Microalbuminuria in Rats Treated with D-Nitroarginine Methyl Ether.

Microalbuminuria is an early symptom and prognostic marker of the progression of renal pathology. The analysis of the role of anionic components of the renal glomeruli in the albumin retention and the development of a model of minimal changes in the glomerular filter leading to the appearance of microalbuminuria are relevant. The effect of organic cations D-arginine methyl esters (D-AME) and D-nitroarginine (D-NAME) on the excretion of albumin by the kidneys in rats was studied. D-AME had no effect on urinary albumin excretion in rats. D-NAME caused microalbuminuria, which persisted for more than a day and sharply increased after injection of vasopressin. The number of anionic sites labeled with polyethyleneimine decreased in the structures of the glomerular filter. D-NAME-induced microalbuminuria can later serve as a model for studying nephroprotective or damaging factors.

In vitro inhibition of phosphodiesterase type 4 enhances rat corpus cavernosum nerve-mediated relaxation induced by gasotransmitters.

AIMS: Nitric oxide (NO) and hydrogen sulfide (H2S) are involved in nerve-mediated corpus cavernosum (CC) relaxation. Expression of phosphodiesterase type 5 (PDE5) and type 4 (PDE4), cyclic guanosine monophosphate (cGMP)- and cyclic adenosine monophosphate (cAMP)-specific, respectively, has been described and PDE5- and PDE4-inhibitors induce cavernous smooth muscle relaxation. Whereas the NO/cGMP signaling pathway is well established in penile erection, the cAMP-mediated mechanism is not fully elucidated. The aim of this study is to investigate the localization and the functional significance of PDE4 in rat CC tone regulation. MAIN METHODS: We performed immunohistochemistry for the detection of the PDE4A isoenzyme. Isometric tension recordings for roflumilast and tadalafil, PDE4 and PDE5 inhibitors, respectively, electrical field stimulation (EFS) and ß-adrenoceptor agonist isoproterenol and endogenous H2S production measurement. KEY FINDINGS: A marked PDE4A expression was detected mainly localized in the nerve cells of the cavernous smooth muscle. Furthermore, roflumilast and tadalafil exhibited strong corpus cavernous relaxations. Endogenous H2S production was decreased by NO and H2S synthase inhibitors and increased by roflumilast. Isoproterenol- and EFS-induced relaxations were increased by roflumilast. SIGNIFICANCE: These results indicate that PDE4A is mainly expressed within the nerves cells of the rat CC, where roflumilast induces a potent corpus cavernous relaxation per se and potentiates the response induced by ß-adrenoceptor activation. The fact that roflumilast enhances H2S production, as well as EFS-elicited responses suggests that PDE4 inhibitors modulate, in a positive feedback fashion, nerve-mediated relaxation induced by gasotransmitters, thus indicating a key role for neuronal PDE4 in penile erection.

Noncanonical transnitrosylation network contributes to synapse loss in Alzheimer's disease.

Here we describe mechanistically distinct enzymes (a kinase, a guanosine triphosphatase, and a ubiquitin protein hydrolase) that function in disparate biochemical pathways and can also act in concert to mediate a series of redox reactions. Each enzyme manifests a second, noncanonical function-transnitrosylation-that triggers a pathological biochemical cascade in mouse models and in humans with Alzheimer's disease (AD). The resulting series of transnitrosylation reactions contributes to synapse loss, the major pathological correlate to cognitive decline in AD. We conclude that enzymes with distinct primary reaction mechanisms can form a completely separate network for aberrant transnitrosylation. This network operates in the postreproductive period, so natural selection against such abnormal activity may be decreased.

Biodegradable amino acid-based poly(ester amine) with tunable immunomodulating properties and their in vitro and in vivo wound healing studies in diabetic rats' wounds.

