We investigated to see whether an altered role of nitric oxide (NO) system is involved in erythropoietin (EPO)-induced hypertension in chronic renal failure (CRF). Male Sprague-Dawley rats were five-sixths nephrectomized to induce CRF. Six weeks after the operation, EPO or vehicle was injected for another 6 weeks. Plasma and urine nitrite/nitrate (NOx) levels were determined. Expression of NO synthase (NOS) proteins in the aortae and kidneys were also determined. In addition, the isometric tension of isolated aorta in response to acetylcholine and nitroprusside was examined. Blood pressure progressively rose in CRF groups, the degree of which was augmented by EPO treatment. Plasma NOx levels did not differ among the groups, while urine NOx levels were lower in CRF groups. Endothelial NOS expression was lower in the kidney and aorta in CRF rats, which was not further affected by EPO-treatment. The inducible NOS expression in the kidney and aorta was not different among the groups. Acetylcholine and sodium nitroprusside caused dose-dependent relaxations of aortic rings, the degree of which was not altered by EPO-treatment. Taken together, EPO-treatment aggravates hypertension in CRF, but altered role of NO system may not be involved.
Acetylcholine/pharmacology ; Anemia/metabolism ; Anemia/etiology ; Anemia/drug therapy* ; Animal ; Aorta, Thoracic/physiology ; Body Weight ; Erythropoietin/pharmacology* ; Hypertension, Renal/metabolism ; Hypertension, Renal/drug therapy ; Isometric Contraction/drug effects ; Kidney/enzymology ; Kidney Failure, Chronic/metabolism* ; Kidney Failure, Chronic/complications ; Male ; Nitrates/urine ; Nitrates/blood ; Nitric Oxide/metabolism* ; Nitric-Oxide Synthase/metabolism ; Nitrites/urine ; Nitrites/blood ; Nitroprusside/pharmacology ; Rats ; Rats, Sprague-Dawley ; Vasoconstriction/drug effects ; Vasoconstrictor Agents/pharmacology ; Vasodilator Agents/pharmacology

Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species (ROS) and hypoxia inducing factor 1 alpha (HIF-1 α) in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg x (-1) sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine (NAC, a reactive oxygen scavenger). DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy (TEM), more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COX I and COXIV mRNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3 (mitophagy marker molecular) increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite (16 mg x L(-1)) increased the potentials of invasion and migration of hepatic cancer SMMC-7721 cells through induction of ROS and HIF-1α mediated mitophagy.
Acetylcysteine ; pharmacology ; Autophagy ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Movement ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Liver Neoplasms ; pathology ; Mitochondrial Degradation ; Neoplasm Invasiveness ; Nitrites ; metabolism ; Reactive Oxygen Species ; metabolism ; Sodium Nitrite ; pharmacology

The present experiments were done to determine the effectiveness of a non-specific nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), on oxidative stress parameters induced by aluminium chloride (AlCl3) intrahippocampal injections in Wistar rats. Animals were sacrificed 3 h and 30 d after treatments, heads were immediately frozen in liquid nitrogen and forebrain cortices were removed. Crude mitochondrial fraction preparations of forebrain cortices were used for the biochemical analyses: nitrite levels, superoxide production, malondialdehyde concentrations, superoxide dismutase (SOD) activities and reduced glutathione contents. AlCl3 injection resulted in increased nitrite concentrations, superoxide anion production, malondialdehyde concentrations and reduced glutathione contents in the forebrain cortex, suggesting that AlCl3 exposure promoted oxidative stress in this brain structure. The biochemical changes observed in neuronal tissues showed that aluminium acted as a pro-oxidant. However, the non-specific nitric oxide synthase (NOS) inhibitor, L-NAME, exerted anti-oxidant actions in AlCl3-treated animals. These results revealed that NO-mediated neurotoxicity due to intrahippocampal AlCl3 injection spread temporally and spatially to the forebrain cortex, and suggested a potentially neuroprotective effect for L-NAME.
Aluminum Compounds/*toxicity ; Animals ; Chlorides/*toxicity ; Glutathione/metabolism ; Male ; Malondialdehyde ; NG-Nitroarginine Methyl Ester/*pharmacology ; Nitric Oxide Synthase/*antagonists & inhibitors ; Nitrites/chemistry/metabolism ; Prosencephalon/*drug effects ; Rats ; Rats, Wistar ; Superoxide Dismutase/metabolism ; Superoxides/metabolism

