Relationship between endothelin 1 and nitric oxide system in the corpus luteum regression

The present study was designed to investigate the relationship between the nitric oxide (NO) system and endothelin 1 (ET-1) in the mechanism of corpus luteum (CL) development and consequently regression in rats. We first evaluated basal ET-1 levels in ovarian tissue from rats with different stages of CL development. An increased ovarian ET-1 content was found during CL regression. In a dose-department response, ET-1 decreased progesterone (P4) and increased prostaglandin (PG) PGF2alpha production. By means of a competitive nitric oxide synthase (NOS) inhibitor: L-nitro arginine methyl ester (L-NAME) and a slow NO releasing: diethyl-aminetriamine (DETA-NONOate), we demonstrated that NO system could be the intermediary in the ET-1 diminishing P4 production. The Western blot analysis revealed an increase on iNOS while eNOS protein expression was diminished. We also found a diminution of total NOS activity after ET-1 treatment. These data suggest the existence of a functional relationship between ET-1 and NOS isoforms leading the regulation of CL functionally.     

Interleukin-1beta, Src- and non-Src tyrosine kinases, and nitric oxide synthase induction in rat aorta in vitro

We studied the potential roles for endogenous interleukin-1beta (IL-1beta) and for several signaling pathways in the spontaneous induction in vitro of inducible nitric oxide synthase (iNOS) in endothelium-denuded rat aorta rings. Added IL-1beta augmented, whereas the IL-1beta receptor antagonist IL-1ra blocked, spontaneous iNOS induction. Furthermore, increases in IL-1beta mRNA preceded those of iNOS mRNA. Mitogen-activated protein kinase kinase and phosphatidyl inositol 3' kinase inhibition did not block iNOS induction, whereas nuclear factor kappaB inhibition did. The sarcoma virus tyrosine kinase (Src) family-selective inhibitor 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) blocked the upregulation of IL-1beta mRNA and the subsequent induction of iNOS but not the induction of iNOS stimulated by exogenously added IL-1beta. In contrast, the non-Src inhibitors TP 47/AG 213 and genistein and the tyrosine phosphatase inhibitor vanadate did not affect the spontaneous upregulation of IL-1beta mRNA but blocked both the IL-1beta-mediated and spontaneous induction of iNOS. We conclude that 1) the upregulation of tissue IL-1beta, via a signaling pathway involving a Src family kinase, plays a key role in rat vascular iNOS induction and 2) non-Src tyrosine kinases play roles downstream from IL-1beta for iNOS induction.     

Interleukin-1 beta induces nitric oxide production and inhibits the activity of aconitase without decreasing glucose oxidation rates in isolated mouse pancreatic islets.

The aim of this investigation was to further characterize the process of interleukin-1 beta (IL-1 beta) induced nitric oxide production in isolated pancreatic islets. It was found that both IL-1 beta and nitroprusside increased islet nitrite production. This effect was paralleled by inhibition of islet aconitase activity and glucose oxidation rates. Neither trifluoroperazinen or aminopterin could prevent the IL-1 beta induced increase in nitrite production, aconitase inhibition and decrease in glucose oxidation rates. In a second series of experiments, isolated mouse pancreatic islets were exposed to IL-1 beta for 24 h and subsequently used for nitrite production, aconitase activity and glucose oxidation determinations. The islets responded to IL-1 beta with an increased nitrite production and a decreased activity of aconitase, whereas the islet glucose oxidation rates were not decreased. It is concluded that IL-1 beta in both rat and mouse islets induces nitric oxide formation and that this induction leads to the inhibition of the Krebs cycle enzyme aconitase. In rat islets this probably leads to an inhibited insulin secretion, whereas IL-1 beta in mouse islets suppresses insulin secretion by a non-mitochondrial mechanism.     

Interleukin-1 beta inhibits proinsulin conversion in rat beta-cells via a nitric oxide-dependent pathway.

