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MK-801, an NMDA receptor antagonist, and scopolamine, a cholinergic receptor blocker, are widely used as tool compounds to induce learning and memory deficits in animal models to study schizophrenia or Alzheimer-type dementia (AD), respectively. Memory impairments are observed after either acute or chronic administration of either compound. The present experiments were performed to study the nitric oxide (NO)-related mechanisms underlying memory dysfunction induced by acute or chronic (14 days) administration of MK-801 (0.3 mg/kg, i.p.) or scopolamine (1 mg/kg, i.p.). The levels of L-arginine and its derivatives, L-citrulline, L-glutamate, L-glutamine and L-ornithine, were measured. The expression of constitutive nitric oxide synthases (cNOS), dimethylaminohydrolase (DDAH1) and protein arginine N-methyltransferases (PMRTs) 1 and 5 was evaluated, and the impact of the studied tool compounds on cGMP production and NMDA receptors was measured. The studies were performed in both the cortex and hippocampus of mice. S-nitrosylation of selected proteins, such as GLT-1, APP and tau, was also investigated. Our results indicate that the availability of L-arginine decreased after chronic administration of MK-801 or scopolamine, as both the amino acid itself as well as its level in proportion to its derivatives (SDMA and NMMA) were decreased. Additionally, among all three methylamines, SDMA was the most abundant in the brain (~70%). Administration of either compound impaired eNOS-derived NO production, increasing the monomer levels, and had no significant impact on nNOS. Both compounds elevated DDAH1 expression, and slight decreases in PMRT1 and PMRT5 in the cortex after scopolamine (acute) and MK-801 (chronic) administration were observed in the PFC, respectively. Administration of MK-801 induced a decrease in the cGMP level in the hippocampus, accompanied by decreased NMDA expression, while increased cGMP production and decreased NMDA receptor expression were observed after scopolamine administration. Chronic MK-801 and scopolamine administration affected S-nitrosylation of GLT-1 transport protein. Our results indicate that the analyzed tool compounds used in pharmacological models of schizophrenia or AD induce changes in NO-related pathways in the brain structures involved in cognition. To some extent, the changes resemble those observed in human samples.

Ghrelin is a novel growth hormone (GH)-releasing and orexigenic peptide with anti-inflammatory activities. However, the role of ghrelin in the colonic inflammation is still controversial. The aim of the present study was: 1) to examine the expression of ghrelin and TNF-alpha mRNA in the inflamed colonic mucosa of patients with ulcerative colitis (UC), 2) to analyze the effect of treatment with exogenous ghrelin on the healing of trinitrobenze sulphonic acid (TNBS)-induced colitis in rats, and 3) to assess the effects of ghrelin treatment on mRNA expression for iNOS and protein expression for COX-2 and PPARalpha in intact colonic mucosa and in that with TNBS-induced colitis. Fifteen patients with UC and fifteen healthy controls were enrolled in this study. Expression of ghrelin and TNF-alpha was assessed by semi-quantitative RT-PCR in the colonic mucosal biopsies from UC patients and healthy controls. In addition, the effect of exogenous ghrelin on healing of TNBS colitis was tested in rats without or with capsaicin-induced functional ablation of sensory nerves. Patients with UC showed a significant upregulation of mRNA for ghrelin and TNF-alpha in colonic mucosa as compared to that observed in healthy controls. The expression of ghrelin correlated with the grade of inflammation and expression of TNF-alpha. In rats the exogenous ghrelin administered daily at a dose of 20 microg/kg i.p. significantly accelerated the healing of TNBS colitis and this effect was accompanied by an increase in mRNA expression for iNOS and protein expression for COX-2 in the colonic mucosa. The protein expression for PPARgamma, which was down-regulated in rat colonic mucosa after exposure to TNBS as compared to that in intact colonic mucosa, was not significantly influenced by ghrelin treatment. We conclude that 1) patients with UC show an increased mucosal expression of mRNA for ghrelin in the colonic mucosa which could trigger protective response in inflamed colon; and 2) exogenous ghrelin accelerates healing of colonic lesions in animal model of ulcerative colitis via increased release of NO and PGE(2) due to an increase in iNOS and COX-2 expression and stimulation of sensory neuropeptides such as CGRP released from sensory afferent endings.

Orexin-A, identified in the neurons and endocrine cells in the gut, has been implicated in control of food intake and sleep behavior but little is known about its influence on gastric secretion and mucosal integrity. The effects of orexin-A on gastric secretion and gastric lesions induced in rats by 3.5 h of water immersion and restraint stress (WRS) or 75% ethanol were determined. Orexin-A (5-80 microg/kg i.p.) increased gastric acid secretion and attenuated gastric lesions induced by WRS and this was accompanied by the significant rise in plasma orexin-A, CGRP and gastrin levels, the gastric mucosal blood flow (GBF), luminal NO concentration and an increase in mRNA for CGRP and overexpression of COX-2 protein and the generation of PGE(2) in the gastric mucosa. Orexin-A-induced protection was abolished by selective OX-1 receptor antagonist, vagotomy and attenuated by suppression of COX-1 and COX-2, deactivation of afferent nerves with neurotoxic dose of capsaicin, pretreatment with CCK(2)/gastrin antagonist, CGRP(8-37) or capsazepine and by inhibition of NOS with L-NNA. This study shows for the first time that orexin-A exerts a potent protective action on the stomach of rats exposed to non-topical ulcerogens such as WRS or topical noxious agents such as ethanol and these effects depend upon hyperemia mediated by COX-PG and NOS-NO systems, activation of vagal nerves and sensory neuropeptides such as CGRP released from sensory nerves probably triggered by an increase in gastric acid secretion induced by this peptide.

