N-Acetyl Neuraminic Acid (NANA) Activates L-Type Calcium Channels on Isolated Tentacle Supporting Cells of the Sea Anemone (Aiptasia pallida).

AbstractSensory receptors control nematocyst discharge on sea anemone tentacles. Micromolar N-acetylated sugars (e.g., N-acetyl neuraminic acid [NANA]) bind chemoreceptors on ectodermal supporting cells and predispose adjacent nematocyst discharge in response to mechanical contact via a cyclic adenosine monophosphate (cAMP)-dependent sensitization pathway, while higher NANA levels dose-dependently desensitize. Recent evidence implicates L-type calcium channels in desensitizing the pathway in aconitate sea anemones Aiptasia pallida (also known as Exaiptasia diaphana). We, therefore, hypothesize that NANA activates calcium influx via L-type calcium channels. We demonstrate a dose-dependent, NANA-activated 45Ca influx into dissociated ectodermal cells isolated from A. pallida tentacles, with maximal influx occurring at desensitizing concentrations of NANA. The L-type calcium channel inhibitors nifedipine, diltiazem, methoxyverapamil, and cadmium blocked NANA-stimulated 45Ca influx. Elevated extracellular KCl levels dose-dependently increased nifedipine-sensitive 45Ca influx to implicate voltage-gated calcium channels. Forskolin, 8-bromo-cAMP, and the protein kinase A inhibitor H-8 affect NANA-stimulated calcium influx in a manner consistent with activated cAMP-dependent pathway involvement. Because NANA chemoreceptors localize to supporting cells of cnidocyte supporting cell complexes, NANA activation of 45Ca influx into isolated tentacle ectodermal cells suggests that L-type calcium channels and NANA chemoreceptors co-localize to supporting cells. Indeed, a fluorescent marker of L-type calcium channels localizes to the apical ectoderm adjacent to nematocysts of live tentacles. We conclude that supporting cell chemoreceptors activate co-localized L-type calcium channels via a cAMP-dependent mechanism in order to initiate desensitization. We suggest that pathway desensitization may conserve nematocysts from excessive discharge during prey capture.

The effects of glucagon-like peptide 1 receptor agonist (exenatide) on memory impairment, and anxiety- and depression-like behavior induced by REM sleep deprivation.

Previous investigations have shown that REM sleep deprivation impairs the hippocampus-dependent memory, long-term potentiation and causing mood changes. The aim of the present study was to explore the effects of exenatide on memory performance, anxiety- and depression like behavior, oxidative stress markers, and synaptic protein levels in REM sleep deprived rats. A total of 40 male Wistar rats were randomly divided to control, exenatide-treated control, sleep deprivation (SD), wide platform (WP) and exenatide-treated SD groups. During experiments, exenatide treatment (0.5 µg/kg, subcutaneously) was applied daily in a single dose for 9 days. Modified multiple platform method was employed to generate REM sleep deprivation for 72 h. The Morris water maze test was used to assess memory performance. Anxiety- and depression-like behaviors were evaluated by open field test (OFT), elevated plus maze (EPM) forced swimming test (FST), respectively 72 h after REMSD. The levels of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and postsynaptic density proteins 95 (PSD95) were measured in tissues of hippocampus and prefrontal cortex. The content of malondialdehyde (MDA) and reduced glutathione (GSH) were also measured. In the present study, an impairment in memory was observed in SD rats at the 24th hour of SD in compare to those of other groups. REMSD increased depression-like behavior in FST as well as the number of rearing and crossing square in OFT. Anxiety is the most common comorbid condition with depressive disorders. Contents of CaMKII and PSD95 decreased in hippocampus of SD rats. Exenatide treatment improved the impaired memory of SD rats and increased CaMKII content in hippocampus There was no difference in MDA and GSH levels among groups. Exenatide treatment also diminished locomotor activity in OFT. In conclusion, treatment with exenatide, at least in part, prevented from these cognitive and behavioral changes possibly through normalizing CaMKII levels in the hippocampus.

