Synthesis,nitric oxide release,and α-glucosidase inhibition of nitric oxide donating apigenin and chrysin derivatives

α-Glucosidase (AG) play crucial roles in the digestion of carbohydrates. Inhibitors of α-glucosidase (AGIs) are promising candidates for the development of anti-diabetic drugs. Here, five series of apigenin and chrysin nitric oxide (NO)-donating derivatives were synthesised and evaluated for their AG inhibitory activity and NO releasing capacity in vitro. Except for 9a–c, twelve compounds showed remarkable inhibitory activity against α-glucosidase, with potency being better than that of acarbose and 1-deoxynojirimycin. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure activity relationship studies indicated that 5-OH, hydrophobic coupling chain, and carbonyl groups of the coupling chain could enhance the inhibitory activity. Apigenin and chrysin derivatives therefore represents a new class of promising compounds that can inhibit α-glucosidase activity and supply moderate NO for preventing the development of diabetic complications.     

Chronic administration of a nitric oxide synthase inhibitor,Nω-nitro-l-arginine,and drug-induced increase in cerebellar cyclic GMP in vivo

N-nitro-l-arginine (N^G-nitro-l-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N-nitro-l-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), andl-glutamic acid- (400 g/10 l/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increase induced by harmaline, picrotoxin, andl-glutamic acid was attentuated in N-nitro-l-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist,l-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N-nitro-l-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N-nitro-l-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.Part of this work was presented at the Experimental Biology 93 FASEB Meeting at New Orleans, March 1993.     

Involvement of the nitric oxide signaling in modulation of naringin against intranasal manganese and intracerbroventricular β-amyloid induced neurotoxicity in rats

Manganese -induced aggregation of the amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease (AD). The current study was designed to investigate the effects of chronic administration of naringin against β-A_1–42 and manganese induced experimental model. Wistar rats received intracerebroventricular (ICV) β-A_1–42 once, intranasal manganese, naringin and nitric oxide modulators for 21 days and behavioral alterations were assessed. Mitochondrial enzymes, oxidative parameters, TNF-α, β-A_1–42 acetylcholinesterase (AChE) levels and manganese concentration were measured. ICV β-A_1–42 and intranasal manganese treated rats showed a memory deficit and significantly increased in β-A_1–42 level and manganese concentration, mitochondrial oxidative damage, AChE level and inflammatory mediator in the hippocampus and cortex. Chronic administration of naringin (40 and 80 mg/kg) significantly improved memory performance and attenuated the oxidative damage and mitochondrial dysfunction in Aβ with Mn treated rats. In addition, naringin also attenuates the pro-inflammatory cytokines like TNF-α, AChE, Amyloid deposition and Mn concentration. Further, pretreatment of N(G)-Nitro-L-arginine methyl ester (L-NAME) with (5 mg/kg) with lower dose of naringin significantly potentiated its protective effect. These results demonstrate that naringin offers protection against ICV β-A_1–42 and intranasal manganese induced memory dysfunction possibly due to its antioxidant, anti-inflammatory, anti-amyloidogenesis therefore, could have a therapeutic potential in Alzheimer's disease.     

Effect of recombinant Mce4A protein of Mycobacterium bovis on expression of TNF-α, iNOS, IL-6, and IL-12 in bovine alveolar macrophages

CD38 is a type II transmembrane protein with 25% of its molecular mass consisting of glycosyl moieties. It has long been predicted that the carbohydrate moieties of glycoproteins play important roles in the physical function and structural stability of the proteins on cell surfaces. To determine the structural/functional significance of glycosylation of the human CD38, the four potential N-linked glycosylation sites asparagine residues, N100, N164, N209 and N219 were mutated. The mutant (CD38mu) and wild-type (CD38wt) were expressed separately in Escherichia coli, HeLa, and MCF-7 cells. SDS-polyacrylamide gel electrophoresis under reducing conditions and western blotting indicated that the molecular mass of the CD38wt is 45 kDa, and that of the CD38mu is 34 kDa in HeLa cells. Importantly, the CD38mu protein expressed in HeLa cells, showed the high molecular weight oligomers in addition to the 34 kDa monomeric form. Similarly, in E. coli, the CD38wt formed dimers and other oligomers besides the monomeric form. Moreover, MCF-7 cells stably transfected with CD38wt cDNA, also revealed the presence of cross-linked oligomers when treated with a N-linked glycosylation inhibitor tunicamycin (TM). These results suggested that the N-linked glycosylation of CD38 plays a crucial role in the structure stability by preventing the formation inter-molecular cross-links. In addition, immunostaining, enzyme activity (cyclase), and western blotting data revealed that the glycosylation of human CD38 protein is not required for its localization to the cell membrane.     

