Nitric oxide mediates laminin-induced neurite outgrowth in PC12 cells

Laminin is a potent stimulator of neurite outgrowth in a variety of primary neurons and neuronal cell lines. Here, we investigate the role of nitric oxide in the signaling mechanism of laminin-mediated neurite outgrowth in the PC12 cell line. Within 8 s of exposure to laminin, PC12 cells produce nitric oxide. Peak laminin-induced nitric oxide levels reach 8 nM within 12 s of exposure to laminin and constitutive nitric oxide production is sustained for 1 min. A neurite outgrowth promoting synthetic peptide (AG73), derived from the laminin-1-alpha globular domain, also stimulated nitric oxide release. The nitric oxide synthase inhibitor, 1-NAME, prevents the formation of nitric oxide and here, 1-NAME inhibited both laminin-mediated and AG73-mediated neurite outgrowth by 88 and 95%, respectively. In contrast, C16, a synthetic peptide derived from the laminin-1-gamma chain, is shown here to promote PC12 cell attachment, but not neurite outgrowth. Interestingly, the C16 peptide did not activate nitric oxide release, suggesting that laminin-induced nitric oxide release in PC12 cells is associated only with neurite outgrowth promoting laminin domains and signals. In addition, the data here show that the nitric oxide released by PC12 cells in response to laminin is required as a part of the mechanism of laminin-mediated neurite outgrowth.     

Metabolism of glutamate and {gamma}-aminobutyrate in cultured tobacco cells

In cultured tobacco cells glutamate-U-¹⁴C administrated wasreadily converted to -aminobutyrate (GABA) by decarboxylation,however, GABA-1-¹⁴C remained unchanged. Glutamate decarboxylasewas found in tobacco cells and reached its maximum activityin the rapidly growing stage during culture. Enzyme activityparalleled formation of GABA from glutamate-U-¹⁴C. A high contentof GABA in tobacco cells seems to be due to the rapid decarboxylationof glutamate by glutamate decarboxylase and a slow turn overof GABA. ¹ Present address: The Okayama Tobacco Experiment Station, JapanMonopoly Corp., Tamashima, Kurashiki, Japan. (Received November 20, 1971; )     

Nitric oxide mediates tumor necrosis factor-alpha cytotoxicity in endothelial cells.

Tumor necrosis factor alpha (TNF-alpha) exerts multiple actions on endothelial cells including among others the expression of pro-coagulant activity and adhesion molecules, and secretion of cytokines. We now show that TNF-alpha induces a time- and dose-dependent cytotoxic effect on cultured bovine aortic endothelial cells. This TNF-induced cytotoxicity, which is preceded by increased production of nitric oxide (NO), is significantly decreased by the NO synthase inhibitor N-iminoethyl-L-ornithine (L-NIO). Dexamethasone, which prevents the expression of cytokine-induced NO synthase in endothelial cells, also inhibits TNF-alpha-dependent cytotoxicity. The results indicate that NO is involved in the cytotoxic effect of TNF-alpha on endothelial cells.     

Nitric oxide synthase I mediates osteoclast activity in vitro and in vivo

Bone resorption is responsible for the morbidity associated with a number of inflammatory diseases such as rheumatoid arthritis, orthopedic implant osteolysis, periodontitis and aural cholesteatoma. Previous studies have established nitric oxide (NO) as a potentially important mediator of bone resorption. NO is a unique intercellular and intracellular signaling molecule involved in many physiologic and pathologic pathways. NO is generated from L-arginine by the enzyme nitric oxide synthase (NOS). There are three known isoforms of NOS with distinct cellular distributions. In this study, we have used mice with targeted deletions in each of these isoforms to establish a role for these enzymes in the regulation of bone resorption in vivo and in vitro. In a murine model of particle induced osteolysis, NOS I-/- mice demonstrated a significantly reduced osteoclast response. In vitro, osteoclasts derived from NOS I-/- mice were larger than wild type controls but demonstrated decreased resorption. Although NOS I has been demonstrated in osteoblasts and osteocytes as a mediator of adaptive bone remodeling, it has not previously been identified in osteoclasts. These results demonstrate a critical role for NOS I in inflammatory bone resorption and osteoclast function in vitro.     

