Streptozotocin-pancreatic damage in the rat: modulatory effect of 15-deoxy delta12,14-prostaglandin j(2) on nitridergic and prostanoid pathway.

15-deoxy-delta (12,14)prostaglandin J(2) (15d-PGJ(2)) has been identified as a natural ligand of the PPARgamma subtype. PPAR activation in nonadipose tissues seems to inhibit iNOS and COX2 expression. Vasoactive compounds like nitric oxide and prostaglandins are increased in pancreatic tissue from streptozotocin-diabetic rats. We hypothesize that 15d-PGJ(2) may regulate the production of these proinflammatory compounds that lead to beta cell destruction in the diabetic pathology. In this work we evaluated Ca(2+)-dependent (cNOS) and Ca(2+)-independent (iNOS) activity, nitrate/nitrite levels, 15-dPGJ(2) and prostaglandin E(2) (PGE(2)) levels in isolated pancreatic islets, and 15d-PGJ(2) levels in plasma from control and streptozotocin-diabetic rats. Our results show that cNOS is predominant in control, while iNOS isoform is increased in the diabetic islets (P < 0.01). 15d-PGJ(2) 10(-5)M inhibits cNOS and iNOS activity both in control and diabetic islets (P < 0.05). Nitrate/nitrite and PGE(2) levels are higher in diabetic than in control islets (P < 0.05 and P < 0.01, respectively). 15d-PGJ(2) 10(-5)M decreases nitrate/nitrite and PGE(2) levels both in control and in diabetic islets. Bisphenol A diglycidyl ether (BADGE), a recently described PPARgamma antagonist, seems to act as a PPARgamma agonist, diminishing nitrate/nitrite and PGE2 levels in control and diabetic islets. 15d-PGJ(2) production is lower in islets from diabetic animals compared to control (P < 0.05). Our observations suggest that 15d-PGJ(2) is able to diminish the production of vasoactive proinflammatory agents in pancreatic islets. The diminished 15d-PGJ(2) levels in the diabetic islets are probably related to the diminished capacity to limit the inflammatory response due to experimental diabetes in the rat.     

Cytotoxic role of methylglyoxal in rat retinal pericytes: Involvement of a nuclear factor-kappaB and inducible nitric oxide synthase pathway

Methylglyoxal (MGO), a cytotoxic metabolite, is produced from glycolysis. Elevated levels of MGO are observed in a number of diabetic complications, including retinopathy, nephropathy and cardiomyopathy. Loss of retinal pericyte, a hallmark of early diabetic retinal changes, leads to the development of formation of microaneurysms, retinal hemorrhages and neovasculization. Herein, we evaluated the cytotoxic role of MGO in retinal pericytes and further investigated the signaling pathway leading to cell death. Rat primary retinal pericytes were exposed to 400 μM MGO for 6 h. Retinal vessels were prepared from intravitreally MGO-injected rat eyes. We demonstrated apoptosis, nuclear factor-kappaB (NF-κB) activation and inducible nitric oxide synthase (iNOS) induction in cultured pericytes treated with MGO and MGO-injected retinal vessels. In MGO-treated pericytes, TUNEL-positive nuclei were markedly increased, and NF-κB was translocalized into the nuclei of pericytes, which paralleled the expression of iNOS. The treatment of pyrrolidine dithiocarbamate (an NF-κB inhibitor) or l-N6-(1-iminoethyl)-lysine (an iNOS inhibitor) prevented apoptosis of MGO-treated pericytes. In addition, in intravitreally MGO-injected rat eyes, TUNEL and caspase-3-positive pericytes were significantly increased, and activated NF-κB and iNOS were highly expressed. These results suggest that the increased expression of NF-κB and iNOS caused by MGO is involved in rat retinal pericyte apoptosis.     

