Ljiljana Šćepanović

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

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.

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.

Myocardial Na+ K+-ATPase and SERCA: Clinical and Pathological Significance From a Cytological Perspective

Structure and functions of Na+/K+-ATPase and SERCA are described with details on their subunits, isoforms, and intracellular localization. Main regulatory mechanisms are summarized. Molecular mechanisms of cell death and heart failure are explained with the analysis of the role of Na+/K+-ATPase and SERCA in these processes. Facts are considered from a cytological, pathological, and clinical perspective with an accent to new therapeutic strategies. The aim of this contribution is an overview of functional results in a structural context.

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.

[Plasma levels of cortisol and opioid peptide beta-endorphin during spontaneous vaginal delivery].

INTRODUCTION Labor pain is very frequent in clinical practice, but the underlying mechanisms as well as numerous neuroendocrine responses activated by such pain have not been fully explained yet. OBJECTIVE The objective of the study was to determine the influence of labor pain on plasma levels of cortisol and opioid peptide beta-endorphin. METHOD Cortisol and beta-endorphin levels were measured in blood plasma of: health, non-pregnant women (group 1, n=8), health pregnant women (group 2, n=8) and in parturitions, through fourth ages (group 3, n=8). Plasma level ofcortisol was measured by radioimmunoassay, and beta-endorphin by enzyme immunoassay. Data were expressed as mean +/- standard error of mean and were analyzed by Students t test and Mann Whitney test. RESULTS Plasma level of cortisol in group 2 was significantly increased compared to the group 1. During labor progression, plasma level of cortisol was rising till the third labor age. Plasma level of cortisol in fourth labor age was not significantly different from the age one and group 1. Plasma level of beta-endorphin was (ng/L): in group 1: 64 +/- 20, group 2: 70 +/- 22, group 3: the first labor age: 75 +/- 15, the second labor age: 193 +/- 54, the third labor age: 346 +/- 97 and the fourth labor age: 114 +/- 31. CONCLUSION These results indicate that both beta-endorphin and cortisol are involved in regulation and modulation of labor pain and stress.