Marija Stojanović

Multidisciplinary approach to nitric oxide signaling: Focus on the gastrointestinal and the central nervous system.

Nitric oxide (NO), with close historical ties to cardiovascular physiology, is an important endogenous mediator, the most potent natural vasorelaxant known, involved in many biological functions . NO acts as an intra/intercellular signalling molecule and is a mediator in almost all organ systems . NO is known to occur in many cells types, including vascular endothelial cells, neurons, and epithelial cells . The transmitter of nonadrenergic, noncholinergic (NANC) inhibitory neurons has been the subject of hundreds of investigations over the past 3 decades. Recent evidence suggests that NO may serve as a NANC inhibitory signaling molecule in the gastrointestinal (GI) tract. NO serves as the primary enteric inhibitory neurotransmitter in GI muscles, and nitrergic neurons regulate gut tone, phasic contractile amplitude and frequency, and inhibitory reflexes . In the central nervous system (CNS) NO is involved in some major processes such as memory through longterm potentiation (LTP) and learning . This gasotransmitter also contributes to a pathogenesis of epilepsy. The role of NO in the generation of epilepsy is contradictory since there is evidence of its proconvulsive and anticonvulsive effects . In this review, we will discuss about the possible role of NO as neurotransmitter in the GI and CNS, with focus on the contribution of NO-mediated signaling pathways in the GI motility and CNS excitability. Physiological functions of nitric oxide

Influence of fatty acid on lipase-catalyzed synthesis of ascorbyl esters and their free radical scavenging capacity

Fatty acid (FA) ascorbyl esters are recently emerging food, cosmetic, and pharmaceutical additives, which can be prepared in an eco‐friendly way by using lipases as catalysts. Because they are amphiphilic molecules, which possess high free radical scavenging capacity, they can be applied as liposoluble antioxidants as well as emulsifiers and biosurfactants. In this study, the influence of a wide range of acyl donors on ester yield in lipase‐catalyzed synthesis and ester antioxidant activity was examined. Among saturated acyl donors, higher yields and antioxidant activities of esters were achieved when short‐chain FAs were used. Oleic acid gave the highest yield overall and its ester exhibited a high antioxidant activity. Optimization of experimental factors showed that the highest conversion (60.5%) in acetone was achieved with 5 g L−1 of lipase, 50 mM of vitamin C, 10‐fold molar excess of oleic acid, and 0.7 mL L−1 of initial water. Obtained results showed that even short‐ and medium‐chain ascorbyl esters could be synthesized with high yields and retained (or even exceeded) free radical scavenging capacity of l‐ascorbic acid, indicating prospects of broadening their application in emulsions and liposomes.

Different pathways involved in the stimulatory effects of homocysteine on rat duodenal smooth muscle

Abstract Recent studies have confirmed that hyperhomocysteinemia is associated with gastrointestinal diseases; however, the direct effect of homocysteine on gastrointestinal reactivity still remains unknown. The aim of this study was to demonstrate how homocysteine may affect nitric oxide mediated duodenal relaxation and whether cholinergic receptors and K+ channels take part in stimulating motility, as well as to explore whether oxidative stress is associated with homocysteine-mediated effects. Experiments were carried out on male rats, body mass 250-300 g. Two groups of animals were treated by i.p. application of saline and D,L-Hcy (0.6 μmol/g bm). After 2h of incubation, the duodenal segments were prepared for biochemical analysis and contractile response measurements in an organ bath with Tyrode’s solution. Effects of TEA (10 mmol/L) and L-NAME (30 μmol/L) on duodenal contractility in the presence of D,L-Hcy (0.6 μmol/g bm) were investigated. Elevated homocysteine levels seem to be of crucial importance for the deterioration of contractility through nitric oxide mediated relaxation, and, in part, by activation of K+ channels. Hcy showed direct promuscarinic effects, since 30 min pretreatment of rat duodenum significantly enhanced the contractile effect of increasing concentrations of ACh (10−9-10−2 mol/L). Catalase activity, superoxide dismutase, glutathione peroxidase and the total antioxidant system were reduced while the thiobarbituric acid-reactive substances level was elevated. Our data showed a consistent profile of gastrointestinal injury elicited by sulfur-containing amino acid-homocysteine. This could contribute to explain, at least in part, the mechanisms involved in human gastrointestinal diseases associated to hyperhomocysteinemia.