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 microm) were isolated by micro-dissection and cultured in 0 (control), 10(-3), 10(-5), 10(-7), and 10(-9) M SNP. To examine the reversible effect of SNP, PFs were cultured with 10(-5) M SNP + 1 mM Nomega-nitro-L-arginine methyl ester (L-NAME) or 1.0 microg hemoglobin (Hb). The results showed that greater concentrations of SNP (10(-3), 10(-5), 10(-7) M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10(-9) M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.
Animals ; *Apoptosis ; Buffaloes/*physiology ; Estradiol/biosynthesis ; Female ; Follicle Stimulating Hormone/metabolism ; NG-Nitroarginine Methyl Ester/pharmacology ; Nitrates/pharmacology ; Nitric Oxide/*metabolism ; Nitric Oxide Donors/pharmacology ; Nitrites/pharmacology ; Nitroprusside/pharmacology ; Oocytes/cytology/drug effects/growth & development/metabolism ; Ovarian Follicle/*cytology/drug effects/growth & development/*metabolism ; Progesterone/biosynthesis

OBJECTIVETo study the effects of potassium salts on direct induction of microtubers from different explants in Pinellia ternata.

METHODLeaves, petioles and tubers were cut and cultured on the medium with different kinds of potassium salts and plant growth regulators.

RESULT AND CONCLUSIONLow concentration of potassium salts, which were lower than 4.02 mmol x L(-1), could promote the microtuber formation in vitro. While high concentration of potassium salts, which were more than 12.06 mmol x L(-1), inhibited the formation of microtubers.

Culture Media ; Dose-Response Relationship, Drug ; Nitrites ; administration & dosage ; pharmacology ; Phosphates ; administration & dosage ; pharmacology ; Pinellia ; growth & development ; Plant Growth Regulators ; pharmacology ; Plant Leaves ; growth & development ; Plant Tubers ; growth & development ; Plants, Medicinal ; growth & development ; Potassium Chloride ; administration & dosage ; pharmacology ; Potassium Compounds ; administration & dosage ; pharmacology ; Tissue Culture Techniques

Cytokines produced by immune cells infiltrating pancreatic islets have been implicated as one of the important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the protective effects of epigallocatechin gallate (EGCG) on cytokine-induced beta-cell destruction were investigated. EGCG effectively protected IL-1beta and IFN-g-mediated cytotoxicity in insulinoma cell line (RINm5F). EGCG induced a significant reduction in IL-1b and IFN-gamma-induced nitric oxide (NO) production and reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein levels on RINm5F cells. The molecular mechanism by which EGCG inhibited iNOS gene expression appeared to involve the inhibition of NF-kB activation. These findings revealed EGCG as a possible therapeutic agent for the prevention of diabetes mellitus progression.
Animals ; Blotting, Western ; Catechin/*analogs & derivatives/*pharmacology ; Cell Line ; Cell Survival/drug effects ; Cytokines/*antagonists & inhibitors/pharmacology ; Interferon Type II/antagonists & inhibitors/pharmacology ; Interleukin-1/antagonists & inhibitors/pharmacology ; Islets of Langerhans/cytology/*drug effects ; Nitric-Oxide Synthase/metabolism ; Nitrites/metabolism ; Tea/*chemistry

BACKGROUNDSalivary nitrate is positively correlated with plasma nitrate and its level is 9 times the plasma level after nitrate loading. Nitrate in saliva is known to be reduced to nitrite by oral bacteria. Nitrate and nitrite levels in saliva are 3 - 5 times those in serum in physiological conditions respectively in our previous study. The biological functions of high salivary nitrate and nitrite are still not well understood. The aim of this in vitro study was to investigate the antimicrobial effects of nitrate and nitrite on main oral pathogens under acidic conditions.