The objective of this study is to design a new family of biodegradable synthetic polymeric biomaterials for providing a tunable inhibition of macrophage's nitric oxide synthase (NOS) pathway. l-Arginine (Arg) is the common substrate for NOS and arginase. Both two metabolic pathways participate in the wound healing process. An impaired wound healing, such as diabetic or other chronic wounds is usually associated with an overproduction of NO by macrophages via the NOS pathway. In this study, a new family of l-nitroarginine (NOArg) based polyester amide (NOArg-PEA) and NOArg-Arg PEA copolymers (co-PEA) were designed and synthesized with different composition ratios. The NOArg-PEA and NOArg-Arg co-PEAs are biodegradable (more than 50% degradation in vitro in 4 days at 37 °C), biocompatible and did not activate the resting macrophage immune response per se. When classically activated or alternatively activated macrophages (CAM/AAM) were incubated with NOArg-PEA and NOArg-Arg co-PEAs, the treatments decreased the NO production of CAM, increased the arginase activity in both CAM and AAM, increased TGF-ß1 production of CAM to various degrees and had no significant effect on TNF-α production. Diabetic rat models were used to evaluate the efficacy of NOArg-PEA and NOArg-Arg co-PEAs on wound healing. Diabetic rats treated with 2-NOArg-4 PEA, 2-NOArg-4-Arg-4 20/80, and 2-NOArg-4-Arg-4 50/50 biomaterials achieved 40%-80% faster-wound healing when compared with the control on day 7. The data from the histological and immunohistochemical analysis showed that the 2-NOArg-4-Arg-4 20/80 and 2-NOArg-4-Arg-4 50/50 treatments led to more AAM phenotypes (CD206) and arginase I production in wound tissue than the control during the first 7 days, i.e., suggesting pro-healing wound microenvironment with improved re-epithelialization of wound healing. A similar trend was retained until day 14. The 2-NOArg-4-Arg-4 20/80 and 2-NOArg-4-Arg-4 50/50 treatments also increased the collagen deposition and angiogenesis in the healing wound between day 7 and day 14. Both in vitro and in vivo data of this study showed that this new family of NOArg-Arg co-PEA biomaterials have the potential as viable alternatives for treating impaired wound healing, such as diabetic or other types of chronic wounds. STATEMENT OF SIGNIFICANCE: Diabetic or other chronic wounds is usually associated with an overproduction of NO and pro-inflammatory signals by macrophages. Arginine supplement or NOS inhibitors administration failed to achieve an expected improved wound healing because of the dynamic complexity of arginine catabolism, the difficulty in transition from pro-inflammatory to pro-healing, and the short-term efficacy. We designed and synthesized a new family of water-soluble and degradable nitroarginine-arginine polyester amides to rebalance NOS/arginase metabolism pathways of macrophages. They showed tunable immunomodulating properties in vitro. The in vivo studies were performed to evaluate their efficacy in accelerating the healing. These new biomaterials have the potential as viable alternatives for treating impaired wound healing. The general audience of Acta Biomaterialia should be interested in these findings.

Type 2 diabetes specifically attenuates purinergic skin vasodilatation without affecting muscarinic and nicotinic skin vasodilatation and sweating.

NEW FINDINGS: What is the central question of this study? It remains to be determined whether type 2 diabetes attenuates muscarinic and nicotinic cutaneous vasodilatation and sweating as well as purinergic cutaneous vasodilatation. What is the main finding and its importance? We show that type 2 diabetes specifically attenuates purinergic cutaneous vasodilatation without influencing muscarinic and nicotinic cutaneous vasodilatation and sweating. Our results provide valuable new information regarding the receptor-specific influence of type 2 diabetes on microvascular and sudomotor function. ABSTRACT: The present study evaluated whether type 2 diabetes (T2D) attenuates muscarinic and/or nicotinic cutaneous vasodilatation and sweating as well as purinergic cutaneous vasodilatation. Cutaneous vascular conductance and sweat rate were evaluated in 12 healthy non-diabetic older adults (Control, 60 ± 8 years) and 13 older adults with T2D (62 ± 10 years) at three intradermal forearm skin sites perfused with the following: (i) methacholine (muscarinic receptor agonist, five doses: 0.0125, 0.25, 5, 100 and 2000 mm); (ii) nicotine (nicotinic receptor agonist, five doses: 1.2, 3.6, 11, 33 and 100 mm); or (iii) ATP (purinergic receptor agonist, five doses: 0.03, 0.3, 3, 30 and 300 mm). Each agonist was administered for 25 min per dose. At the end of the protocol, 50 mm sodium nitroprusside was administered to all skin sites to elicit maximal cutaneous vasodilatation. Cutaneous vascular conductance during methacholine and nicotine administration did not differ between groups (all P > 0.05). In contrast, cutaneous vascular conductance during administration of 30 mm (42 ± 28 versus 63 ± 26% maximum, P ≤ 0.05) and 300 mm ATP (56 ± 24 versus 71 ± 20% maximum, P ≤ 0.05) was attenuated in individuals with T2D in comparison to the Control participants. Furthermore, cutaneous vascular conductance during administration of 50 mm sodium nitroprusside was lower in individuals with T2D relative to Control subjects (P = 0.04). Methacholine- and nicotine-induced sweating was similar between groups (all P > 0.05). Thus, T2D attenuates purinergic cutaneous vasodilatation without affecting muscarinic and nicotinic cutaneous vascular and sweating responses.

