Intestinal Bacteria Maintain Adult Enteric Nervous System and Nitrergic Neurons via Toll-like Receptor 2-induced Neurogenesis in Mice.

BACKGROUND & AIMS: The enteric nervous system (ENS) exists in close proximity to luminal bacteria. Intestinal microbes regulate ENS development, but little is known about their effects on adult enteric neurons. We investigated whether intestinal bacteria or their products affect the adult ENS via toll-like receptors (TLRs) in mice. METHODS: We performed studies with conventional C57/BL6, germ-free C57/BL6, Nestin-creERT2:tdTomato, Nestin-GFP, and ChAT-cre:tdTomato. Mice were given drinking water with ampicillin or without (controls). Germ-free mice were given drinking water with TLR2 agonist or without (controls). Some mice were given a blocking antibody against TLR2 or a TLR4 inhibitor. We performed whole gut transit, bead latency, and geometric center studies. Feces were collected and analyzed by 16S ribosomal RNA gene sequencing. Longitudinal muscle myenteric plexus (LMMP) tissues were collected, analyzed by immunohistochemistry, and levels of nitric oxide were measured. Cells were isolated from colonic LMMP of Nestin-creERT2:tdTomato mice and incubated with agonists of TLR2 (receptor for gram-positive bacteria), TLR4 (receptor for gram-negative bacteria), or distilled water (control) and analyzed by flow cytometry. RESULTS: Stool from mice given ampicillin had altered composition of gut microbiota with reduced abundance of gram-positive bacteria and increased abundance of gram-negative bacteria, compared with mice given only water. Mice given ampicillin had reduced colon motility compared with mice given only water, and their colonic LMMP had reduced numbers of nitrergic neurons, reduced neuronal nitric oxide synthase production, and reduced colonic neurogenesis. Numbers of colonic myenteric neurons increased after mice were switched from ampicillin to plain water, with increased markers of neurogenesis. Nestin-positive enteric neural precursor cells expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from enteric neural precursor cells to approximately 10%, compared with approximately 0.01% in cells incubated with the TLR4 agonist or distilled water. Mice given an antibody against TLR2 had prolonged whole gut transit times; their colonic LMMP had reduced total neurons and a smaller proportion of nitrergic neurons per ganglion, and reduced markers of neurogenesis compared with mice given saline. Colonic LMMP of mice given the TLR4 inhibitor did not have reduced markers of neurogenesis. Colonic LMMP of germ-free mice given TLR2 agonist had increased neuronal numbers compared with control germ-free mice. CONCLUSIONS: In the adult mouse colon, TLR2 promotes colonic neurogenesis, regulated by intestinal bacteria. Our findings indicate that colonic microbiota help maintain the adult ENS via a specific signaling pathway. Pharmacologic and probiotic approaches directed towards specific TLR2 signaling processes might be developed for treatment of colonic motility disorders related to use of antibiotics or other factors.

Sacral nerve stimulation increases gastric accommodation in rats: a spinal afferent and vagal efferent pathway.

Impaired gastric accommodation (GA) has been frequently reported in various gastrointestinal diseases. No standard treatment strategy is available for treating impaired GA. We explored the possible effect of sacral nerve stimulation (SNS) on GA and discovered a spinal afferent and vagal efferent mechanism in rats. Sprague-Dawley rats (450-500 g) with a chronically implanted gastric cannula and ECG electrodes were studied in a series of sessions to study: 1) the effects of SNS with different parameters on gastric tone, compliance, and accommodation using a barostat device; two sets of parameters were tested as follows: parameter 1) 5 Hz, 500 µs, 10 s on 90 s off; 90% motor threshold and parameter 2) same as parameter 1 but 25 Hz; 2) the involvement of spinal afferent pathway via detecting c-fos immunoreactive (IR) cells in the nucleus of the solitary tract (NTS) of the brain; 3) the involvement of vagal efferent activity via the spectral analysis of heart rate variability derived from the ECG; and 4) the nitrergic mechanism, Nω-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase (NOS) inhibitor, was given before SNS at 5 Hz. Compared with sham-SNS: 1) SNS at 5 Hz inhibited gastric tone and increased gastric compliance and GA. No difference was noted between the stimulation frequencies of 5 and 25 Hz. 2) SNS increased the expression of c-fos in the NTS. 3) SNS increased cardiac vagal efferent activity and decreased the sympathovagal ratio. 4) l-NAME blocked the relaxation effect of SNS. In conclusion, SNS with certain parameters relaxes gastric fundus and improves gastric accommodation mediated via a spinal afferent and vagal efferent pathway.NEW & NOTEWORTHY Currently, there is no adequate medical therapy for impaired gastric accommodation, since medications that relax the fundus often impair antral peristalsis and thus further delay gastric emptying that is commonly seen in patients with functional dyspepsia or gastroparesis. The advantage of the potential sacral nerve stimulation therapy is that it improves gastric accommodation by enhancing vagal activity, and the enhanced vagal activity would lead to enhanced antral peristalsis rather than inhibiting it.

