l‐NAME‐Induced Dispersion of Melanosomes in Melanophores Activates PKC,MEK and ERK1

Melanosome movement represents a good model of cytoskeleton‐mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nω‐nitro‐l ‐arginine methyl ester (l ‐NAME) induced dispersion in melanophores pre‐aggregated with melatonin. Activation of cyclic adenosine 3′,5′‐monophosphate (cAMP)‐dependent protein kinase (PKA) or calcium‐dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal‐regulated kinase (MEK)‐ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of l ‐NAME‐induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in l ‐NAME‐dispersed melanophores. l ‐NAME also caused dispersion in latrunculin‐B‐treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the l ‐NAME‐induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.     

Involvement of Nitric Oxide in UVB‐Induced Pigmentation in Guinea Pig Skin

Ultraviolet (UV) B irradiation evokes erythema and delayed pigmentation in skin, where a variety of toxic and modulating events are known to be involved. Nitric oxide (NO) is generated from l ‐arginine by NO synthases (NOS). Production of NO is enhanced in response to UVB‐stimulation and has an important role in the development of erythema. NO has recently been demonstrated as a melanogen which stimulates melanocytes in vitro, however, no known in vivo data has been reported to support this finding. In this study, we investigated the contribution of NO with UV‐induced pigmentation in an animal model using an NOS inhibitor. UVB‐induced erythema in guinea pig skin was reduced when an NOS inhibitor, l ‐NAME (N‐nitro‐ l ‐arginine methylester hydrochloride), was topically applied to the skin daily, beginning 3 days before UVB‐irradiation. Delayed pigmentation and an increased number of DOPA‐positive melanocytes in the skin were markedly suppressed by sequential daily treatment with l ‐NAME. Furthermore, melanin content 13 days after UVB‐irradiation was significantly lower in skin treated with l ‐NAME than in the controls. In contrast, d ‐NAME (N‐nitro‐ d ‐arginine methylester hydrochloride), an ineffective isomer of l ‐NAME, demonstrated no effect on these UV‐induced skin responses. These results suggest that NO production may contribute to the regulation of UVB‐induced pigmentation.     

Fluoxetine induces vasodilatation of cerebral arterioles by co‐modulating NO/muscarinic signalling

Ischaemic stroke patients treated with Selective Serotonin Reuptake Inhibitors (SSRI) show improved motor, cognitive and executive functions, but the underlying mechanism(s) are incompletely understood. Here, we report that cerebral arterioles in the rat brain superfused with therapeutically effective doses of the SSRI fluoxetine showed consistent, dose‐dependent vasodilatation (by 1.2 to 1.6‐fold), suppressible by muscarinic and nitric oxide synthase (NOS) antagonists [atropine, NG‐nitro‐l ‐arginine methyl ester (l ‐NAME)] but resistant to nicotinic and serotoninergic antagonists (mecamylamine, methylsergide). Fluoxetine administered 10–30 min. following experimental vascular photo‐thrombosis increased arterial diameter (1.3–1.6), inducing partial, but lasting reperfusion of the ischaemic brain. In brain endothelial b.End.3 cells, fluoxetine induced rapid muscarinic receptor‐dependent increases in intracellular [Ca²⁺] and promoted albumin‐ and eNOS‐dependent nitric oxide (NO) production and HSP90 interaction. In vitro, fluoxetine suppressed recombinant human acetylcholinesterase (rhAChE) activity only in the presence of albumin. That fluoxetine induces vasodilatation of cerebral arterioles suggests co‐promotion of endothelial muscarinic and nitric oxide signalling, facilitated by albumin‐dependent inhibition of serum AChE.     

Physical training prevents oxidative stress in L‐NAME‐induced hypertension rats

The present study investigated the effects of a 6‐week swimming training on blood pressure, nitric oxide (NO) levels and oxidative stress parameters such as protein and lipid oxidation, antioxidant enzyme activity and endogenous non‐enzymatic antioxidant content in kidney and circulating fluids, as well as on serum biochemical parameters (cholesterol, triglycerides, urea and creatinine) from Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME)‐induced hypertension treated rats. Animals were divided into four groups (n = 10): Control, Exercise, L‐NAME and Exercise L‐NAME. Results showed that exercise prevented a decrease in NO levels in hypertensive rats (P < 0·05). An increase in protein and lipid oxidation observed in the L‐NAME‐treated group was reverted by physical training in serum from the Exercise L‐NAME group (P < 0·05). A decrease in the catalase (CAT) and superoxide dismutase (SOD) activities in the L‐NAME group was observed when compared with normotensive groups (P < 0·05). In kidney, exercise significantly augmented the CAT and SOD activities in the Exercise L‐NAME group when compared with the L‐NAME group (P < 0·05). There was a decrease in the non‐protein thiols (NPSH) levels in the L‐NAME‐treated group when compared with the normotensive groups (P < 0·05). In the Exercise L‐NAME group, there was an increase in NPSH levels when compared with the L‐NAME group (P < 0·05). The elevation in serum cholesterol, triglycerides, urea and creatinine levels observed in the L‐NAME group were reverted to levels close to normal by exercise in the Exercise L‐NAME group (P < 0·05). Exercise training had hypotensive effect, reducing blood pressure in the Exercise L‐NAME group (P < 0·05). These findings suggest that physical training could have a protector effect against oxidative damage and renal injury caused by hypertension. Copyright © 2012 John Wiley & Sons, Ltd.     

