Iranian

---

Atropine has been used to block cholinergic neurotransmission in basic research. Large doses of atropine cause vasodilation of the blood vessels in the skin. This effect is apparently unconnected with the antimuscarinic activity of atropine and seems to be due to a direct action on the blood vessels. It has been suggested that atropine blocks muscarinic receptors at low doses and it induces the release of endothelium derived relaxing factor (EDRF) at large doses. This study examined the effects of atropine on isolated rat pulmonary artery rings with or without endothelium intact in the absence and presence of nitric oxide synthase inhibitors, N-omega nitro-L-arginine methyl ester (L-NAME) or N-omega nitro-L-arginine (L-NOARG) that precontracted with phenylephrine (PHE), 5-hydroxy-tryptamine (5-HT) or KCl. Atropine (1 nM) blocked the vasorelaxant effect of acethylcholine (1 µM) in pulmonary artery rings precontracted with PHE (100 nM). Atropine (10 nM-5 μM) also produced concentration dependent relaxation in these rings precontracted with PHE or 5-HT, but did not relax rings precontracted with KCl. The vasorelaxant effects of atropine were partially inhibited by the mechanical remove of endothelium or pretreatment the rings with L-NAME or L-NOARG, although they were not statistically significant. These results suggest that the ability of atropine to relax pulmonary artery rings may be dependent upon the mechanism of action of the precontracting agonist and also, the vasorelaxant effect of atropine is not wholly mediated by the release of NO (nitric oxide)/EDRF.

---

Estrogen Replacement Therapy (ERT) in postmenopausal women may decrease the risk of Coronary Artery Diseases (CAD). We hypothesized that Nitric Oxide (NO) releasing due to ERT may be the essential factor by which endothelial permeability decreases. Four groups of ovariectomized rabbits were under investigation for five weeks. Groups 1 & 4 received high cholesterol diet and other two groups (2 & 3) had normal diet. Estradiol valerate (5 mg) was injected weekly in groups 1 & 2. Blood samples were taken before and after the experiment. Finally, the animals were sacrificed for endothelial permeability determination and pathological investigation of aortae. After five weeks, the total cholesterol, triglycerides, HDL and LDL were significantly different between high cholesterol‑fed and normal diet groups (P<0.05). In cholesterol-fed groups, triglycerides concentration was also different significantly (P<0.05). Nitrite concentration was increased significantly in group 1, and it was different from other groups (P<0.05). A considerable decrease of aorta permeability was obtained in group 1 but it was not significantly different from group 4 (P<0.1). The considerable existence of fatty streaks was observed in the animals aortae of group 4, and it was significantly different from group 1 (P<0.05). It suggests that prevention of intimal collection of foam cells and fatty streak in aorta by estrogen may be exerted by NO production.

---

Clinical studies have shown that in pathological conditions such as endometriosis and reproductive tract infection (male and female) there is an activation status of macrophages that produce large quantities of nitric oxide (NO) in addition to other effector molecules. Large amounts of NO may have embryotoxic roles and produce infertility. This study was designed to evaluate the effect of different concentrations of NO on mouse pre-implantation embryo development in vitro. Mouse embryos (2-cell stage) were cultured in media containing different concentrations of sodium nitroprusside (SNP), an NO donor, or L-arginine methyl ester (L-NAME), an NO syntase (NOS) inhibitor. At the end of culture, cell apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. The results showed that development of preimplantation embryos were inhibited by high concentration of SNP (1 and 10 μM). In contrast, 0.1 μM of SNP stimulated the embryo development. Similarly, the inhibition of NO by NOS inhibitor resulted in the dose-dependent inhibition of embryo development, but the addition of 0.1 μM SNP with L-NAME reversed the inhibitory effect of L-NAME. TUNEL technique showed that high concentration of NO could induce apoptosis in the embryo, but at low concentration, it decreased apoptotic cell death

---

Angiotensin-converting enzyme (ACE) inhibitors including quinapril could exert a protective effect on cardiovascular system through endothelial system in normoglycemic and diabetic rats. The present experimental work was designed to study the vascular reactivity of aortic ring segments isolated from streptozotocin (STZ)-diabetic rats treated for 4 weeks with nitro-L-arginine-methyl ester (L-NAME 50 mg/100 ml) or L-NAME plus quinapril (10 mg/100 ml) in drinking water. The results showed that quinapril treatment significantly attenuated the augmented contractile response to phenylephrine and KCl in diabetic rats. In addition, quinapril treatment partially restored the reduced contractile response in diabetic animals treated chronically with L-NAME. It can be concluded that quinapril could partly counteract the effect of long-term L-NAME administration on vascular reactivity in STZ-diabetic rats