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.

Activity within specific enteric neurochemical subtypes is correlated with distinct patterns of gastrointestinal motility in the murine colon.

The enteric nervous system in the large intestine generates two important patterns relating to motility: 1) propagating rhythmic peristaltic smooth muscle contractions referred to as colonic migrating motor complexes (CMMCs) and 2) tonic inhibition, during which colonic smooth muscle contractions are suppressed. The precise neurobiological substrates underlying each of these patterns are unclear. Using transgenic animals expressing the genetically encoded calcium indicator GCaMP3 to monitor activity or the optogenetic actuator channelrhodopsin (ChR2) to drive activity in defined enteric neuronal subpopulations, we provide evidence that cholinergic and nitrergic neurons play significant roles in mediating CMMCs and tonic inhibition, respectively. Nitrergic neurons [neuronal nitric oxide synthase (nNOS)-positive neurons] expressing GCaMP3 exhibited higher levels of activity during periods of tonic inhibition than during CMMCs. Consistent with these findings, optogenetic activation of ChR2 in nitrergic neurons depressed ongoing CMMCs. Conversely, cholinergic neurons [choline acetyltransferase (ChAT)-positive neurons] expressing GCaMP3 markedly increased their activity during the CMMC. Treatment with the NO synthesis inhibitor Nω-nitro-l-arginine also augmented the activity of ChAT-GCaMP3 neurons, suggesting that the reciprocal patterns of activity exhibited by nitrergic and cholinergic enteric neurons during distinct phases of colonic motility may be related.NEW & NOTEWORTHY Correlating the activity of neuronal populations in the myenteric plexus to distinct periods of gastrointestinal motility is complicated by the difficulty of measuring the activity of specific neuronal subtypes. Here, using mice expressing genetically encoded calcium indicators or the optical actuator channelrhodopsin-2, we provide compelling evidence that cholinergic and nitrergic neurons play important roles in mediating coordinated propagating peristaltic contractions or tonic inhibition, respectively, in the murine colon.

The normoglycaemic biobreeding rat: a spontaneous model for impaired gastric accommodation.

OBJECTIVE: Impaired gastric accommodation is reported in patients with functional dyspepsia (FD). Previous findings in postinfectious patients with FD suggest that low-grade inflammation and dysfunction of nitrergic nerves play a role in impaired accommodation. To date, spontaneous animal models to study the relationship between these changes are lacking. We hypothesise that the normoglycaemic BioBreeding diabetes-prone (BB-DP) rat provides an animal model of inflammation-induced impaired gastric motor function. DESIGN: Control diabetes-resistant biobreeding, normoglycaemic and hyperglycaemic BB-DP rats were sacrificed at the age of 30, 70 and 220 days and gastric fundus tissue was harvested to study nitrergic motor control, inflammation and expression of neuronal isoform of nitric oxide synthase (nNOS) and inducible isoform of nitric oxide synthase (iNOS). Nutrient-induced changes in intragastric pressure (IGP) were measured in normoglycaemic BB-DP rats to study accommodation. RESULTS: No differences in nitrergic function and inflammation were observed between BB-DP and control rats at 30 days. The nitrergic component of the fundic muscle relaxation was reduced in BB-DP rats of 70 and 220 days. This was accompanied by a significant loss of nNOS proteins. IGP significantly increased during nutrient infusion in BB-DP rats of 220 days, indicating impaired accommodation. Infiltration of polymorphonuclear cells, increased myeloperoxidase activity and increased expression of iNOS was observed in the fundic mucosa and muscularis propria of 70-day-old and 220-day-old BB-DP rats. CONCLUSIONS: BB-DP rats of 220 days display altered fundic motor control and impaired accommodation, which is least partially explained by loss of nitrergic function. This may be related to inflammatory changes in the neuromuscular layer, suggesting that normoglycaemic BB-DP rats provide a spontaneous model for inflammation-induced impaired gastric accommodation.