The dynamic distribution of NO and NADPH–diaphorase activity during IBA-induced adventitious root formation

Nitric oxide (NO) is a multifunctional molecule involved in numerous physiological processes in plants. In this study, we investigate the spatiotemporal changes in NO levels and endogenous NO‐generating system in auxin‐induced adventitious root formation. We demonstrate that NO mediates the auxin response, leading to adventitious root formation. Treatment of explants with the auxin indole‐3‐butyric acid (IBA) plus the NO donor sodium nitroprusside (SNP) together resulted in an increased number of adventitious roots compared with explants treated with SNP or IBA alone. The action of IBA was significantly reduced by the specific NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO), and the nitric oxide synthase (NOS, enzyme commission 1.14.13.39) inhibitor, N^G‐nitro‐l ‐arg‐methyl ester (l ‐NAME). Detection of endogenous NO by the specific probe 4,5‐diaminofluorescein diacetate and survey of NADPH–diaphorase activity (commonly employed as a marker for NOS activity) by histochemical staining revealed that during adventitious root formation, NO and NADPH–diaphorase signals were specifically located in the adventitious root primordia in the basal 2‐mm region (as zone I) of both control and IBA‐treated explants. With the development of root primordia, NO and NADPH–diaphorase signals increased gradually and were mainly distributed in the root meristem. Endogenous NO and NADPH–diaphorase activity showed overall similarities in their tissue localization. Distribution of NO and NADPH–diaphorase activity similar to that in zone I were also observed in the basal 2–4‐mm region (zone II) of IBA‐treated explants, but neither NO nor NADPH–diaphorase signals were detected in this region of the control explants. l ‐NAME and c‐PTIO inhibited the formation of adventitious roots induced by IBA and reduced both NADPH–diaphorase staining and NO fluorescence. These results show the dynamic distribution of endogenous NO in the developing root primordia and demonstrate that NO plays a vital role in IBA‐induced adventitious rooting. Also, the production of NO in this process may be catalyzed by a NOS‐like enzyme.     

Effects of propolis,caffeic acid phenethyl ester,and pollen on renal injury in hypertensive rat: An experimental and theoretical approach

The objective of this study was to evaluate the antioxidant effects of propolis, caffeic acid phenethyl ester (CAPE; active compound in propolis), and pollen on biochemical oxidative stress biomarkers in rat kidney tissue inhibited by N^ω‐nitro‐L‐arginine methyl ester (L‐NAME). The biomarkers evaluated were paraoxonase (PON1), oxidative stress index (OSI), total antioxidant status (TAS), total oxidant status (TOS), asymmetric dimethylarginine (ADMA), and nuclear factor kappa B (NF‐κB). TAS levels and PON1 activity were significantly decreased in kidney tissue samples in the L‐NAME‐treated group (P < 0.05). The levels of TAS and PONI were higher in the L‐NAME plus propolis, CAPE, and pollen groups compared with the L‐NAME‐treated group. TOS, ADMA, and NF‐κB levels were significantly increased in the kidney tissue samples of the L‐NAME‐treated group (P < 0.05). However, these parameters were significantly lower in the L‐NAME plus propolis, CAPE, and pollen groups (P < 0.05) compared with rats administered L‐NAME alone (P < 0.05). Furthermore, the binding energy of CAPE within catalytic domain of glutathione reductase (GR) enzyme as well as its inhibitory mechanism was determined using molecular modeling approaches. In conclusion, experimental and theoretical data suggested that oxidative alterations occurring in the kidney tissue of chronic hypertensive rats may be prevented via active compound of propolis, CAPE administration.     