Nitric oxide inhibits cAMP-dependent CFTR trafficking in intestinal epithelial cells

Nitric oxide is produced during intestinal inflammation and inhibits the epithelial responsiveness to cAMP-dependent secretagogues. The effect is presumably due to inhibition of activation of the CFTR. However, because insertion of CFTR into the epithelial apical membrane is also a cAMP-dependent process, we tested the hypothesis that NO could inhibit cAMP-dependent CFTR trafficking. SCBN intestinal epithelial cells were treated with forskolin to activate adenylate cyclase activity. The cells were fixed at various times and immunostained for CFTR. Some cells were pretreated with the nitric oxide donor PAPA-NONOate, the protein kinase A inhibitor H89, or the microtubule blocker nocodazole. Cross sections of epithelial monolayers were then studied under fluorescence, and the ratio of apical to basolateral CFTR immunoreactivity was determined. Stimulation of adenylate cyclase activity caused an increase in the apical-to-basolateral ratio of CFTR within 30 s. This effect was transient and preceded changes in short-circuit current in SCBN monolayers mounted in Ussing chambers. PAPA-NONOate, H89, and nocodazole all reduced forskolin-stimulated CFTR trafficking. The inhibitory effect of the NO donor was not affected by pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. PAPA-NONOate reduced forskolin-stimulated increases in intracellular cAMP. The data suggest that a portion of the inhibitory effect of nitric oxide donors on cAMP-dependent chloride secretion is through the inhibition of cAMP-dependent insertion of CFTR into the apical plasma membrane. These data provide insight into the mechanism of secretory dysfunction in inflammatory diseases of the gut where mucosal nitric oxide is elevated.     

Analysis of binding sites in human C-reactive protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by site-directed mutagenesis

Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998-4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.     

Nitric oxide mediates intestinal pathology but not immune expulsion during Trichinella spiralis infection in mice

The relationship between intestinal pathology and immune expulsion of gastrointestinal (GI) nematodes remains controversial. Although immune expulsion of GI helminth parasites is usually associated with Th2 responses, the effector mechanisms directly responsible for parasite loss have not been identified. We have previously shown that while the intestinal pathology accompanying the expulsion of the GI parasite Trichinella spiralis may be dependent on IL-4 and mediated by TNF, parasite loss is independent of TNF. In contrast, intestinal pathology in other disease models has been attributed to Th1 cytokines, although it closely resembles that seen in helminth infections. Whereas production of inducible NO synthase (iNOS) in the gut is important for both homeostasis of the epithelial layer and in protection against pathogenic microorganisms, overproduction of NO has been implicated in the pathogenesis of a number of inflammatory conditions. We therefore investigated the role of NO in T. spiralis infection using iNOS-deficient mice. iNOS-/- and iNOS-/+ mice were infected with T. spiralis, and parasite expulsion and intestinal pathology were followed. Parasite expulsion proceeded similarly in both groups of animals, but significant intestinal pathology was only observed in the heterozygous mice. Thus it appears that, although the protective effects of Th2 responses in GI helminth infection do not require NO, this mediator contributes substantially to the associated enteropathy. NO may therefore be an important mediator of enteropathy in both Th1- and Th2-inducing conditions.     

Nitric oxide mediates gravitropic bending in soybean roots

Plant roots are gravitropic, detecting and responding to changes in orientation via differential growth that results in bending and reestablishment of downward growth. Recent data support the basics of the Cholodny-Went hypothesis, indicating that differential growth is due to redistribution of auxin to the lower sides of gravistimulated roots, but little is known regarding the molecular details of such effects. Here, we investigate auxin and gravity signal transduction by demonstrating that the endogenous signaling molecules nitric oxide (NO) and cGMP mediate responses to gravistimulation in primary roots of soybean (Glycine max). Horizontal orientation of soybean roots caused the accumulation of both NO and cGMP in the primary root tip. Fluorescence confocal microcopy revealed that the accumulation of NO was asymmetric, with NO concentrating in the lower side of the root. Removal of NO with an NO scavenger or inhibition of NO synthesis via NO synthase inhibitors or an inhibitor of nitrate reductase reduced both NO accumulation and gravitropic bending, indicating that NO synthesis was required for the gravitropic responses and that both NO synthase and nitrate reductase may contribute to the synthesis of the NO required. Auxin induced NO accumulation in root protoplasts and asymmetric NO accumulation in root tips. Gravistimulation, NO, and auxin also induced the accumulation of cGMP, a response inhibited by removal of NO or by inhibitors of guanylyl cyclase, compounds that also reduced gravitropic bending. Asymmetric NO accumulation and gravitropic bending were both inhibited by an auxin transport inhibitor, and the inhibition of bending was overcome by treatment with NO or 8-bromo-cGMP, a cell-permeable analog of cGMP. These data indicate that auxin-induced NO and cGMP mediate gravitropic curvature in soybean roots.     