Recent advances in research on nitrergic nerve-mediated vasodilatation.

Cerebral vascular resistance and blood flow were widely considered to be regulated solely by tonic innervation of vasoconstrictor adrenergic nerves. However, pieces of evidence suggesting that parasympathetic nitrergic nerve activation elicits vasodilatation in dog and monkey cerebral arteries were found in 1990. Nitric oxide (NO) as a neurotransmitter liberated from parasympathetic postganglionic neurons decreases cerebral vascular tone and resistance and increases cerebral blood flow, which overcome vasoconstrictor responses to norepinephrine liberated from adrenergic nerves. Functional roles of nitrergic vasodilator nerves are found also in peripheral vasculature, including pulmonary, renal, mesenteric, hepatic, ocular, uterine, nasal, skeletal muscle, and cutaneous arteries and veins; however, adrenergic nerve-induced vasoconstriction is evidently greater than nitrergic vasodilatation in these vasculatures. In coronary arteries, neurogenic NO-mediated vasodilatation is not clearly noted; however, vasodilatation is induced by norepinephrine released from adrenergic nerves that activates ß1-adrenoceptors. Impaired actions of NO liberated from the endothelium and nitrergic neurons are suggested to participate in cerebral hypoperfusion, leading to brain dysfunction, like that in Alzheimer's disease. Nitrergic neural dysfunction participates in impaired circulation in peripheral organs and tissues and also in systemic blood pressure increase. NO and vasodilator peptides, as sensory neuromediators, are involved in neurogenic vasodilatation in the skin. Functioning of nitrergic vasodilator nerves is evidenced not only in a variety of mammals, including humans and monkeys, but also in non-mammals. The present review article includes recent advances in research on the functional importance of nitrergic nerves concerning the control of cerebral blood flow, as well as other regions, and vascular resistance. Although information is still insufficient, the nitrergic nerve histology and function in vasculatures of non-mammals are also summarized.

Gut microbial products regulate murine gastrointestinal motility via Toll-like receptor 4 signaling.

BACKGROUND & AIMS: Altered gastrointestinal motility is associated with significant morbidity and health care costs. Toll-like receptors (TLR) regulate intestinal homeostasis. We examined the roles of TLR4 signaling in survival of enteric neurons and gastrointestinal motility. METHODS: We assessed changes in intestinal motility by assessing stool frequency, bead expulsion, and isometric muscle recordings of colonic longitudinal muscle strips from mice that do not express TLR4 (Tlr4(Lps-d) or TLR4(-/-)) or Myd88 (Myd88(-/-)), in wild-type germ-free mice or wild-type mice depleted of the microbiota, and in mice with neural crest-specific deletion of Myd88 (Wnt1Cre(+/-)/Myd88(fl/fl)). We studied the effects of the TLR4 agonist lipopolysaccharide (LPS) on survival of cultured, immortalized fetal enteric neurons and enteric neuronal cells isolated from wild-type and Tlr4(Lps-d) mice at embryonic day 13.5. RESULTS: There was a significant delay in gastrointestinal motility and reduced numbers of nitrergic neurons in TLR4(Lps-d), TLR4(-/-), and Myd88(-/-) mice compared with wild-type mice. A similar phenotype was observed in germ-free mice, mice depleted of intestinal microbiota, and Wnt1Cre(+/-)/Myd88(fl/fl) mice. Incubation of enteric neuronal cells with LPS led to activation of the transcription factor nuclear factor (NF)-κB and increased cell survival. CONCLUSIONS: Interactions between enteric neurons and microbes increases neuron survival and gastrointestinal motility in mice. LPS activation of TLR4 and NF-κB appears to promote survival of enteric neurons. Factors that regulate TLR4 signaling in neurons might be developed to alter gastrointestinal motility.