L‐NAME co‐treatment prevent oxidative damage in the lung of adult Wistar rats treated with vitamin A supplementation

Based on the fact that vitamin A in clinical doses is a potent pro‐oxidant agent to the lungs, we investigated here the role of nitric oxide (NO^?) in the disturbances affecting the lung redox environment in vitamin A‐treated rats (retinol palmitate, doses of 1000–9000 IU·kg^?1·day^?1) for 28 days. Lung mitochondrial function and redox parameters, such as lipid peroxidation, protein carbonylation and the level of 3‐nytrotyrosine, were quantified. We observed, for the first time, that vitamin A supplementation increases the levels of 3‐nytrotyrosine in rat lung mitochondria. To determine whether nitric oxide (NO ?) or its derivatives such as peroxynitrite (ONOO‐) was involved in this damage, animals were co‐treated with the nitric oxide synthase inhibitor L‐NAME (30 mg·kg^?1, four times a week), and we analysed if this treatment prevented (or minimized) the biochemical disturbances resulting from vitamin A supplementation. We observed that L‐NAME inhibited some effects caused by vitamin A supplementation. Nonetheless, L‐NAME was not able to reverse completely the negative effects triggered by vitamin A supplementation, indicating that other factors rather than only NO? or ONOO‐ exert a prominent role in mediating the redox effects in the lung of rats that received vitamin A supplementation. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of N‐acetylarginine,a metabolite accumulated in hyperargininemia,on parameters of oxidative stress in rats: protective role of vitamins and L‐NAME

In the present investigation, we initially evaluated the in vitro effect of N‐acetylarginine on thiobarbituric acid‐reactive substances (TBA‐RS), total sulfhydryl content and on the activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) in the blood, kidney and liver of rats. Results showed that N‐acetylarginine, at a concentration of 5.0 μM, decreased the activity of CAT in erythrocytes, enhanced TBA‐RS in the renal cortex, decreased CAT and SOD activities in the renal medulla and decreased CAT and increased SOD and GSH‐Px activities in the liver of 60‐day‐old rats. Furthermore, we tested the influence of the antioxidants, trolox and ascorbic acid, as well as of the N^ω‐nitro‐L ‐arginine methyl ester (L‐NAME) on the effects elicited by N‐acetylarginine on the parameters tested. Antioxidants and L‐NAME prevented most of the alterations caused by N‐acetylarginine on the oxidative stress parameters evaluated. Data indicate that oxidative stress induction is probably mediated by the generation of NO and/or ONOO^? and other free radicals because L‐NAME and antioxidants prevented the effects caused by N‐acetylarginine in the blood, renal tissues and liver of rats. Our findings lend support to a potential therapeutic strategy for this condition, which may include the use of appropriate antioxidants for ameliorating the damage caused by N‐acetylarginine. Copyright © 2014 John Wiley & Sons, Ltd.     

4,5‐diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia

We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5‐Diaminofluoroscein diacetate, DAF‐2 DA. Following DAF‐2 loading, ganglia showed cell‐specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L ‐NAME prevented much of the initial development of DAF‐2 fluorescence, whereas the inactive isomer D ‐NAME had no effect. Washout of preincubated L ‐NAME caused increased cell‐specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L ‐arginine also resulted in an increase of DAF‐2 fluorescence in a cell‐specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF‐2 fluorescence increases in most cells. We therefore presume that the cell‐specific pattern of DAF‐2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF‐2 staining pattern and previously published studies of NOS activity are discussed. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 361–369, 2002     

Human Leptin Stimulates Systemic Nitric Oxide Production in the Rat

Objective: Apart from having an effect on energy balance, leptin is also involved in cardiovascular regulation and in the pathogenesis of obesity‐associated hypertension. We investigated the effect of leptin on nitric oxide (NO) production. Research Methods and Procedures: Wistar rats were placed in metabolic cages, and urine was collected in 2‐hour periods. After the control period, leptin (1 mg/kg intraperitoneal) was administered, and urine collection was continued for up to 6 hours. Blood was obtained 0.5, 1, 2, 4, and 6 hours after hormone injection. Results: Leptin increased plasma concentrations of NO metabolites (nitrates + nitrites, NO_x) by 32.5%, 58.0%, and 29.7% at 1, 2, and 4 hours, respectively. Urinary NO_x excretion increased by 28.8% in the first and by 20.1% in the second 2‐hour period after injection. The plasma concentration of the NO second messenger, cyclic guanosine 3′,5′‐monophosphate (cGMP), increased by 83% and 50.6% at 2 and 4 hours after leptin administration, respectively. Urinary excretion of cyclic GMP increased by 36.1% in the first and by 43.1% in the second 2‐hour period. Leptin had no effect on the plasma concentration of atrial natriuretic peptide (ANP). The effect of leptin on plasma and urinary NO_x was abolished by the NO synthase inhibitor, N^G‐nitro‐l ‐arginine methyl ester (l ‐NAME) (30 mg/kg intraperitoneal) administered 15 minutes before leptin injection. l ‐NAME alone caused a 32.2% increase in systolic blood pressure, but this increase was not observed in rats receiving l ‐NAME and leptin. Discussion: The results indicate that leptin stimulates systemic NO production; leptin prevents blood pressure elevation induced by acute NO blockade, suggesting that leptin also triggers additional hypotensive mechanisms; and ANP is not involved in renal and vascular effects of leptin.     