Exposure of pancreatic beta-cells to interleukin-1 beta (IL-1 beta) alters their protein expression and phenotype. Previous work has shown that IL-1 beta inhibited proinsulin conversion in rat islets, but the mechanism of this inhibition remained unknown. To investigate this phenomenon further, we examined purified rat beta-cells for IL-1 beta-induced inhibition of proinsulin conversion and nitric oxide (NO)-dependency of this inhibitory process. Rat beta-cells were cultured for 24 h with or without IL-1 beta and the inducible-nitric-oxide-synthase (iNOS) inhibitor N(G)-methyl-L-arginine (NMA). Exposure to IL-1 beta suppressed proinsulin-1 and proinsulin-2 synthesis by more than 50 %. Conversion of both proinsulin isoforms was also delayed. The suppressive effects of IL-1 beta on proinsulin synthesis and conversion were prevented by addition of NMA. Exposure to IL-1 beta also decreased the expression of the proinsulin convertase (PC) PC2. This decrease in PC2 expression was NO-dependent. In conclusion, IL-1 beta inhibition of proinsulin conversion in rat beta-cells occurs via an NO-mediated pathway.     

Luteal phospholipase A2 activity increases during functional and structural luteolysis in pregnant rats.

We determined cytosolic phospholipase A2 activity of the corpus luteum during luteolysis in pregnant and post-partum rats. Phospholipase A2 activity and its metabolite prostaglandin F2alpha in the corpus luteum remarkably increased just before parturition and further rose transiently during post-partum structural luteolysis. The absence of a pups' suckling stimulus delayed corpus luteum involution, being associated with an altered fluctuation in phospholipase A2 activity and depressed prostaglandin F2alpha levels. Exogenous prolactin had a reversal effect. Pharmacological and immunochemical characterization suggests multiple isoforms of phospholipase A2 in a pregnant corpus luteum. These results show the increased phospholipase A2 activity and its possible implication in luteolysis in pregnant rats.     

IL-1β对精氨酸升压素诱导的大鼠心肌成纤维细胞INOS-NO系统活性的影响

目的：探讨白细胞介素-1β（IL-1β）对精氨酸升压素（AVP）诱导下大鼠心肌成纤维细胞（CFs）诱导型一氧化氮合酶（iNOS）-一氧化氮（NO）系统活性的影响。方法：胰酶消化法分离培养SD仔鼠CFs,硝酸还原酶法、分光光度法和逆转录-聚合酶链式反应（RT—PCR）分别测定不同浓度IL-1β与AVP协同作用下CFs的NO含量、NOS活性和iNOSmRNA表达。结果：AVP诱导下CFs iNOSmRNA表达、NOS活性和NO合成均显著增加（P〈0.05）。一定浓度范围内IL-1β与AVP协同作用,剂量依赖性地增加AVP对CFs iNOS-NO系统活性的提高作用,其中AVP＋3ng／ml和AVP＋5ng／ml IL-1β组的iNOS mRNA表达、NOS活性和NO合成均显著高于AVP组（P〈0。05）,但IL-1β浓度增加至5ng／ml时,CFs的iNOSmRNA表达、NOS活性和NO合成不再继续升高,反而有所下降。结论：在一定浓度范围内IL-1β可与AVP协同提高CFs iNOS-NO系统活性。     

Role of nitric oxide in the cerebrovascular and thermoregulatory response to interleukin-1 beta

Central administration of interleukin-1 beta (IL-1 beta) increases cerebral blood flow (CBF) and body temperature, in part, through the production of prostaglandins. In previous studies, the temporal relationship between these effects of IL-1 beta have not been measured. In this study, we hypothesized that the increase in CBF occurs before any change in brain or body temperature and that the cerebrovascular and thermoregulatory effects of IL-1 beta would be attenuated by inhibiting the production of nitric oxide (NO). Adult male rats received 100 ng intracerebroventricular (icv) injection of IL-1 beta, and cortical CBF (cCBF) was measured by laser-Doppler in the contralateral cerebral cortex. A central injection of IL-1 beta caused a rapid increase in cCBF to 133 +/- 12% of baseline within 15 min and to an average of 137 +/- 12% for the remainder of the 3-h experiment. Brain and rectal temperature increased by 0.4 +/- 0.2 and 0.5 +/- 0.2 degrees C, but not until 45 min after IL-1 beta administration. Pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME; 5 mg/kg iv) completely prevented the changes in cCBF and brain and rectal temperature induced by IL-1 beta. L-Arginine (150 mg/kg iv) partially reversed the effects of L-NAME and resulted in increases in both cCBF and temperature. These findings suggest that the vasodilatory effects of IL-1 beta in the cerebral vasculature are independent of temperature and that NO plays a major role in both the cerebrovascular and thermoregulatory effects of centrally administered IL-1 beta.     

