The distribution of A1 adenosine receptor and 5′-nucleotidase in pig brain cortex subcellular fractions

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [³H]R-PIA binding, the activity of adenosine metabolizing enzymes (5-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A₁ adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A₁ adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.     

Dual mechanism of laminin modulation of ecto-5′-nucleotidase activity

The myoblast cell surface activity of ecto-5′-nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto-5′-nucleotidase. This increase was related to a higher expression of ecto-5′-nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′₁ and E₈ modified the AMPase activity of the ecto-5′-nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E₈, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto-5′-nucleotidase. By contrast, the large fragment derived from the short arms, designated E′₁, inhibited the AMPase activity. Furthermore, the monoclonal anti-ecto-5′-nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E₈ on the AMPase activity of ecto-5′-nucleotidase but did not reverse the inhibitory effect of fragment E′₁. In conclusion, laminin stimulates the AMPase activity of ecto-5′-nucleotidase by two mechanisms: inducing the expression of ecto-5′-nucleotidase to the cell surface and direct modulation of the enzymatic activity.     

Properties of Wilms' tumor line (TuWi) and pig kidney line (LLC-PK1) typical of normal kidney tubular epithelium

Summary Two stable epithelial-like cell lines, the pig kidney strain (LLC-PK₁) and a Wilms' tumor line (TuWi), previously established in other laboratories, were found to exhibit a number of properties characteristic of kidney proximal tubular epithelium. Electron micrographs of LLC-PK₁ monolayers revealed cells forming rosettes reminiscent of tubules. Numerous elongated microvilli and an amorphous basal laminar material surrounded the cell membranes. Cell junctions were located between cell membranes at regions adjacent to the patent lumens. Wilms' cells in culture were similar in appearance to the pig kidney cells; they exhibited numerous microvilli, a thin basal laminar coating on the membrane, and desmonsomes between cells. No rosette formation was evident. Neither cell line was found to produce extracellular reticulin fibers when grown in the presence ofl-ascorbic acid for 1 week. Absence of stainable reticulin in cell monolayer culture after ascorbicacid treatment has been noted only in cell lines of apparent epithelial origin. Histochemically, both lines reacted positively for activities of a number of enzymes found in high amounts in normal kidney tubular epithelium. Pig kidney cells were highly positive for γ-glutamyl transpeptidase activity and moderately active for acid phosphatase and leucine aminopeptidase activities. Wilms' tumor cells were markedly active for γ-glutamyl transpeptidase, 5′-nucleotidase, ATPase, glucose-6-phosphatase, and acid phosphatase activities. These findings in conjunction with the ultrastructural observations indicate that these two lines in culture maintain many of the properties typical of proximal kidney tubular epithelium.     

Initial Studies to Define the Physiologic Role of cN-II

IMP preferring cytosolic 5 ′-nucleotidase II (cN-II) is a widespread enzyme whose amino acid sequence is highly conserved among vertebrates. Fluctuations of its activity have been reported in some pathological conditions and its mRNA levels have been proposed as a prognostic factor for poor outcome in patients with adult acute myeloid leukemia. As a member of the oxypurine cycle, cN-II is involved in the regulation of intracellular concentration of 5′-inosine monophosphate (IMP), 5′-guanosine monophosphate (GMP), and also 5-phosphoribose 1-pyrophosphate (PRPP) and is therefore involved in the regulation of purine and pyrimidine de novo and salvage synthesis. In addition, several studies demonstrated the involvement of cN-II in pro-drug metabolism. Notwithstanding some publications indicating that cN-II is essential for the survival of several cell types, its role in cell metabolism remains uncertain. To address this issue, we built two eucaryotic cellular models characterized by different cN-II expression levels: a constitutive cN-II knockdown in the astrocytoma cell line (ADF) by short hairpin RNA (shRNA) strategy and a cN-II expression in the diploid strain RS112 of Saccharomyces cerevisiae. Preliminary results suggest that cN-II is essential for cell viability, probably because it is directly involved in the regulation of nucleotide pools. These two experimental approaches could be very useful for the design of a personalized chemotherapy.     

Differences in biochemical properties of two 5′-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions

Deep-sea Shewanella violacea 5′-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions.

Abbreviations: NTase: 5′-nucleotidase; SANTase: Shewanella amazonensis 5′-nucleotidase; SVNTase: Shewanella violacea 5′-nucleotidase; CD: circular dichroism     

NTPDase and 5′-Nucleotidase Activities in Synaptosomes of Hippocampus and Serum of Rats Subjected to Homocysteine Administration

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.     

ATPase and Adpase activities in synovial membrane of equine metacarpophalangeal joint

ATPase and ADPase activities capable of hydrolyzing nucleoside di- and triphosphates in the presence of Ca2+ are present in synovial membrane of metacarpophalangeal joint mainly associated to membrane fractions. These hydrolytic activities have been considered involved in the inflammatory process where ATP and ADP are inflammatory mediators while adenosine counteracts this effect. Both, subcellular localization and kinetic properties of these nucleotidase activities, suggest that could correspond to single enzyme called ATP-diphosphohydrolase or apyrase. The comparison of the activity on ATP-Ca and ADP-Ca from normal and pathological equine synovial membrane did not show significant differences either in the subcellular fraction distribution or in the enrichment of each subcellular fraction. Neither differences on 5'-nucleotidase activity present in the microsomal fraction were observed.     

Changes in ectonucleotidase activities in rat Sertoli cells during sexual maturation

Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P₂X and P₂Y and PA₁. The signals to purinoceptors are usually terminated by the action of ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre pubertal, mid pubertal and young adult (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.     

Localisation and functional studies on the 5′ -nucleotidase of the cattle tick Boophilus microplus

The ecto-5-nucleotidase from the cattle tick Boophilus microplus is an unusual enzyme, hydrolysing a variety of nucleoside mono-, di- and triphosphates to release the free nucleoside. The gene has been sequenced and the recombinant protein expressed as a functional, active enzyme. Nevertheless, the function of the enzyme in the tick remains obscure. The enzyme is present throughout the life cycle, but in largest amounts in unfed larvae and adult ticks. The tissue location has been studied in adult female ticks by Western blotting, RT-PCR and immunofluorescence. All methods show the enzyme to be principally in the Malpighian tubules, though significant amounts are also present on the surface of ovaries and in detectible amounts in other tissues. This, together with the known specificity of the enzyme, suggests a role in purine salvage pathways. Sensitivity of ticks to allopurinol, an inhibitor of hypoxanthine-guanine-phosphoribosyltransferase, supports the importance of purine salvage in this tick and the potential role of nucleotidase in this pathway.