---

It is a well-established fact that adenosine and its receptor subtypes (A 1 and A ) are involved in changes of contractility, heart rate and coronary blood flow (CBF) under different circumstances. This study was conducted to evaluate the role of nitric oxide and prostaglandins in development of these changes. For this purpose, Nitro-L-Arginine methyl ester (L-NAME), and indomethacin as inhibitors of nitric oxide and prostaglandins synthesis were used respectively. In this respect, guinea pig isolated hearts were randomly divided into control (receiving adenosine) and groups II and III which received L-NAME (100 µM) and indomethacin (50 nM) before adenosine application, respectively, using isolated heart setup. The results showed that adenosine increased CBF and decreased heart rate and contractility in control group. In the presence of L-NAME, adenosine was less effective in enhancing the CBF and decreasing cardiac contractility. Furthermore, no significant change was observed in the presence of indomethacin (regarding all of parameters). It can be concluded that nitric oxide (and not prostaglandins) is essential for the effect of adenosine on CBF and cardiac contractility. Iran. Biomed. J. 9 (4): 177-180, 2005 2

---

It has been shown that nitric oxide is synthesized in the central nervous system as well as in vascular endothelial cells. Recently, it was reported that nitric oxide was involved in central cardiovascular regulation, baroreflex modulation, and involved in a reciprocal release with excitatory amino acids in the nucleus tractus solitarii of rats. The purpose of the present study was to investigate the possible interaction of nitric oxide and glucose in the nucleus tractus solitarii on blood pressure regulation. Male Wistar stereptozotocin induced diabetic rats were anesthetized with urethane. A cannula was inserted above the nucleus tractus solitarii and blood pressure was monitored intra-arterially. Unilateral microinjection of L-glutamate (2.3 nmol/60 nL) into the nucleus produced a decrease in blood pressure in diabetic rats. Microinjection of lidocaine (0.5 µl %2) increased blood pressure. Unilateral microinjection of sodium nitroprusside (100 mmol/60 nL) into the nucleus increased blood pressure in diabetic rats. After microinjection of sodium nitroprusside, the depressive responses to glutamate were significantly attenuated. These results demonstrated the probable role of glucose on blood pressure regulation in diabetic animals affecting on nitric oxidergic neurons and so it implicates an interaction between nitric oxide and glucose in central cardiovascular regulation. Iran. Biomed. J. 10 (1): 15-19, 2006

---

It is well established that the esophageal distention (ED) leads to gastric relaxation, partly by vago-vagal reflex, but till now, the effect of ED on gastric acid secretion has not been investigated. The aim of this study was to investigate the effect of ED on basal and stimulated gastric acid secretion. Methods: Adult male Wistar rats (200-240 g) were deprived of food but not the water 24 h before the experiments. Under urethane anesthesia (1.2 g/kg, i.p.), animals underwent tracheostomy and laparotomy. A catheter was inserted in the stomach through duodenum for gastric distention and gastric washout and the esophagus was cannulated with a distensible balloon orally to distend esophagus (0.3 ml, 10 min). Gastric acid secretion was stimulated by gastric distention, carbachol (4 µg/kg, i.p.) or histamine (5 mg/kg, s.c.). Effects of vagotomy, NG–nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v.) and also hexamethonium were investigated. Results: Basal and gastric distention- and carbachol, histamine-stimulated acid secretion were reduced by the ED (P<0.05, P<0.0001, P<0.01 and P<0.02, respectively). L-NAME (10 mg/kg, i.v.) elevated the acid output (P<0.002). Vagotomy reduced the inhibitory effect of the esophagus distention on gastric distention-induced acid secretion (P<0.01). Conclusion: These results indicate that the vagus nerves are involved in the inhibitory effect of the ED on the basal and stimulated gastric acid secretion. Furthermore, nitric oxide could be involved.

---

Evidence supports the involvement of nitric oxide (NO) in a Varity of male reproductive processes such as spermatogenesis, spermiogenesis, sperm motion, sperm metabolism and sperm capacitation. However, Low concentration of NO is essential in biology and physiology of spermatozoa, but high amounts of NO is toxic and has negative effects on sperm functions. On the other hand, it is established that high amounts of NO have detrimental effects on DNA. The integrity of sperm DNA is an important factor in successful fertility and embryo development. It is hypothesized that supra physiological concentrations of NO in seminal plasma cause sperm DNA damage. The aim of this study was to determine sperm DNA damage by comet assay and its correlation with NO level in seminal plasma of fertile and infertile men. Methods: Semen samples were collected from 45 patients and 70 healthy donors. The stable metabolites of NO (nitrite and nitrate) in seminal plasma were measured by Griess assay and DNA damage was determined using single cell gel electrophoresis (comet) assay method. Results: The NO concentration in the seminal plasma of infertile males was significantly higher than fertile males (5.74 ± 1.01 &muM/L vs. 3.88 ± 0.53 &muM/L). There was a significant positive correlation between the NO concentration and sperm DNA comet value in infertile males (P<0.01, R = 0.598). Conclusion: These results indicate that the overproduction of NO in genital tract of infertile males has a potential pathogenetic role in the reduction of sperm DNA integrity.

