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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 8, 2004 as doi:10.1096/fj.03-0596fje. |
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,2
* Departamento de Farmacología and
Unidad Mixta CNIC-UVEG, Facultad de Medicina, Universidad de Valencia, Valencia, Spain; and
Cytometry Unit, Fundación Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
2Correspondence: Department of Pharmacology, Faculty of Medicine, University of Valencia, Avd. Blasco Ibáñez 15, 46010 Valencia, Spain. E-mail: dolores.barrachina{at}uv.es
SPECIFIC AIMS
NO synthesized by the enteric nervous system plays a physiological role in relaxation of the stomach, while the induction of iNOS by the immune system is involved in changes in gastrointestinal motor function associated with infection or injury. Following bacterial infection, the host responds acutely by activating neuroendocrine, metabolic, and behavioral processes that attempt to eliminate the challenge and restore homeostasis. Diminution in gastric motor function constitutes an early event in endotoxemia and we hypothesized that a neural mechanism mediated by nitric oxide (NO) synthesis in enteric motor neurones is involved in this event.
PRINCIPAL FINDINGS
1. Activation of inhibitory postganglionic myenteric neurons, by extrinsic neural control of the gut, mediates the decrease in gastric motor function during early endotoxemia
Thirty minutes after systemic administration to rats of low doses of endotoxin, the stomach was removed and gastric hypocontractility to carbachol observed in isolated fundal strips (Fig. 1
). Local inhibition of neural transmission with the neurotoxin tetrodotoxin (TTX) or blockade of ganglia nicotinic receptors with hexamethonium significantly prevented the inhibitory effect of endotoxin (Fig. 1A
). In vitro incubation with endotoxin (100 ng/mL or 1 µg/mL) for 30 min, 3 h, or 8 h failed to significantly modify (P>0.05) carbachol-induced fundus contraction.
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2. nNOS-derived NO synthesis, but not VIP or ATP, is involved in the diminution in gastric motor function associated with early endotoxemia
Blockade of NO synthesis by incubation of fundal strips with L-NOARG or the selective nNOS inhibitor TRIM prevented the hypocontractility induced by endotoxin (Fig. 1B
). However, the effect of endotoxin was not significantly modified (P>0.05) by incubation with the VIP antagonist VIP 10-28 (10 µM) or antagonism of P2-purinoceptors with suramin (100 µM) plus PPADS (100 µM). No changes in nNOS mRNA or protein content were observed in gastric tissue 1 h after endotoxin.
3. During early endotoxemia, NO synthesis in the gastric wall is specifically localized in postganglionic myenteric neurons
We analyzed the cellular source of NO synthesis by confocal microscopy in living gastric tissue loaded with DAF-FM, a dye that upon binding to NO in the presence of oxygen results in irreversible fluorescence. Fundus whole mounts from endotoxin-treated rats exhibited an intense DAF-FM fluorescent signal in the myenteric plexus (Fig. 2
D). Moving along the Z axis of the fundus whole mount, DAF-FM fluorescence was observed localized in enteric nerve cell bodies and nerve fibers within myenteric ganglia (Fig. 2D, J
) as well as in nerve fibers running parallel to smooth muscle cells (Fig. 2E
). Image analysis of deep circular smooth muscle layer failed to show any DAF-FM fluorescent signal (Fig. 2F
). Preincubation with L-NAME (1 mM, Fig. 2G, J
), TTX (1 µM; Fig. 2H, J
) or hexamethonium (100 µM; Fig. 2I, J
) prevented the appearance of DAF-FM fluorescence in strips from endotoxin-treated rats, confirming the NO-specificity of the fluorochrome and neural cellular origin of DAF-FM fluorescence, and suggesting the location of NO synthesis in postganglionic myenteric neurons.
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Fundus whole mount from saline-treated rats showed a weak DAF-FM signal (Fig. 2B
) that was not modified by preincubation with L-NAME (Fig. 2C
), TTX, or hexametonium; (Fig. 2J
).
4. NO-induced relaxation of gastric fundus is mediated via guanylyl cyclase and small conductance Ca2+-sensitive K+ channels
Inhibition of the activity of soluble guanylyl cyclase (sGC) with ODQ or blockade of the small conductance Ca2+ activated K+ (CaK+) channels by apamin, prevented the hypocontractility observed in strips from endotoxin-treated rats (Fig. 1C
).