Involvement of DDAH/ADMA/NOS pathway in nicotine-induced endothelial dysfunction

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is a key contributor for endothelial dysfunction. Decrease in activity of dimethylarginine dimethylaminohydrolase (DDAH), a major hydrolase of ADMA, causes accumulation of ADMA under cardiovascular abnormalities. The study was to determine whether nicotine-induced endothelial dysfunction is related to modulating DDAH/ADMA/NOS pathway. Four-week oral nicotine treatment (5 mg/kg/day) significantly increased the plasma level of ADMA and decreased aortic DDAH expression as well as impaired endothelial function in Sprague-Dawley rats. Similarly, the medium levels of both ADMA and lactate dehydrogenase were markedly elevated in umbilical vein endothelial cells (HUVECs) treated with nicotine (10 microM) for 48 h. Nicotine-induced endothelial damages were markedly attenuated by L-arginine or overexpression of DDAH-II. Nicotine greatly downregulated both mRNA and protein levels of DDAH-II, and decreased DDAH activity in HUVECs. HUVECs express alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), whose antagonists could block these effects of nicotine mentioned above. Intracellular Ca2+ chelator did not affect nicotine-induced decrease in DDAH-II mRNA level. In conclusion, nicotine modulates DDAH/ADMA/NOS pathway of endothelial cell via activation of alpha7 nAChR, which may be involved in endothelial dysfunction associated to smoking.     

Overexpression of inducible nitric oxide synthase and cyclooxygenase-2 in rat zinc-deficient lung: Involvement of a NF-kappaB dependent pathway.

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of pulmonary diseases. The goal of this study was to measure the response of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 enzymes (COX-2) in lung with moderate zinc deficiency. Adult male Wistar rats were divided into two groups receiving (1) a zinc-deficient diet (ZD) or (2) a zinc-adequate control diet. After 2 months of treatment, the zinc-deficient group showed a significant pulmonary edema. This was associated to a reduction of protein thiols and to a significant increase of metallothionein and glutathione disulfide levels. In addition, a higher serum and lung NO production in ZD group was positively related to the higher activity and expression of iNOS and COX-2 found in lungs. Western blot analysis revealed increased IkappaBalpha degradation, an indicator of NF-kappaB activation in ZD lungs. Anatomopathologic analysis of ZD lungs showed an increase of connective tissue fibers with an influx of polymorphonuclear cells. These cells and type II cells from the alveoli showed specific immunohistochemical signals for iNOS. The conclusion is that, during the development of zinc-deficiency, iNOS activity increases in lung and contributes to lung injury. Zinc deficiency implications must be taken into account to design therapies and public health interventions involving targeted zinc supplementation for high-risk subjects or certain diseases, such as asthma.     

Involvement of recQ in the ultraviolet damage repair pathway in Deinococcus radiodurans

Deinococcus radiodurans is a bacterium which can survive extremely DNA damage. To investigate the relationship between recQ and the ultraviolet radiation (UV) damage repair pathway, we created a four mutant strain by constructing recQ knockout mutants in uvrA1, uvrA2, and uvsE backgrounds. Using the rpoB/Rif^r system, we measured the mutation frequencies and rates in wild type, recQ (MQ), uvsE uvrA1 uvrA2 (TNK006), and uvsE uvrA1 uvrA2 recQ (TQ). We then isolated Rif^r mutants of these strains and sequenced the rpoB gene. The mutation frequency of TQ was 6.4, 10.1, and 2.43 times that of wild type, MQ, and TNK006, respectively, and resulted in rates of 4.7, 6.71, and 2.15 folds higher than that of wild type, MQ, and TNK006, respectively. All the strains demonstrated specific mutational hotspots. Furthermore, the TQ strain showed a transversion bias that was different from the other three strains. The results indicate that recQ is involved in the ultraviolet damage repair pathway via the interaction between recQ and uvrA1, uvrA2, and uvsE in D. radiodurans.     

