Cyclical upregulated iNOS and long-term downregulated nNOS are the bases for relapse and quiescent phases in a rat model of IBD (original) (raw)

Porras, M., M. T. Martín, R. Torres, and P. Vergara. Cyclical upregulated iNOS and long-term downregulated nNOS are the bases for relapse and quiescent phases in a rat model of IBD..—We previously reported that indomethacin induces a chronic intestinal inflammation in the rat where the cyclical characteristic phases of Crohn's disease are manifested with a few days' interval and lasting for several months: active phase (high inflammation, hypomotility, bacterial traslocation) and reactive phase (low inflammation, hypermotility, no bacterial traslocation). In this study, we investigated the possible role of both constitutive and inducible isoforms of nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) in the cyclicity of active and reactive phases in rats with chronic intestinal inflammation. Rats selected at either active or reactive phases and from 2 to 60 days after indomethacin treatment were used. mRNA expression of both consti-tutive and inducible NOS and COX isoforms in each phase was evaluated by RT-PCR and cellular enzyme localization by immuno-histochemistry. The effects of different COX and NOS inhibitors on the intestinal motor activity were tested. mRNA expression of COX-1 was not modified by inflammation, whereas mRNA expression of neuronal NOS was reduced in all indomethacin-treated rats. In contrast , NOS and COX inducible forms showed a cyclical oscillation. mRNA expression and protein of both iNOS and COX-2 increased only during active phases. The intestinal hypomotility associated with active phases was turned into hypermotility after the administration of selective iNOS inhibitors. Sustained downregulation of constitutive NOS caused hypermotility, possibly as a defense mechanism. However , this reaction was masked during the active phases due to the inhibitory effects of NO resulting from the increased levels of the inducible NOS isoform. nitric oxide; prostaglandins; intestinal inflammation; dysmotility; in-ducible nitric oxide synthase; neuronal nitric oxide synthase INFLAMMATORY BOWEL DISEASE (IBD) includes two chronic pa-thologies characterized by alternation of active and quiescent phases of inflammation: ulcerative colitis and Crohn's disease (9). Although its pathogenesis has not been well established, it has been suggested that some clinical manifestations frequently observed in IBD patients, such as abdominal pain, nausea, vomiting, ileus, or diarrhea, could be attributed to the deranged gastrointestinal motility associated with inflammation (5). Furthermore , an overproduction of nitric oxide (NO) and prosta-glandins has been detected in inflamed intestinal samples in both human and experimental IBD (1, 7). In a healthy state, constitutive neuronal NO synthase (nNOS) produces basal levels of NO to maintain the physiological tonic inhibition of the intestine (6). Moreover, NO derived from nNOS also plays a role in the control of small intestinal motor parameters, i.e., inducing the conversion of fasting motility into a postprandial pattern (23). Prostaglandins produced by constitutive COX (COX-1) also modulate some gastrointestinal functions under physiological conditions, inducing contractibility of smooth muscles and neurotransmitter release (10). During inflammation, inducible isoforms of NOS (iNOS) and COX (COX-2) are produced in response to different stimuli, such as LPS and proinflammatory cytokines (8, 13). Although several studies (14, 15, 26) have focused on the relationship between overproduction of NO and prostaglandins derived from iNOS and COX-2 and the disturbed motor activity related to intestinal inflammation, their specific pathophys-iological effect on gut motility is still controversial. By modifying a previously reported protocol (24), we have obtained a new chronic model of IBD induced by indomethacin and characterized by spontaneous cyclical alternation of active and reactive phases of inflammation, which are correlated with long-lasting motor disturbances (21). In this model, active phases of inflammation were related to high blood leukocyte and TNF concentration as well as a reduced motor activity, bacterial overgrowth, and bacterial translocation into the intestinal wall. In contrast, reactive phases were related to a reduction of inflammatory parameters, hypermotility, and normal bacterial load. This reactive phase is equivalent to the quies-cent periods in IBD disease. Because of the relevant role of NO during inflammatory states, we hypothesized that hypomotility associated with the active inflammatory phases of our model is related to an overproduction of NO derived from increased levels of iNOS. However, because coexpression of iNOS and COX-2 has been reported in intestinal inflammation, a putative role of prosta-glandins in the disturbed motor activity cannot be dismissed. Thus the aims of the present study were 1) to evaluate the mRNA expression of the constitutive and inducible isoforms of both NOS and COX by RT-PCR in both active and reactive phases of inflammation; 2) to confirm and localize iNOS and COX-2 protein by immunohistochemistry; and 3) to determine the specific contribution of these enzymes on the deranged motor activity by testing the effects of different NOS and COX enzyme inhibitors on the intestinal motility of anesthetized rats.