Under NANC conditions, nicotine (10 µM) -induced relaxation (96.6±3.6%) of 5-HT (3 µM) -precontracted strips was prevented (P<0.05) by preincubation with L-NOARG (27.1±8.3%; 100 µM), ODQ (17.0±4.9%; 10 µM), or apamin (56.4±14.5%; 1 µM).
Preincubation with the NO donor, DETA NONOate (100 µM) induced hypocontractility (52.3±4.3% of contraction) to carbachol (0.1 µM). This effect was prevented (P<0.05) in the presence of ODQ (116.2±9.0%) or apamin (79.3±7.6%). In a similar manner, addition of the NO-independent sGC activator BAY 41-2272 (3 µM) reduced the contraction to carbachol (32.8±4.4%), and this effect was prevented (P<0.05) by preincubation with ODQ (92.9±10.1%) or apamin (54.9±4.0%).
5. iNOS isoform is not involved in the decrease in gastric motor function during early endotoxemia
In vivo pretreatment with dexamethasone (5 mg/kg, s.c., twice) failed to significantly (P>0.05) modify the response to carbachol in saline (Emax 5.3±0.7 g) or endotoxin-treated rats (2.4±0.4 g). In addition, no changes in iNOS mRNA expression, protein content and enzyme activity have been observed in gastric tissue one hour after the administration of endotoxin.
CONCLUSIONS AND SIGNIFICANCE
In the present study we have shown for the first time the synthesis of NO in rat gastric postganglionic myenteric neurons. This synthesis is observed in fundus from endotoxin-treated rats and seems to be involved in the associated hypocontractility.
Systemic administration of endotoxin to rats results in the hypocontractility to carbachol of isolated fundal strips. This effect is rapid in onset and independent of endotoxin-induced protein synthesis since in vivo pretreatment with dexamethasone did not prevent its effects, thus suggesting a neural mechanism. This is reinforced by the fact that both tetrodotoxin and hexamethonium prevented the hypocontractility. Failure of endotoxin to modify gastric contractility when directly added to the organ bath suggests that the effects of systemic endotoxin are a consequence of an extrinsic regulation of motor neurons rather than a direct action of endotoxin on enteric neurons or smooth muscle cells. These observations, together with previous studies showing activation of capsaicin-sensitive afferent vagal neurons and c-fos expression in the dorsal vagal complex of the brainstem by endotoxin, suggest a vago-vagal reflex involved in the diminution of gastric motor function observed in early endotoxemia (Fig. 3
). Such a mechanism could be involved in the associated delay in gastric emptying.
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NO synthesis mediates physiological relaxation of the fundus. However, the present study is the first to demonstrate a nontranscriptional regulation of NO synthesis via nNOS in the acute diminution of gastric motor function induced by endotoxin. In parallel confocal microscopy studies, we also demonstrate that in vivo pretreatment with endotoxin induces an intense DAF-FM fluorescence in the fundal myenteric plexus, indicative of NO synthesis. In addition, this fluorescence seems to be specifically localized in postganglionic myenteric neurons since pretreatment with TTX or hexamethonium significantly prevented it. Taking into account that nNOS is primarily expressed in neurons, these results suggest that NO synthesis localized in postganglionic myenteric neurones is involved in fundus relaxation induced by endotoxin (Fig. 3)
.
Further analysis of intracellular mechanisms showed that activation of sGC and small conductance CaK+ channels mediates endotoxin-induced fundal relaxation. Furthermore, activation of these channels seems to be cGMP-dependent because gastric relaxation induced by an NO-independent sGC activator depends on their activity. Since a direct action of endotoxin on these targets has been ruled out, our results suggest that NO activates sGC, and subsequent increase in cGMP levels is involved in the activation of these channels and relaxation of the tissue (Fig. 3)
.
Changes in gastrointestinal motor function during endotoxemia have been linked to iNOS-induction. However, our results do not support a role for iNOS-derived NO synthesis in the decrease in gastric tone induced by endotoxin, since in vivo pretreatment with dexamethasone failed to modify its effects. In addition, we observed no significant changes in iNOS mRNA expression, iNOS protein content or Ca2+-independent NOS activity in strips from endotoxin-treated rats.
We hypothesize that in early endotoxemia the host tries to maintain homeostasis by activating neural mechanisms which maximize the activity of its constitutive resources (nNOS), before the immune response is fully functioning (iNOS).
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0596fje; 
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5). *P < 0.05 for Emax vs. all experimental groups in each graph.
