Ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio)

Adenosine, a well-known neuromodulator, may be formed intracellularly in the CNS from degradation of AMP and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. This study reports the enzymatic properties of an ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for AMP hydrolysis in a pH range of 7.0-7.5 in the presence of Mg(2+). The enzyme presented a maximal activity for AMP hydrolysis at 37 degrees C. The apparent K(m) and V(max) values for Mg(2+)-AMP were 135.3+/-16 microM and 29+/-4.2 nmol Pi.min(-1).mg(-1) protein, respectively. The enzyme was able to hydrolyze both purine and pyrimidine monophosphate nucleotides, such as UMP, GMP and CMP. Levamisole and tetramisole (1 mM), specific inhibitors of alkaline phosphatases, did not alter the enzymatic activity. However, a significant inhibition of AMP hydrolysis (42%) was observed in the presence of 100 microM alpha,beta-methylene-ADP, a known inhibitor of ecto-5'-nucleotidase. Since 5'-nucleotidase represents the major enzyme responsible for the formation of extracellular adenosine, the enzymatic characterization is important to understand its role in purinergic systems and the involvement of adenosine in the regulation of neurotransmitter release.     

Production of endothelin-1 by rat cultured mesangial cells

We investigated whether ET-1 is synthesized by and released from mesangial cells. ET-1-like immunoreactivity (LI) released into medium increased time-dependently under a serum-free condition. The amounts of ET-1-LI released into the medium was augmented in the presence of fetal calf serum. Reverse-phase HPLC profile of the conditioned media revealed a major component coeluting with standard ET-1. Northern blot analysis of poly(A) +RNA extracted from mesangial cells showed a single major band corresponding to the size of preproET-1 mRNA (2.3 kb). These findings demonstrate that ET-1 is synthesized by and released from rat mesangial cells and suggest a possibility that it acts on their own cells as an autocrine factor.     

Ecto-5'-nucleotidase: a candidate virulence factor in Streptococcus sanguinis experimental endocarditis

Streptococcus sanguinis is the most common cause of infective endocarditis (IE). Since the molecular basis of virulence of this oral commensal bacterium remains unclear, we searched the genome of S. sanguinis for previously unidentified virulence factors. We identified a cell surface ecto-5'-nucleotidase (Nt5e), as a candidate virulence factor. By colorimetric phosphate assay, we showed that S. sanguinis Nt5e can hydrolyze extracellular adenosine triphosphate to generate adenosine. Moreover, a nt5e deletion mutant showed significantly shorter lag time (P<0.05) to onset of platelet aggregation than the wild-type strain, without affecting platelet-bacterial adhesion in vitro (P?=?0.98). In the absence of nt5e, S. sanguinis caused IE (4 d) in a rabbit model with significantly decreased mass of vegetations (P<0.01) and recovered bacterial loads (log(10)CFU, P?=?0.01), suggesting that Nt5e contributes to the virulence of S. sanguinis in vivo. As a virulence factor, Nt5e may function by (i) hydrolyzing ATP, a pro-inflammatory molecule, and generating adenosine, an immunosuppressive molecule to inhibit phagocytic monocytes/macrophages associated with valvular vegetations. (ii) Nt5e-mediated inhibition of platelet aggregation could also delay presentation of platelet microbicidal proteins to infecting bacteria on heart valves. Both plausible Nt5e-dependent mechanisms would promote survival of infecting S. sanguinis. In conclusion, we now show for the first time that streptococcal Nt5e modulates S. sanguinis-induced platelet aggregation and may contribute to the virulence of streptococci in experimental IE.     