Effects of Acute Administration of D,L-Homocysteine Thiolactone on the Antioxidative Status of Rat Intestine and Liver

Abstract Oxidative stress appears to play a role in the pathogenesis of several inflammatory gastrointestinal diseases. Increased homocysteine levels may play a role in the pathogenesis of Chron’s disease and ulcerative colitis. The aim of this study was to examine the influence of homocysteine on the antioxidant status of rat intestine and liver. The levels of thiobarbituric acid reactive substances (TBARS), activity of catalase (CAT) and total antioxidant status (TAS) were investigated in the isolated gut and liver of young male rats in the control group (8 rats) and after 3-hоur incubation in high doses of D, L-homocysteine thionolactone (Hcy) (10 μmol/L) (8 rats). Samples of duodenum, ileum, colon and liver were homogenized in sodium phosphate buffer (1:10). Homogenates were centrifuged at 10000 for 10 min at 4° C and the supernatant was taken for biochemical assays. Our results showed that high D, L-homocysteine thionolactone concentration reduced enzymatic catalase activity in homogenates of the isolated segments of duodenum (27.04%) p<0.01; ileum (37.27%), colon (34.17%) and liver (67.46%) p<0.001. Exposition to high D,L-homocysteine thiolactone concentration significantly increased TBARS levels in the duodenum (106.05%), ileum (47.24%), colon (112.75%) and liver (32.07%) (p<0.01). Homocysteine also modifi ed the total antioxidant status of homogenates from the duodenum, ileum, colon and liver, increasing by 20.68% (duodenum), 24.74% (ileum), 14.88% (colon) and 19.35% (liver) (p<0.001). Homocysteine induced a consistent oxidative stress in rat’s intestine and liver (reduced activity of catalase and increased level of TBARS), but the elevated activity of TAS in our experiments could be explained as an adaptive response to the generated free radicals which indicates the failure of the total antioxidant defense mechanism to protect the tissues from damage caused by homocysteine.

Strain differences in thymic atrophy in rats immunized for EAE correlate with the clinical outcome of immunization

An accumulating body of evidence suggests that development of autoimmune pathologies leads to thymic dysfunction and changes in peripheral T-cell compartment, which, in turn, perpetuate their pathogenesis. To test this hypothesis, thymocyte differentiation/maturation in rats susceptible (Dark Agouti, DA) and relatively resistant (Albino Oxford, AO) to experimental autoimmune encephalomyelitis (EAE) induction was examined. Irrespective of strain, immunization for EAE (i) increased the circulating levels of IL-6, a cytokine causally linked with thymic atrophy, and (ii) led to thymic atrophy reflecting partly enhanced thymocyte apoptosis associated with downregulated thymic IL-7 expression. Additionally, immunization diminished the expression of Thy-1, a negative regulator of TCRαβ-mediated signaling and activation thresholds, on CD4+CD8+ TCRαβlo/hi thymocytes undergoing selection and thereby impaired thymocyte selection/survival. This diminished the generation of mature CD4+ and CD8+ single positive TCRαβhi thymocytes and, consequently, CD4+ and CD8+ recent thymic emigrants. In immunized rats, thymic differentiation of natural regulatory CD4+Foxp3+CD25+ T cells (nTregs) was particularly affected reflecting a diminished expression of IL-7, IL-2 and IL-15. The decline in the overall thymic T-cell output and nTreg generation was more pronounced in DA than AO rats. Additionally, differently from immunized AO rats, in DA ones the frequency of CD28- cells secreting cytolytic enzymes within peripheral blood CD4+ T lymphocytes increased, as a consequence of thymic atrophy-related replicative stress (mirrored in CD4+ cell memory pool expansion and p16INK4a accumulation). The higher circulating level of TNF-α in DA compared with AO rats could also contribute to this difference. Consistently, higher frequency of cytolytic CD4+ granzyme B+ cells (associated with greater tissue damage) was found in spinal cord of immunized DA rats compared with their AO counterparts. In conclusion, the study indicated that strain differences in immunization-induced changes in thymopoiesis and peripheral CD4+CD28- T-cell generation could contribute to rat strain-specific clinical outcomes of immunization for EAE.