The effects of S-nitrosoglutathione on intestinal ischemia reperfusion injury and acute lung injury in rats: Roles of oxidative stress and NF-κB.

BACKGROUND: Intestinal ischemia and reperfusion (I/R) induces oxidative stress, inflammatory response, and acute lung injury. S-nitrosoglutathione (GSNO), a nitric oxide donor, has been documented to have protective effects on experimental ischemia models. AIM: The aim of this study was to examine the effect of GSNO on I/R-induced intestine and lung damage and detect the potential mechanisms emphasizing the protective role of GSNO. METHODS: Intestinal I/R was induced by occluding the superior mesenteric artery for 30 min followed by reperfusion for 180 min. GSNO was administered intravenously before reperfusion period (0.25 mg/kg). The levels of lipid peroxidation, reduced glutathione, and myeloperoxidase (MPO), histopathological evaluation and immunohistochemical expressions of both nuclear factor KappaB (NF-κB) and inducible nitric oxide (iNOS) in intestine and lung tissues were assessed. RESULTS: Histolopathologic evaluation demonstrated that intestinal I/R induced severe damages in the intestine and the lung tissues. Histopathological scores decreased with GSNO treatment. GSNO treatment reduced lipid peroxidation and MPO levels and inhibited expression of NF-κB and iNOS in the intestine. CONCLUSION: Our results suggest that GSNO treatment may ameliorate the intestinal and lung injury in rats, at least in part, by inhibiting inflammatory response and oxidative stress.

Agmatine attenuates intestinal ischemia and reperfusion injury by reducing oxidative stress and inflammatory reaction in rats.

AIMS: Oxidative stress and inflammatory response are major factors causing several tissue injuries in intestinal ischemia and reperfusion (I/R). Agmatine has been reported to attenuate I/R injury of various organs. The present study aims to analyze the possible protective effects of agmatine on intestinal I/R injury in rats. MAIN METHODS: Four groups were designed: sham control, agmatine-treated control, I/R control, and agmatine-treated I/R groups. IR injury of small intestine was induced by the occlusion of the superior mesenteric artery for half an hour to be followed by a 3-hour-long reperfusion. Agmatine (10mg/kg) was administered intraperitoneally before reperfusion period. After 180min of reperfusion period, the contractile responses to both carbachol and potassium chloride (KCl) were subsequently examined in an isolated-organ bath. Malondialdehyde (MDA), reduced glutathione (GSH), and the activity of myeloperoxidase (MPO) were measured in intestinal tissue. Plasma cytokine levels were determined. The expression of the intestinal inducible nitric oxide synthase (iNOS) was also assessed by immunohistochemistry. KEY FINDINGS: The treatment with agmatine appeared to be significantly effective in reducing the MDA content and MPO activity besides restoring the content of GSH. The treatment also attenuated the histological injury. The increases in the I/R induced expressions of iNOS, IFN-γ, and IL-1α were brought back to the sham control levels by the treatment as well. SIGNIFICANCE: Our findings indicate that the agmatine pretreatment may ameliorate reperfusion induced injury in small intestine mainly due to reducing inflammatory response and oxidative stress.

Reduction of Acute Lung Injury by Administration of Spironolactone After Intestinal Ischemia and Reperfusion in Rats.