Interleukin-1beta-induced nitric oxide production and inhibition of insulin secretion in rat islets of langerhans is dependent upon the nitric oxide synthase cofactor tetrahidrobiopterin

Interleukin (IL)-1 beta-induced inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans is mediated in part by nitric oxide (NO). The NO synthase cofactor 5,6,7,8-tetrahydrobiopterin (BH(4)) supports NO synthesis in many cell types and IL-1 beta-induced NO generation and inhibition of insulin secretion have been previously correlated with intracellular BH(4 )levels in rat insulinoma cells. Using rat islets and the beta cell line BRIN-BD11, we have investigated whether synthesis of BH(4) limits IL-1beta-induced NO generation and inhibition of glucose-induced insulin secretion. IL-1 beta-induced NO generation by BRIN cells and islets was reduced by 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of de novo BH(4) synthesis. Sepiapterin, the substrate for salvage pathway BH(4) synthesis, reversed this inhibitory effect of DAHP in islets but not BRIN cells. DAHP reversed IL-1 beta-induced inhibition of islet insulin secretion, an effect prevented by sepiapterin. We conclude that BH(4) generation is necessary for IL-1 beta-induced NO generation in rat islets and BRIN cells. While a contribution of non-NO mediators cannot be excluded, our results support the proposal that IL-1 beta-induced, NO-mediated inhibition of insulin secretion in rat islets is dependent on the NOS cofactor BH(4).     

Interleukin-1beta and depressive states

Administration of Interleukin-1 beta (IL-1 beta) in pyrogenic and subpyrogenic doses induced a depression of social and exploratory behaviour in rats. A reduction in locomotor activity only occurred with pyrogenic doses of the IL-1 beta. The low dose induced the reduction whereas the high dose the increase of anxiety in elevated plus maze. The opposite effects of two doses of IL-1 beta were observed also in a test with saccharine.     

Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus

The intracellular localization and activity of the nitric oxide synthase (NOS) isoforms were investigated in rat brown adipocytes. Immunohistochemistry showed cytoplasmic and nuclear staining for the endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms; accordingly, anti-L-citrulline antibody, a marker of NOS activity, immunostained both the cytoplasm and the nucleus. The presence of metabolically active NOS in the nucleus was further confirmed by immunoblotting analyses of subcellular fractions of homogenates from cultured brown adipocytes and by measurements of NOS activity in the cytosol and nucleus. Sympathetic stimulation in vivo (i.e. cold exposure or beta(3)-adrenergic agonist treatment) and in vitro (i.e. noradrenaline treatment of cultured cells) significantly increased both cytosolic and nuclear eNOS and iNOS expression and activities. By contrast, the number of iNOS-positive, but not eNOS-positive, nuclei was significantly lower in the functionally impaired brown fat of genetically obese Zucker fa/fa rats. These data suggest the existence of a noradrenaline-modulated functional NOS system in the nucleus of brown adipocytes.     

Administration of a nitric oxide synthase inhibitor counteracts prostaglandin F2-induced luteolysis in cattle

The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2alpha (PGF2alpha)-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2alpha-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nomega-nitro-l-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2alpha (aPGF2alpha; 100 microg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2alpha increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2alpha were higher (P < 0.05) than in animals injected only with aPGF2alpha. The PGF2alpha analogue shortened the cycle length compared with that of saline (17.5 +/- 0.22 days vs. 21.5 +/- 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2alpha (22.6 +/- 1.07 days vs. 17.5 +/- 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2alpha.     

Interaction of nitric oxide with catalase: structural and kinetic analysis

We present the structures of bovine catalase in its native form and complexed with ammonia and nitric oxide, obtained by X-ray crystallography. Using the NO generator 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, we were able to generate sufficiently high NO concentrations within the catalase crystals that substantial occupation was observed despite a high dissociation rate. Nitric oxide seems to be slightly bent from the heme normal that may indicate some iron(II) character in the formally ferric catalase. Microspectrophotometric investigations inline with the synchrotron X-ray beam reveal photoreduction of the central heme iron. In the cases of the native and ammonia-complexed catalase, reduction is accompanied by a relaxation phase. This is likely not the case for the catalase NO complex. The kinetics of binding of NO to catalase were investigated using NO photolyzed from N,N'-bis(carboxymethyl)-N,N'-dinitroso-p-phenylenediamine using an assay that combines catalase with myoglobin binding kinetics. The off rate is 1.5 s(-1). Implications for catalase function are discussed.     

The interaction of nitric oxide with soybean lipoxygenase-1.

