ABSTRACT
Title
Involvement of neuronal and inducible nitric oxide synthases in NO formation during acute in vitro ischemia/reperfusion in the guinea pig ileum
Authors
E. Carpanese1,S. Marchet1, E. Moro2, P. Moretto3, G. Cipolla2, C. Giaroni1, F. Crema2,S. Lecchini1, G.Frigo2.
Dept of Clinical Medicine, University of Insubria1; Dept. of Internal Medicine and Therapeutics, University of Pavia2; Dept. of Biomedical Clinical Science University of Insubria3
Dept of Clinical Medicine, University of Insubria1; Dept. of Internal Medicine and Therapeutics, University of Pavia2; Dept. of Biomedical Clinical Science University of Insubria3
Abstract
Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) represents one of the main inhibitory transmitter in the enteric nervous system, exerting several functional effects in the gastrointestinal tract, which include modulation of the descending reflex during peristalsis and accommodation (Holzer et al., 1997). There are reports suggesting that NO produced by nNOS may participate also to enteric neuronal derangements occurring during an ischemic/reperfusion damage in the gut, that may contribute to gastrointestinal dysmotility (Rivera et al., 2009). In these conditions, activation of inducible NO synthase (iNOS) may also be detrimental to the intestinal motor function after a metabolic insult (Naito et al., 2004). In the present study, we further investigated the possible participation of both nNOS and iNOS in NO formation in the gastrointestinal tract during an ischemic/reperfusion insult induced in vitro in the guinea pig ileum. Conditions of ischemia/reperfusion were obtained by perfusing isolated longitudinal muscle-myenteric plexus preparations with a physiological Tyrode’s solution deprived of oxygen and glucose for 60 min, followed by 60 min of reperfusion with an oxygenated medium containing glucose. NO formation in the absence and presence of nNOS and iNOS inhibitors was quantified in the superfusate via the nitrite/nitrate method, based on the colorimetric Griess reaction. Expression of ileal nNOS and iNOS was evaluated by immunoblotting and immunohistochemistry. Under normal conditions, NO2- and NO3- concentrations were 3.60±0.25 µmol g-1 min-1 (n=36) and 14.84±1.33 µmol g-1 min-1 (n=31), respectively, and were stable for 120 min. NO2- Holzer P et al. (1997). J Pharmacol Exp Ther. 280, 154-161. Rivera et al. (2009) Acta Neuropathol. 118, 261-270. Naito et al. (2004) Nitric Oxide 10, 170-177. |
concentration moderately increased after in vitro ischaemia and raised significantly above control values 5’ after reperfusion (P<0.01). NO3- concentration significantly increased 5’ after in vitro ischemia (P<0.05) and raised moderately above control values during reperfusion. The non selective NOS blocker, L-NAME (100 µM), the nNOS selective blocker 3-bromo-7-nitroindazole (10 µM), and the iNOS selective blocker, 1400W (10 µM), significantly (p<0.0001) reduced enhancement of both NO2- and NO3- concentrations after ischemia/reperfusion. Protein levels of both nNOS and iNOS decreased 5' after inducing in vitro ischemia and their expression remained downregulated thereafter. In ileal whole mounts preparations of control animals the number of myenteric neurons staining for nNOS was 27.70±2.90% (n=4) and did not significantly change after in vitro ischemia/reperfusion. In these experimental conditions also the number of myenteric neurons, as indicated by labelling with HUC/D, used as a neuronal cell marker, remained unchanged with respect to control preparations. On the whole the present data suggest that, in the guinea pig ileum, both nNOS and iNOS participate to a transient enhancement of NO formation during in vitro ischemia/reperfusion. Such contribution do not seem to depend upon increased expression of both synthases.