---

Background: The assessment of altered nitric oxide (NO) availability is of potentially important diagnostic and prognostic significance. The present study is aimed to investigate the effect of L-arginine (as a natural NO donor) supplementation on NO metabolite in a rabbit model of hypercholesterolemia to find a reliable marker for endothelial NO production. Methods: White male rabbits (n = 30) randomly assigned to 2 groups. Rabbits were fed 1% high-cholesterol diet (HC group, n = 15), or HC diet with oral L-arginine (3% in drinking water) (HC + L-arginine group, n = 15) for 4 weeks. The serum levels of lipids, L-arginine, total NO metabolites (NOx), nitrite and nitrate were measured before and after the study. Results: In this study, L-arginine supplementation led to a significant increased plasma level of L-arginine. The serum level of nitrite was significantly higher in L-arginine treated group while serum level of nitrate and NOx was significantly lower than HC group. Conclusion: As the result of our study showed, nitrite is a useful marker of endogenous endothelial NO production and although frequently used, neither nitrate nor NOx are reliable markers of acute changes in endothelial NO synthase activity.

---

Background: Role of nitric oxide (NO) in morphine-induced conditioned place preference (CPP) has already been proposed in the rat medial septum (MS), but no molecular evidence has been provided to clear this fact. Methods: Effects of intraseptal injections of L-arginine and/or NG-nitro-L-arginine methyl ester (L-NAME) on morphine place conditioning in Wistar rats were examined. Morphine (2.5-7.5 mg/kg) was injected s.c. using a three-day schedule of an unbiased place preference. All of the brain samples were examined histochemically by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), the main marker for NO activation. Results: Morphine induced a significant CPP in the rats. Single injections of L-arginine or L-NAME (0.3, 1.0 and 3.0 µg/rat) did not induce CPP. In addition, co-administration of morphine (5.0 mg/kg) with L-arginine or L-NAME (0.3, 1.0 and 3.0 µg/rat) did not affect morphine response. However, administration of L-arginine (0.3, 1.0 and 3.0 µg/rat) prior to morphine conditioning testing enhanced the expression of morphine response. Moreover, pre-injection of L-NAME (0.3, 1.0 and 3.0 µg/rat) to L-arginine (0.3 µg/rat) did not reverse the response to the agent. The expression of NADPH-d was observed in the rat brain samples treated by L-arginine. A decreased expression of NADPH-d was also observed in rats pre-injected by L-NAME. Conclusion: This finding strongly suggests that NO system in the rat MS has an impact on the expression of morphine rewarding, and that the NO participates in place conditioning induced of morphine.

---

Background: Nitric oxide synthase (NOS) activity is increased during hypertension and cerebral ischemia. NOS inactivation reduces stroke-induced cerebral injuries, but little is known about its role in blood-brain barrier (BBB) disruption and cerebral edema formation during stroke in acute hypertension. Here, we investigated the role of NOS inhibition in progression of edema formation and BBB disruptions provoked by ischemia/reperfusion injuries in acute hypertensive rats. Methods: Rats were made acutely hypertensive by aortic coarctation. After 7 days, the rats were randomly selected for the recording of carotid artery pressure, or regional cerebral blood flow (rCBF) using laser Doppler. Ishcemia induced by 60-min middle cerebral artery occlusion (MCAO), followed by 12-h reperfusion. A single i.p. dose of L-NAME (1 mg/kg) was injected before MCAO. After evaluation of neurological disabilities, rats were slaughtered under deep anesthesia to assess cerebral infarction volume, edema, or BBB disruption. Results: A 75-85% reduction in rCBF was occurred during MCAO which returned to pre-occluded levels during reperfusion. Profound neurological disabilities were evidenced after MCAO alongside with severe cerebral infarctions (628 ± 98 mm3), considerable edema (4.05 ± 0.52%) and extensive BBB disruptions (Evans blue extravasation, 8.46 ± 2.03 µg/g). L-NAME drastically improved neurological disabilities, diminished cerebral infarction (264 ± 46 mm3), reduced edema (1.49 ± 0.47%) and BBB disruption (2.93 ± 0.66 µg/g). Conclusion: The harmful actions of NOS activity on cerebral microvascular integrity are intensified by ischemia/reperfusion injuries during acute hypertension. NOS inactivation by L-NAME preserved this integrity and diminished cerebral edema.