METHODSSix common oral pathogens including Streptococcus mutans NCTC 10449, Lactobacillus acidophilus ATCC 4646, Porphyromonas gingivalis ATCC 33277, Capnocytophaga gingivalis ATCC 33624, Fusobacterium nucleatum ATCC 10953, and Candida albicans ATCC 10231 were cultured in liquid medium. Sodium nitrate or sodium nitrite was added to the medium to final concentrations of 0, 0.5, 1, 2, and 10 mmol/L. All of the microorganisms were incubated for 24 to 48 hours. The optical densities (OD) of cell suspensions were determined and the cultures were transferred to solid nutrient broth medium to observe the minimum inhibitory concentration and minimum bactericidal/fungicidal concentration for the six tested pathogens.

RESULTSNitrite at concentrations of 0.5 to 10 mmol/L had an inhibitory effect on all tested organisms at low pH values. The antimicrobial effect of nitrite increased with the acidity of the medium. Streptococcus mutans NCTC 10449 was highly sensitive to nitrite at low pH values. Lactobacillus acidophilus ATCC 4646 and Candida albicans ATCC 10231 were relatively resistant to acidified nitrite. Nitrate at the given concentrations and under acidic conditions had no inhibitory effect on the growth of any of the tested pathogens.

CONCLUSIONNitrite, at a concentration equal to that in human saliva, is both cytocidal and cytostatic to six principal oral pathogens in vitro, whereas nitrate at a similar concentration has no antimicrobial effect on these organisms.

Anti-Infective Agents ; pharmacology ; Candida albicans ; drug effects ; Fusobacterium nucleatum ; drug effects ; Hydrogen-Ion Concentration ; Lactobacillus acidophilus ; drug effects ; Microbial Sensitivity Tests ; Mouth ; microbiology ; Nitrates ; analysis ; blood ; pharmacology ; Nitrites ; analysis ; blood ; pharmacology ; Porphyromonas gingivalis ; drug effects ; Saliva ; chemistry ; Streptococcus mutans ; drug effects

OBJECTIVETo observe the effect of nitric oxide (NO) on the survival of a random pattern skin flap.

METHODSCaudal based random skin flaps (9 cm x 3 cm) were raised on the back of Wistar rats. Six methods were used in the experiment to observe the effect of NO synthase inhibitor L-NAME and NO synthase substrate L-arginine on flaps: image analysis technology; light and electron microscopic studies; enzyme histochemistry of NOS in flaps; concentration of NO2-/NO3- in plasma and wet/dry ratio of the flap tissue.

RESULTSSurvival area of flap in the L-arginine-treated group significantly increased (67.06 +/- 5.65)% (p < 0.01) whereas the area in the L-NAME-treated group significantly decreased (35.17 +/- 1.87)% (p < 0.01) compared with the control group (53.25 +/- 3.24)% at seven days after the operation. General and microscopic observations showed that pathological changes in the L-arginine-treated group were fewer. Abundant capillaries and fewer inflammatory cells were noticed in the L-arginine-treated group. Transmission Electron Microscopy (TEM) studies find endothelial swelling, thrombosis-formation and endothelial loss of contact with the basement membrane in the L-NAME treated group. Before operation, the serum NO concentrations were not significantly different in three groups (p > 0.05). After operation, NO concentration of the control group began to increase and reached to the top at the third day. L-Arg kept serum NO concentration in a higher level than the control. Enzyme histochemistry of NOS in flaps: microvessel intima in dermis, hair follicles, sweat glands and inflammatory cells showed oxford blue, more positive in flaps of the L-Arg treated group than the control group at the third day after operation. The flaps of L-NAME-treated group demonstrated negative or weak positive. Wet/dry ratio: twenty-four hours after flap elevation wet/dry weight ratios increased significantly in all regions of the flap of the L-arginine-treated rats compared with saline-treated rats. The ratios of the flaps of L-NAME-treated rats were reduced compared with saline-treated rats.

