Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

Expression of Notch1 and Jagged2 in the enteric nervous system.

The Notch signaling pathway is a vitally important pathway in regulating brain development. To explore the involvement of the Notch pathway in neuronal cells of adult rat gut, we investigated the expression of Notch1 and Jagged2 by in situ hybridization (ISH) and immunohistochemistry (IHC). In the enteric nervous system, Notch1 and Jagged2 were expressed in ganglia of the submucosal and myenteric plexus. Notch1 was preferentially expressed in cholinergic neurons lacking calretinin or nitric oxide synthase (NOS), whereas Jagged2 was present in most neuron subtypes. We propose that Notch1 and Jagged2 have a continuing role in the maintenance and function of neuronal cells in the adult enteric nervous system.     

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Abstract. The snail Helix lucorum has been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substrate l ‐arginine (l ‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca²⁺, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis in H. lucorum has a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.     

Expression of nitric oxide synthase isoforms in the mouse kidney: cellular localization and influence by lipopolysaccharide and Toll-like receptor 4

We determined the cellular mRNA expression of all intrarenal nitric oxide (NO)-producing NO synthase (NOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS) and inducible NOS (iNOS) in kidneys from wild-type mice (WT) and immune deficient Toll-like receptor 4 (TLR4) mutant mice, during normal physiological conditions and during a short-term (6–16 h) endotoxic condition caused by systemically administered lipopolysaccaride (LPS). Investigations were performed by means of in situ hybridization and polymerase chain reaction amplification techniques. In WT, LPS altered the expression rate of all intrarenal NOS isoforms in a differentiated but NOS-isoform coupled expression pattern, with iNOS induction, and up- and down-regulation of the otherwise constitutively expressed NOS isoforms, e.g. eNOS and nNOS and an iNOS isotype. In TLR4 mutants, LPS caused none or a lowered iNOS induction, but altered the expression rate of the constitutive NOS isoforms. It is concluded that the intrarenal spatial relation of individual NOS-isoforms and their alteration in expression provide the basis for versatile NO-mediated renal actions that may include local interactions between NOS isoforms and their individual NO-target sites, and that the NOS-isoform dependent events are regulated by TLR4 during endotoxic processes. These regulatory mechanisms are likely to participate in different pathophysiological conditions affecting NO-mediated renal functions.     

CaMKII Is Essential for the Function of the Enteric Nervous System

Background

Ca²⁺/calmodulin-dependent protein kinases (CaMKs) are major downstream mediators of neuronal calcium signaling that regulate multiple neuronal functions. CaMKII, one of the key CaMKs, plays a significant role in mediating cellular responses to external signaling molecules. Although calcium signaling plays an essential role in the enteric nervous system (ENS), the role of CaMKII in neurogenic intestinal function has not been determined. In this study, we investigated the function and expression pattern of CaMKII in the ENS across several mammalian species.

Methodology/Principal Findings

CaMKII expression was characterized by immunofluorescence analyses and Western Blot. CaMKII function was examined by intracellular recordings and by assays of colonic contractile activity. Immunoreactivity for CaMKII was detected in the ENS of guinea pig, mouse, rat and human preparations. In guinea pig ENS, CaMKII immunoreactivity was enriched in both nitric oxide synthase (NOS)- and calretinin-containing myenteric plexus neurons and non-cholinergic secretomotor/vasodilator neurons in the submucosal plexus. CaMKII immunoreactivity was also expressed in both cholinergic and non-cholinergic neurons in the ENS of mouse, rat and human. The selective CaMKII inhibitor, KN-62, suppressed stimulus-evoked purinergic slow EPSPs and ATP-induced slow EPSP-like response in guinea pig submucosal plexus, suggesting that CaMKII activity is required for some metabotropic synaptic transmissions in the ENS. More importantly, KN-62 significantly suppressed tetrodotoxin-induced contractile response in mouse colon, which suggests that CaMKII activity is a major determinant of the tonic neurogenic inhibition of this tissue.