Nitric oxide,actin reorganization and vacuoles change are involved in PEG 6000‐induced stomatal closure in Vicia faba

Water deficit and the resulting osmotic stress affect stomatal movement. There are two types of signals, hydraulic and chemical signals, involving in the regulation of stomatal behavior responses to osmotic stress. Compared with the chemical signals, little has been known about the hydraulic signals and the corresponding signal transduction network and regulatory mechanisms. Here, using an epidermal‐strip bioassay and laser‐scanning confocal microscopy, we provide evidence that nitric oxide (NO) generation in Vicia faba guard cells can be induced by hydraulic signals. We used polyethylene glycol (PEG) 600 to simulate hypertonic conditions. This hydraulic signal led to stomatal closure and rapid promotion of NO production in guard cells. The effects were decreased by NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5, 5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO) and NO synthase (Enzyme Commission 1.14.13.39) inhibitor N^G‐nitro‐ l ‐Arg‐methyl ester (l ‐NAME). These results indicate that PEG 6000 induces stomatal closure by promoting NO production. Cytochalasin B (CB) inhibited stomatal closure induced by PEG 6000 but did not prevent the increase of endogenous NO levels, indicating that microfilaments polymerization participate in stomatal closure induced by PEG 6000, and may act downstream of NO signaling. In addition, big vacuoles split into many small vacuoles were observed in response to PEG 6000 and sodium nitroprusside (SNP) treatment, and CB inhibited these changes of vacuoles, the stomatal closure was also been inhibited. Collectively, these results suggest that the stomatal closure induced by PEG 6000 may be intimately associated with NO levels, reorganization of actin filaments and the changes of vacuoles, showing a crude outline of guard‐cells signaling process in response to hydraulic signals.     

Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt‐NOS signaling

Ischemia/reperfusion (I/R) injury in cardiomyocytes is related to excess reactive oxygen species (ROS) generation and can be modulated by nitric oxide (NO). We have previously shown that grape seed proanthocyanidin extract (GSPE), a naturally occurring antioxidant, decreased ROS and may potentially stimulate NO production. In this study, we investigated whether GSPE administration at reperfusion was associated with cardioprotection and enhanced NO production in a cardiomyocyte I/R model. GSPE attenuated I/R‐induced cell death [18.0 ± 1.8% (GSPE, 50 µg/ml) vs. 42.3 ± 3.0% (I/R control), P < 0.001], restored contractility (6/6 vs. 0/6, respectively), and increased NO release. The NO synthase (NOS) inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 200 µM) significantly reduced GSPE‐induced NO release and its associated cardioprotection [32.7 ± 2.7% (GSPE + L‐NAME) vs. 18.0 ± 1.8% (GSPE alone), P < 0.01]. To determine whether GSPE induced NO production was mediated by the Akt‐eNOS pathway, we utilized the Akt inhibitor API‐2. API‐2 (10 µM) abrogated GSPE‐induced protection [44.3% ± 2.2% (GSPE + API‐2) vs. 27.0% ± 4.3% (GSPE alone), P < 0.01], attenuated the enhanced phosphorylation of Akt at Ser473 in GSPE‐treated cells and attenuated GSPE‐induced NO increases. Simultaneously blocking NOS activation (L‐NAME) and Akt (API‐2) resulted in decreased NO levels similar to using each inhibitor independently. These data suggest that in the context of GSPE stimulation, Akt may help activate eNOS, leading to protective levels of NO. GSPE offers an alternative approach to therapeutic cardioprotection against I/R injury and may offer unique opportunities to improve cardiovascular health by enhancing NO production and increasing Akt‐eNOS signaling. J. Cell. Biochem. 107: 697–705, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of Chronic Nitric Oxide Inhibition on the Renal Excretory Response to Leptin