PURPOSE: Multiple organ failure, including acute lung injury, is a common complication of intestinal ischemia and reperfusion (I/R) injury and contributes to its high mortality rate. Activated polymorphonuclear neutrophils and reactive oxygen species contribute to the lung injury caused by intestinal I/R. Mineralocorticoid receptor antagonist spironolactone has a protective effect against I/R injury in animal models of retina, kidney, heart, and brain. The aim of the present study is to investigate the effect of aldosterone receptor blocker spironolactone on lung injury induced by intestinal I/R. METHODS: Wistar albino rats were divided into four groups: (1) sham control; (2) intestinal I/R (30 min of ischemia by superior mesenteric artery occlusion followed by 3 h of reperfusion); (3) spironolactone pretreatment (20 mg/kg) + I/R; and, (4) spironolactone pretreatment without I/R. Spironolactone was given orally 3 days prior to intestinal I/R. A marker for lipid peroxidation (malondialdehyde; MDA), an indicator or oxidation state (reduced glutathione; GSH), an index of polymorphonuclear neutrophil sequestration (myeloperoxidase; MPO), inducible nitric oxide synthase (iNOS) immunoreactivity, and the histopathology of the lung tissue were analyzed. RESULTS: Spironolactone pretreatment markedly reduced intestinal I/R-induced lung injury as indicated by histology and MDA and MPO levels. Moreover, the pretreatment decreased the iNOS immunoreactivity. CONCLUSION: The present study strongly suggests that spironolactone pretreatment decreased neutrophil infiltration, iNOS induction, oxidative stress, and histopathological injury in an experimental model of intestinal I/R induced-lung injury of rats.

Neuroprotective efficacy of the peroxisome proliferator-activated receptor-γ ligand in chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion can cause learning and memory impairment and neuronal damage resembling the effects observed in vascular dementia. PPAR-γ agonists were shown to modulate inflammatory response and neuronal death following cerebral ischemia. The present study was designed to evaluate possible neuroprotective effects of rosiglitazone, a PPAR-γ agonist, in rat model of chronic cerebral hypoperfusion. Cerebral hypoperfusion was induced by permanent bilateral occlusion of the common carotid arteries. Oral administration of rosiglitazone (1.5, 3, and 6 mg/kg/day) or vehicle was carried out for 5 weeks, starting one week before the surgery. Cognitive performance was assessed using the Morris water maze. The density of S100B protein-immunoreactive astrocytes and the OX-42-labeled microglial activation were estimated. Synaptogenesis was also evaluated by the measurement of synaptophysin, the pre-synaptic vesicular protein, level via western blotting technique. Cerebral hypoperfusion for 30 days induced a significant cognitive impairment along with hyperactivation of both microglial and astroglial cells, and reduction of synaptophysin level. Rosiglitazone treatment (3 and 6 mg/kg) not only suppressed the activation of astrocytes and microglia markedly but also alleviated the impairment of memory and increased the synaptophysin level. In conclusion, our results suggest that the chronic administration of rosiglitazone significantly prevents chronic cerebral hypoperfusion-induced brain damage, at least, partly through suppressing glial activation and preserving synaptic plasticity. Thus, it appears that rosiglitazone may be a promising pharmacological agent in the development of therapeutic approaches for the prevention or treatment of cerebrovascular diseases.

Protective effects of L-arginine on rat terminal ileum subjected to ischemia/reperfusion.

OBJECTIVES: Studies have shown that nitric oxide (NO) may play a major role in sustaining mucosal integrity; however, NO has been also implicated in the pathogenesis of ischemia/reperfusion (I/R)-related tissue injury. We investigated the effects of L-arginine and NG-nitro L-arginine methyl ester (L-NAME) on the acetylcholine-induced contractile response of ileum and the levels of malondialdehyde (MDA) and reduced glutathione (GSH). Histopathological changes were also evaluated in ileal preparations. MATERIALS AND METHODS: Male Wistar Albino rats were subjected to mesenteric ischemia (30 min) followed by reperfusion (3 hours). Four groups were designed: sham-operated control; I/R; I/R and L-arginine pretreatment; and I/R and L-NAME pretreatment. After reperfusion, ileum specimens were collected to determine the parameters mentioned above. RESULTS: Following reperfusion, a significant decrease in acetylcholine-induced contractile response, an increase in lipid peroxidation, a decrease in GSH content, and mucosal damage of the ileal preparations were observed. We showed that decreased contractility, increased lipid peroxidation, and reduced GSH content have been reversed by L-arginine but not by L-NAME. Mucosal injury was significantly lowered in the L-arginine group. CONCLUSIONS: Treatment with L-arginine exerted a protective effect in intestinal I/R injury, which was mediated in part by regulating MDA and GSH levels, consequently ameliorating impaired contractile response and mucosal injury.

