Parts of this study have already been published in abstract form.1–3
Pieter Vanden Berghe PhD, Centre for Gastroenterological Research K.U. Leuven, Herestraat 49, 3000 Leuven, Belgium. Tel: +32 16 345762; fax: +32 16 345939; e-mail: email@example.com
Abstract Previously, we demonstrated that intestinal inflammation leads to a postinflammatory loss of nitric oxide synthase (NOS)-expressing myenteric neurones and motility disturbances. Here, we investigated whether high NO concentrations could be responsible for the decrease in NOS neurones. Myenteric neurone cultures, prepared from guinea-pig small intestine, were incubated with NO donors [sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1)]. After fixation, NOS neurones were identified by NADPH diaphorase staining and neurone-specific enolase (NSE)-positive neuronal content was assessed with an enzyme-linked immunosorbent assay (ELISA)-based method. Twenty-four hours incubation with SIN-1 (10−3 mol L−1) or SNP (10−4 mol L−1 or higher) reduced the number of NADPH diaphorase-positive neurones. SNP incubation did not affect the NSE-positive neuronal content. Shorter incubations (SNP: 4 and 12 h) had no significant effect. The SNP-induced reduction was reversed by glutathione (GSH), but not by NO- or O-scavengers, whereas GSH depletion enhanced the decrease. The NO-dependent guanylate cyclase-blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) did not affect the SNP effect. This reduction can be explained by either specific apoptosis of NOS neurones or downregulation of NOS activity. However, TdT-mediated X-dUTP nick end labelling (TUNEL®) stainings argue in favour of the latter. In conclusion, the NO donor SNP decreases the number of NOS-expressing myenteric neurones time and concentration dependently, without affecting the amount of neuronal material. Glutathione plays an important protective role.
Besides its function in normal cell physiology, nitric oxide (NO), if generated in large amounts, plays an important role in pathophysiological conditions, such as inflammation, where it can have both beneficial and adverse effects. Factors that determine the net effect of NO signalling include the presence of other reactants, such as superoxide (O) and the reaction rate between NO and other molecules, such as haeme-containing proteins.4
In the enteric nervous system, NO is a major inhibitory neurotransmitter that controls in part gut relaxation.5 Therefore, changes in NO release might have an important impact on gastrointestinal motility. Trinitrobenzene sulfonic acid (TNBS)-induced inflammation of guinea-pig small intestine and rat colon has been shown to result in inducible nitric oxide synthase (iNOS) upregulation.6,7 The NOS activity was also increased in rectal and colonic biopsy specimens from patients with inflammatory bowel disease.8,9 In addition, several groups have shown a decreased neuronal NOS (nNOS) activity or nNOS expression in myenteric neurones after inflammation10,11 or as a result of exogenous NO application.12 Upregulation of iNOS and downregulation of nNOS was observed in inflamed and healthy mucosa of patients with ulcerative colitis and the ratio of iNOS/nNOS activity in healthy mucosa seemed to be predictive for disease extent.13 Also in our group, a loss of myenteric nNOS immunoreactivity was shown after TNBS-induced inflammation of rat jejunum which was accompanied by motility disturbances (I. Demedts, unpublished data). Recently, we have shown that dysfunction of myenteric nitrergic neurones underlies impaired accommodation in patients with presumed postinfectious functional dyspepsia.14 Preliminary results from a guinea-pig ileitis model point towards a causal link between inflammation, iNOS induction and reduced nNOS expression.15 In rat forebrain slices, downregulation of nNOS, after oxygen-glucose deprivation, was attributed to NO released by upregulated iNOS and was mimicked by exposure to a NO donor.16
In this study, we aimed to investigate whether the high NO concentrations, present after inflammation-induced iNOS upregulation, could be responsible for the decreased numbers of nNOS-expressing neurones. To test this hypothesis, we incubated myenteric neurone cultures with NO donors in the absence or presence of free radical scavengers and antioxidants and found that there is indeed a decrease of NOS-expressing neurones, partially prevented by glutathione (GSH).