IMmunosuppressants and TGF-β1 accelerated and prolonged the nitric oxide/oxyradicals-dependent suppression by dexamethasone in paw edema of mice

Dexamethasone (Dex, 0.3 mg/Kg, S.c.) did not suppress histamine and ischemic paw edema of mice up to 1 hr. However, given TGF-β1 (0.3 μ g/Kg, I.p.), Dex suppression appeared early as 30 min (36% and 42%). When Dex (0.1 mg/Kg, S.c.) was injected 6 hr before the assay, Dex alone, TGF-β1 ± Dex, FK506 (10 mg/kg, oral)± Dex, cyclosporin A (CsA, 30 mg/kg, oral) ± Dex, rapamycin (Rapa, 10 mg/Kg, I.p.) ± Dex, deoxyspergualin (DSP, 10 mg/Kg, I.p.) ± Dex, did not suppress the edemata (less than 11 %). Nevertheless, if Dex and TGF-β1 were dosed together with one of these immunosuppressants, suppressions of histamine and ischemic edema were 53%, 45% (FK506), 45%, 49% (CsA), 44%, 48% (Rapa) and 39%, 51% (DSP), respectively. Glucocorticoid (GC) receptor (GR) complex contains heat shock proteins such as hsp56 (or CsA-binding protein: CyP-40), hsp70 and hsp90. FK506, Rapa and TGF-β1 receptor I (TR-I) bind FK-binding protein-12 (FKBP-12). FK506 and Rapa bind also hsp56. CsA binds CyP-40. DSP binds hsp70 and/or hsp90. These bindings might change or stabilize the conformation of GR complex resulting in edema suppressions. Nitric oxide synthase (NOS) inhibitors, superoxide dismutase (SOD), catalase, mannitol and cycloheximide, reversed the edema suppressions by TGF-β1 ± immunosuppressant at 30 min and 6 hr after Dex. Endogenous NO, Or and/or · OH seemed to be essential for edema suppressions. Our demonstration in vivo may offer a theoritical support for clinicians to adopt combination therapy of immunosuppressant(s) and GC.     

B56α subunit of protein phosphatase 2A mediates retinoic acid-induced decreases in phosphorylation of endothelial nitric oxide synthase at serine 1179 and nitric oxide production in bovine aortic endothelial cells

We previously showed that all-trans retinoic acid (atRA) decreased nitric oxide (NO) production through Akt-mediated decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS-Ser¹¹⁷⁹) in bovine aortic endothelial cells (BAEC). Since protein phosphatase 2A (PP2A) was also reported to decrease eNOS-Ser¹¹⁷⁹ phosphorylation, we investigated using BAEC whether PP2A mediates atRA-induced eNOS-Ser¹¹⁷⁹ dephosphorylation and subsequent decreased NO production. Treatment with okadaic acid (5 nM), a selective PP2A inhibitor, or ectopic expression of small interference RNA (siRNA) of PP2A catalytic subunit α (PP2A Cα) significantly increased eNOS-Ser¹¹⁷⁹ phosphorylation and NO production. Each treatment also significantly reversed atRA-induced observed effects, suggesting a role for PP2A. We also found that atRA significantly increased cellular PP2A activity. However, Western blot analysis revealed that atRA did not increase the expression of PP2A Cα, although it significantly increased the level of B56α of PP2A regulatory B subunit (PP2A B56α), but not PP2A B55α and PP2A B56δ. Real-time PCR assay confirmed a significant increase in PP2A B56α mRNA expression in atRA-treated cells. Ectopic expression of siRNA of PP2A B56α significantly reversed atRA-induced inhibitory effects on eNOS-Ser¹¹⁷⁹ phosphorylation and NO production, suggesting a role for PP2A B56α. Our study demonstrates for the first time that atRA decreases eNOS-Ser¹¹⁷⁹ phosphorylation and NO release at least in part by increasing PP2A B56α-mediated PP2A activity in BAEC.     

Discovery of non-peptide, small molecule antagonists of α9α10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain

A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of α9α10 nAChRs and greater selectivity over α7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

Effect of corticosteroids on nitric oxide production in inflammatory bowel disease: are leukocytes the site of action?