Effect of (-)-epicatechin, a flavonoid on the NO and NOS activity of Raillietina echinobothrida.

(-)-Epicatechin, a natural flavonoid reportedly has huge pharmacological properties. In this study the cestocide effect of (-)-epicatechin is demonstrated in Raillietina echinobothrida. Although the antiparasitic activity of (-)-epicatechin has been demonstrated against protozoa, helminths and ectoparasites, in the present study the cestocide activity of (-)-epicatechin is shown to be related to a decrease in nitric oxide synthase (NOS) activity and nitric oxide (NO) production. On exposure to 0.53mg/ml each of epicatechin, reference drug praziquantel and Ñ  Nitro-l- Arginine Methyl Ester (NOS inhibitor), the parasites attained paralysis at 10.15, 0.27 and 11.21h followed by death at 30.15, 1.21 and 35.18h respectively. Biochemical analysis showed a significant decrease in activity of NOS (57.360, 36.040 and 44.615%) and NO (41.579, 19.078 and 24.826%) in comparison to the controls. NADPH-diaphorase histochemical staining (a selective marker for NOS in neuronal tissue) demonstrated a pronounced decline in the visible staining activity in the tegument, subtegument and the peripheral nerve regions following exposure to the treatments. Strong binding affinity of (-)-epicatechin with NOS protein was also revealed through docking studies. The results strongly define the probable anthelmintic activity of our compound through its influence on the NOS activity.

The interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in mouse corpus cavernosum.

The aim of this study was to investigate the possible interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). l-cysteine (endogenous H2S substrate; 10-6-10-3 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10-6-10-3 M) and acetylcholine (10-9-10-4 M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H2S were reduced by non-selective mAChR antagonist atropine (5 × 10-5 M), selective M1 mAChR antagonist pirenzepine (5 × 10-5 M) and selective M3 mAChR antagonist 4-DAMP (10-7 M) but not by selective M2 mAChR antagonist AF-DX 116 (10-6 M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor d,l-propargylglycine (PAG, 10-2 M) and cystathionine-ß-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10-3 M). l-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H2S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by l-cysteine but not by exogenous H2S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M1 and M3 mAChRs contributes to relaxant effect mediated by endogenous H2S but at same time l-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded.

[Effect of methamphetamine exposure on S-nitrosylation of protein disulphide isomerase in PC12 cells].

OBJECTIVE: To study the effect of methamphetamine (METH) exposure on S-nitrosylation of protein disulphide isomerase and the neurotoxicity of METH in PC12 cells. METHODS: PC12 cells were exposed to different concentrations of METH, and the cell viability was assessed using the cell-counting kit-8. PC12 cells exposed to METH in the presence of the NOS inhibitor N-nitro-L-arginine (L-NNA) were examined for cell viability and S-nitrosylation of protein disulphide isomerase using the biotin-switch method, and the changes in cell morphology were examined with HE staining. RESULTS: METH exposure obviously decreased the cell viability and increased S-nitrosylation of protein disulphide isomerase, and the effect of METH was obviously inhibited by L-NNA treatment. CONCLUSION: METH can cause obvious neurotoxicity and promote S-nitrosylation of protein disulphide isomerase in PC12 cells.

Regional specific modulation of neuronal activation associated with nitric oxide synthase inhibitors in an animal model of antidepressant activity.