Expression of cyclooxygenase-2 (COX-2) is involved in the chronic inflammation-related development of Barrett's adenocarcinoma and the use of selective COX-2 inhibitors (coxibs) might provide new chemoprevention strategy for Barrett's adenocarcinoma (BA). Despite an excellent gastrointestinal (GI) safety profile of coxibs, their use is limited because of the possible cardiovascular complications. The coupling of NSAIDs with a NO-donating moiety has led to the birth of a new class of anti-inflammatory drugs, called the COX-inhibiting nitric oxide donators (CINODs). The member of this group, NO-aspirin (NO-ASA) retains the anti-inflammatory properties of traditional aspirin (ASA), but the release of NO accounts for anti-thromboembolic effect and better GI safety profile. The role of NO-ASA in the prevention of Barrett's adenocarcinoma (BA) has not been studied so far. Therefore, the aim of the present study was: 1) to analyse the expression of COX-2 in the biopsies obtained from BE; 2) to compare the effect of NO-ASA with that of ASA on proliferation rate in Barrett''s adenocarcinoma cell line (OE-33 cells); 3) to determine the effect of both compounds on the apoptosis rate using FACS analysis and expression of 32-kDa procaspase-3 and active proapoptotic 20-kDa caspase-3 in OE-33 cell line. The expression of COX-2 was assessed in biopsies obtained from the Barrett's mucosa and normal squamous epithelial esophageal mucosa from 20 BE patients by RT-PCR and Western blot analysis, respectively. The BA cell line (OE-33) was incubated with NO-ASA or ASA (10-1000 microM). The cell proliferation and apoptosis rate was measured by BrdU and FACS-analysis, respectively. The expression of caspase-3 (active and inactive form) was analyzed by Western blot. In Barrett's mucosa a significant up-regulation of COX-2 was observed. Compared with traditional ASA, NO-ASA caused a significantly stronger induction of apoptosis (dose-dependently). Inhibition of cell proliferation in OE-33 cells observed under NO-ASA treatment was due to the apoptosis induction. The increase in apoptotic rate was accompanied by the upregulation of active 20-kDa caspase-3. At the highest concentration (1000 microM), a necrotic death of OE-33 cells was observed under NO-ASA treatment. We conclude that: NO-ASA caused induction of apoptosis in BA cell line and slight growth inhibition. These results indicate that this compound may represent a promising chemopreventive agent for Barrett's adenocarcinoma.

Ghrelin has been recently identified as an endogenous ligand for growth hormone secretagogue receptor that regulates growth hormone secretion, increases appetite and contributes to energy homeostasis. Although this peptide is predominantly produced by the fasted stomach, little is known about its influence on the gastric mucosal integrity. The aim of the present study was (1) to investigate the effect of acylated ghrelin on the formation and healing of acute gastric mucosal lesions induced by ischemia-reperfusion and gastric mucosal blood flow in rats; (2) to analyse the effects of the deactivation of afferent sensory nerves with capsaicin and of the inhibition of nitric oxide (NO)-synthase by NG-nitro-l-arginine (l-NNA) on the ghrelin-induced protection; (3) to examine the influence of ghrelin on nuclear factor-kappa B (NF-kappaB) activation and on release of proinflammatory cytokines, such as tumor necrosis factor-alpha, (4) to assess the effect of ghrelin on the mRNA expression of constitutive nitric oxide synthase (cNOS), calcitonin gene related peptide (CGRP) and angiogenesis related proteins such as hypoxia inducible factor-1 alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF), and (5) to determine the effect of ischemia/reperfusion on the gastric mucosa expression of ghrelin in rats without and with administration of exogenous hormone. Wistar rats were exposed to 30 min of ischemia followed by 3 h of reperfusion. Ghrelin was administered in dose of 5, 10 or 20 mug/kg intraperitoneally (i.p.) 30 min prior exposure to ischemia/reperfusion and at 3 h after the end of ischemia, the mean lesion area was measured by planimetry and the changes in gastric blood flow were determined by hydrogen (H2)-gas clearance method. The healing of ischemia/reperfusion induced lesions was evaluated at 24 h or 6 days after the end of standard ischemia/reperfusion. The expression of cNOS, CGRP, HIF-1alpha, VEGF and ghrelin was evaluated by reverse transcription polymerase chain reaction or Western blot. Ghrelin significantly attenuated the ischemia/reperfusion-induced gastric lesions and accelerated the healing of these lesions while significantly raising the gastric blood flow. Deactivation of sensory nerves with capsaicin or inhibition of cNOS by L-NNA significantly attenuated the protective activity of ghrelin and accompanying increase in the GBF. Exogenous ghrelin significantly inhibited the activation of NF-kappaB and plasma TNF-alpha levels. The ghrelin-enhanced acceleration of healing of ischemia/perfusion induced lesions was accompanied by enhanced expression of mRNA for HIF-1alpha and by diminution of the ischemia/reperfusion induced increase in mRNA expression for TNF-alpha. We conclude that ghrelin exerts a potent protective action on the gastric mucosa and accelerates the healing of ischemia/reperfusion-induced lesions and these effects depend upon activation of sensory nerves, hyperemia mediated by NO, increased angiogenesis due to expression of YEGF and anti-inflammatory properties of this peptide.