---

Nitric oxide (NO) is a simple ubiquitous signaling molecule and plays important roles in almost every biological system. Recent evidences suggest that NO may act as an endocrine molecule. The aim of this review is considering available literature on endocrine roles of NO and/or its metabolites, i.e. nitrite and nitrate. Existing data suggest the idea that NO is a hormone that after production in tissues, it is stabilized and transported as nitrite and/or S-nitrosothiols in the blood to target cells.

---

Background: Neuroinflammation, as a major outcome of microglia activation, is an important factor for progression of neurodegenerative disorders including Alzheimer's disease and Parkinson's disease. Microglial cells, as the first-line defense in the central nervous system, act as a source of neurotoxic factors such as nitric oxide (NO), a free radical which is involved in neuronal cell death. The aim of this study was to inhibit production of NO in activated microglial cells in order to decrease neurological damages that threat the central nervous system. Methods: An in vitro model of a newborn rat brain cell culture was used to examine the effect of betaine on the release of NO induced by lipopolysaccharide (LPS). Briefly, primary microglial cells were stimulated by LPS and after 2 minutes, they were treated by different concentrations of betaine. The production of NO was assessed by the Griess assay while cell viability was determined by the MTT assay. Results: Our investigations indicated that LPS-induced NO release was attenuated by betaine, suggesting that this compound might inhibit NO release. The effects of betaine on NO production in activated microglial cells after 24 h were "dose-dependent". It means that microglial cells which were treated with higher concentrations of betaine, released lowers amount of NO. Also our observations showed that betaine compound has no toxic effect on microglial cells. Conclusion: Betaine has an inhibitory effect on NO release in activated microglial cells and may be an effective therapeutic component to control neurological disorders.

---

Background: Skin flap grafting is a popular approach for reconstruction of critical skin and underlying soft tissue injuries. In a previous study, we demonstrated the beneficial effects of two 5α-reductase inhibitors, azelaic acid and finasteride, on tissue survival in a rat model of skin flap grafting. In the current study, we investigated the involvement of nitric oxide and inducible nitric oxide synthase (iNOS) in graft survival mediated by these agents. Methods: A number of 42 male rats were randomly allocated into six groups: 1, normal saline topical application 2, azelaic acid (100 mg/flap) 3, finasteride (1 mg/flap) 4, injection of L-NG-nitroarginine methyl ester (L-NAME) (i.p., 20 mg/kg) 5, L-NAME (20 mg/kg, i.p.) + azelaic acid (100 mg/flap, topical) 6, L-NAME (20 mg/kg, i.p.) + finasteride (1 mg/flap, topical). Tissue survival, level of nitric oxide, and iNOS expression in groups were measured. Results: Our data revealed that azelaic acid and finasteride significantly increased the expression of iNOS protein and nitric oxide (NO) levels in graft tissue (P < 0.05). These increases in iNOS expression and NO level were associated with higher survival of the graft tissue. Conclusion: It appears that alterations of the NO metabolism are implicated in the azelaic acid- and finasteride-mediated survival of the skin flaps.

---

Background: Microglial cells act as the sentinel of the central nervous system .They are involved in neuroprotection but are highly implicated in neurodegeneration of the aging brain. When over-activated, microglia release pro-inflammatory factors, such as nitric oxide (NO) and cytokines, which are critical in eliciting neuroinflammatory responses associated with neurodegenerative diseases. This study examined whether bromelain, the pineapple-derived extract, may exert an anti-inflammatory effect in primary microglia and may be neuroprotective by regulating microglial activation. Methods: Following the isolation of neonatal rat primary microglial cells, the activation profile of microglia was investigated by studying the effects of bromelain (5, 10, 20, and 30 µg/ml) on the levels of NO, inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-&kappaB) in microglia treated with lipopolysaccharide (LPS) (1 µg/ml). Data were analyzed using Student's t-test. P values less than 0.05 were considered to be statistically significant, compared with the LPS-treated group without bromelain. Results: Results showed that pretreatment of rat primary microglia with bromelain, decreased the production of NO induced by LPS (1 µg/ml) treatment in a dose-dependent manner. Bromelain (30 µg/ml) also significantly reduced the expression of iNOS at mRNA level and NF-&kappaB at protein level. Moreover, the study of mitochondrial activity in microglia indicated that bromelain had no cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to cell death. Conclusion: Bromelain can be of potential use as an agent for alleviation of symptoms in neurodegenerative diseases.

---