Expression of nitric oxide synthase isoforms (NOS II and NOS III) in adult rat lung in hyperoxic pulmonary hypertension

Breathing air with a high oxygen tension induces an inflammatory response and injures the microvessels of the lung. The resulting development of smooth muscle cells in these segments contributes to changes in vasoreactivity and increased pulmonary artery pressure. This in vivo study determines the temporal and spatial expression of endogenous endothelial nitric oxide synthase (NOS III) and inducible NOS (NOS II), important enzymes regulating vasoreactivity and inflammation, in the adult rat lung during the development of experimental pulmonary hypertension induced by oxidant injury. We analyzed the cellular distribution of these NOS isoforms, using specific antibodies, and assessed enzyme activity at baseline and after 1-28 days of hyperoxia (FIO₂ 0.87). The number of NOS III-immuno-positive endothelial cells increased early in hyperoxia and then remained high. By day 28, the relative number of these cells had increased from 40% in proximal vessels and 13-16% in distal alveolar vessels of the normal lung to 73-86% and 40-59%, respectively, in hyperoxia. Pulmonary alveolar macrophages (PAMs), normally few in number and only weakly immunopositive for NOS II or III in the normal lung, increased in number in hyperoxia and were strongly immunopositive for each isoform. These morphological data were supported by a temporal increase in total and calcium-independent NOS activity. Thus NOS expression and activity significantly increased in hyperoxia as pulmonary hypertension developed, and NOS III expression increased selectively in vascular endothelial cells, while both NOS isoforms were expressed by the PAM population. We conclude that this increase in expression of a potent vasodilator, an antiproliferative agent for smooth muscle cells, and an antioxidant molecule represents an adaptive response to protect the lung from oxidant-induced vascular and epithelial injury.     

Genetic and physical interactions between DPB11 and DDC1 in the yeast DNA damage response pathway

DPB11 is essential for DNA replication and S/M checkpoint control in Saccharomyces cerevisiae. The Dpb11 protein contains four BRCT domains, which have been proposed to be involved in protein-protein interactions. To further investigate the regulation and function of Dpb11, a yeast two-hybrid screen was carried out to identify proteins that physically interact with Dpb11. One positive clone isolated from the screen encoded a carboxyl-terminal fragment of Ddc1 (339-612 aa). Ddc1 is a DNA damage checkpoint protein, which, together with Mec3 and Rad17, has been proposed to form a PCNA-like complex and acts upstream in the DNA damage checkpoint pathways. We further determined that the carboxyl region of Dpb11 is required for its interaction with Ddc1. DDC1 and DPB11 also interact genetically. The Deltaddc1 dpb11-1 double mutant is more UV and MMS sensitive than the Deltaddc1 or the dpb11-1 single mutants. Furthermore, the double mutant is more hydroxyurea sensitive and displayed a lower restrictive temperature than dpb11-1. These results suggest that DPB11 and DDC1 may function in the same or parallel pathways after DNA damage and that DDC1 may play a role in responding to replication defects.     

Involvement of inducible costimulator-B7 homologous protein costimulatory pathway in murine lupus nephritis

Inducible costimulator (ICOS)-B7 homologous protein (B7h) is a new member of the CD28-B7 family of costimulatory molecules that regulates T cell-dependent humoral immune responses. In this study, we examined the involvement of this costimulatory pathway in the development and progression of lupus in NZB/W F(1) mice. Expression of ICOS on T cells was enhanced with disease progression, whereas B7h expression on B cells was down-regulated. Administration of anti-B7h mAb before the onset of renal disease significantly delayed the onset of proteinuria and prolonged survival. Blockade of B7h effectively inhibited all subclasses of IgG autoantibody production and accumulation of both Th1 and Th2 cells. Hypercellularity and deposition of IgG and C3 in glomeruli were significantly reduced. B7h blockade after the onset of proteinuria prevented the disease progression and improved the renal pathology. Our results demonstrated the involvement of the ICOS-B7h costimulatory pathway in the pathogenesis of lupus nephritis, and the blockade of this pathway may be beneficial for the treatment of human systemic lupus erythematosus.     

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms

Nitric oxide (NO), which is produced from l-arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two-cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two-cell embryos/mouse collected after fertilization in vivo (P<0.01), but the rate of blastocyst formation from two-cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS-deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P<0.001). While all three types of NOS do not seem to play a significant role in pre-ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.     