Conclusion

ENS neurons across multiple mammalian species express CaMKII. CaMKII signaling constitutes an important molecular mechanism for controlling intestinal motility and secretion by regulating the excitability of musculomotor and secretomotor neurons. These findings revealed a fundamental role of CaMKII in the ENS and provide clues for the treatment of intestinal dysfunctions.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Structural diversity of NADPH diaphorase-reactive enteral networks in Stylommatophora (Gastropoda, Pulmonata)

Abstract. In this work we investigated the involvement of putative nitric oxide (NO)-forming neurons in enteric plexuses of stylommatophoran gastropods. The nitric oxide synthase (NOS)-containing cells were detected by NADPH diaphorase (NADPHd) histochemistry in the entreral nervous systems of several stylommatophoran species (Achatinacea: Achatina fulica , Helicacea: Cepaea hortensis, Cepaea nemoralis, Discus rotundatus, Helicella obvia, Helix lucorum, Helix lutescens, Monachoides umbrosa, Trichia hispida, Zebrina detrita , Succineacea: Succinea putris , Vertiliginacea: Clausilia dubia , Zonitacea: Arion ater, Arion subfuscus, Limax maximus ). We detected the NO synthesis of isolated midintestinal segments by Griess's quantification of nitrite, one end product of NO. Effects of the NOS substrate L-arginine and the NOS inhibitor Nω-nitro-L-arginine (NOARG) were also tested on nitrite production. We found NADPHd-reactive neurons and extrinsic nerves with NADPHd-stained fibers within the myenteric and submucosal networks of the midintestine of investigated members of Helicacea, Succineacea, and Vertiliginacea families. These networks innervated the midintestinal musculature and several nerve cells of the myenteric and submucosal plexi. In investigated members of Achatinacea and Zonitacea, NADPHd-stained networks were not detectable within the digestive tract. Administration of 1 mM L-arginine elevated, whereas 2 mM of NOARG diminished, the nitrite levels of the NADPHd-stained networks containing midintestine in C. nemoralis and H. lucorum . Enteral NADPHd staining was not detected in A. ater and L. maximus , and the nitrite production was not affected by L-arginine. Our results indicate a possible, but evolutionarily not conserved, NO-mediated enteral transmission in stylommatophoran gastropods.     

Acetylcholine inhibits nitric oxide (NO) synthesis in the gastropod nervous system

Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals. We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine. Nitrite production can be elevated by L-arginine but inhibited by N_ω-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine, acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails. The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory regulation of neuronal NO synthesis by ACh in an invertebrate species. This article is dedicated to Dr. Gábor Hollósi on the 50th anniversary of his graduation and being a teacher at the University of Debrecen.     

cGMP production is coupled to Ca-dependent nitric oxide generation in rabbit parotid acinar cells

We investigated the mechanism of guanosine 3′,5′-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a β-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca²⁺ mobilization, because intracellular Ca ²⁺ elevating reagents, such as thapsigargin and the Ca²⁺ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca²⁺ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by N^G-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca²⁺. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca²⁺ mobilization.     

Distribution and morphological features of nitrergic neurons in the porcine large intestine

The distribution of nitric oxide synthase (NOS), an enzyme involved in the synthesis of the presumed non-adrenergic noncholinergic inhibitory neurotransmitter nitric oxide (NO), was demonstrated in the enteric nervous system of the porcine caecum, colon and rectum. Techniques used were NOS-immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-histochemistry. Throughout the entire large intestine, NOS-immunoreactive (IR) and NADPHd-positive neurons were abundant in the myenteric and outer submucous plexus. In the inner submucous plexus, only a small number of positive neurons were found in the caecum and colon, while a moderate number was observed in the rectum. The nitrergic neurons in the porcine enteric nerve plexuses were of a range of sizes and shapes, with a small proportion showing immunostaining for vasoactive intestinal polypeptide. Varicose and non-varicose NOS-IR and NADPHd-positive nerve fibres were present in the ganglia and connecting strands of all three plexuses. Nerve fibres were also numerous in the circular muscle layer, scarce in the longitudinal muscle coat and negligible in the mucosal region. The abundance of NOS/NADPHd in the intrinsic innervation of the caecum, colon and rectum of the pig implicates NO as an important neuronal messenger in these regions of the gastrointestinal tract.     