Objective: Previous investigations have demonstrated that leptin promotes natriuresis with a renal tubular effect. However, the mechanisms involved in this response are unclear. The present study was designed to examine the hypothesis that the natriuretic response to leptin in normotensive Sprague‐Dawley rats is regulated by nitric oxide (NO). Research Methods and Procedures: The hemodynamic and renal excretory effects of intravenous bolus administration of pharmacological doses of synthetic murine leptin were examined in groups of control Sprague‐Dawley rats (n = 8), Sprague‐Dawley rats treated for 4 days with the NO synthase inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME) (n = 8), and Sprague‐Dawley rats treated for 4 days with l‐NAME followed by acute treatment with sodium nitroprusside (n = 8). Results: In the control group (n = 8), an intravenous bolus of leptin, 400 μg/kg body weight, increased urinary sodium excretion 4‐ to 6‐fold. In the Sprague‐Dawley rats chronically administered l‐NAME (n = 8), an intravenous bolus of 400 μg/kg of leptin did not increase sodium excretion. Acute sodium nitroprusside infusion to Sprague‐Dawley rats chronically treated with l‐NAME (n = 8) was associated with partial restoration of the sodium excretory response to leptin administration. Discussion: Collectively, these results are interpreted to suggest that the natriuretic and diuretic responses to leptin observed in the Sprague‐Dawley rat require a functional NO system.     

Reduced L‐arginine transport contributes to the pathogenesis of myocardial ischemia‐reperfusion injury

Myocardial injury due to ischemia‐reperfusion (I‐R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I‐R process induces a state of insufficient L ‐arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I‐R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia‐reoxygenation significantly decreased L ‐arginine uptake and NO production (42 ± 2% and 71 ± 4%, respectively, both P < 0.01), maximal after 2 h reoxygenation. In parallel, mitochondrial membrane potential significantly decreased and ROS production increased (both P < 0.01). NVCMs infected with adenovirus expressing the L ‐arginine transporter, CAT1, and NVCMs supplemented with L ‐arginine both exhibited significant (all P < 0.05) improvements in NO generation and mitochondrial membrane potentials, with a concomitant significant fall in ROS production and lactate dehydrogenase release during hypoxia‐reoxygenation. In contrast, L ‐arginine deprived NVCM had significantly worsened responses to hypoxia‐reoxygenation. In isolated perfused mouse hearts, L ‐arginine infusion during reperfusion significantly improved left ventricular function after I‐R. These improved contractile responses were not dependent on coronary flow but were associated with a significant decrease in nitrotyrosine formation and increases in phosphorylation of both Akt and troponin I. Collectively, these data strongly implicate reduced L ‐arginine availability as a key factor in the pathogenesis of I‐R injury. Increasing L ‐arginine availability via increased CAT1 expression or by supplementation improves myocardial responses to I‐R. Restoration of L ‐arginine availability may therefore be a valuable strategy to ameliorate I‐R injury. J. Cell. Biochem. 108: 156–168, 2009. © 2009 Wiley‐Liss, Inc.     

Staphylococcus aureus lactate‐ and malate‐quinone oxidoreductases contribute to nitric oxide resistance and virulence

Staphylococcus aureus is a Gram‐positive pathogen that resists many facets of innate immunity including nitric oxide (NO·). Staphylococcus aureus NO‐resistance stems from its ability to evoke a metabolic state that circumvents the negative effects of reactive nitrogen species. The combination of l ‐lactate and peptides promotes S. aureus growth at moderate NO‐levels, however, neither nutrient alone suffices. Here, we investigate the staphylococcal malate‐quinone and l ‐lactate‐quinone oxidoreductases (Mqo and Lqo), both of which are critical during NO‐stress for the combined utilization of peptides and l ‐lactate. We address the specific contributions of Lqo‐mediated l ‐lactate utilization and Mqo‐dependent amino acid consumption during NO‐stress. We show that Lqo conversion of l ‐lactate to pyruvate is required for the formation of ATP, an essential energy source for peptide utilization. Thus, both Lqo and Mqo are essential for growth under these conditions making them attractive candidates for targeted therapeutics. Accordingly, we exploited a modelled Mqo/Lqo structure to define the catalytic and substrate‐binding residues.We also compare the S. aureus Mqo/Lqo enzymes to their close relatives throughout the staphylococci and explore the substrate specificities of each enzyme. This study provides the initial characterization of the mechanism of action and the immunometabolic roles for a newly defined staphylococcal enzyme family.     

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Abstract. The snail Helix lucorum has been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substrate l ‐arginine (l ‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca²⁺, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis in H. lucorum has a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.     

Conformations of helical Aib peptides containing a pair of l‐amino acid and d‐amino acid

A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, ¹H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 3₁₀‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 3₁₀‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10^?9, 1×10^?8, 1×10^?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10^?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10^?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10^?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10^?8 and 10^?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.