The interaction of nitric oxide with the non-heme iron dioxygenase lipoxygenase is reported. This apparently resulted in a novel type of complex where an electron is donated to the NO molecule. In addition a new position for an EPR transition from iron was discovered which, it is suggested results from high spin ferric iron in a field of axial symmetry characterised by a very low value for D.     

Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide.

Bacterial denitrification reverses nitrogen fixation in the global N-cycle by transforming nitrate or nitrite to dinitrogen. Both nitrite and nitric oxide (NO) are considered as the chemical species within the denitrification pathway, that precede nitrous oxide (N2O), the first recognized intermediate with N,N-bonds antecedent to N2. Molecular cloning of the structural genes for NO reductase from Pseudomonas stutzeri has allowed us to generate the first mutants defective in NO utilization (Nor- phenotype) by marker exchange of the norCB genes with a gene cassette for gentamicin resistance. Nitric oxide reductase was found to be an indispensable component for denitrification; its loss constituted a conditionally lethal mutation. NO as the sole product accumulated from nitrite by mutant cells induced for nitrite respiration (denitrification). The Nor- mutant lost the capability to reduce NO and did not grow anymore anaerobically on nitrate. A Nir-Nor- double mutation, that inactivated also the respiratory nitrite reductase cytochrome cd1 rendered the bacterium again viable under anaerobiosis. Our observations provide evidence for a denitrification pathway in vivo of NO2(-)----NO----N2O, and N,N-bond formation catalyzed by NO reductase and not by cytochrome cd1.     

Interleukin-1beta causes different levels of nitric oxide-mediated depression of contractility in different positions of rat thoracic aorta.

Interleukin-1beta (IL-1beta) can be synthesized by macrophages, endothelial cells and vascular smooth muscle cells when stimulated by bacterial lipopolysaccharide (endotoxin) during septic shock. The IL-1beta levels in the blood vessel wall are also elevated in atherosclerosis. IL-1beta can cause induction of inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cells and produce vasorelaxation, hypotension and ultimately tissue damage. We studied the depressions of vascular smooth muscle contractions at 3 hours after exposure to IL-1beta in different positions of rat thoracic aorta. The data show that the aortic rings from the cranial end of rat thoracic aorta had little response to IL-1beta (0.5 and 1.0 ng/ml) while those from the caudal end of thoracic aorta had larger depressant response. S-methylisothiourea sulfate (SMT), an iNOS inhibitor, completely blocked the depression of contraction caused by IL-1beta in intact aortic rings. If the endothelium was removed from the aortic rings before exposure to IL-1beta, all rings from different parts of the thoracic aorta showed an equal amount of vasodepression. Thus, the difference in the depressant response of IL-1beta in different portions of thoracic aorta is endothelium-dependent and involves induction of NOS.     

Production and functional roles of nitric oxide in the proximal tubule

A significant role for nitric oxide (NO) in proximal tubule physiology and pathophysiology has been revealed by a series of in vivo and in vitro studies. Whether the proximal tubule produces NO under basal conditions is still controversial; however, evidence suggests that the proximal tubule is constantly exposed to NO that might include NO from nonproximal tubule sources. When challenged with a variety of stimuli, including hypoxia, the proximal tubule is able to produce large quantities of NO. In vivo studies generally indicate that NO inhibits fluid and sodium reabsorption by the proximal tubule. However, the final effect of NO on proximal tubular reabsorption appears to depend on the concentration of NO and involve interaction with other regulatory mechanisms. NO regulates Na(+)-K(+)-ATPase, Na(+)/H(+) exchangers, and paracellular permeability of proximal tubular cells, which may contribute to its effect on proximal tubular transport. Enhanced production of NO, perhaps depending on macrophage type inducible NO synthase, participates in hypoxic/ischemic proximal tubular injury. In conclusion, NO plays a fundamental role in both physiology and pathophysiology of the proximal tubule.     

Interleukin-1beta stimulates cyclic GMP efflux in brain astrocytes.

In rat brain astroglia-enriched cultures long-term treatment with interleukin-1beta induces NO release and stimulation of soluble guanylyl cyclase. The cGMP formed is recovered in the extracellular medium but not in the cell monolayer. The interleukin-1beta effect is mediated by type I receptor and potentiated by interferon-gamma. In cells treated with bacterial endotoxin a larger NO-dependent cGMP accumulation occurs only intracellularly, however a significant cGMP egression is observed when cells are co-treated with interleukin-1beta. The non-selective anion transport inhibitors probenecid and verapamil block cGMP efflux, indicating that interleukin-1beta stimulates a cGMP transporter.