Nitric oxide mediates 17beta-estradiol-stimulated human and rodent osteoblast proliferation and differentiation

Oestradiol can stimulate osteoblast activity. Osteoblast function is thought to be regulated by nitric oxide (NO). We hypothesised that the effect of 17beta-oestradiol (17beta-E(2)) on osteoblast activity is mediated by NO. This hypothesis was tested using osteoblasts isolated from human trabecular bone, calvariae of rats, endothelial NO synthase (eNOS) gene-deficient mice, and their wild-type counterparts. Our results show that 17beta-E(2) dose-dependently stimulated proliferation and differentiation of primary human, rat and wild-typeosteoblasts. The presence of N(G)-monomethyl-l-arginine (10(-3) M), an inhibitor of NOS activity, blocked the 17beta-E(2)-(10(-7) M)-induced increases in thymidine incorporation (P < 0.01), alkaline phosphatase activity (P < 0.01) and bone nodule formation (P < 0.01) of wild-type, human and rat osteoblasts, respectively. Moreover, 17beta-E(2) did not induce a response in eNOS gene-deficient osteoblasts. 17beta-E(2) also increased total eNOS enzyme expression in rat osteoblasts. These findings indicate 17beta-E(2) modulates osteoblast function by NO-dependent mechanisms mediated via the eNOS isoform.     

III. Nitric oxide mediates the action of lipoteichoic acid on the function of human urothelial cells

Gram-positive bacteria are recognized pathogens in urinary tract infections. Lipoteichoic acids, major components of the cell wall of gram-positive bacteria, are important virulence attributes, but their mechanism of action is not well understood. We have postulated that infection-induced altered function of progenitors of urothelial cells (UT) residing in the basal layer is likely to have long-lasting effects on the architecture and function of the urothelium. Our earlier in vitro studies in UT of basal type, grown under growth restricting conditions, have shown that (1) treatment with lipoteichoic acid from Streptococcus faecalis (LT-2) stimulates a subpopulation of progenitors of urothelial cells to proliferate, and (2) resulting large colonies differentiated at an increased rate under conditions simulating those in the basal layer of the urothelium. The hypothesis underlying the present studies was that nitric oxide (NO) mediated LT-2 action on these functions of UT. Immunocytochemical studies using an antibody against inducible nitric oxide synthase (iNOS) confirmed expression of iNOS in LT-2-treated UT. Our hypothesis was tested by treating UT grown under growth restricting conditions (0.005% bovine pituitary extract) with LT-2 (25 μg/ml), in the presence or absence of inhibitors of NOS (1 mM N^G-nitro-L-arginine methyl ester [L-NAME]; 1 μM dexamethasone [DEXA]) or 25 μM hemoglobin, a potent inactivator of NO. Treatment with LT-2 in the presence of these agents prevented the following effects of LT-2 alone: (1) the stimulatory effect on proliferation of single cells, as well as within the resulting large colonies; (2) the subsequent differentiation of large colonies resulting from this proliferative activity, as indicated by distribution of β₁ subunit-containing integrins to cell-cell contacts; (3) the inhibitory effect on the subsequent ability of LT-2-treated UT to attach to extracellular matrix proteins. These studies suggest that induction of NOS by LT-2, initially aimed at restricting the replication of infectious agents, may have potential cost of damage to the host bladder by interfering with urothelial differentiation. © 1996 Wiley-Liss, Inc.     

Nitric oxide mediates hypoxia-induced cerebral vasodilation in humans.