Autoimmunity in CD73/Ecto-5'-nucleotidase deficient mice induces renal injury

Extracellular adenosine formed by 5'-ectonucleotidase (CD73) is involved in tubulo-glomerular feedback in the kidney but is also known to be an important immune modulator. Since CD73(-/-)mutant mice exhibit a vascular proinflammatory phenotype, we asked whether long term lack of CD73 causes inflammation related kidney pathologies. CD73(-/-)mice (13 weeks old) showed significantly increased low molecule proteinuria compared to C57BL6 wild type controls (4.8 ≥ 0.52 vs. 2.9 ± 0.54 mg/24 h, p<0.03). Total proteinuria increased to 5.97 ± 0.78 vs. 2.55 ± 0.35 mg/24 h at 30 weeks (p<0.01) whereas creatinine clearance decreased (0.161 ± 0.02 vs. 0.224 ± 0.02 ml/min). We observed autoimmune inflammation in CD73(-/-)mice with glomerulitis and peritubular capillaritis, showing glomerular deposition of IgG and C3 and enhanced presence of CD11b, CD8, CD25 as well as GR-1-positive cells in the interstitium. Vascular inflammation was associated with enhanced serum levels of the cytokines IL-18 and TNF-α as well as VEGF and the chemokine MIP-2 (CXCL-2) in CD73(-/-)mice, whereas chemokines and cytokines in the kidney tissue were unaltered or reduced. In CD73(-/-)mice glomeruli, we found a reduced number of podocytes and endothelial fenestrations, increased capillaries per glomeruli, endotheliosis and enhanced tubular fibrosis. Our results show that adult CD73(-/-)mice exhibit spontaneous proteinuria and renal functional deterioration even without exogenous stress factors. We have identified an autoimmune inflammatory phenotype comprising the glomerular endothelium, leading to glomeruli inflammation and injury and to a cellular infiltrate of the renal interstitium. Thus, long term lack of CD73 reduced renal function and is associated with autoimmune inflammation.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Ecto-5'-nucleotidase can provide the total purine requirements of mitogen-stimulated human T cells and rapidly dividing human B lymphoblastoid cells

The ability of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells to drive their total purine requirements from inosine 5'-monophosphate, inosine, or hypoxanthine was compared. Inosine 5'-monophosphate first must be converted to inosine by the action of the enzyme ecto-5'-nucleotidase before it can be transported into the cell; inosine and hypoxanthine, however, can be transported directly. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and to make the cells dependent on an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 30 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA or [3H]leucine incorporation into protein at rates equal to that of untreated control cultures. Similar results were found when azaserine was used to inhibit purine synthesis de novo, and thus DNA synthesis. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells, and suggest that this enzyme may be important for purine salvage when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.     

Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis

The extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host's response to sepsis. In this study, we investigated the role of the adenosine-generating ectoenzyme, ecto-5'-nucleotidase (CD73), in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury, as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased cleavage of caspase-3 and poly(ADP-ribose) polymerase and increased activation of NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and increased cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using α, β-methylene ADP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.     

Calcium-activated, phospholipid-dependent protein kinase in cultured rat mesangial cells

The analysis of the 100 000 X g supernatant fraction of cultured rat glomerular mesangial cells with DEAE-cellulose ion-exchange chromatography revealed a large peak showing the activity of a protein kinase (protein kinase C) which depended on phospholipid and diolein as well as Ca2+. Furthermore, it was shown that angiotensin II (AII) (10(-6)M) induced rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate, leading to production of diacylglycerol rich in arachidonic acid, in the cultured rat mesangial cells. These results suggest that activation of protein kinase C resulting from enhancement of phosphoinositide metabolism may be important as an intracellular regulatory mechanism of AII upon cultured mesangial cells.     

Ecto-5'-nucleotidase (eN, CD73) is coexpressed with metastasis promoting antigens in human melanoma cells

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: alpha5-, beta1-, beta3-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. alphav-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.     

Ecto-5'-nucleotidase activity in lymphoblastoid cell lines derived from heterozygotes for congenital X-linked agammaglobulinemia

Lymphoblastoid cell lines were established from female relatives of patients with congenital X-linked-agammaglobulinemia by Epstein-Barr virus transformation of their peripheral B lymphocytes. Cell lines derived from presumed carriers were characterized by low ecto-5'-nucleotidase activity and a reduced percentage of surface immunoglobulin-bearing cells. Measurement of ecto-5'-nucleotidase activity in newly established lymphoblastoid cell lines may provide a means for the identification of heterozygotes for congenital X-linked agammaglobulinemia.     

Heterogeneity of protein kinase C in cultured rat mesangial cells.

Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity. Upon hydroxyapatite chromatography (HPLC), the PKC activity was eluted as a main peak at 150 mM potassium phosphate with a shoulder at 180 mM. Both peaks corresponded to the type III PKC from rat brain and were identified as PKC alpha isoform by immunoblot analysis. In contrast with what was observed using histone H1, the increased phosphorylation of endogenous proteins in the presence of a mixture of Ca2+/PS, plus either DAG or PMA, was only partly reduced by EGTA. Moreover, the level of the PKC activity detected in the presence of EGTA was comparable to the level of kinase activity, measured in the presence of PS alone or associated with DAG or PMA. This suggests that mesangial cells contain PKC activity which does not absolutely require calcium. Polyacrylamide gel electrophoresis revealed that patterns of phosphorylated mesangial cell proteins are different depending on whether calcium was added or not. In the presence of calcium, PKC strongly phosphorylated the proteins of 53,000 molecular weight, a doublet of 37,000-39,000, the 24,000 and the triplet of 17,000-20,000-22,000 molecular weight. The addition of EGTA to the assays suppressed completely the labelling of most proteins; only the 20,000 molecular weight protein remained strongly labelled, while the 39,000 molecular weight band was only faintly visible. The same patterns of phosphorylations were obtained after omission of calcium in the assays containing only PS and DAG (or PMA). So, the main substrates of calcium-dependent PKC are proteins of 53,000, 39,000, 37,000, 22,000, 24,000 and 17,000 molecular weight while the protein of 20,000 molecular weight appears to be the main substrate of calcium-independent PKC. The existence in mesangial cells of at least two forms of PKC, which phosphorylate specific endogenous proteins, emphasizes the complexity of the phospholipid-dependent regulatory cascade and raises the possibility that actions of different regulators may be transduced through distinct PKC isozymes.     

Signal transduction mechanism of interleukin 6 in cultured rat mesangial cells

Interleukin 6 (IL-6) is one of the potent autocrine growth factors for mesangial cells. We investigated the signal transduction mechanism of IL-6 in cultured rat mesangial cells. IL-6 induced a transient increase of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) followed by a transient and sustained increase of intracellular calcium concentration, suggesting that IL-6 stimulates phosphoinositide turnover. IL-6 also stimulated prostaglandin E2 (PGE2) production. The IL-6-concentration dependency in PGE2 production was similar to that in Ins 1,4,5-P3 production. We concluded that the action of IL-6 on mesangial cells is exerted at least partially through the enhancement of phosphoinositide turnover and PGE2 production.     

Morin hydrate protects cultured rat glomerular mesangial cells against oxyradical damage

Cultured rat glomerular mesangial cells were damaged when exposed to oxyradicals generated either from xanthine oxidase plus hypoxanthine, or by superoxide radicals formed from menadione. Morin hydrate is an antioxidant extracted from yellow Brazil wood. When morin hydrate was added to cultured rat glomerular mesangial cells which were attacked by oxyradicals generated by xanthine oxidase plus hypoxanthine, the survival time of the cells was doubled. However, this protective effect of morin hydrate was less marked when the cells were attacked by menadione. Note that the protective effects of Trolox which is a polar analogue of vitamin E were miniscule relative to those of morin hydrate with both oxidants.     

Ecto-5'-nucleotidase expression during human B cell development. An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia

Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in human B cells at different stages of development. Ecto-5'-NT activity of B cell preparations from fetal spleen and cord blood was 5.08 and 5.59 +/- 2.8 nmol/hr/10(6) cells, respectively; that of B cell preparations from adult peripheral blood, spleen, or lymph node was fivefold to sixfold higher (27.9 +/- 12, 29.2 and 33.8 nmol/hr/10(6) cells, respectively). The increased enzyme activity in B cell preparations from adult peripheral blood as compared with cord blood paralleled increased percentages of 5'-NT+ cells (69 +/- 12% vs 32 +/- 17%) and an average of twice as much enzyme activity per positive cell. Small, resting B cells that cannot synthesize Ig in vitro in response to pokeweed mitogen (PWM) were isolated from adult peripheral blood by mouse erythrocyte rosetting. Total ecto-5'-NT activity and the percentage of 5'-NT+ cells were equivalent in total B cells and the mouse erythrocyte rosette-positive subpopulation. Thus, ecto-5'-NT activity is acquired before B cells gain the ability to differentiate into Ig-secreting plasma cells in response to PWM. Ecto-5'-NT activity was also measured in B cell preparations from eight patients with common variable immunodeficiency. Six had reduced ecto-5'-NT activity (2.83 to 15.4 nmol/hr/10(6) cells), and two had normal activity (34.7 and 58.2 nmol/hr/10(6) cells). B cells from all six patients with low ecto-5'-NT activity failed to synthesize Ig when cultured with PWM and normal irradiated T cells. Of the two patients with normal B cell ecto-5'-NT activity, one also had B cells unresponsive to PWM, but B cells from the other patient appeared to more normal, in that they synthesized IgM and IgG when cultured with PWM plus irradiated allogeneic T cells. Thus, measurement of B cell ecto-5'-NT activity allows the subclassification of patients who have a common inability to synthesize immunoglobulin in vitro response to PWM. B cells with low ecto-5'-NT activity are presumably blocked at an earlier stage in development than B cells with normal ecto-5'-NT activity. Evaluation of ecto-5'-NT activity along with the expression of other B cell surface antigens should aid in the definition of discrete stages of B cell development.     