OBJECTIVE: The regional specific modulation of neuronal activation following drug administration is of interest to determine brain areas involved in the behavioural effects of experimental test compounds. In the current investigation the effects of the L-arginine related NOS inhibitor Nω-l-nitroarginine (L-NA) and the structurally unrelated selective neuronal NOS inhibitor 1-(2-Trifluoro-methyl-phenyl) imidazole (TRIM) were assessed in the rat for changes in regional c-FOS immunoreactivity, a marker of neuronal activation, upon exposure to the forced swimming test (FST). Behaviour and regional FOS and FosB/ΔFosB expression was assessed in naive animals and in animals exposed to stress with central serotonin-depletion which exhibit a stress related phenotype in the FST. METHODS: Male Sprague-Dawley rats (n=5- 6 per group) were treated with the irreversible tryptophan hydroxylase inhibitor, DL-4-p-chlorophenylalanine (pCPA, 150mg/kg, i.p.), to achieve central serotonin-depletion followed by repeated exposures to restraint stress and were then subjected to the FST. 24, 5 and 1h prior to the test, animals were treated with either L-NA (10mg/kg, i.p.), TRIM (50mg/kg, i.p.) or saline vehicle (1mg/kg i.p). RESULTS: pCPA treatment coupled with restraint stress increased immobility in the FST compared to naïve controls. Both NOS inhibitors decreased immobility time in 5-HT depleted and stressed animals only in keeping with their antidepressant-like properties. Brain regions analyzed for c-FOS immunoreactivity included the pre-limbic cortex, lateral septum (LS), nucleus accumbens, paraventricular hypothalamic nucleus (PVN), central amygdala, hippocampus (dorsal dentate gyrus and ventral CA1), and the dorsal raphe nucleus (DRN). Exposure to the FST increased c-FOS immunoreactivity in the LS, PVN, dentate gyrus, vCA1 and the DRN when compared to non-FST exposed controls. FST-induced c-FOS immunoreactivity was further increased in the LS following treatment with L-NA or TRIM when compared to vehicle-treated FST controls. By contrast, FST-induced c-FOS immunoreactivity was reduced in dorsal dentate gyrus, vCA1 and the DRN following treatment with L-NA or TRIM when compared to vehicle-treated FST controls. There was no difference observed in FST-induced expression of c-FOS between naïve animals and animals exposed to pCPA and restraint stress. This combination however provoked an increase in FosB/ΔFosB immunoreactivity in the infra-limbic cortex and nucleus accumbens with a concomitant reduction in the lateral septum, suggesting alterations to long-term, adaptive neuronal activation. CONCLUSION: This study identified a pattern of enhanced and reduced FST-related c-FOS immunoreactivity indicative of a NO-regulated network where inhibition of NO leads to activation of the septum with concomitant inhibition of the hippocampus, and the DRN. No link between FST-induced regional expression of c-FOS and increased immobility in the FST was observed in animals exposed to pCPA and stress. However, the 5-HT depletion regime combined with restraint stress provoked regional changes in the expression of ΔFosB which may relate to increased immobility in the FST.

Nitrergic Pathway Is the Main Contributing Mechanism in the Human Gastric Fundus Relaxation: An In Vitro Study.

BACKGROUND: Human gastric fundus relaxation is mediated by intrinsic inhibitory pathway. We investigated the roles of nitrergic and purinergic pathways, two known inhibitory factors in gastric motility, on spontaneous and nerve-evoked contractions in human gastric fundus muscles. METHODS: Gastric fundus muscle strips (12 circular and 13 longitudinal) were obtained from patients without previous gastrointestinal motility disorder who underwent gastrectomy for stomach cancer. Using these specimens, we examined basal tone, peak, amplitude, and frequency of spontaneous contractions, and peak and nadir values under electrical field stimulation (EFS, 150 V, 0.3 ms, 10 Hz, 20 s). To examine responses to purinergic and nitrergic inhibition without cholinergic innervation, atropine (muscarinic antagonist, 1 µM), MRS2500 (a purinergic P2Y1 receptor antagonist, 1 µM), and N-nitro-L-arginine (L-NNA, a nitric oxide synthase inhibitor, 100 µM) were added sequentially for spontaneous and electrically-stimulated contractions. Tetrodotoxin was used to confirm any neuronal involvement. RESULTS: In spontaneous contraction, L-NNA increased basal tone and peak in both muscle layers, while amplitude and frequency were unaffected. EFS (up to 10 Hz) uniformly induced initial contraction and subsequent relaxation in a frequency-dependent manner. Atropine abolished initial on-contraction and induced only relaxation during EFS. While MRS2500 showed no additional influence, L-NNA reversed relaxation (p = 0.012 in circular muscle, and p = 0.006 in longitudinal muscle). Tetrodotoxin abolished any EFS-induced motor response. CONCLUSIONS: The relaxation of human gastric fundus muscle is reduced by nitrergic inhibition. Hence, nitrergic pathway appears to be the main mechanism for the human gastric fundus relaxation.

Administration of prostacyclin modulates cutaneous blood flow but not sweating in young and older males: roles for nitric oxide and calcium-activated potassium channels.