Pivotal role of the interstitial cells of Cajal in the nitric oxide signaling pathway of rat small intestine. Morphological evidence

The nitric oxide (NO) signaling pathway is a major nonadrenergic-noncholinergic transmitter mechanism in the enteric nervous system. Our aim was to localize the enzymes in question, i.e., neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and cGMP-dependent kinase type I (cGK-I) in rat small intestine by indirect immunofluorescence. nNOS staining was found in neurons of the myenteric plexus and in varicose nerve fibers mainly in the circular muscle layer. The cells positive for neurokinin-1 (NK-1) receptor and c-kit (interstitial cells of Cajal, ICC) in the deep muscular plexus (DMP) did not show nNOS reactivity, but nNOS-positive nerve fibers were directly adjacent to them. sGC was found in flattened cells surrounding myenteric ganglia (periganglionic cells, PGC), in ICC of the DMP, faintly in smooth muscle cells (SMC), and in cells perivascularly scattered throughout the circular muscle layer. cGK-I immunoreactivity was found abundantly in PGC (which presumably are ICC), in ICC of DMP, in SMC of the innermost circular and longitudinal muscle layers, but less intensively in the outer circular layer. Weak cGK-I staining occurred in nerve cells within the myenteric and submucosal plexus. Conclusively the key enzymes of the NO signaling pathway are differentially distributed: Occurrence of nNOS exclusively in neurons and the presence of sGC and cGK-I predominantly in ICC suggest a sequence of neuronal NO release, activation of ICC, and consecutive smooth muscle relaxation. ICC of the DMP seem to be the primary targets for neurally released NO.     

Cyclical upregulated iNOS and long-term downregulated nNOS are the bases for relapse and quiescent phases in a rat model of IBD

We previously reported that indomethacin induces a chronic intestinal inflammation in the rat where the cyclical characteristic phases of Crohn's disease are manifested with a few days' interval and lasting for several months: active phase (high inflammation, hypomotility, bacterial translocation) and reactive phase (low inflammation, hypermotility, no bacterial translocation). In this study, we investigated the possible role of both constitutive and inducible isoforms of nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) in the cyclicity of active and reactive phases in rats with chronic intestinal inflammation. Rats selected at either active or reactive phases and from 2 to 60 days after indomethacin treatment were used. mRNA expression of both constitutive and inducible NOS and COX isoforms in each phase was evaluated by RT-PCR and cellular enzyme localization by immunohistochemistry. The effects of different COX and NOS inhibitors on the intestinal motor activity were tested. mRNA expression of COX-1 was not modified by inflammation, whereas mRNA expression of neuronal NOS was reduced in all indomethacin-treated rats. In contrast, NOS and COX inducible forms showed a cyclical oscillation. mRNA expression and protein of both iNOS and COX-2 increased only during active phases. The intestinal hypomotility associated with active phases was turned into hypermotility after the administration of selective iNOS inhibitors. Sustained downregulation of constitutive NOS caused hypermotility, possibly as a defense mechanism. However, this reaction was masked during the active phases due to the inhibitory effects of NO resulting from the increased levels of the inducible NOS isoform.     

Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum

In the present paper we show the inhibitory effect of melatonin on rat cerebellar nitric oxide synthase (NOS) activity. NO production was monitored by the stoichiometric conversion of L-arginine to L-citrulline. The inhibitory effect of melatonin was dose-dependent, with an IC₅₀ value of about 0.1 mM. However, a significant inhibition of enzyme activity (> 22%) was observed at 1 nM melatonin which is in the range of the physiological serum concentration of the hormone at night. The inhibitory effect of melatonin was observed exclusively in the presence of Ca⁺⁺. Results suggest a new and important role of the pineal hormone melatonin on central nervous system processes, i.e., by modulating NO production.     