Attenuation of contractile dysfunction by atorvastatin after intestinal ischemia reperfusion injury in rats.

Growing number of studies implicate that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, have beneficial effects on ischemia/reperfusion injury that are unrelated to their cholesterol-lowering action. In the present study, we aimed to evaluate possible effects of atorvastatin on oxidative stress, neutrophil accumulation, and contractile response of terminal ileum segments in rats subjected to intestinal ischemia/reperfusion. Intestinal ischemia/reperfusion model was generated by clamping the superior mesenteric artery for 30 min followed by reperfusion for 3 h. Oral administration of atorvastatin at a dose of 10 mg/kg/day lasted 3 days just before induction of intestinal ischemia. At the end of reperfusion period, terminal ileum samples were removed to determine the concentrations of malondialdehyde, reduced glutathione, and myeloperoxidase. Samples were collected also to assess histopathological alterations and contractile response to agonists. Ischemia/reperfusion significantly decreased contractile responses, and this decrease was attenuated by atorvastatin. Pretreatment with atorvastatin caused remarkable decrease in both oxidative stress and neutrophil accumulation. Atorvastatin appeared to be restoring amount of reduced glutathione back to about control level. Furthermore, the pretreatment lowered mucosal damage at histopathological level. Our results suggested that pretreatment with atorvastatin attenuated intestinal muscle dysfunction associated with ischemia/reperfusion. This remarkable effect of atorvastatin is accomplished at least by decreasing oxidative stress and neutrophil accumulation as well as preventing the depletion of reduced glutathione.

Pretreatment with adenosine and adenosine A1 receptor agonist protects against intestinal ischemia-reperfusion injury in rat.

AIM: To examine the effects of adenosine and A1 receptor activation on reperfusion-induced small intestinal injury. METHODS: Rats were randomized into groups with sham operation, ischemia and reperfusion, and systemic treatments with either adenosine or 2-chloro-N(6)-cyclopentyladenosine, A1 receptor agonist or 8-cyclopentyl-1,3-dipropylxanthine, A1 receptor antagonist, plus adenosine before ischemia. Following reperfusion, contractions of ileum segments in response to KCl, carbachol and substance P were recorded. Tissue myeloperoxidase, malondialdehyde, and reduced glutathione levels were measured. RESULTS: Ischemia significantly decreased both contraction and reduced glutathione level which were ameliorated by adenosine and agonist administration. Treatment also decreased neutrophil infiltration and membrane lipid peroxidation. Beneficial effects of adenosine were abolished by pretreatment with A1 receptor antagonist. CONCLUSION: The data suggest that adenosine and A1 receptor stimulation attenuate ischemic intestinal injury via decreasing oxidative stress, lowering neutrophil infiltration, and increasing reduced glutathione content.

Erythropoietin stimulates wound healing and angiogenesis in mice.

Erythropoietin exerts hematopoietic effects by stimulating proliferation of early erythroid precursors. Nonhematopoietic effects of erythropoietin have also been shown. It may act as a new angiogenic factor in wound healing. This study aimed to investigate the effect of systemic administration of recombinant human erythropoietin on wound healing in mice. Dorsal incisional wounds were performed in mice, which were then divided into two groups; a group treated for 7 days with recombinant human erythropoietin, and a control group. Sacrificing animals on day 7, the wound tissues were collected for analysis of wound breaking strength, malondialdehyde, a marker of lipid peroxidation, hydroxyproline, an index of reparative collagen deposition, reduced glutathione levels, and for histological evaluation. The immunohistochemical determination of vascular endothelial growth factor (VEGF) which is believed to be the most prevalent angiogenic factor throughout the skin repair process, was also studied. The treatment significantly increased wound breaking strength by decreasing malondialdehyde and increasing hydroxyproline levels on day 7 after wounding. No statistically meaningful change was observed in reduced glutathione content. VEGF was immunostained significantly more on wound tissue of treated animals compared to the control group. Recombinant human erythropoietin treatment may be effective in wound healing due to inhibition of lipid peroxidation, deposition of collagen, and VEGF expression in wound area.