KEY POINTS: In young adults, cyclooxygenase (COX) contributes to the heat loss responses of cutaneous vasodilatation and sweating, and this may be mediated by prostacyclin-induced activation of nitric oxide synthase (NOS) and calcium-activated potassium (KCa) channels. This prostacyclin-induced response may be diminished in older relative to young adults because ageing is known to attenuate COX-dependent heat loss responses. We observed that, although prostacyclin does not mediate sweating in young and older males, it does modulate cutaneous vasodilatation, although the magnitude of increase is similar between groups. We also found that, although NOS and KCa channels contribute to prostacyclin-induced cutaneous vasodilatation in young males, these contributions are diminished in older males. Our findings provide new insight into the mechanisms governing heat loss responses and suggest that the age-related diminished COX-dependent heat loss responses reported in previous studies may be a result of the reduced COX-derived production of prostanoids (e.g., prostacyclin) rather than the decreased sensitivity of prostanoid receptors. ABSTRACT: Cyclooxygenase (COX) contributes to the regulation of cutaneous vasodilatation and sweating; however, the mechanism(s) underpinning this response remain unresolved. We hypothesized that prostacyclin (a COX-derived product) may directly mediate cutaneous vasodilatation and sweating through nitric oxide synthase (NOS) and calcium-activated potassium (KCa) channels in young adults. However, these responses would be diminished in older adults because ageing attenuates COX-dependent cutaneous vasodilatation and sweating. In young (25 ± 4 years) and older (60 ± 6 years) males (nine per group), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites: (i) control; (ii) 10 mm NG -nitro-l-arginine (l-NNA), a non-specific NOS inhibitor; (iii) 50 mm tetraethylammonium (TEA), a non-specific KCa channel blocker; and (iv) 10 mm l-NNA + 50 mm TEA. All four sites were coadministered with prostacyclin in an incremental manner (0.04, 0.4, 4, 40 and 400 µm each for 25 min). Prostacyclin-induced increases in CVC were similar between groups (all concentrations, P > 0.05). l-NNA and TEA, as well as their combination, lowered CVC in young males at all prostacyclin concentrations (P ≤ 0.05), with the exception of l-NNA at 0.04 µm (P > 0.05). In older males, CVC during prostacyclin administration was not influenced by l-NNA (all concentrations), TEA (4-400 µm) or their combination (400 µm) (P > 0.05). No effect on sweat rate was observed in either group (all concentrations, P > 0.05). We conclude that, although prostacyclin does not mediate sweating, it modulates cutaneous vasodilatation to a similar extent in young and older males. Furthermore, although NOS and KCa channels contribute to the prostacyclin-induced cutaneous vasodilatation in young males, these contributions are diminished in older males.

KCa 3.1 upregulation preserves endothelium-dependent vasorelaxation during aging and oxidative stress.

Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium-dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial KCa 3.1, which contributes to EDR, is upregulated by H2 O2 . We investigated whether KCa 3.1 upregulation compensates for diminished EDR to NO during aging-related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1-phosphate were increased in aged wild-type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild-type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age-matched wild-type mice. Increased H2 O2 levels induced Fyn and extracellular signal-regulated kinases (ERKs) phosphorylation and KCa 3.1 upregulation. Catalase/GPX1 double knockout (catalase(-/-) /GPX1(-/-) ) upregulated KCa 3.1 in MAECs. NO production was decreased in aged wild-type, CerS2 null, and catalase(-/-) /GPX1(-/-) MAECs. However, KCa 3.1 activation-induced, N(G) -nitro-l-arginine-, and indomethacin-resistant EDR was increased without a change in acetylcholine-induced EDR in aortic rings from aged wild-type, CerS2 null, and catalase(-/-) /GPX1(-/-) mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1-phosphate induced similar changes in levels of the antioxidant enzymes and upregulated KCa 3.1. Our findings suggest that, during aging-related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H2 O2 and thereby upregulate KCa 3.1 expression and function via a H2 O2 /Fyn-mediated pathway. Altogether, enhanced KCa 3.1 activity may compensate for decreased NO signaling during vascular aging.

The Protective Role of Carbon Monoxide (CO) Produced by Heme Oxygenases and Derived from the CO-Releasing Molecule CORM-2 in the Pathogenesis of Stress-Induced Gastric Lesions: Evidence for Non-Involvement of Nitric Oxide (NO).

