Interaction between NMDA glutamatergic and nitrergic enteric pathways during in vitro ischemia and reperfusion

Nitric oxide (NO) and glutamate, via N-methyl-D-aspartate (NMDA) receptors, participate to changes in neuromuscular responses after ischemic/reperfusion (I/R) injury in the gut. In the present study we investigated the existence of a possible interplay between nitrergic and NMDA receptor pathways in the guinea pig ileum after in vitro I/R injury, resorting to functional and biomolecular approaches. In normal metabolic conditions NMDA concentration-dependently enhanced both glutamate (analyzed by high performance liquid chromatography with fluorimetric detection) and NO (spectrophotometrically quantified as NO2- and NO3- spontaneous overflow from isolated ileal segments. Both effects were reduced by the NMDA antagonists, (-)-AP5 (10 mu M) and 5,7-diCl-kynurenic acid (10 mu M, 5,7-diCl-KYN). N-omega-propyl-L-arginine (1 mu M, NPLA) and 1400W (10 mu M), respectively, nNOS and iNOS inhibitors, reduced NMDA-stimulated glutamate overflow. After in vitro l/R, glutamate overflow increased, and returned to control values in the presence of NPLA and 1400W. NO2- and NO3- levels transiently increased during l/R and were reduced by both (-)-AP5 and 5,7-diCl-KYN. In longitudinal muscle myenteric plexus preparations, iNOS mRNA and protein levels increased after in vitro I/R; both parameters were reduced to control values by (-)-AP5 and 5,7-diCl-KYN. Both antagonists were also able to reduce ischemia-induced enhancement of nNOS mRNA levels. Protein levels of GluN1, the ubiquitary subunit of NMDA receptors, increased after l/R and were reduced by both NPLA and 1400W. On the whole, this data suggests the existence of a cross-talk between NMDA receptor and nitrergic pathways in guinea pig ileum myenteric plexus, which may participate to neuronal rearrangements occurring during I/R. (C) 2015 Elsevier B.V. All rights reserved.