Ultrastructural localization of carbonic anhydrase activity in the rat choroid plexus epithelial cell

Summary Localization of carbonic anhydrase activity was studied electron microscopically on cells of the rat choroid plexus epithelium. For the ultracytochemical detection of these activities, Yokota's technique (1969), which is the modification of Hansson's method (1967) was employed. Numerous electron dense reaction products were observed in the microvilli of the choroidal epithelial cell. The reaction deposits were also remarkably present in the infoldings of the basal plasmalemma but to a lesser extent than in the microvilli. The localization sites were mainly on the plasma membrane, but some reaction products were also observed in the cytoplasm near the plasma membrane. Hardly any reaction product was found in the intracellular organelles except for the mitochondria in which reaction products were occasionally observed on the cristae. These activities were completely inhibited by acetazolamide. As the carbonic anhydrase activity was histochemically seen in the microvilli and the basal infoldings, it is likely that carbonic anhydrase is related to an active transport process in the secretion of cerebrospinal fluid as is Na⁺, K⁺-ATPase (Masuzawa et al. 1980).     

Asymmetric localization of Notch2 on the microvillous surface in choroid plexus epithelial cells

Notch family molecules are transmembrane receptors that play various roles in contact-dependent cell–cell interactions in a wide range of organs. In the brain, Notch2, but not the other members of Notch, is expressed in the choroid plexus at an exceptionally high level. We immunohistochemically examined the cellular and subcellular localization of Notch2 protein in the choroid plexus using confocal and electron microscopy. Unexpectedly, Notch2 was asymmetrically localized on the microvillous surface of epithelial cells in the choroid plexus of both postnatal and adult rats. This localization pattern of Notch2 suggests its novel and unknown role independent of contact with adjacent cells in the choroid plexus. In organotypic cultures of the choroid plexus, the addition of anti-Notch2 antibody resulted in deformation of microvilli in epithelial cells, which suggests a role of Notch2 in the maintenance of the microvillous structure in choroid plexus epithelial cells.     

Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells

The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution.     

Intraepithelial lymphocytes coinduce nitric oxide synthase in intestinal epithelial cells

The study of mucosal immunity has revealed that complex reciprocal interactions occur between intestinal intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC). The present study focuses on the induction of inducible nitric oxide (NO) synthase in cocultures of freshly isolated rat IEL and the rat epithelial cell line IEC-18 after the addition of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, or lipopolysaccharide. When IEL and IEC were separated using Transwell chambers, NO synthesis was not induced, indicating that cell-cell contact was required. Culture of IEC-18 with IEL, even in the absence of inflammatory stimuli such as IL-1beta, resulted in upregulation of class I and II antigens on IEC-18, due to the interferon-gamma (IFN-gamma) that is constitutively produced by IEL. Addition of anti-IFN-gamma antibody to the NO-producing cocultures resulted in inhibition of NO synthesis as well as the upregulation of class I and II antigen expression. These data indicate that IFN-gamma production by IEL conditions IEC for the expression of other components of the inflammatory process.     

Immunohistochemical approach reveals involvement of inducible nitric oxide synthase in rat late development

To better understand the role of nitric oxide (NO) in mammal development, specifically in the transition of the fetal stages at birth, we studied the timing of cell-specific expression of inducible NO synthase (iNOS) isoform during gestational periods of rats, mainly at the late stages of intra-uterine development. Before experimentation, the samples were collected (from 17th to 21st gestational days), fixed in 10% buffered formalin and embedded in paraffin for histological procedures. Hereafter, the sections (5 μm thickness) obtained from different embryos were immunostained by avidin–biotin–immunoperoxidase technique, by using antibody against iNOS isoform. The most of cell immunopositive was suggestive of granulocyte-like cells and those cells were resident close to the blood vessels in different organs, such as: lung, liver or bone marrow environment. Sometimes we noted immunopositive cells in the blood flow, as reported in the thymus. In agreement, iNOS expression, obtained by western blotting analysis, showed the same profile. Together, our data shows that iNOS expression increased gradually during the late stages of rat development (from E17 to E21) and it was executed by cells close to blood vessels. Thus, we can clearly to predict that this expression was finely modulated and it contributes for time-line dependent NO production during rat late development.     