The NOS/sGC pathway in the rat central nervous system: a microdialysis overview

It is now well established that nitric oxide is involved in a variety of physiopathological processes in the central nervous system, which mainly result from the interaction of this gaseous molecule with the heme group of soluble guanylyl cyclase and the elevation of intracellular cGMP in target neurons. During the last decade, several studies have monitored extracellular cGMP, by means of intracerebral microdialysis, to investigate in vivo the functioning and modulation of this neurochemical pathway under different experimental conditions and in various brain regions. In this review, we summarise some of the most relevant results obtained in this research field.     

Relative contributions of NOS isoforms during experimental colitis: endothelial-derived NOS maintains mucosal integrity

The role of nitric oxide (NO) in inflammatory bowel diseases has traditionally focused on the inducible form of NO synthase (iNOS). However, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms may also impact on colitis, either by contributing to the inflammation or by regulating mucosal integrity in response to noxious stimuli. To date, studies examining the roles of the NOS isoforms in experimental colitis have been conflicting, and the mechanisms by which these enzymes exert their effects remain unclear. To investigate and clarify the roles of the NOS isoforms in gut inflammation, we induced trinitrobenzenesulfonic acid colitis in eNOS, nNOS, and iNOS knockout (KO) mice, assessing the course of colitis at early and late times. Both eNOS and iNOS KO mice developed a more severe colitis compared with wild-type mice. During colitis, iNOS expression dramatically increased on epithelial and lamina propria mononuclear cells, whereas eNOS expression remained localized to endothelial cells. Electron and fluorescence microscopy identified bacteria in the ulcerated colonic mucosa of eNOS KO mice, but not in wild-type, iNOS, or nNOS KO mice. Furthermore, eNOS KO mice had fewer colonic goblet cells, impaired mucin production, and exhibited increased susceptibility to an inflammatory stimulus that was subthreshold to other mice. This susceptibility was reversible, because the NO donor isosorbide dinitrate normalized goblet cell numbers and ameliorated subsequent colitis in eNOS KO mice. These results identify a protective role for both iNOS and eNOS during colitis, with eNOS deficiency resulting in impaired intestinal defense against lumenal bacteria and increased susceptibility to colitis.     

Specificity of interactions between mDia isoforms and Rho proteins

Formins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily. Binding of these molecular switch proteins to the regulatory N-terminal mDia(N), including the GTPase-binding domain, leads to the release of auto-inhibition. From the three mDia isoforms, mDia1 is activated only by Rho (RhoA, -B, and -C), in contrast to mDia2 and -3, which is also activated by Rac and Cdc42. Little is known about the determinants of specificity. Here we report on the interactions of RhoA, Rac1, and Cdc42 with mDia1 and an mDia1 mutant (mDia(N)-Thr-Ser-His (TSH)), which based on structural information should mimic mDia2 and -3. Specificity is analyzed by biochemical studies and a structural analysis of a complex between Cdc42.Gpp(NH)p and mDia(N)-TSH. A triple NNN motif in mDia1 (amino acids 164-166), corresponding to the TSH motif in mDia2/3 (amino acids 183-185 and 190-192), and the epitope interacting with the Rho insert helix are essential for high affinity binding. The triple N motif of mDia1 allows tight interaction with Rho because of the presence of Phe-106, whereas the corresponding His-104 in Rac and Cdc42 forms a complementary interface with the TSH motif in mDia2/3. We also show that the F106H and H104F mutations drastically alter the affinities and thermodynamics of mDia interactions.     

Epigenetic deregulation of the COX pathway in cancer

Inflammation is a major cause of cancer and may condition its progression. The deregulation of the cyclooxygenase (COX) pathway is implicated in several pathophysiological processes, including inflammation and cancer. Although, its targeting with nonsteroidal antiinflammatory drugs (NSAIDs) and COX-2 selective inhibitors has been investigated for years with promising results at both preventive and therapeutic levels, undesirable side effects and the limited understanding of the regulation and functionalities of the COX pathway compromise a more extensive application of these drugs. Epigenetics is bringing additional levels of complexity to the understanding of basic biological and pathological processes. The deregulation of signaling and biosynthetic pathways by epigenetic mechanisms may account for new molecular targets in cancer therapeutics. Genes of the COX pathway are seldom mutated in neoplastic cells, but a large proportion of them show aberrant expression in different types of cancer. A growing body of evidence indicates that epigenetic alterations play a critical role in the deregulation of the genes of the COX pathway. This review summarizes the current knowledge on the contribution of epigenetic processes to the deregulation of the COX pathway in cancer, getting insights into how these alterations may be relevant for the clinical management of patients.     