Vasa nervorum in rat major pelvic ganglion are innervated by nitrergic nerve fibers.

INTRODUCTION: The vasa nervorum comprises a network of small diameter blood vessels that provide blood supply to nerves and ganglia. The cell bodies of autonomic nerves innervating the urogenital organs are housed in the major pelvic ganglia (MPG) in rats. The vasa nervorum of rat MPG have not been characterized previously, and it is not known whether these blood vessels are innervated by neuronal nitric oxide synthase (nNOS) containing nitrergic nerves. AIM: To characterize the blood vessels in and around the rat MPG and to assess their nitrergic innervation. MAIN OUTCOME MEASURES: Characterization of small blood vessels in and around the rat MPG and expression of nNOS in nerve fibers around those blood vessels. METHODS: MPG were obtained from healthy Sprague Dawley rats, fixed in paraformaldehyde, frozen and sectioned using a cryostat. The blood vessels and their nitrergic innervation were assessed with immunohistochemistry using antibodies against alpha-smooth muscle actin (smooth muscle marker), CD31 (endothelial marker), collagen IV (basal membrane marker) and nNOS. The immunofluorescence was imaged using a laser scanning confocal microscope. RESULTS: The neuronal cell bodies were contained within a capsule in the MPG. Blood vessels were observed within the capsule of the MPG as well as outside the capsule. The blood vessels inside the capsule were CD31-positive capillaries with no smooth muscle staining. Outside the capsule capillaries, arterioles and venules were observed. The extra-capsular arterioles and venules, but not the capillaries were innervated by nNOS-positive nerve fibers. CONCLUSIONS: This study, to our knowledge, is the first to demonstrate the blood vessel distribution pattern and their nitrergic innervation in the rat MPG. While similar studies in human pelvic plexus are warranted, these results suggest that the blood flow in the MPG may be regulated by nitrergic nerve fibers and reveal a reciprocal relationship between nerves and blood vessels.

Knockouts reveal overlapping functions of M(2) and M(4) muscarinic receptors and evidence for a local glutamatergic circuit within the laterodorsal tegmental nucleus.

Cholinergic neurons in the laterodorsal tegmental (LDT) and peduncolopontine tegmental (PPT) nuclei regulate reward, arousal, and sensory gating via major projections to midbrain dopamine regions, the thalamus, and pontine targets. Muscarinic acetylcholine receptors (mAChRs) on LDT neurons produce a membrane hyperpolarization and inhibit spike-evoked Ca(2+) transients. Pharmacological studies suggest M(2) mAChRs are involved, but the role of these and other localized mAChRs (M(1-)-M(4)) has not been definitively tested. To identify the underlying receptors and to circumvent the limited receptor selectivity of available mAChR ligands, we used light- and electron-immunomicroscopy and whole cell recording with Ca(2+) imaging in brain slices from knockout mice constitutively lacking either M(2), M(4), or both mAChRs. Immunomicroscopy findings support a role for M(2) mAChRs, since cholinergic and noncholinergic LDT and pedunculopontine tegmental neurons contain M(2)-specific immunoreactivity. However, whole cell recording revealed that the presence of either M(2) or M(4) mAChRs was sufficient, and that the presence of at least one of these receptors was required for these carbachol actions. Moreover, in the absence of M(2) and M(4) mAChRs, carbachol elicited both direct excitation and barrages of spontaneous excitatory postsynaptic potentials (sEPSPs) in cholinergic LDT neurons mediated by M(1) and/or M(3) mAChRs. Focal carbachol application to surgically reduced slices suggest that local glutamatergic neurons are a source of these sEPSPs. Finally, neither direct nor indirect excitation were knockout artifacts, since each was detected in wild-type slices, although sEPSP barrages were delayed, suggesting M(2) and M(4) receptors normally delay excitation of glutamatergic inputs. Collectively, our findings indicate that multiple mAChRs coordinate cholinergic outflow from the LDT in an unexpectedly complex manner. An intriguing possibility is that a local circuit transforms LDT muscarinic inputs from a negative feedback signal for transient inputs into positive feedback for persistent inputs to facilitate different firing patterns across behavioral states.