Carbon monoxide (CO) produced by heme oxygenase (HO)-1 and HO-2 or released from the CO-donor, tricarbonyldichlororuthenium (II) dimer (CORM-2) causes vasodilation, with unknown efficacy against stress-induced gastric lesions. We studied whether pretreatment with CORM-2 (0.1-10 mg/kg oral gavage (i.g.)), RuCl3 (1 mg/kg i.g.), zinc protoporphyrin IX (ZnPP) (10 mg/kg intraperitoneally (i.p.)), hemin (1-10 mg/kg i.g.) and CORM-2 (1 mg/kg i.g.) combined with N(G)-nitro-l-arginine (l-NNA, 20 mg/kg i.p.), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 mg/kg i.p.), indomethacin (5 mg/kg i.p.), SC-560 (5 mg/kg i.g.), and celecoxib (10 mg/kg i.g.) affects gastric lesions following 3.5 h of water immersion and restraint stress (WRS). Gastric blood flow (GBF), the number of gastric lesions and gastric CO and nitric oxide (NO) contents, blood carboxyhemoglobin (COHb) level and the gastric expression of HO-1, HO-2, hypoxia inducible factor 1α (HIF-1α), tumor necrosis factor α (TNF-α), cyclooxygenase (COX)-2 and inducible NO synthase (iNOS) were determined. CORM-2 (1 mg/kg i.g.) and hemin (10 mg/kg i.g.) significantly decreased WRS lesions while increasing GBF, however, RuCl3 was ineffective. The impact of CORM-2 was reversed by ZnPP, ODQ, indomethacin, SC-560 and celecoxib, but not by l-NNA. CORM-2 decreased NO and increased HO-1 expression and CO and COHb content, downregulated HIF-1α, as well as WRS-elevated COX-2 and iNOS mRNAs. Gastroprotection by CORM-2 and HO depends upon CO's hyperemic and anti-inflammatory properties, but is independent of NO.

P2Y(1) receptors mediate purinergic relaxation in the equine pelvic flexure.

In the equine large intestine, the knowledge of the basic mechanisms underlying motility function is crucial to properly treat motility disorders. P2Y1 receptors are responsible for mediating purinergic colonic relaxation in several species. In vitro experimental studies of the circular muscle from the equine pelvic flexure (n = 6) were performed to characterize inhibitory and excitatory neuromuscular transmission. Electrophysiological studies showed that electrical field stimulation (EFS) evoked biphasic inhibitory junction potentials (IJPs) in smooth muscle cells: a fast IJP (IJPf) followed by a sustained IJP (IJPs). IJPs was sensitive to L-NNA 1 mM (a nitric oxide synthase inhibitor) (P <0.01), while IJPf was abolished by MRS2500 1 µM (a P2Y1 receptor antagonist) (P <0.001). EFS (5 Hz for 2 min) in the organ bath inhibited rhythmic contractions to 3.0 ± 2.5% of basal area under the curve (P <0.0001). EFS under MRS2500 1 µM or L-NNA 1 mM incubation inhibited contractions to 6.0 ± 2.8% (P <0.05) and 24.4 ± 11.3% respectively (P <0.05). Combination of MRS2500 1 µM and L-NNA 1 mM completely reversed the EFS-induced inhibition of colonic motility. Non-nitrergic, non-purinergic conditions were used to reveal voltage-dependent EFS-induced contractions sensitive to atropine 1 µM (P <0.001) and, therefore, cholinergic. In conclusion, nerve-mediated relaxation and contraction in the equine pelvic flexure involve the same mechanisms as those observed in the human colon. P2Y1 receptors mediate purinergic relaxations and are potential targets for the treatment of equine colonic motor disorders.

Enhanced serelaxin signalling in co-cultures of human primary endothelial and smooth muscle cells.