Novel action of estrone on vascular tissue: regulation of NOS and COX activity

The hypothesis tested in the present work is that estrone non-genomically regulates aortic nitric oxide synthase (NOS) and cyclooxygenase (COX) activities in female rats, and that such regulation depends on ovarian function. We found that physiological concentrations of estrone (E(1)) (0.1-10nM) significantly increased nitric oxide (NO) production (133 and 163% above control). The stimulatory action of E(1) on NOS activity was independent of calcium influx since the increase in NO elicited by the hormone was not affected by EGTA or verapamil. When COX activity was measured, we observed that estrone enhanced thromboxane (TXB(2)) production and prostacyclin (PGI(2)) release, but not prostaglandin (PGF(2), PGD(2), and PGE(2)) synthesis. Finally we demonstrated that the hormonal effect on NOS activity was not detected in rat aortic strips (RAS) isolated from animals deprived of ovarian activity (FR(-)) or ovariectomized rats (OVX). These results suggest that estrone exerts a direct, non-genomic action on rat aortic metabolism, which involves NOS and COX activation and depends on ovarian activity.     

Involvement of calpain in hypoxia-induced damage in rat retina in vitro

Our previous study suggested that calpain isoforms played an important role in retinal ganglion cell death induced by ischemia-reperfusion in rats [Curr. Eye Res. 21 (2000) 571]. The purpose of the present study was to further establish the direct involvement of calpain in hypoxia-induced damage by administering calpain inhibitor SJA6017 to oxygen-starved, cultured retinas. Retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. To induce a hypoxic condition, retinas were incubated with 95% N2/5% CO2. Leakage of LDH in the medium was measured to assess retinal cell damage. Activation of calpain and proteolysis of calpain substrate alpha-spectrin were analyzed by casein zymography and immunoblotting. Large amounts of LDH leaked into the medium from retinas under hypoxic conditions for 12 h, and SJA6017 significantly reduced LDH leakage. Caseinolytic activity of mu- and m-calpains decreased with hypoxia for 5 and 12 h, suggesting calpain activation followed by autolytic degradation. SJA6017 partially inhibited decreased calpain activities. Proteolysis of 230 kDa alpha-spectrin to 150 and 145 kDa breakdown products was observed in retinas with hypoxia. SJA6017 completely inhibited production of the 145 kDa breakdown product and partially inhibited production of the 150 kDa breakdown product. These results confirm the direct involvement of calpains in retinal cell damage induced by hypoxia in vitro.     

Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes

The ability of l-methionine to support glutathione biosynthesis has been investigated in isolated rat hepatocytes under conditions of normal and depleted glutathione status. The addition of l-[³⁵S]methionine or [l-[³⁵S]homocysteine to incubation media containing hepatocytes results in the incorporation of ³⁵S into intracellular glutathione. Additionally both l-methionine and l-homocysteine are capable of supporting the resynthesis of glutathione in isolated hepatocytes after prior depletion with diethyl maleate. The inclusion in the incubation medium of 1 mm propargylglycine, which is an irreversible inhibitor of the terminal enzyme of the cystathionine pathway, substantially blocks the incorporation of ³⁵S from methionine and l-homocysteine into cellular glutathione. Propargylglycine treatment of hepatocytes in the presence of [³⁵S]methionine is shown to result in the intracellular accumulation of [³⁵S]cystathionine. These results strongly support the conclusion that in rat hepatocytes the cystathionine pathway enables methionine to provide a significant source of l-cysteine for the support of glutathione biosynthesis, under both normal and glutathione-depleted conditions.