Impairment of gastric nitrergic and NRF2 system in apolipoprotein E knockout mice.

BACKGROUND AND AIM: Gastric motility dysfunction is most commonly seen in diabetic and idiopathic gastroparesis patients. Recently we reported that impaired nitrergic relaxation and a reduced NO (nitric oxide) bioavailability were responsible for gastric motility dysfunction in diabetic female rats. One of the main factors involved in the inactivation of the nitrergic system is oxidative stress commonly seen in diabetic patients. Hyperlipidemia may also be one of the detrimental causes for impaired gastric motility associated with diabetes. In the current study, we investigated whether apolipoprotein E knockout mice (ApoE-KO), an oxidative stress animal model with a hyperlipidemia burden, also displays an impaired nitrergic system. To test this, nitrergic relaxation (AUC/mg tissue) was measured at 2 Hz through electric field stimulation using gastric pyloric strips prepared from C57BL WT or ApoE-KO female mice. Protein expression was determined by Western blots. RESULTS: Nitrergic relaxation was reduced in gastric strips from ApoE-KO versus WT mice. Protein levels of nNOS (neuronal nitric oxide synthase), GCH-1 (GTP cyclohydrolase 1), Nrf2 (nuclear factor E-2 related factor 2) and GCSc (glutamate-cysteine ligase catalytic) were also reduced in ApoE-KO compared to controls, with no significant change in GCSm (glutamate-cysteine ligase modifier) and HO-1 (heme oxygenase 1). The activities of DHFR (dihydrofolate reductase) and antioxidant enzymes were also reduced in ApoE-KO mice. CONCLUSIONS: This novel study is the first to reveal that a deficiency in ApoE impairs gastric motility functions, and that hyperlipidemia and the suppression of selective antioxidants may be an underlying mechanism for this pathological change.

Immunohistochemical study on the development of cholinergic and nitrergic nerve structures in the bovine esophageal groove.

The proper functioning of the perinatal sucking reflex in calves is essential for the prevention of milk leakage into the rumen. The complex process behind its regulation is mediated at the gut level via multiple excitatory and inhibitory neurotransmitters, of which acetylcholine and nitric oxide are of fundamental importance. The aim of our study was to depict age-related alterations in the cholinergic and nitrergic innervation of the esophageal groove (EG) using immunohistochemistry and Real-Time PCR methods. We found out that the highest number of cholinergic nerve cells was present in the second trimester fetuses. From this developmental stage onward, their amount was gradually decreasing and reached the lowest value in 4-year-old cows. The same developmental pattern was observed for nitrergic nerve structures with the highest percentage of nitrergic neurons in the third trimester fetuses. Our observations prove that both neuronal populations are crucial for a proper closure of EG in calves. Therefore, their contribution to a general neuronal activity in the ENS diminishes with age as the high motility of a gastric groove is not necessarily required in older cattle.

Role of enteric dopaminergic neurons in regulating peristalsis of rat proximal colon.

BACKGROUND: Constipation is commonly seen in patients with Parkinson's disease associated with a loss of dopaminergic neurons in both central and enteric nervous systems. However, the roles of enteric dopaminergic neurons in developing constipation remain to be elucidated. Here, we investigated the roles of enteric dopaminergic neurons in the generation of colonic peristalsis. METHODS: Cannulated segments of rat proximal colon were situated in the organ bath, abluminally perfused with physiological salt solution and luminally perfused with 0.9% saline. Drugs were applied in the abluminal solution. Changes in diameter along the length of the colonic segment were captured by a video camera and transformed into spatio-temporal maps. Fluorescence immunohistochemistry was also carried out. KEY RESULTS: Blockade of nitrergic neurotransmission prevented oro-aboral propagation of peristaltic waves and caused a colonic constriction without affecting ripples, non-propagating myogenic contractions. Blockade of cholinergic neurotransmission also prevented peristaltic waves but suppressed ripples with a colonic dilatation. Tetrodotoxin (0.6 µM) abolished peristaltic waves and increased ripples with a constriction. SCH 23390 (20 µM), a D1 -like dopamine receptor antagonist, slowed the peristaltic waves and caused a constriction, while GBR 12909 (1 µM), a dopamine reuptake inhibitor, diminished the peristaltic waves with a dilatation. Bath-applied dopamine (3 µM) abolished the peristaltic waves associated with a colonic dilation in an SCH 23390 (5 µM)-sensitive manner. D1 receptor immunoreactivity was co-localized to nitrergic and cholinergic neurons. CONCLUSIONS AND INFERENCES: Dopaminergic neurons appear to facilitate nitrergic neurons via D1 -like receptors to stabilize asynchronous contractile activity resulting in the generation of colonic peristalsis.