Material and methods
Culturing of myenteric neurones
Primary cultures of myenteric neurones were prepared from guinea-pig (300–600 g) small intestine.17,18 The longitudinal muscle/myenteric plexus preparations were dissected in ice-cold sterile Krebs solution (120.9 mmol L−1 NaCl, 5.9 mmol L−1 KCl, 1.2 mmol L−1 MgCl2, 1.2 mmol L−1 NaH2PO4, 14.4 mmol L−1 NaHCO3, 2.5 mmol L−1 CaCl2, 11.5 mmol L−1 glucose; all purchased from Merck®, Overijse, Belgium) and enzymatically digested [30 min, 37 °C, protease: 1 mg mL−1, collagenase: 1.25 mg mL−1, bovine serum albumin (BSA): 0.4% w/v]. The suspension was then cooled and centrifuged (350 g, 4 °C); pellets were resuspended and washed and ganglia were selected under a binocular microscope. NUNCTM 24-well plates (VWR International, Leuven, Belgium) were inoculated (50 μL per well) and incubated in a humidified CO2 incubator (37 °C). Cell culture medium was neurone-specific Medium 199 enriched with 10% (v/v) fetal bovine serum (FBS) and 50 ng mL−1 nerve growth factor (7sNGF; Alomone Labs®, Jerusalem, Israel); glucose concentration was 30 mmol L−1 and penicillin (100 U mL−1)/streptomycin (100 μg mL−1) was present to prevent bacterial growth. Antibiotics, FBS and Medium 199 were purchased from Invitrogen® (Merelbeke, Belgium). Collagenase and protease were from Sigma-Aldrich® (Bornem, Belgium) and BSA from Serva® (Heidelberg, Germany).
Incubation with NO donors
Neurone cultures were incubated with the NO donors sodium nitroferricyanide, known as sodium nitroprusside (SNP; 10−6 to 10−3 mol L−1; 4–24 h) and 3-morpholinosydnonimine (SIN-1; 10−4 to 10−3 mol L−1; 24 h). Potassium ferricyanide [K3Fe(CN)6] was used to verify that the SNP-mediated effect was caused by the NO produced. The following chemicals were added to the cell cultures to influence the SNP effect: superoxide dismutase (SOD; 10−4 mol L−1), diethyldithiocarbamate (DETC; 5 × 10−4 mol L−1), 2-phenyl-4,4,5,5,-tetramethyllimidazoline-3-oxyde-1-oxyl (PTIO; 10−4 mol L−1), oxyhaemoglobin (10−5 mol L−1), d,l-buthionine-(S,R)-sulfoximine (BSO; 10−4 mol L−1), reduced GSH ethyl ester (10−4 mol L−1), ascorbic acid (10−4 mol L−1) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10−5 mol L−1). These chemicals were always added 24 h before fixation, unless mentioned otherwise. All chemicals were purchased from Sigma® except (oxy)-haemoglobin from Serva® (Heidelberg, Germany), ascorbic acid from Merck® and SNP from DBL® (Warwick, UK).
NADPH diaphorase staining
Neurones were fixed (4% paraformaldehyde; 1 h) and permeabilized (0.5% Triton X-100; 0.1 mol L−1 PO; 2 h). To identify NOS-expressing neurones, we performed a NADPH diaphorase staining (30 min, 37 °C, 1 mg mL−1β-NADPH, 0.2 mg mL−1 tetrazolium blue, 0.5% Triton X-100, 0.1 mol L−1 PO; chemicals from Sigma). NADPH diaphorase-positive (NADPH-d+) neurones were counted per well and expressed as percentages of controls. To confirm colocalization of NADPH diaphorase reactivity and NOS immunoreactivity, we used a rabbit antibody against nNOS (1/400; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in combination with the secondary antibody goat antirabbit IgG Alexa Fluor® 488 (1/500; Invitrogen). The reactive cells were photographed and processed subsequently for NADPH diaphorase. As we found a perfect match between NOS and NADPH diaphorase activity (Fig. 1A), NADPH diaphorase stainings were used throughout the study, because it is less labour intensive (one-step staining) and can easily be viewed on a transmitted light microscope. The terms ‘NOS-positive’ and ‘NADPH diaphorase-positive’ will therefore be used interchangeably throughout the manuscript.