Ultrastructural immunogold localization of nitric oxide synthase isoforms in rat and human eosinophils

The involvement of nitric oxide (NO) as both pro and anti-inflammatory agent in allergic, airway inflammatory, and asthmatic diseases and the active participation of eosinophils in such ailments have been previously suggested. NO modulates eosinophil number, migration and their survival. The microenvironment of NO synthase (NOS) in subcellular organelles determines its rate and efficiency of catalysis, which in turn influences NO generation at distinct intracellular locales. The present study was undertaken to assess the intracellular distribution of NOS isoforms by transmission electron microscopy followed by morphometric analysis in human and rat eosinophils. Rat eosinophils were explored in parallel, and since they are widely used as model systems to mimic human diseases, a comparative study on NOS localization patterns might provide useful information in deciphering NO role in diverse aspects of eosinophil-related inflammatory ailments. The results demonstrated predominance of neuronal NOS (nNOS) in the eosinophilic granules and even distribution of inducible NOS (iNOS) and nNOS in the cytoplasm and nucleus of human eosinophils. In rat eosinophils, however, iNOS was mainly localized in the eosinophilic granules and nucleus, while nNOS was distributed evenly in cytoplasm and nucleus. Distribution of endothelial NOS (eNOS) in eosinophils was scanty. Differences in NOS isoforms and their localization in human and rat cells might have implications in differential mode of catalysis and functional contribution to eosinophil physiology and pathology, warranting detailed investigations. The present study highlights species-specific differences in the relative abundance and distribution pattern of NOS isoforms in rat and human eosinophils, which should be considered cautiously in interpreting the rat data to humans.     

Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

The proteomic analysis of mouse choroid plexus secretome reveals a high protein secretion capacity of choroidal epithelial cells

Choroid plexuses (CP) are involved in multiple functions related to their unique architecture and localization at the interface between the blood and cerebrospinal fluid compartments. These include the release by choroidal epithelial cells (CEC) of biologically active molecules, such as polypeptides, which are distributed globally to the brain. Here, we have used a proteomic approach to get an unbiased overview of the proteins that are secreted by primary cultures enriched in epithelial cells from mice CP. We identified a total of 43 proteins secreted through the classical vesicular pathway in CEC -conditioned medium. They include transport proteins, collagen subunits and other cell matrix proteins, proteases, protease inhibitors and neurotrophic factors. Treating CEC cultures with lipopolysaccharide, increased the secretion of four protein species and induced the release of two additional proteins. Our study also reveals a higher protein secretion capacity of CECs compared with other CP cells or cultured astrocytes. In conclusion, this study provides for the first time the characterization of the major proteins that are secreted by CECs. These proteins may play a critical role in neuronal growth, differentiation and function as well as in brain pathologies.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Immunohistochemical detection of apoptosis, proliferation and inducible nitric oxide synthase in rat urothelium damaged by cyclophosphamide treatment

The present study was conducted to investigate cell death, proliferation and inducible nitric oxide synthase (iNOS) immunoreactivity in rat urothelium within 24 h after a single intraperitoneal dose of cyclophosphamide (CP). Necrotic cells were identified predominantly in the superficial cell layer from 1 h until 6 h after CP injection, most of them desquamating from the urothelium into the lumen of the urinary bladder. Active caspase-3 immunohistochemistry revealed apoptotic cells from 12 h until 24 h after CP injection. The apoptotic index reached a peak at 18 h and then rapidly dropped. Simultaneously with the decrease of apoptosis, the proliferation index increased from 18 h until 24 h after CP treatment. Immunoreaction to iNOS was first detected at 6 h in basal and intermediate cells. Later, iNOS immunoreactivity became stronger and was present in all cell layers. Our results suggest that the destruction of rat urothelium during 24 h after CP administration is due not only to necrosis, but also to apoptosis. The first 6 h are characterised by necrotic changes and no iNOS immunoreactivity. Thereafter, apoptosis and iNOS immunoreactivity are observed within the damaged urothelium. At 24 h after CP injection, iNOS immunoreactivity is still present, but proliferation prevails over cell death, enabling the urothelium to start regeneration.     

Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were seen in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes.     

Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta

Nitric oxide (NO) production in therat placenta was monitored and quantified by electron paramagneticresonance (EPR) spectroscopy with hemoglobin and anFe-N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trappingreagents. Expression of nitric oxide synthase (NOS) isoformswas also examined by quantitative RT-PCR analysis. The EPR spectrum ofthe placenta with hemoglobin trapping showed a three-line hyperfinestructure (g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOSinhibitor L-NAME was administered to pregnant rats.Therefore, the specific signal was definitely identified as beingderived from endogenous NO spin-trapped by hemoglobin, and the EPRspectrum showed that the NO adduct existed as a pentacoordinate -NOheme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal (g = 2.038) derived from anNO-Fe-DTCS complex. The height of the triplet signal did not varysignificantly with gestational stage during the last few days ofgestation. At the gestational stages examined, the level of NOS II mRNAexpression was significantly higher than that of NOS III mRNA. NOS IIexpression in term (day 21.5) placenta was significantlyincreased compared with that in preterm (day 19.5) placenta(P < 0.01, n = 4 or 5). These resultssuggest that NOS II is the predominant producer of NO in the placentaand that NOS II-generated NO plays significant roles in the maintenanceof placental functions immediately before birth.

     

Up-regulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: possible role of interferon-gamma in polarized nitric oxide secretion

Background. Nitric oxide (NO) generated by nitric oxide synthase (NOS) is known to be an important modulator of the mucosal inflammatory response. In this study, we questioned whether Helicobacter pylori infection could up‐regulate the epithelial cell inducible NOS (iNOS) gene expression and whether NO production could show polarity that can be regulated by immune mediators. Materials and Methods. Human gastric epithelial cell lines were infected with H. pylori, and the iNOS mRNA expression was assessed by quantitative RT‐PCR. NO production was assayed by determining nitrite/nitrate levels in culture supernatants. To determine the polarity of NO secretion by the H. pylori‐infected epithelial cells, Caco‐2 cells were cultured as polarized monolayers in transwell chambers, and NO production was measured. Results. iNOS mRNA levels were significantly up‐regulated in the cells infected with H. pylori, and expression of iNOS protein was confirmed by Western blot analysis. Increased NO production in the gastric epithelial cells was seen as early as 18 hours postinfection, and reached maximal levels by 24 hours postinfection. The specific MAP kinase inhibitors decreased H. pylori‐induced iNOS and NO up‐regulation. After H. pylori infection of polarized epithelial cells, NO was released predominantly into the apical compartment, and IL‐8 was released predominantly into basolateral compartment. The addition of IFN‐γ to H. pylori‐infected polarized epithelial cells showed a synergistically higher apical and basolateral NO release. Conclusion. These results suggest that apical NO production mediated by MAP kinase in H. pylori‐infected gastric epithelial cells may influence the bacteria and basolateral production of NO and IL‐8 may play a role in the tissue inflammation.     

Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca(2+) and kinase signaling pathways. Using UAEC derived from pregnant ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187, we nonetheless observed more p-S1179 with ATP than with A23187 (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation.     

Down-regulation of inducible nitric oxide synthase by lysophosphatidic acid in human respiratory epithelial cells

Viral infection generally results in the activation of inducible nitric oxide synthase (iNOS or NOS2) in respiratory epithelial cells by inflammatory cytokines. Activated NOS2 catalyzes synthesis of nitric oxide (NO), which in excess can cause cellular injury. On the other hand, lysophosphatidic acid (LPA), a lipid mediator released from epithelial cells, platelets, and fibroblasts in injured tissue, functions in repair of cell injury. However, details of the mechanism for repair by LPA remain unknown. We demonstrated one effect of LPA favoring repair, specifically inhibition by LPA of cytokine-induced NOS2 protein and mRNA expression by human respiratory epithelial cells in vitro. NO production by LPA-treated, cytokine-stimulated cells was also reduced. These decreases were prevented by Rho kinase inhibition with Y-27632. Thus, down-regulation by LPA of cytokine-induced increases in NOS2 activity is likely to involve a Rho-dependent signaling pathway. Harmful biologic effects of NO in viral respiratory infection might be modified by therapeutic manipulations involving LPA or Rho.