Induction of human high K(M) 5'-nucleotidase in cultured 293 cells

Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.     

Platelet-activating factor stimulates multiple signaling pathways in cultured rat mesangial cells.

We have previously reported that platelet-activating factor (PAF) elevates cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded glomerular mesangial cells. To confirm that this increase in [Ca2+]i is a result of receptor-mediated activation of phospholipase C, we investigated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) in PAF-treated mesangial cells. PAF (10(-7) M) stimulated a rapid and transient formation of inositol trisphosphate. In concomitant experiments, PAF stimulated a biphasic accumulation of 3H-arachidonate-labeled 1,2-diacylglycerol (DAG). The secondary elevation in DAG was coincident with a rise in 3H-phosphorylcholine (PC) and 3H-phosphorylethanolamine (PE) suggesting that PAF stimulates delayed phospholipase activities which hydrolyze alternate phospholipids besides the polyphosphoinositides. This PAF-stimulated elevation in 3H-water soluble phosphorylbases was seen at 5 min but not at 15 sec suggesting that the initial rise in DAG as well as the initial elevation in [Ca2+]i are due primarily to PtdIns-4,5-P2 hydrolysis. PAF also stimulated PGE2 as well as 3H-arachidonic acid and 3H-lyso phosphatidylcholine (PtdCho) formation. We suggest that arachidonate released specifically from PtdCho via phospholipase A2 is a source of this PAF-elevated PGE2. It has been postulated that anti-inflammatory prostaglandins may antagonize the contractile and proinflammatory effects of PAF via activation of adenylate cyclase. Surprisingly, exogenous PAF reduced basal and receptor-mediated cAMP concentration indicating that PAF-stimulated transmembrane signaling pathways may oppose receptor-mediated activation of adenylyl cyclase. We have taken advantage of the different sensitivities of phospholipases A2 and C(s) to PMA, EGTA, and pertussis toxin to dissociate phospholipase A2 and C activities. Acute PMA-treatment enhanced PAF-stimulated PGE2 formation, reduced PAF-induced elevations in [Ca2+]i and had no effect upon PAF-stimulated 3H-PE. We have also demonstrated that phospholipase A2, but not PtdIns-specific phospholipase C, was sensitive to external calcium concentration. The role of a GTP-binding protein to couple PAF-receptors to the PtdIns-specific phospholipase C was confirmed as GTP gamma S synergistically elevated PAF-stimulated inositol phosphate formation. We also demonstrated that pertussis toxin ADP-ribosylates a single protein of an apparent 42 kD mass and that PAF pretreatment reduced subsequent ADP-ribosylation in a time-dependent manner. However, pertussis toxin had no effect upon phospholipase C-generated water soluble phosphorylbases or inositol phosphates. In contrast, PAF-stimulated phospholipase A2 and PAF-inhibited adenylyl cyclase activities were sensitive to pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)     

肾素(原)受体在大鼠肾小球系膜细胞和肾脏的表达

近年发现的肾素（原）受体（renin／prorenin receptor,RnR）已被证明具有生物学功能,在心、肾及多种细胞表达。本文旨在观察RnR在体外培养的大鼠肾小球系膜细胞（mesangial cells,MCs）和肾脏中是否表达,及其表达的细胞部位,并用RnR的多肽阻断剂肾素原“柄区肽”（handle region peptide,HRP）与RnR结合后观察受体复合物进入细胞的过程与定位。结果显示,RnR主要存在于大鼠肾脏皮质肾小球系膜区和体外培养的MCs的细胞核周围胞浆和细胞膜。将FITC标记的HRP（FITC-HRP）加入细胞培养液后30S到30min期间,可观察到FITC-HRP由培养液转移到胞浆内并进入细胞核。用免疫荧光和激光共聚焦技术观察到,HRP与RnR的共定位主要位于细胞膜和细胞核周围胞浆;在30min时,一部分HRP已进入细胞核,而RnR没有进入细胞核内,仍主要位于细胞核周围胞浆。上述结果提示,RnR与其配基结合后进入细胞内并发挥生物学效应。