Semiquantitative measurement of NSE-positive neuronal content and TUNEL staining
Culture dishes (96-well) were inoculated with 30 μL ganglia suspension. To validate our assay, some wells received culture medium with 2% FBS instead of 10% with or without arabinose-C-furanoside (ARA-C; 10−6 mol L−1; Sigma). SNP (10−5 and 10−4 mol L−1) was administered on day 7 for 24 h. After fixation (4% paraformaldehyde; 30 min), the cells were processed for permeabilization and blocking of non-specific binding sites [2 h; 0.1 mol L−1 phosphate-buffered saline (PBS), 0.5% Triton X-100 and 4% normal goat serum]. The primary antibody, rabbit antineurone-specific enolase (NSE, 1/2000; Polysciences®, Warrington, PA, USA) was added (24 h) to label neuronal cell bodies and fibres. Incubation (2 h) with the secondary antibody, biotinylated goat antirabbit IgG (1/400; Jackson ImmunoResearch Laboratories®, Inc., West Grove, PA, USA), was followed by successive incubation with 1μg mL−1 streptavidine in 0.1 mol L−1 PBS (10 min; 37 °C) and with 3 μg mL−1 biotinylated horseradish-peroxidase (HRP) in 0.1 mol L−1 PBS (10 min; 37 °C). Finally, 100 μL per well citrate buffer (Na-citrate 0.05 mol L−1, Na-phosphate 0.05 mol L−1, pH 5), 1 mg mL−1o-phenylenediamine (OPD) and 1 μL mL−1 H2O2 were added. The reaction was stopped by adding oxalic acid (50 μL per well, 1 mol L−1). Absorbance of the reaction product was measured spectrophotometrically (λ = 492 nm) and reflects the amount of neuronal material present in each well.
In other dishes, we assessed neuronal apoptosis with a TdT-mediated X-dUTP nick end labelling (TUNEL®) kit (Roche Diagnostics®, Vilvoorde, Belgium).
All results were analysed using graphpad software. Data were expressed as mean percentages (±SEM). The n-values are the number of wells. The data were compared using anova (with Bonferroni post hoc test) or non-parametric anova (with Dunn's post hoc test) depending on the outcome of a Barlett's test for equal standard deviations.
The effect of NO donors on the number of NADPH diaphorase-positive neurones
To study the effect of NO on the number of NADPH-d+ neurones, cell cultures were incubated with SNP (10−4 mol L−1) during 4, 12 and 24 h. The number of NADPH-d+ neurones was significantly (P < 0.05) reduced after a 24 h incubation (74 ± 11%, 84 ± 14%, 42 ± 14%vs 100 ± 14%; for respectively 4, 12, 24 h vs control; Fig. 1B). As 24 h incubation was required to obtain a measurable effect at 10−4 mol L−1, we used this exposure time to evaluate different SNP concentrations. We observed a dose-dependent reduction (P < 0.01; Fig. 1C) with an EC50 value near 10−4 mol L−1. The SNP [Na2Fe(CN)5NO·2H2O] induced effect was due to its NO-moiety, as K3Fe(CN)6 (10−4 mmol L−1) was ineffective (85 ± 13%, n = 13). Similar effects were also seen with another NO donor, SIN-1, albeit at higher concentrations (94 ± 24% at 5 × 10−4 mol L−1 and 18 ± 8% at 10−3 mol L−1, P < 0.01). However, as the EC50 for SIN-1 was higher and because SIN-1 concomitantly releases superoxide,19 we used the more efficient SNP as NO donor throughout the rest of the study at a dose of 10−4 mol L−1.
