Levator ani muscle innervation: Anatomical study in human fetus.

To characterize the nature and function of the levator ani muscle innervation pathways and to perform a comprehensive three-dimensional reconstruction of female pelvic innervation. A computer-assisted anatomical dissection protocol was applied to seven female human fetuses, after approval from the national biomedicine agency. Specimens were serially sectioned and immunostained for overall (antibody against protein S100), somatic (antibody against peripheral myelin protein 22), adrenergic (antibody against tyrosine hydroxylase), cholinergic (antibody against vesicular acetylcholine transferase), and nitrergic (antibody against the neural isoform of nitric oxide synthase) nerve fibers. Slides were digitized for three-dimensional reconstructions using WinSurf®. Three main nerve pathways to the levator ani muscle were observed: the levator ani nerve, the pudendal nerve, and the inferior hypogastric plexus. The pudendal nerve was both somatic and autonomic, located below the levator ani muscle (infralevator pathway), supplying innervation to the inferior aspect of the levator ani muscle. The levator ani nerve was solely somatic, located above the levator ani muscle (supralevator pathway), supplying innervation to the superior aspect of the levator ani muscle. The inferior hypogastric plexus nerve fibers were solely autonomic, located in between the levator ani muscle and pelvic organs (endolevator pathway), supplying innervation to the medial portion of the levator ani muscle. Our study provides a new representation of levator ani muscle innervation with three nerve pathways, and the levator ani muscle itself as an anatomical landmark.

Bacterial toxins activation of abbreviated urea cycle in porcine cerebral vascular smooth muscle cells

Nitric oxide (NO) overproduction via induction of inducible nitric oxide synthase (iNOS) is implicated in vasodilatory shock in sepsis, leading to septic encephalopathy and accelerating cerebral ischemic injury. An abbreviated urea-cycle (l-citrulline-l-arginine-NO cycle) has been demonstrated in cerebral perivascular nitrergic nerves and endothelial cells but not in normal cerebral vascular smooth muscle cell (CVSMC). This cycle indicates that argininosuccinate synthase (ASS) catalyzes l-citrulline (l-cit) conversion to form argininosuccinate (AS), and subsequent AS cleavage by argininosuccinate lyase (ASL) forms l-arginine (l-arg), the substrate for NO synthesis. The possibility that ASS enzyme in this cycle was induced in the CVSMC in sepsis was examined. Blood-vessel myography technique was used for measuring porcine isolated basilar arterial tone. NO in cultured CVSMC and in condition mediums were estimated by diaminofluorescein (DAF)-induced fluorescence and Griess reaction, respectively. Immunohistochemical and immunoblotting analyses were used to examine iNOS and ASS induction. l-cit and l-arg, which did not relax endothelium-denuded normal basilar arteries precontracted by U-46619, induced significant vasorelaxation with increased NO production in these arteries and the CVSMCs following 6-hour exposure to 20μg/ml lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Pre-treatment with pyrrolidine dithiocarbamate (PDTC) and salicylate (SAL) (NFκB inhibitors), aminoguanidine (AG, an iNOS inhibitor), and nitro-l-arg (NLA, a non-specific NOS inhibitor) blocked NO synthesis in the CVSMC and attenuated l-cit- and l-arg-induced relaxation of LPS- and LTA-treated arteries. Furthermore, immunohistochemical and immunoblotting studies demonstrated that expression of basal iNOS and ASS in the smooth muscle cell of arterial segments denuded of endothelium and the cultured CVSMCs was significantly increased following 6-hour incubation with LPS or LTA. This increased iNOS- and ASS-proteins expression in both preparations was inhibited by SAL, but was further increased by AG. These results indicate that LPS and LTA induce the l-cit-l-arg-NO cycle via induction of iNOS and ASS in the CVSMCs, accounting for massively increased NO-production and cerebral vasodilation in septic shock. Simultaneous inhibition of both pathways and NFκB-activation may be necessary to efficiently decrease or normalize NO production in the CVSMCs in this disease condition, and/or prevention and treatment of cerebral vessel-related brain dysfunctions. Our results further suggest to avoid using iNOS inhibitors alone which may cause upregulation of iNOS and ASS resulted from feedback-inhibition of iNOS activity. Accordingly, combined treatments with specific iNOS-activity inhibitor and inhibitor for iNOS genomic expression may provide a strategy in optimally managing brain sepsis and related encephalopathy associated with enhanced iNOS expression and NO overproduction.