Differential blockade by huperzine A and donepezil of sympathetic nicotinic acetylcholine receptor-mediated nitrergic neurogenic dilations in porcine basilar arteries.

Nicotinic acetylcholine receptor activation on the perivascular sympathetic nerves via axo-axonal interaction mechanism causes norepinephrine release, which triggers the neurogenic nitrergic relaxation in basilar arteries to meet the need of a brain. Donepezil and huperzine A, which are the cholinesterase inhibitors used for Alzheimer's disease therapy, exert controversial effects on nicotinic acetylcholine receptors. Therefore, we investigated how donepezil and huperzine A via the axo-axonal interaction regulate the neurogenic vasodilation of isolated porcine basilar arteries and define their action on different subtypes of the nicotinic acetylcholine receptor by using blood vessel myography, calcium imaging, and electrophysiological techniques. Both nicotine (100 µM) and transmural nerve stimulation (TNS, 8 Hz) induce NO-mediated dilation in the arteries. Nicotine-induced vasodilations were concentration-dependently inhibited by huperzine A and donepezil, with the former being 30 fold less potent than the latter. Both cholinesterase inhibitors weakly and equally decreased TNS-elicited nitrergic vasodilations. Neither huperzine A nor donepezil affected isoproterenol (a ß adrenoceptor-agonist)- or sodium nitroprusside (a NO donor)-induced vasodilation. Further, huperzine A was less potent than donepezil in inhibiting nicotine-elicited calcium influxes in rodent superior cervical ganglionic neurons and inward currents in α7- and α3ß2-nicotinic acetylcholine receptor-expressing Xenopus oocytes. In conclusion, huperzine A may exert less harmful effect over donepezil on maintaining brainstem circulation and on the nicotinic acetylcholine receptor-associated cognition deficits during treatment for Alzheimer's disease.

Inhibition by lithium of the nitrergic relaxation of rat anococcygeus muscle.

In the present study, we evaluated the effect of lithium on the nitric oxide (NO)-mediated nonadrenergic noncholinergic (NANC) relaxation of rat anococcygeus muscle. The isolated precontracted (phenylephrine, 7.5 microM) rat anococcygeus muscle were relaxed via electrical field stimulation (5 Hz) in the absence or presence of lithium (0.5, 1, and 5mM) or in tissues excised from ex vivo lithium (600 mg/L in drinking water for 30 days)-treated animals. Effects of the NO synthase (NOS) inhibitor L-NAME (0.03 and 100 microM) or guanylyl cyclase inhibitor ODQ (1 microM) and NO precursor L-arginine (1mM) on relaxations were investigated. Effect of either in vitro (1 and 5mM) or ex vivo lithium treatment on relaxation to the NO donor sodium nitroprusside (SNP; 0.1-1000 microM) was also investigated on phenylephrine-contracted strips. The NANC relaxation was significantly reduced by in vitro (1 and 5mM; up to P<0.01) and ex vivo lithium treatment (P<0.001). L-NAME (100 microM and 1mM) and ODQ (1 and 10 microM) significantly inhibited NANC relaxations in either control or lithium-treated strips. Combination of lithium (0.5mM) with L-NAME (0.03 microM) significantly (P<0.001) reduced the NANC relaxation. Although 1mM l-arginine had no effect on relaxations, it prevented their inhibition by both in vitro (1 and 5mM) and ex vivo lithium of relaxations. SNP produced concentration-dependent relaxation in precontracted rat anococcygeus muscle which was not altered by lithium treatment. Reverse transcription polymerase chain reaction (RT-PCR) revealed a significant increase in the neuronal NOS expression in the anococcygeus muscle of ex vivo lithium-treated animals compared with controls. Our experiments suggested that both ex vivo and in vitro lithium administration attenuated the NO-mediated neurogenic relaxation of isolated rat anococcygeus muscle.