The effect of SNP on total amount of NSE-positive neuronal material and apoptosis
So far it was unclear whether SNP had an overall neurotoxic effect. Therefore, we developed an NSE-enzyme-linked immunosorbent assay (ELISA) technique to measure the total amount of NSE-positive neuronal material, which was not changed after 24 h incubation with SNP (107 ± 5%, 97 ± 4%vs 100 ± 3% respectively with 10−5 and 10−4 mol L−1vs control; Fig. 2A). However, the amount of NSE-positive neuronal material was decreased significantly to 61 ± 4% when neurones were starved by reducing the concentration of FBS from 10% to 2% (P < 0.01). The effect of serum deprivation was amplified (NSE amount down to 42 ± 4%) by addition of 10μ mol L−1 ARA-C, which blocks proliferation of dividing cells-like fibroblasts and glial cells.20 Rather than neuronal cell division, which is unlikely to occur in adult guinea-pig cultures, this reflects the importance of neuronal support by these cells during serum starvation.
As the total NSE amount does not take into consideration different neuronal sizes or different levels of NSE expression, a TUNEL kit was used to further investigate whether neuronal apoptosis occurred. The TUNEL-positive cells were rare in control and SNP conditions and there was no indication as would 10−4 mol L−1 SNP induce apoptosis in the cultured cells (Fig. 2B).
Taken together, these results indicate that although our technique is able to detect changes in the amount of neuronal material, SNP does not have an overall toxic effect.
The influence of NO- and O-scavengers
The effect of SNP on the number of NADPH-d+ neurones (later referred to as ‘the SNP effect’) was further examined by using NO- and O-scavengers (Table 1). The PTIO21,22 and oxyhaemoglobin23,24 were added to scavenge the NO released from SNP. Oxyhaemoglobin (10−5 mol L−1) was not able to reverse the SNP effect. Incubation with SNP alone or in combination with oxyhaemoglobin caused a significant reduction in the number of NADPH-d+ neurones, which did not differ significantly. Similar to oxyhaemoglobin, PTIO (10−4 mol L−1) also failed to reverse the SNP effect. Moreover, after simultaneous incubation with PTIO and SNP, many neurones had swollen cell bodies with a rough appearance due to vacuole formation and fibres showed signs of ‘blebbing’. The TUNEL staining confirmed that in this condition massive apoptosis was ongoing (data not shown). PTIO alone caused a small but non-significant increase in the number of NADPH-d+ neurones under basal conditions.
Table 1. The influence of the NO-scavengers oxyhaemoglobin and PTIO and the O-scavenger SOD on the number of NADPH-d+ neurones
oxyHb (10−5 mol L−1)
PTIO (10−4 mol L−1)
SOD (250 U mL−1)
*Significant differences from control [P < 0.01 (non)parametric anova with Dunn's/Bonferroni post hoc test].
As NO-scavengers were unable to reverse the effect of SNP, we assumed that another NO-related species might be responsible for the observed effects. Peroxynitrite, formed by the reaction of NO with superoxide, has been suggested to account for the adverse effects of NO.25 We therefore studied the effect of Cu/Zn SOD, which catalyses the conversion of cytotoxic superoxide to oxygen and hydrogen. Exogenous SOD did not reverse the SNP effect. SNP (10−4 mol L−1) incubation alone or simultaneously with SOD (250 U mL−1), caused the same significant reduction in the number of NADPH-d+ neurones (Table 1). However, inhibition of endogenous Cu/Zn SOD by adding DETC (5 ×10−4 mol L−1) significantly enhanced the SNP effect (55 ± 15%, 57 ± 11%, 0 ± 0%; respectively for DETC, SNP, SNP + DETC, P < 0.05; Fig. 3), probably due to the presence of superoxide radicals.
The role of antioxidant defence
The tripeptide-reduced GSH is a major antioxidant that is abundantly present (low mmol L−1 concentrations) in the nervous system.26 It functions mainly through the elimination of free radical species by providing a reducing milieu within cells.27 To investigate the importance of antioxidant defence in the SNP effect on NADPH-d+ neurones, experiments were carried out in the absence or presence of GSH.