L-Arginine and melatonin interaction in rat intestinal ischemia--reperfusion.

We investigated the combinative effects of L-arginine and melatonin on the contractile responses of terminal ileum after the intestinal ischemia-reperfusion (I/R), in vivo. Male rats were subjected to mesenteric ischemia (30 min) followed by reperfusion (180 min). We have observed a dramatic decrease in spontaneous basal activity and Ach-induced contractile response. Our data clearly showed that the contractility decrease was ameliorated by L-arginine but not by L-NAME. Melatonin has reversed the inhibition of contractility caused by I/R injury in part. We did not observe an augmentation in the contractility of ileum when we use melatonin and L-arginine in combination, in fact, melatonin decreased the protective effect of L-arginine in intestinal I/R injury.

Protective effect of melatonin on contractile activity and oxidative injury induced by ischemia and reperfusion of rat ileum.

Free radicals derived from molecular oxygen have been reported to be responsible for changes in motility and mucosal damage observed in intestinal ischemia-reperfusion injury. Melatonin has been considered as an antioxidant that prevents injuries resulted from I/R in various tissues. The present study was designed to determine the effect of melatonin on the contractile responses of acetylcholine (Ach) and KCl, on malondialdehyde (MDA), a product of lipid peroxidation, and reduced glutathione (GSH) levels and to assess histopathological changes in the smooth muscle of terminal ileum subjected to ischemia-reperfusion. The intestinal ischemia-reperfusion was induced by occlusion of superior mesenteric artery of rat for 30 min, followed by a period of reperfusion for 3 h. Melatonin at doses of 10 or 50 mg/kg was administered via the tail vein in 5 min prior to reperfusion. Following reperfusion, segments of terminal ileum were rapidly taken and transferred into isolated organ bath and responses to Ach and KCl were recorded. Samples of terminal ileum were also taken for measuring the MDA and GSH levels. EC50 values of these contracting substances were seriously reduced in the ischemia-reperfusion group compared to that of the sham-operated control group. The decreased contraction response to Ach and KCl was significantly ameliorated by a dosage of 50 mg/kg of melatonin, while not by a dosage of 10 mg/kg. Similar pattern of the effect was observed in the tissue levels of MDA and GSH as well as in histological improvement. Melatonin appeared to be restoring the amounts of tissue MDA and GSH back to about control levels. These results suggest that the high dose of melatonin not only physiologically but also biochemically and morphologically could be useful to normalize contractility injured by oxidative stress in intestinal ischemia-reperfusion.

Beneficial effects of melatonin on reperfusion injury in rat sciatic nerve.

Studies have shown that ischemia-reperfusion (I/R) produces free radicals leading to lipid peroxidation and to damage of the nervous tissue. Melatonin, a main secretory product of the pineal gland, has free radical scavenging and antioxidant properties and has been shown to diminish I/R injury in many tissues. There are a limited number of studies related to the effects of melatonin on I/R injury in the peripheral nervous system. Therefore, in the present study, the protective effect of melatonin was investigated in rats subjected to 2 hr of sciatic nerve ischemia followed by 3 hr of reperfusion. Following reperfusion, nerve tissue samples were collected for quantitative assessment of malondialdehyde (MDA), an oxidative stress marker, and superoxide dismutase (SOD), a principal antioxidant enzyme. Samples were further evaluated at electron microscopic level to examine the neuropathological changes. I/R elevated the concentration of MDA significantly while there was a reduction at SOD levels. Melatonin treatment reversed the I/R-induced increase and decrease in MDA and SOD levels, respectively. Furthermore, melatonin salvaged the nerve fibers from ischemic degeneration. Histopathologic findings in the samples of melatonin-treated animals indicated less edema and less damage to the myelin sheaths and axons than those observed in the control samples. Our results suggest that administration of melatonin protects the sciatic nerve from I/R injury, which may be attributed to its antioxidant property.