Nitric oxide (NO) production is increased in the human colonic mucosa in intestinal inflammation. We examined the effect of corticosteroids and the role of mononuclear cells in this production. Colonic biopsies from patients with ulcerative colitis and normal controls were cultured with either budesonide or prednisolone in the presence of proinflammatory cytokines. Human mixed mononuclear cells (MMCs) were cocultured with HT-29 cells stimulated with IFN-gamma and LPS in the presence or absence of corticosteroids. Nitrite production was measured in supernatants by a modification of the Griess reaction, and inducible NO synthase (iNOS) mRNA expression was studied in colonic tissue by RT-PCR. Both steroids significantly suppressed the nitrite production and iNOS mRNA expression in inflamed colonic biopsies from ulcerative colitis patients and in cytokine-stimulated normal colonic biopsies but not in cytokine-stimulated HT-29 cells. Nitrite production by HT-29 cells was significantly increased (P < 0.01) in cocultures with MMCs stimulated with IFN-gamma and LPS. The presence of either prednisolone or budesonide significantly (P < 0.01) suppressed nitrite production from cocultures of HT-29 cells and MMCs but not from cultures of HT-29 cells stimulated with conditioned media from activated MMCs. Interestingly, stimulation of HT-29 with conditioned media from MMCs pretreated with steroids before stimulation with LPS and IFN-gamma induced a significantly (P < 0.01) lower nitrite production. These results suggest that the inhibitory effect of corticosteroids on the NO production in the intestinal inflammation might be via the inhibition of MMC-produced mediators responsible for NO production by colonic epithelial cells.     

Characteristics of β-adrenergic receptors in bovine corneal epithelium: Comparison of fresh tissue and cultured cells

The characteristics of the β-adrenergic receptors in homogenates of fresh tissue and cultured bovine corneal epithelium were compared using [³H]dihydroalprenolol. High affinity, specific binding sites were observed in both preparations. Fresh tissue exhibited a higher binding site density (165 fmol/mg protein) than did cells in culture (57 fmol/mg protein). Studies with various β-adrenergic agonists and antagonists indicated that binding characteristics were typical of β-adrenergic receptors, predominantly of the β₂ subtype. These results demonstrate that β-adrenergic receptors exist in both fresh and cultured bovine corneal epithelium and that these receptors are qualitatively and quantitatively similar.     

Mechanism of action of TNF-α-stimulated prostaglandin production in cultured bovine luteal cells

Tumor necrosis factor-alpha (TNF-α) influences hormone synthesis of many ovarian cell types and can also exert cytotoxic effects, possibly by increasing the synthesis of prostaglandins. The purpose of the present study was to characterize the mechanism of TNF-α-stimulated prostaglandin F_2α (PGF_2α) production in cultured bovine luteal cells. Inhibitors of RNA and protein synthesis (actinomycin D and cycloheximide, respectively) completely blocked TNF-α-stimulated PGF_2α production. The phospholipase A₂ inhibitor, aristolochic acid, prevented TNF-α-stimulated, but not basal, PGF_2α production, whereas the phospholipase C inhibitor, compound , was without effect. The addition of arachidonic acid to cultures did not overcome the inhibitory effects of cycloheximide or aristolochic acid. In conclusion, TNF-α-stimulated prostaglandin production by bovine luteal cells is dependent upon the stimulation of phospholipase A₂ through mechanisms which require synthesis of RNA and protein.     

Effect of an inhibitor of nitric oxide production on Cu−Zn/SOD and its cofactors in diabetic rats

The aim of this study was to investigate the effect of an inhibitor of nitric oxide production, N(omega)-nitro-L-arginine methyl esther (L-NAME) on Cu-Zn/SOD (superoxide dismutase) enzyme activity and copper and zinc concentrations in diabetes-induced rats. The control group consisted of 12 male albino Sprague-Dawley rats, 10-12 wk of age and weighing 300 g. Twenty-six albino Sprague-Dawley rats, 10-12 wk of age and weighing 315 g, constituted the experimental group. The experimental group was divided into two groups. The first group (n=12) constituted streptozotocininduced (55 mg/kg, intraperitoneally) diabetic rats and the second group (n=14) was administered L-NAME (1 mg/kg/d) after streptozotocin induction. For determination of Cu-Zn/SOD activity, spectrophotometry was used. Zinc and copper concentrations were determined by atomic absorption spectrophotometry. Results showed that Cu-Zn/SOD activity was increased significantly in both experimental groups compared to controls, and the increase in the second group was higher than in the first group (p<0.01, p<0.01, p<0.05). Plazma zinc concentration was increased in the second group when compared with controls (p<0.05). Plasma copper was decreased significantly in the second group compared to controls and the first group (p<0.001, p<0.001). Red cell copper concentration was decreased significantly in the first group compared to controls (p<0.05). This study showed that L-NAME administration has ensured an additive effect on the antioxidant defense system, which was proved by the increase in Cu-Zn/SOD activity. This increase might have a protective effect against tissue damage in the acute period, with corresponding changes in zinc and copper concentrations.     