Role of nitrergic nerves in the regulation of motility of the omasum and abomasum in healthy sheep (Ovis aries).

We examined the physiological role of nitrergic nerves in the regulation of omasal and abomasal motility in conscious healthy sheep and omasal muscle specimens. Nitric oxide (NO)-donor, S-nitroso-acethyl-dl-penicillamine (SNAP, 3-30 nmol/kg per min, i.v.) significantly inhibited omasal electromyographic (EMG) activity, whereas it did not alter EMG activity in the abomasal antrum. However, NO synthase inhibitor, Nomega-nitro-l-arginine-methyl ester (L-NAME, 0.3-3.0 micromol/kg per min, i.v.) did not alter EMG activity of the omasum and abomasum. In the in vitro experiments, SNAP application (6-200 micromol/l) significantly inhibited bethanechol (10 micromol/l)-induced contraction of longitudinal and circular muscles of the omasum. L-NAME application (0.03-3.0 mmol/l) enhanced electric field stimulation-induced contractions of the circular muscles. The results suggest that the omasal muscles are responsive to exogenous NO and that nitrergic nerves innervate the circular muscle layer of the omasum, however, nitrergic nerves are not or scarcely involved the physiological regulation of omasal and possibly abomasal motility in healthy sheep.

DOES THE RHEUMATOID ARTHRITIS AFFECT THE ENTERIC NERVOUS SYSTEM?

BACKGROUND: Few studies regarding arthritic diseases have been performed to verify the presence of the neurodegeneration. Given the increased oxidative stress and extra-articular effects of the rheumatoid arthritis, the gastrointestinal studies should be further investigated aiming a better understanding of the systemic effects the disease on enteric nervous system. OBJECTIVE: To determine whether the rheumatoid arthritis affects the nitrergic density and somatic area of the nNOS- immunoreactive (IR) myenteric neurons, as well as the morphometric areas of CGRP and VIP-IR varicosities of the ileum of arthritic rats. METHODS: Twenty 58-day-old male Holtzmann rats were distributed in two groups: control and arthritic. The arthritic group received a single injection of the Freund's Complete Adjuvant in order to induce arthritis model. The whole-mount preparations of ileum were processed for immunohistochemistry to VIP, CGRP and nNOS. Quantification was used for the nitrergic neurons and morphometric analyses were performed for the three markers. RESULTS: The arthritic disease induced a reduction 6% in ileal area compared to control group. No significant differences were observed in nitrergic density comparing both groups. However, arthritic group yielded a reduction of the nitrergic neuronal somatic area and VIP-IR varicosity areas. However, an increase of varicosity CGRP-IR areas was also observed. CONCLUSION: Despite arthritis resulted in no alterations in the number of nitrergic neurons, the retraction of ileal area and reduction of nitrergic somatic and VIP-IR varicosity areas may suggest a negative impact the disease on the ENS.

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

ETHNOPHARMACOLOGICAL RELEVANCE: Perivascular 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 STUDY: To investigate the effects of BBR on the diabetes-induced changes in nitrergic and adrenergic function in mesenteric and iliac arteries. MATERIALS AND METHODS: In 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. RESULTS: EFS 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. CONCLUSIONS: In 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.

Balanced Caloric Restriction Minimizes Changes Caused by Aging on the Colonic Myenteric Plexus.