BACKGROUND AND PURPOSE: In the phase III clinical trial, RELAX-AHF, serelaxin caused rapid and long-lasting haemodynamic changes. However, the cellular mechanisms involved are unclear in humans. EXPERIMENTAL APPROACH: This study examined the effects of serelaxin in co-cultures of human primary endothelial cells (ECs) and smooth muscle cells (SMCs) on cAMP and cGMP signalling. KEY RESULTS: Stimulation of HUVECs or human coronary artery endothelial cells (HCAECs) with serelaxin, concentration-dependently increased cGMP accumulation in co-cultured SMCs to a greater extent than in monocultures of either cell type. This was not observed in human umbilical artery endothelial cells (HUAECs) that do not express the relaxin receptor, RXFP1. Treatment of ECs with l-N(G) -nitro arginine (NOARG; 30 µM, 30 min) inhibited serelaxin-mediated (30 nM) cGMP accumulation in HUVECs, HCAECs and co-cultured SMCs. In HCAECs, but not HUVECs, pre-incubation with indomethacin (30 µM, 30 min) also inhibited cGMP accumulation in SMCs. Pre-incubation of SMCs with the guanylate cyclase inhibitor ODQ (1 µM, 30 min) had no effect on serelaxin-mediated (30 nM) cGMP accumulation in HUVECs and HCAECs but inhibited cGMP accumulation in SMCs. Serelaxin stimulation of HCAECs, but not HUVECs, increased cAMP accumulation concentration-dependently in SMCs. Pre-incubation of HCAECs with indomethacin, but not l-NOARG, abolished cAMP accumulation in co-cultured SMCs, suggesting involvement of prostanoids. CONCLUSIONS AND IMPLICATIONS: In co-cultures, treatment of ECs with serelaxin caused marked cGMP accumulation in SMCs and with HCAEC also cAMP accumulation. Responses involved EC-derived NO and with HCAEC prostanoid production. Thus, serelaxin differentially modulates vascular tone in different vascular beds.

Pharmacological modulation of ureteric peristalsis in a chronically instrumented conscious pig model: effect of adrenergic and nitrergic modulation.

PURPOSE: To evaluate the role of adrenergic and nitrergic signaling on ureteric peristaltic frequency and contraction force in vivo using a large animal model. METHODS: Twelve female pigs (72 ± 4 kg) were chronically instrumented with an electronic pressure-monitoring catheter in the right ureter. Nephrostomy, cystostomy, and arterial and venous catheters were left in situ. Ureteral peristalsis was recorded before and after the administration of propranolol, isoprenaline, doxazosin, urapidil, phenylephrine, LNNA (Nω-nitro-L-arginine), and L-arginine. RESULTS: α1-Adrenergic receptor stimulation resulted in an increased P max and peristaltic frequency. However, α1-inhibition decreased P max alone. Similarly, ß-adrenergic stimulation decreased P max and peristaltic frequency, whereas ß-inhibition increased only P max. LNNA administration increased P max in the distal ureter and hydrostatic pressure in the pyelocalyceal system. L-Arginine did not affect P max or frequency, but resulted in a significantly higher diuresis. Either agonist or antagonist of NO did not affect peristaltic frequency and length of contraction. CONCLUSIONS: Activation of α- and ß-adrenergic receptors, respectively, stimulates and inhibits ureteric peristalsis. The biological effect of NO on ureteric motility is regionally determined and corresponds to the distribution of NOS-positive nerves. Inhibition of NOS activity increases P max in the distal ureter and tonic activity of the ureteric muscle resulting in higher hydrostatic pressure in the renal pelvis.

Effect of Neutrophils on Nitric Oxide Production from Stimulated Macrophages.

BACKGROUND: During the initial phase of an infection, there is an upregulation of inducible nitric oxide synthase in the macrophages for the production of nitric oxide. This is followed by the recruitment of polymorphonuclear leukocytes (neutrophils) which release arginase. Arginase competes with inducible nitric oxide synthase for a common substrate L-arginine. OBJECTIVE: To investigate whether the entry of neutrophils and release of arginase can interfere with nitric oxide production from stimulated mouse macrophages. METHODS: Neutrophils were isolated from human blood and stimulated with cytodex-3 beads. Cultured macrophages were stimulated with lipopolysaccharide and interferon gamma with or without N (G)-nitro-L-arginine methyl ester or N (omega)-hydroxy-nor-L-arginine. Measurement of NO2-/NO3- and urea were done using the spectrophotometer. RESULTS: A significantly higher level of nitric oxide production from stimulated macrophages was observed compared to control. There was a decrease in nitric oxide production when stimulated macrophages were treated with the supernatant from activated neutrophils (p<0.05). CONCLUSION: Arginase from neutrophils can modulate nitric oxide production from activated macrophages which may affect the course of infection by intracellular bacteria.

Puerarin enhances proliferation and osteoblastic differentiation of human bone marrow stromal cells via a nitric oxide/cyclic guanosine monophosphate signaling pathway.