Ménage à trois: Aldosterone,sodium and nitric oxide in vascular endothelium

Aldosterone, a mineralocorticoid hormone mainly synthesized in the adrenal cortex, has been recognized to be a regulator of cell mechanics. Recent data from a number of laboratories implicate that, besides kidney, the cardiovascular system is an important target for aldosterone. In the endothelium, it promotes the expression of epithelial sodium channels (ENaC) and modifies the morphology of cells in terms of mechanical stiffness, surface area and volume. Additionally, it renders the cells highly sensitive to small changes in extracellular sodium and potassium. In this context, the time course of aldosterone action is pivotal. In the fast (seconds to minutes), non-genomic signalling pathway vascular endothelial cells respond to aldosterone with transient swelling, softening and insertion of ENaC in the apical plasma membrane. In parallel, nitric oxide (NO) is released from the cells. In the long-term (hours), aldosterone has opposite effects: The mechanical stiffness increases, the cells shrink and NO production decreases. This leads to the conclusion that both the physiology and pathophysiology of aldosterone action in the vascular endothelium are closely related. Aldosterone, at concentrations in the physiological range and over limited time periods can stabilize blood pressure and regulate tissue perfusion while chronically high concentrations of this hormone over extended time periods impair sodium homeostasis promoting endothelial dysfunction and the development of tissue fibrosis.     

Reaction between nitric oxide,glutathione, and oxygen in the presence and absence of protein: How are S-nitrosothiols formed?

The reaction between NO, thiols, and oxygen has been studied in some detail in vitro due to its perceived importance in the mechanism of NO-dependent signal transduction. The formation of S-nitrosothiols and thiol disulfides from this chemistry has been suggested to be an important component of the biological chemistry of NO, and such subsequent thiol modifications may result in changes in cellular function and phenotype. In this study we have reinvestigated this reaction using both experiment and simulation and conclude that: (i) S-nitrosation through radical and nonradical pathways is occurring simultaneously, (ii) S-nitrosation through direct addition of NO to thiol does not occur to any meaningful extent, and (iii) protein hydrophobic environments do not catalyze or enhance S-nitrosation of either themselves or of glutathione. We conclude that S-nitrosation and disulfide formation in this system occur only after the initial reaction between NO and oxygen to form nitrogen dioxide, and that hydrophobic protein environments are unlikely to play any role in enhancing and targeting S-nitrosothiol formation.     

Lipid synthesis in macrophages during inflammation in vivo: Effect of agonists of peroxisome proliferator activated receptors α and γ and of retinoid X receptors

The effects of peroxisome proliferator activated receptors α and γ (PPAR-α and PPAR-γ) and retinoid X receptor (RXR) agonists upon synthesis and accumulation of lipids in murine C57B1 macrophages during inflammation induced by injection of zymosan and Escherichia coli lipopolysaccharide (LPS) have been studied. It is significant that intraperitoneal injection of zymosan (50 mg/kg) or LPS (0.1 mg/kg) in mice led to a dramatic increase of [¹⁴C]oleate incorporation into cholesteryl esters and triglycerides and [¹⁴C]acetate incorporation into cholesterol and fatty acids in peritoneal macrophages. Lipid synthesis reached its maximum rate 18–24 h after injection and was decreased 5–7 days later to control level after LPS injection or was still heightened after zymosan injection. In macrophages obtained in acute phase of inflammation (24 h), degradation of ¹²⁵I-labeled native low density lipoprotein (NLDL) was 4-fold increased and degradation of ¹²⁵I-labeled acetylated LDL (AcLDL) was 2–3-fold decreased. Addition of NLDL (50 μg/ml) or AcLDL (25 μg/ml) into the incubation medium of activated macrophages induced 9–14-and 1.25-fold increase of cholesteryl ester synthesis, respectively, compared with control. Addition of NLDL and AcLDL into the incubation medium completely inhibited cholesterol synthesis in control macrophages but had only slightly effect on cholesterol synthesis in activated macrophages. Injection of RXR, PPAR-α, or PPAR-γ agonists—9-cis-retinoic acid (5 mg/kg), bezafibrate (10 mg/kg), or rosiglitazone (10 mg/kg), respectively—30 min before zymosan or LPS injection led to significant decrease of lipid synthesis. Ten hour preincubation of activated in vivo macrophages with the abovementioned agonists (5 μM) decreased cholesteryl ester synthesis induced by NLDL and AcLDL addition into the cell cultivation medium. The data suggest that RXR, PPAR-α, or PPAR-γ agonists inhibited lipid synthesis and induction of cholesteryl ester synthesis in inflammatory macrophages caused by capture of native or modified LDL. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 364–374.     