Aging can promote significant morphofunctional changes in the gastrointestinal tract (GIT). Regulation of GIT motility is mainly controlled by the myenteric neurons of the enteric nervous system. Actions that aim at decreasing the aging effects in the GIT include those related to diet, with caloric restriction (CR). The CR is achieved by controlling the amount of food or by manipulating the components of the diet. Therefore, the objective of this study was to evaluate different levels of CR on the plasticity of nicotinamide adenine dinucleotide phosphate- (NADPH-) reactive myenteric neurons in the colon of Wistar rats during the aging process using ultrastructural (transmission electron microscopy) and morphoquantitative analysis. Wistar male rats (Rattus norvegicus) were distributed into 4 groups (n = 10/group): C, 6-month-old animals; SR, 18-month-old animals fed a normal diet; CRI, 18-month-old animals fed a 12% CR diet; CRII, 18-month-old animals fed a 31% CR diet. At 6 months of age, animals were transferred to the laboratory animal facility, where they remained until 18 months of age. Animals of the CRI and CRII groups were submitted to CR for 6 months. In the ultrastructural analysis, a disorganization of the periganglionar matrix with the aging was observed, and this characteristic was not observed in the animals that received hypocaloric diet. It was observed that the restriction of 12.5% and 31% of calories in the diet minimized the increase in density and cell profile of the reactive NADPH neurons, increased with age. This type of diet may be adapted against gastrointestinal disturbances that commonly affect aging individuals.

Monosodium glutamate impairs the contraction of uterine visceral smooth muscle ex vivo of rat through augmentation of acetylcholine and nitric oxide signaling pathways.

The aim of the study was to examine the toxic effects of Monosodium glutamate (MSG), an extensively used food additive, on the contraction of uterine visceral smooth muscle (UVSM) in rat and to elucidate the probable neurocrine mechanism involved in it. MSG produced significant potentiation of the force and inhibition of frequency of uterus recorded ex vivo in chronic MSG exposure and in single dose acute experiments. MSG also produced significant potentiation of force of acetylcholine induced contraction and no alterations in atropine induced contraction of uterus. Further, MSG produced significant increase in force and frequency of contraction of neostigmine incubated uterus. We have found significant potentiation of the post pause force of contraction of uterus when MSG was applied in adrenaline incubated uterus. MSG also produced significant decrease in frequency of contraction of sodium nitroprusside incubated uterus; increase in frequency of N-ω-Nitro-l-Arginine Methyl Ester incubated uterus and no significant changes in frequency of contraction of methylene blue incubated uterus. These results indicate that MSG potentiates the force of contraction of UVSM predominantly by augmenting the activity of cholinergic intrinsic efferents and inhibits the frequency of contraction probably by augmenting the activity of nitrergic efferents. In conclusion, MSG potentiates the force and inhibits the frequency of contraction of UVSM, and the MSG induced effect is probably mediated through the augmentation of acetylcholine and nitric oxide signaling pathways.

Cirrhosis induced by bile duct ligation alleviates acetic acid intestinal damages in rats: Involvements of nitrergic and opioidergic systems.

BACKGROUND: Colitis, a colonic inflammatory condition, showed a linkage with hepatobiliary disorders such as cirrhosis. It has been reported that both endogenous opioids and nitric oxide (NO) play critical roles in colitis pathogenesis. Moreover, opioid and NO levels showed elevation in patients with cirrhosis. The aim of this study was to evaluate the effect of cirrhosis on the experimental model of colitis and the possible involvement of opioidergic/nitrergic systems in rats. METHODS: Colitis was induced by acetic acid 28days after bile duct ligation (BDL). L-NAME, as an inhibitor of NO synthase and naltrexone, as an antagonist of opioid receptors were administered intraperitoneally to animals during 3days after induction of colitis. Macroscopic colitis lesion area, inflammatory mediators change, NO metabolite levels, and colon microscopic injuries were assessed 3days after induction. RESULTS: Cirrhosis significantly reduced the severity of damages to the colon. Administration of L-NAME (10mg/kg), naltrexone (10mg/kg) and co-administration of L-NAME (1mg/kg) and naltrexone (5mg/kg) significantly decreased the protective effect of BDL on colitis. Nitrite elevated levels in BDL rats were significantly diminished in L-NAME- and naltrexone-treated animals. Histopathology parameters and cytokines level alterations in the colon of acetic acid-treated animals after BDL was reversed after injection of L-NAME, naltrexone, and co-administration of L-NAME (1mg/kg) + naltrexone (5mg/kg). CONCLUSION: Cirrhosis improved the intestinal damages induced by acetic acid in rats which may be mediated through interaction of nitrergic and opioidergic systems.