CONCLUSIONNO could improve microcirculation of the flap and increase its survival rates. The mechanism might be that NO could accelerate flap vascularization and protect flaps from ischemia-reperfusion injury.

Animals ; Arginine ; pharmacology ; Dermatologic Surgical Procedures ; Enzyme Inhibitors ; pharmacology ; Female ; Graft Survival ; drug effects ; Immunohistochemistry ; Male ; Microscopy, Electron ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitrates ; blood ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; antagonists & inhibitors ; metabolism ; Nitrites ; blood ; Rats ; Rats, Wistar ; Skin ; enzymology ; ultrastructure ; Skin Transplantation ; Surgical Flaps ; physiology

OBJECTIVETo study the effects of different solvent extractions of Mori Ramulus on platelet aggregation, vascular tension, and nitrite production from macrophages stimulated by lipopolysaccharides and interferon-gamma.

METHODThe components of Mori Ramulus were extracted by EtoAc, n-BuOH and chloroform respectively. Platelet aggregation was induced by ADP, arachidonic acid and collagen in vitro; nitrite production of activated macrophages was measured by Griess assay, and the vasodilatory effects of three extractions were investigated by isometric tension changes of aortic rings.

RESULTChloroform extraction concentration-dependently inhibited platelet aggregation by arachidonic acid, and reduced vascular tension of PE preconstricted aorta rings with or without endothelium. On the other hand, extractions of EtoAc and n-BuOH demonstrated dose-dependent inhibition on macrophage NO production stimulated by LPS/IFN-gamma.

CONCLUSIONPharmacological activities of Mori Ramulus depend on solvent specific components. Chloroform extraction of Mori Ramulus may benefit cardiovascular diseases through its properties of anti-platelet aggregation and vasodilatation. The inhibition of macrophage activity by EtoAc and n-BuOH extractions suggested an anti-inflammation effect of the compound.

Animals ; Aorta ; drug effects ; physiopathology ; Cell Line ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; In Vitro Techniques ; Macrophages ; drug effects ; metabolism ; Male ; Mice ; Morus ; chemistry ; Nitrites ; metabolism ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; isolation & purification ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Vasodilation ; drug effects ; Vasodilator Agents ; isolation & purification ; pharmacology

Tumor target-derived soluble secretary factor has been known to influence macrophage activation to induce nitric oxide (NO) production. Since heme oxigenase-1 (HO-1) is induced by a variety of conditions associated with oxidative stress, we questioned whether soluble factor from tumor cells induces HO-1 through NO-dependent mechanism in macrophages. We designated this factor as a tumor-derived macrophage-activating factor (TMAF), because of its ability to activate macrophages to induce iNOS. Although TMAF alone showed modest activity, TMAF in combination with IFN-gamma significantly induced iNOS expression and NO synthesis. Simultaneously, TMAF induced HO-1 and this induction was slightly augmented by IFN-gamma. Surprisingly, however, induction of HO-1 by TMAF was not inhibited by the treatment with the highly selective iNOS inhibitor, 1400 W, indicating that TMAF induces the HO-1 enzyme by a NO-independent mechanism. While rIFN-gamma alone induced iNOS, it had no effect on HO-1 induction by itself. Collectively, the current study reveals that soluble factor from tumor target cells induces HO-1 enzyme in macrophages. However, overall biological significance of this phenomenon remains to be determined.
Animals ; Antineoplastic Agents/pharmacology ; Bladder Neoplasms/metabolism/pathology ; Cell Line ; Drug Interactions ; Gene Expression Regulation, Enzymologic/drug effects ; Heme Oxygenase (Decyclizing)/analysis/*genetics ; Human ; Interferon Type II/pharmacology ; Macrophage Activation/drug effects ; Macrophages, Peritoneal/*metabolism ; Mice ; Mice, Inbred C57BL ; Nitric Oxide/biosynthesis/*metabolism ; Nitric-Oxide Synthase/genetics/metabolism ; Nitrites/analysis ; Tumor Cells, Cultured