Cholecystokinin activates specific enteric neurons in the rat small intestine

Cholecystokinin (CCK) is a peptide hormone released from the I-cells of the upper small intestine. CCK evokes a variety of physiological responses, such as stimulation of pancreatic secretion, reduction of food intake and inhibition of gastric emptying. Previously, we reported that CCK activates enteric neurons in the rat. However the specific subpopulations of enteric neurons activated by CCK have not been identified. In the work reported here, we utilized immunohistochemical detection of nuclear Fos, a marker for neuronal activation, and selected phenotypic markers to identify some of the neuronal subpopulations activated by CCK. The phenotypic markers that we examined were: nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calbindin (Cal), Calretinin (Calr), and neurofilament-M (NF-M). We found that in the myenteric plexus of the rat duodenum and jejunum, CCK activated NOS immunoreactive neurons. In the submucosal plexus of duodenum and jejunum, CCK activated Cal, Calr and NF-M immunoreactive neurons. CCK failed to activate NK-1R immunoreactive neurons in either plexus. Our results indicate that CCK activates distinct enteric neurons in the rat upper small intestine. Furthermore the fact that NOS immunoreactive neurons were activated suggests that CCK modulates the activity of inhibitory motor neurons in the myenteric plexus. Expression of Fos immunoreactivity in Calr and Cal immunoreactive neurons is consistent with a role for CCK in modulation of intrinsic sensory and/or secretomotor neuronal activity in the submucosal plexus.     

急性力竭运动对大鼠胃肠传输速率及回肠肌间神经丛氮能神经的影响

目的：研究力竭运动对大鼠胃肠动力的影响及其肠神经机制。方法：24只大鼠随机分成对照组和急性力竭运动组,建立力竭运动大鼠模型,测定胃肠传输速率,用酶组织化学方法和计算机图像分析技术对两组大鼠回肠肌间神经丛内氮能神经元的数目和一氧化氮合酶（NOS）的表达进行测定。结果：急性力竭运动组大鼠胃肠传输速率明显延迟,回肠肌间神经丛内氮能神经元的数目明显增多和NOS的表达显著增强（P〈0．05和P〈0．01）。结论：大鼠力竭运动后小肠肌问神经丛内氮能神经元的数目增多和NOS的表达增强可能是导致胃肠传输速率延迟的重要原因之一。     

Cyclic GMP and Nitric Oxide Synthase in Aging and Alzheimer's Disease

Cyclic guanosine monophosphate (cGMP) is an important secondary messenger synthesized by the guanylyl cyclases which are found in the soluble (sGC) and particular isoforms. In the central nervous system, the nitric oxide (NO)-sensitive sGC isoform is the major enzyme responsible for cGMP synthesis. Phosphodiesterases (PDEs) are enzymes for hydrolysis of cGMP in the brain, and they are mainly isoforms 2, 5, and 9. The NO/cGMP signaling pathway has been shown to play an important role in the process underlying learning and memory. Aging is associated with an increase in PDE expression and activity and a decrease in cGMP concentration. In addition, aging is also associated with an enhancement of neuronal NO synthase, a lowering of endothelial, and no alteration in inducible activity. The observed changes in NMDA receptor density along with the Ca²⁺/NO/cGMP pathway underscore the lower synaptic plasticity and cognitive performance during aging. This notion is in agreement with last data indicating that inhibitors of PDE2 and PDE9 improve learning and memory in older rats. In this review, we focus on recent studies supporting the role of Ca²⁺/NO/cGMP pathway in aging and Alzheimer's disease.     

Transient expression of neuronal nitric oxide synthase by neurons of the submucous plexus of the mouse small intestine

Although neurons containing neuronal nitric oxide synthase (NOS) are abundant in the myenteric plexus of the small intestine of all mammalian species examined to date, NOS-containing neurons are sparse in the submucous plexus, and there does not appear to be an innervation of the mucosa by nerve fibres containing NOS. In this study, we used immunohistochemical techniques to examine the presence of neuronal NOS in the mouse intestine during development. At embryonic day 18 and postnatal day 0 (P0), about 50% of the neurons in the submucous plexus of the small intestine showed strong immunoreactivity to NOS, and NOS-immunoreactive nerve fibres were present in the mucosa. By P7, there was a gradation in the intensity of NOS immunostaining exhibited by submucosal neurons, varying from intense to extremely weak. During subsequent development, the proportion of submucous neurons showing NOS immunoreactivity decreased, and immunoreactive nerve fibres were no longer observed in the mucosa. In adult mice, NOS neurons comprised only 3% of neurons in the submucous plexus, which is significantly less than at P0. In contrast to the submucous plexus, the percentage of neurons that showed NOS immunoreactivity in the myenteric plexus did not change significantly during development.     

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.