Puerarin, a major active isoflavone extracted from the Traditional Chinese Medicine Radix Puerariae, has been studied for its comprehensive biological effects. However, to date, its effect on bone formation and the underlying mechanism of action have not been well investigated. The present study investigated the effect of puerarin on cell proliferation and osteoblastic maturation in cultured human bone marrow stromal cells (hBMSC) in vitro. Puerarin (2.5-100 µM) increased hBMSC growth in a dose-dependent manner, as indicated by an MTT assay, and stimulated osteoblastic maturation as indicated by assessment of alkaline phosphatase (ALP) activity, as well as calcium deposition into the extracellular matrix detected by alizarin red S staining. Furthermore, polymerase chain reaction analysis showed that the expression of osteoblastic markers, including Runt-related transcription factor 2/core-binding factor alpha 1, osterix and osteocalcin, were increased in hBMSCs following incubation with puerarin. Further experiments indicated that puerarin increased the nitric oxide (NO) production and cyclic guanosine monophosphate (cGMP) content in hBMSCs. The effects of puerarin were mimicked by 17ß-estrodiol (10(-8) M) and were abolished in the presence of estrogen receptor antagonist ICI182780 (10(-7) M). A NO synthase inhibitor, Nx-nitro-L-arginine methylester (6 x 10(-3) M), significantly attenuated puerarin-induced increases in NO production and cGMP content, in parallel with a reduction of cell proliferation and osteoblastic differentiation as well as the expression of osteoblastic markers. These results suggested that puerarin may prevent osteoporosis by exerting stimulatory effects on bone formation and the NO/cGMP pathway, which has an important role in puerarin-induced hBMSC proliferation and osteoblastic differentiation.

Beneficial effects of diminished production of hydrogen sulfide or carbon monoxide on hypertension and renal injury induced by NO withdrawal.

BACKGROUND AND PURPOSE: Whether NO, carbon monoxide (CO) and hydrogen sulfide (H2 S) compensate for each other when one or more is depleted is unclear. Inhibiting NOS causes hypertension and kidney injury. Both global depletion of H2 S by cystathionine γ-lyase (CSE) gene deletion and low levels of exogenous H2 S cause hypertension. Inhibiting CO-producing enzyme haeme oxygenase-1 (HO-1) makes rodents hypersensitive to hypertensive stimuli. We hypothesized that combined inhibition of NOS and HO-1 exacerbates hypertension and renal injury, but how combined inhibition of NOS and CSE affect hypertension and renal injury was unclear. EXPERIMENTAL APPROACH: Rats were treated with inhibitors of NOS (L-nitroarginine; LNNA), CSE (DL-propargylglycine; PAG), or HO-1 (tin protoporphyrin; SnPP) singly for 1 or 4 weeks or in combinations for 4 weeks. KEY RESULTS: LNNA always reduced NO, decreased H2 S and increased CO after 4 weeks. PAG abolished H2 S, always enhanced CO and reduced NO, but not when used in combination with other inhibitors. SnPP always increased NO, enhanced H2 S and inhibited CO after 1 week. Rats treated with LNNA, but not PAG and SnPP, rapidly developed hypertension followed by renal dysfunction. LNNA-induced hypertension was ameliorated and renal dysfunction prevented by all additional treatments. Renal HO-1 expression was increased by LNNA in injured tubules and increased in all tubules by all other treatments. CONCLUSIONS AND IMPLICATIONS: The amelioration of LNNA-induced hypertension and renal injury by additional inhibition of H2 S and/or CO-producing enzymes appeared to be associated with secondary increases in renal CO or NO production.

S-Nitrosylating protein disulphide isomerase mediates α-synuclein aggregation caused by methamphetamine exposure in PC12 cells.

Methamphetamine (METH) belongs to Amphetamine-type stimulants, METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). However, there are still no effective treatments to METH-induced neurodegeneration because its mechanism remains unknown. In order to investigate METH's neurotoxic mechanism, we established an in vitro PD pathology model by exposing PC12 cells to METH. We found the expression of nitric oxide synthase (NOS), nitric oxide (NO) and α-synuclein (α-syn) was significantly increased after METH treatment for 24h, in addition, the aggregattion of α-syn and the S-nitrosylation of protein disulphideisomerase(PDI) were also obviously enhanced. When we exposed PC12 cells to the NOS inhibitor N-nitro-L-arginine(L-NNA) with METH together, the L-NNA obviously inhibited these changes induced by METH. While when we exposed PC12 cells to the precursor of NO L-Arginine together with METH, the L-Arginine resulted in the opposite effect compared to L-NNA. And when we knocked down the PDI gene, the L-NNA did not have this effect. Therefore, PDI plays a significant role in neurological disorders related to α-syn aggregation, and it suggests that PDI could be as a potential target to prevent METH-induced neurodegeneration.