The role of oxidative stress as a risk factor for rupture of posterior inferior cerebellar artery aneurysms

Cerebral aneurysm affects 2–5% of the population and posterior inferior cerebellar artery (PICA) aneurysms account for 1–3% of all intracranial aneurysms. Oxidative stress is known to contribute to the progression of cerebrovascular disease and it may be increased by inflammation, a key contributor to cerebral aneurysm development and rupture. The aim of this study was to examine the role of overall oxidative stress as a risk factor for rupture of PICA aneurysms. This study included 29 patients with PICA aneurysms: 18 ruptured and 11 unruptured. We determined catalase, malondialdehyde, myeloperoxidase and carbonyl groups in homogenates of excised aneurysm tissue after surgery and plasma levels of C reactive protein and fibrinogen. The patient’s age and sex, size of aneurysms, multiplicity, history of previous subarachnoidal hemorrhage (SAH) and risk factors for oxidative stress such as hypertension and smoking were compared between unruptured and ruptured aneurysms. Maximal diameter and SAH history were independent predictors for aneurysm rupture. Activity of catalase was decreased while activity of myeloperoxidase, levels of malondialdehyde, carbonyl groups in aneurismal tissue and plasma levels of C reactive protein and fibrinogen were increased in patients with ruptured aneurysms. Plasma levels of C reactive protein and fibrinogen showed positive correlation with myeloperoxidase, malondialdehyde, carbonyl groups and PHASES score and negative correlation with catalase. These findings suggest that oxidative stress may contribute importantly to rupture of PICA aneurysms and plasma levels of C reactive protein and fibrinogen correlate with oxidative stress markers in tissue.

The Effects of Subchronic Methionine Overload Administered Alone or Simultaneously with L-cysteine or N-acetyl-L-cysteine on Body Weight, Homocysteine Levels and Biochemical Parameters in the Blood of Male Wistar Rats

Abstract Hyperhomocysteinemia (HHC), both basal and after methionine load, may occur due to genetic disorders or deficiencies of nutrients that affect the remethylation or trans-sulphuration pathways during methionine metabolism. HHC is involved in the pathogenesis of many illnesses as a result of its prooxidative effect and its impairment of antioxidative protection. The aim was to examine the effects of subchronic methionine overload on the body weight and standard biochemical parameters in rat serum and to examine whether simultaneous subchronic intraperotoneal administration of methionine alone or together with L-cysteine or N-acetyl-cysteine resulted in a change in the body weight and biochemical parameters in the rat serum. The research was conducted during a three-week period (male Wistar albino rats, n=36, body weight of approximately 160 g, age of 15-20 days), and the animals were divided into a control group and three experimental groups of 8-10 animals each: a) control group (0.9% sodium chloride 0.1-0.2 ml/day); b) methionine (0.8 mmol/kg/bw/day) (MET group); c) methionine (0.8 mmol/kg/bw/day) + L-cysteine (7 mg/kg/bw/day) (L-cys+MET group); and d) methionine (0.8 mmol/kg/bw/day) + N-acetyl-L-cysteine (50 mg/kg/bw/day) (NAC+MET group). In addition to the body weight monitoring, the levels of total homocysteine and the standard biochemical parameters in blood samples (plasma or serum) were determined. The results indicated that monitoring the homocysteine levels and standard biochemical parameters in blood could be used for analysis and could provide an excellent guideline for distinguishing between toxic and non-toxic doses of methionine intake, which may be meaningful for clinical applications.

Protective impact of N-acetyl-L-cysteine on methionine stimulatory effects in rat colon

Introduction: Methionine is an essential sulfuric amino acid, which, according to numerous studies, has a role in the reduction of inflammation, caused by changes in colon, and inhibits important pathways in the development of numerous gastrointestinal disorders. However, subchronic methionine loading leads to oxidative stress, which is a potential cause of disease of numerous organs, digestive tract being one of them. N-acetyl-L-cysteine is a natural antioxidant, which affects some of the important signaling pathways and has a potential therapeutic role in treatment of colon disorders. Aim: To examine the influence of N-acetyl-L-cysteine on histological parameters of rat colon, after subchronic methionine load. Material and methods: Experiments were conducted on 15-20 days old male (Wistar albino) rats, weighing 160 g. There were used 24 animals in the experiment, divided into 3 groups, with eight animals per group. First, control group (K), received NaCl, second group received methionine (M group), while third group received methionine + N-acetyl-L-cysteine (N group). The rats were sacrificed after 21 day and tissue samples of colon were obtained. In all of the three groups, changes of histological parameters of rat colon were analyzed. Results: Methionine load leads to statistically significant increase (p < 0.01) in crypt depth of rat colon, lamina muscularis mucosae thickness, the mucosal thickness and increase in lamina propria cell density compared to control, while N-acetyl- L-cysteine application showed statistically significant decrease (p < 0.01) of these histological parameters compared to M group. Conclusion: N-acetyl-L-cysteine has a significantly protective effect on methionine load induced changes in rat colon.