Role of κ1 opioid receptors and cAMP in regulation of cardiac tolerance to ischemia and reperfusion

Cardioprotective, inotropic, and antiarrhythmic effects of the selective agonist of κ₁ opioid receptors (κ₁-ORs) U-50.488H have been studied after 45-min global ischemia and 30-min reperfusion of isolated perfused rat hearts. The heart κ₁-ORs were stimulated by adding 0.1 or 1 μmol/l U-50.488H to the perfusion solution. The opioid did not affect the frequency of reperfusion arrhythmias. At a concentration of 0.1 μmol/l, it induced a twofold decrease in the reperfusion release of creatine phosphokinase (CPK), which positively correlated with a decrease in the myocardial cAMP level (r = 0.89, p < 0.01). Application of U-50.488H at a final concentration of 1 μmol/l did not change the cAMP level and CPK release. These results suggest that the cardioprotective effect of U-50.488H is due to a decrease in the level of cAMP in cardiomyocytes. Activation of κ₁-ORs decreased the frequency and force of myocardial contractions. It has been shown that the negative inotropic and chronotropic effects of U-50.488H are independent of changes in the myocardial cAMP level. A hypothesis is proposed that the absence of cardioprotective effect of 1μM U-50.488H is a result of activation of nonopioid receptors in cardiomyocytes.     

Production of TNF-α, nitric oxide and hydrogen peroxide by macrophages from mice with paracoccidioidomycosis that were fed a linseed oil-enriched diet

Omega-3 polyunsaturated fatty acids (n-3 PUFA) can modulate the immune system and their primary effect is on macrophage function. Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America that is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). Macrophages are the main defence against this pathogen and have microbicidal activity that is dependent on interferon-Γ and tumour necrosis factor (TNF)-α. These cytokines stimulate the synthesis of nitric oxide (NO) and hydrogen peroxide (H?O?), leading to the death of the fungus. To study the effect of n-3 PUFA on the host immune response during experimental PCM, macrophages that were obtained from animals infected with Pb18 and fed a diet enriched by linseed (LIN) oil were cultured and challenged with the fungus in vitro. The macrophage function was analysed based on the concentrations of TNF-α, NO and H?O?. LIN oil seems to influence the production of TNF-α during the development of disease. A diet enriched with LIN oil influences the microbicidal activity of the macrophages by inducing the production of cytokines and metabolites such as NO and H?O?, predominantly in the chronic phase of infection.     

NG-methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in vivo?

Nitric oxide is a major endothelium-derived vascular smooth muscle relaxing factor; its synthesis from L-arginine is selectively inhibited by L-NG-methylarginine. To assess whether basal nitric oxide release contributes to blood pressure regulation in vivo, we have investigated the cardiovascular effects of L-NG-methylarginine in the anesthetized guinea pig. L-NG-methylarginine (0.1-10 mg/kg, i.v. bolus) elicited a sustained, dose-dependent, increase in arterial pressure and a moderate bradycardia. L-arginine (30 mg/kg i.v.) prevented or reversed the pressor effect of L-NG-methylarginine, while atropine (2 mg/kg) abolished the associated bradycardia. In contrast, L-arginine did not attenuate the pressor effect of norepinephrine or angiotensin. Our findings suggest that basal nitric oxide production is sufficient to modulate peripheral vascular resistance; hence nitric oxide may play a role in arterial pressure homeostasis.