Title

 

Authors

S. Marchet¹, E. Carpanese¹, E. Moro², F. Crema², C. Giaroni¹, S. Lecchini¹, G. Frigo².
 
Dept. of Clinical Medicine, University of Insubria¹; Dept. of Internal Medicine, University of Pavia². 

 

Abstract

Glutamatergic and nitrergic pathways play a key role in neuronal injury during an ischemic/reperfusion damage (Mehta et al., 2007). In these conditions, in the gut, both glutamate, via NMDA receptor activation, and nitric oxide (NO) have been shown to induce changes in several neurotransmitter systems (i.e, cholinergic, glutamatergic, tachikyninergic) as well as in the motor function, which, on the whole, might contribute to gastrointestinal dysmotility (Calcina et al., 2005; Giuliani et al., 2006).In this view, a possible cross-talk between enteric glutamatergic and nitrergic pathways during ischemia/reperfusion has been proposed (Calcina et al., 2005). However, the mechanism/s that may underlie such interaction remain to be elucidated. The aim of the present study was to further investigate the interplay between enteric NMDA receptor pathways and nitric oxide (NO), during in vitro-induced hypoxia and hypoglycemia (in vitro ischemia) followed by reperfusion in the guinea pig ileum, resorting to functional and molecular approaches. Conditions of hypoxia/hypoglycemia were obtained by perfusing isolated longitudinal muscle-myenteric plexus preparations with a physiological Tyrode’s solution deprived of oxygen and glucose for 60 min; reperfusion was attained by returning to a normal glucose containing oxygenated Tyrode's solution. NO formation from the guinea pig ileum, in the absence and presence of NMDA receptor antagonists, ^_AP5 (NMDA receptor) and diCl-kynurenic acid (glycine site associated to NMDA receptor), was quantified in the superfusate via the nitrite/nitrate method based on the colorimetric Griess reaction The antagonists, chelerythrine (protein kinase C, PKC) and H-89 (protein kinase A, PKA) were added in order to evaluate the possible mechanisms modulating NO formation in the ischemic/reperfused ileum. Nitrite concentration moderately increased after in vitro ischemia and raised significantly above control values 5’ after reperfusion (P<0.01). Nitrate concentration significantly increased 5’ after in vitro ischaemia (P<0.05) and raised moderately above control values during reperfusion.^_AP5 (10mM) and diCl-kynurenic acid (10mM) significantly (p<0.0001) reduced nitrite and nitrate concentrations after ischemia/reperfusion. Chelerythrine (1 mM) also significantly (p<0.0001) reduced enhancement of both NO metabolite concentrations after ischemia/reperfusion. On opposite, H-89 (1 mM) did not modify nitrite/nitrate concentrations during both the metabolic insult and after reperfusion. The protein level of NR1 receptor and of its phosphorylated form on Ser896 significantly increased in myenteric neurons 5 min after induction of in vitro ischemia/reperfusion and remained elevated thereafter, such enhancement was abolished in the presence of chelerythrine (1 mM). The protein level of NR1 phosphorylated on Ser897 site did not change at any time after in vitro ischemia/reperfusion. The levels of pan PKC significantly (p<0.05) increased after ischemia/reperfusion both in the cytosolic and soluble fractions of myenteric neurons. On the whole the present data suggest that glutamate participates to NO formation via NMDA receptor activation during in vitro ischemia/reperfusion. The effect of chelerythrine puts forward the hypothesis that PKC is part of the signalling machinery that regulates NOS activation in the guinea pig ileum during a metabolic damage. Such regulation may occur, at least in part, via phosphorylation of the NR1 subunit of the NMDA receptors.
 
Mehta et al. (2007). Brain Res. Rev. 54, 34-66.
Giuliani et al. (2006). Neurochem Int. 48, 191-200.
Calcina et al. (2005). Neuroscience 134:39-49.