The GSH depletion by BSO (10−4 mol L−1), a selective and potent inhibitor of the enzyme γ-glutamylcysteine synthetase, influenced both the concentration- and time-dependent effect of SNP on NADPH-d+ neurones. It shifted the SNP dose–response curve to the left (P < 0.01; Fig. 4A), while BSO itself was ineffective (78 ± 10%, ns). A pretreatment with BSO (20 h) resulted in a significant decrease of the number of NADPH-d+ neurones after only 8 h of incubation with SNP (31 ± 8%; P < 0.01; Fig. 4B). Moreover, the SNP effect was prevented by the addition of reduced GSH ethyl ester, which acts as a cellular GSH delivery agent (102 ± 16%, 46 ± 8%, 95 ± 16%; respectively for GSH, SNP, GSH + SNP; P < 0.05; Fig. 5).
The antioxidative effect seemed to be specific to GSH because ascorbic acid (10−4 mol L−1) did not alter the effect of SNP (95 ± 14%, 40 ± 8%, 50 ± 11%; respectively for ascorbic acid, SNP, ascorbic acid + SNP, n = 12; ns).
Guanylate cyclase inhibition
In these experiments, neurone cultures were incubated with ODQ (10−5 mol L−1), a NO-sensitive guanylate cyclase inhibitor, to investigate whether the reduction in the number of NADPH-d+ neurones was cGMP-dependent, similar to the relaxing action of NO on smooth muscle cells. Culture wells incubated with SNP (10−4 mol L−1) or with ODQ and SNP, showed a similar significant reduction in the number of NADPH-d+ neurones (43 ± 5% and 52 ± 14% respectively; P < 0.05). Incubation with ODQ in basal conditions induced a non-significant increase in the number of NADPH-d+ neurones (158 ± 17%; ns; Fig. 6).
In this study, we aimed to investigate the mechanism for changes in NOS-expressing myenteric neurones, as evidenced during and after intestinal inflammation. More precisely, we investigated whether high NO concentrations, occurring after inflammation induced iNOS upregulation, could explain the loss of nNOS expression in myenteric neurones. This hypothesis was tested by adding NO donors to primary cultures of myenteric neurones, and by counting the number of NADPH-d+ neurones28–30 per well.
Using the NO donor SNP, we observed a dose-dependent reduction in the number of NADPH-d+ neurones after 24 h incubation. A similar effect was seen with a second NO donor, SIN-1, at slightly higher concentrations.
Cultured neurones incubated with SNP had a normal light microscopic appearance when compared with controls. Semiquantitative measurement of NSE-positive neuronal content in the cultures also showed that incubation with SNP did not affect the total amount of neuronal material. Further, a TUNEL staining showed no difference in the amount of ongoing apoptosis in SNP-treated vs control cells. From these results, we excluded an overall non-specific toxic effect of SNP. The fact that a structurally related compound lacking the NO-moiety (K3Fe(CN)6) was ineffective, corroborates the findings and specifically attributes the effect of SNP to NO or a NO metabolite.
Oxyhaemoglobin and PTIO, two NO-scavengers, were ineffective in reversing the effect of SNP on the number of NADPH-d+ neurones. This could be explained by the fact that NO release from SNP in biological systems is dependent on a membrane-associated enzyme.31 The NO-scavengers would only be effective if NO is released extracellularly, which has been shown to occur in the vasculature via a transmembrane NADH oxidoreductase electron transport system.32 It is also conceivable that the scavenger concentration was too low compared with the levels of NO released from SNP. Finally, NO may rapidly react with other reactive molecules, resulting in the generation of NO metabolites, which are no longer affected by these scavengers. One of the most important NO metabolites is peroxynitrite (ONOO−), which is produced when NO is released in the vicinity of O. Peroxynitrite is a powerful agent that causes oxidative stress to cells.25 To investigate whether our results could be explained by the formation of peroxynitrite, we used the O-scavenger Cu/Zn SOD and the SOD inhibitor DETC. Although SOD did not have any protective effect, simultaneous incubation with DETC and SNP further decreased the number of NADPH-d+ neurones. These apparently contrasting results can be explained in several ways. Firstly and most likely, SOD does not cross the membrane, making it impossible to scavenge intracellular superoxide anions. Secondly, DETC and NO might exert their effects on NADPH-d+ neurones via distinct parallel mechanisms, as DETC itself tended to reduce the number of NADPH-d+ neurones. This is consistent with earlier findings that a functionally active Cu/Zn SOD protects nitrergic neurones in the peripheral nervous system.33 Although we cannot draw any firm conclusions about the involvement of peroxynitrite in the SNP-induced effect on nitrergic neurones, the finding that SNP and DETC have an additive effect on the reduction of NADPH-d+ neurones, argues in favour of involvement of oxidative stress.