Nitric oxide (NO) plays a pivotal role in the regulation of peripheral vascular tone. Its role in the regulation of cerebral vascular tone in humans remains to be elucidated. This study investigates the role of NO in hypoxia-induced cerebral vasodilatation in young healthy volunteers. The effect of the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on the cerebral blood flow (CBF) was assessed during normoxia and during hypoxia (peripheral O(2) saturation 97 and 80%, respectively). Subjects were positioned in a magnetic resonance scanner, breathing normal air (normoxia) or a N(2)-O(2) mixture (hypoxia). The CBF was measured before and after administration of L-NMMA (3 mg/kg) by use of phase-contrast magnetic resonance imaging techniques. Administration of L-NMMA during normoxia did not affect CBF. Hypoxia increased CBF from 1,049 +/- 113 to 1,209 +/- 143 ml/min (P < 0.05). After L-NMMA administration, the augmented CBF returned to baseline (1,050 +/- 161 ml/min; P < 0.05). Similarly, cerebral vascular resistance declined during hypoxia and returned to baseline after administration of L-NMMA (P < 0.05 for both). Use of phase-contrast magnetic resonance imaging shows that hypoxia-induced cerebral vasodilatation in humans is mediated by NO.     

Id2 reverses cell cycle arrest induced by {gamma}-irradiation in human HaCaT keratinocytes

Id2 plays a key role in epithelial cells, regulating differentiation, the cell cycle, and proliferation. Because human skin constantly renews itself and is the first target of irradiation, it is of primary interest to evaluate whether such a gene may be regulated in keratinocytes exposed to ionizing radiation. We show here that Id2 is induced in response to gamma-irradiation and have investigated the consequence of this regulation on cell fate. Using RNA interference, we observed that Id2 extinction significantly reduces cell growth in human keratinocytes through the control of the G(1)-S transition of the cell cycle. We have investigated whether the impact of Id2 on the cell cycle may have a physiological role on the cell's ability to cope with radiative stress. Indeed, when Id2 is down-regulated through interfering RNA, cells are more sensitive to irradiation. Conversely, when Id2 is overexpressed, this somehow protects the cell. We propose that Id2 favors reentering the cell cycle after radiation-induced cell cycle arrest to permit the recovery of keratinocytes exposed to ionizing radiation.     

Efflux of sphingoid bases by P-glycoprotein in human intestinal Caco-2 cells

The aim of this study was to determine whether sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 microM digoxin for 48 h caused up-regulation of multidrug-resistance (MDR)1 mRNA and decreased the accumulation of sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive tract.     

{alpha}-Galactosyl (Gal{alpha}1-3Gal{beta}1-4GlcNAc-R) epitopes on human cells: synthesis of the epitope on human red cells by recombinant primate {alpha}1,3galactosyltransferase expressed in E.coli

Developing methods for in vitro synthesis of the carbohydratestructure Gal     

TFF3 modulates NF-{kappa}B and a novel negative regulatory molecule of NF-{kappa}B in intestinal epithelial cells via a mechanism distinct from TNF-{alpha}

Trefoil factor 3 (intestinal trefoil factor) is a cytoprotective factor in the gut. Herein we compared the effect of trefoil factor 3 with tumor necrosis factor- on 1) activation of NF-B in intestinal epithelial cells; 2) expression of Twist protein (a molecule essential for downregulation of nuclear factor-B activity in vivo); and 3) production of interleukin-8. We showed that Twist protein is constitutively expressed in intestinal epithelial cells. Tumor necrosis factor- induced persistent degradation of Twist protein in intestinal epithelial cells via a signaling pathway linked to proteasome, which was associated with prolonged activation of NF-B. In contrast to tumor necrosis factor, trefoil factor 3 triggered transient activation of NF-B and prolonged upregulation of Twist protein in intestinal epithelial cells via an ERK kinase-mediated pathway. Unlike tumor necrosis factor-, transient activation of NF-B by trefoil factor 3 is not associated with induction of IL-8 in cells. To examine the role of Twist protein in intestinal epithelial cells, we silenced the Twist expression by siRNA. Our data showed that trefoil factor 3 induced interleukin-8 production after silencing Twist in intestinal epithelial cells. Together, these observations indicated that 1) trefoil factor 3 triggers a diverse signal from tumor necrosis factor- on the activation of NF-B and its associated molecules in intestinal epithelial cells; and 2) trefoil factor 3-induced Twist protein plays an important role in the modulation of inflammatory cytokine production in intestinal epithelial cells. trefoil factor 3; signal transduction