We therefore investigated the effect of antioxidants. Glutathione, abundantly present in the nervous system, protects cells against free radicals and reactive oxygen species. Therefore, we studied the role of this antioxidant in the SNP effect. Glutathione depletion with BSO further decreased the number of NADPH-d+ neurones, confirming the importance of GSH in protecting cells against oxidative insults. Conversely, GSH substitution through administration of reduced GSH ethyl ester protected the NADPH-d+ neurones against the SNP effect. Taken together, these results suggest that SNP exhibits its effect through an oxidative pathway in which GSH plays an important role as antioxidant. Further in the central nervous system, GSH is one of the molecules protecting against NO toxicity.34–36 Alternatively, GSH can also counteract the NO-mediated inhibition of NOS.37 The protective effect of antioxidants seemed to be GSH-specific, because ascorbic acid did not alter the SNP effect.
Finally, as most NO effects are mediated through the activation of guanylate cyclase and subsequent increase in intracellular cGMP levels, the effect of the NO-dependent guanylate cyclase-blocker ODQ was studied. The reduction in the number of NADPH-d+ neurones was, however, not influenced by ODQ.
The reduction in the number of NADPH-d+ neurones can be the result of nNOS downregulation, reduced nNOS activity or selective apoptosis of nNOS neurones. Downregulation of nNOS, caused by iNOS induction and subsequent release of large NO amounts, has already been shown in the central nervous system,16 although an excess of NO can also lead to neuronal apoptosis.38–40 Until now, little is known about the pathway that leads to loss of NOS neurones in the enteric nervous system, although the TUNEL labelling does not suggest apoptosis. Kurjak et al. reported that NO donor administration to enteric synaptosomes caused inhibition of nNOS activity.12 Mizuta et al. demonstrated reduced activity and synthesis of nNOS in dextran sulphate sodium-induced colitis in rats.10 Similarly, TNBS-induced ileitis leads to a long-lasting loss of rat nitrergic myenteric neurones and subsequent motility disturbances (I. Demedts, unpublished data). In contrast to guinea-pig colon where an indiscriminate loss of neurones was reported,41 in rat inflamed ileum a specific decrease in the number of nNOS+/VIP− and nNOS+/VIP+ neurones was shown. As the nNOS−/VIP+ population was not increased, these data suggest either parallel downregulation of nNOS and VIP or selective apoptosis of nNOS neurones. The apparent discrepancy between these studies might reflect a region-specific effect. Differences between this study and observations in vivo may also depend on the lack of non-neuronal cells in the cultures, which are likely to be the predominant source of scavenging and rescue mechanisms.
Administration of NO donors to myenteric neurone cultures caused a significant decrease in the number of NADPH-d+ neurones, without affecting the total amount of neuronal material. The SNP effect was independent of guanylate cyclase and could not be reversed by NO or superoxide scavengers, but was significantly elevated when endogenous SOD was blocked. Together with the protective role of GSH, this points towards the involvement of oxidative stress. Although the exact pathways remain to be determined, it is clear that large amounts of NO alter nitrergic neurotransmission dramatically and thus influence gastrointestinal motility to an important extent.
PVB is a postdoctoral fellow and JT a Senior Clinical Investigator of the ‘Fonds voor Wetenschappelijk Onderzoek’ (F.W.O., Fund for Scientific Research), Flanders, Belgium.