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.     

The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ecto-enzymes ecto-5′-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamole-sensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and α-smooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5′-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by high-performance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5′-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5′-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5′-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.     

Insulin induces changes in the subcellular distribution of actin and 5′-nucleotidase

The present study utilized a cultured adult myocardial cell model to examine the arachidonic acid metabolism under different cell-damaging and normoxic conditions. Cell injury was caused by short-time hypoxia, calcium ionophore A 23187-triggered cell-damage under hypoxia and cell disruption by freezing and thawing. The current study demonstrates that under the cell-damaging conditions cultured adult heart myocytes resemble myocardial cells under normoxic conditions in metabolizing arachidonic acid into triacylglycerols and phospholipids as the major route (a), in formation of ETYA-inhibitable indomethacin-resistant lipid metabolites in minor amounts (b) and in being independent of calcium overload in the metabolic pathways of arachidonic acid metabolism (c). The ETYA-inhibitable components were resolved by HPLC. There was no evidence in formation of lipoxygenase products. The results were supported by negative hybridisation experiments of the total mRNA isolated from adult myocardial cells with a cDNA probe of a red-cell-specific lipoxygenase mRNA. We conclude from these observations that cell injury does not result in expression of lipoxygenase activities in heart myocytes.Abbreviations HETE Hydroxyeicosatetraenoic acid - DiHETE Dihydroxyeicosatetraenoic acid - ETYA 5.8.11.14-Eicosatetraynoic acid - TLC Thin-Layer Chromatography - NP-HPLC Normal Phase-High Performance Liquid Chromatography - RBC Red Blood Cell - LOX Lipoxygenase     

5′-Nucleotidase and adenosine deaminase in developing fetal guinea pig brain and the effect of maternal hypoxia

The activity of key enzymes of adenosine metabolism was studied in the developing fetal guinea pig brain. The activities of 5-nucleotidase and adenosine deaminase were determined in the brains of fetal guinea pigs at 30, 35, 40, 45, 50, 55, and 60 days of gestation. The level of 5-nucleotidase activity was extremely low at 30 and 35 days of gestation but increased rapidly during the 40 to 60 day period. The enzyme activity increased in the presence of Mg²⁺ with the Mg²⁺-dependent activation increasing with the age of gestation. This Mg²⁺-dependent activity was primarily associated with the membrane fraction. Prenatal hypoxia significantly increased the fetal brain M²⁺-independent 5-nucleotidase activity at 45 days of gestational age and beyond. Prior to this age, no effect was evident. Furthermore, following hypoxia, the Mg²⁺-dependent activation of 5-nucleotidase activity was lost. The activity of adenosine deaminase was present at 30 days of gestation and, unlike 5-nucleotidase, it remained at the same level until 60 days. The results indicate that the term fetal guinea pig brain has the enzymatic mechanisms of adenosine metabolism and thus the potential for adenosine-mediated regulation of cerebrovasculature during hypoxia.     

Distribution of 5′-nucleotidase in the renal interstitium of the rat

Summary The hydrolysis of 5-AMP by 5-nucleotidase is the main source of adenosine. In various tissues adenosine is a local mediator adjusting the organ work to the available energy. In the kidney it regulates renal hemodynamics, glomerular filtration rate and renin release via specific receptors of the arteriolar walls. By immunocytochemistry we identified interstitial and tubular sites of 5-nucleotidase in the rat kidney. In the interstitium the enzyme was detected only in the cortical labyrinth, the compartment that comprises all arteriolar vessels besides other putative targets of adenosine. The 5-nucleotidase-positive cells of the interstitium were identified as fibroblasts. The fibroblasts are in close contact with the tubules as well as with the vessels. Thus, any 5-AMP released by the tubules into the interstitial space would be converted to adenosine in the direct vicinity of its assumed targets. Adenosine produced by tubular cells would hardly have access to its known targets, since 5-nucleotidase is restricted to the luminal cell surface. Pathological events affecting the fibroblasts might influence renal function by modifying the interstitial adenosine production.     

Fibroblast growth factor 2 upregulates ecto-5′-nucleotidase and adenosine deaminase via MAPK pathways in cultured rat spinal cord astrocytes

Purinergic Signalling - Adenosine triphosphate (ATP) and adenosine are neurotransmitters and neuromodulators in the central nervous system. Astrocytes regulate extracellular concentration of...     

Properties of 5′-nucleotidase in rat heart sarcolemma

The activity of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5′-nucleotidase had a high affinity for AMP (K_m 35 μM), and ATP was a potent competitive inhibitor. In contrast, the 5′-nucleotidase displayed by fraction S showed a low substrate affinity (K_m 130 μM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5′-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5′-nucleotidase at 200 μM relative to 50 μM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5′-nucleotidase activity in both membrane preparations at a concentration of 2 μM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5′-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5′-nucleotidase are present at the intra and extracellular surface of the rat heart sarcolemma.     

Light and electron microscopical immunocytochemistry of 5′-nucleotidase in rat cerebellum

Summary 5-Nucleotidase in nervous tissue has so far not been localised at the ultrastructural level using immunocytochemical techniques. We have now applied monoclonal antibodies and a polyclonal antiserum raised against this ecto-enzyme and describe the distribution of 5-nucleotidase antigenicity in rat cerebellum both at the light and electron microscopic levels. Within all cerebellar layers, 5-nucleotidase immunoreactivity was found on plasma membranes of glial elements, i.e. Bergmann glial cell processes crossing the molecular layer, astrocytic end-feet around blood vessels and glial cell extensions surrounding single Purkinje cells. In the granular layer, 5-nucleotidase immunoreactivity was present on glial membranes interposed between granule cells. Neuronal cells or processes were devoid of immunoreactivity. The immunocytochemical results were compared with conventional 5-nucleotidase histochemistry. Both techniques showed the same ecto-localisation of the enzyme and favour the view of 5-nucleotidase being predominantly situated at glial plasma membranes.     

Distribution of ecto-5′-nucleotidase in the rat liver: effect of anaemia

In the kidney a striking parallel exists between the expression of ecto-5-nucleotidase and of erythropoietin by renal fibroblasts. It was therefore hypothesized that the expression of ecto-5-nucleotidase in fibroblasts might be controlled by oxygen tension. In order to test this hypothesis, we examined the distribution of the enzyme in a tissue which displays a defined zonation in respect to oxygen tension, namely in the liver; anaemia was used in order to exaggerate this zonation. The distribution of ecto-5-nucleotidase was investigated by light and electron microscopy using enzyme and immunohistochemical methods in the livers of healthy and of anaemic rats. Anaemia was produced by haemolysis combined with X-ray irradiation. The enzyme was detected in the bile canaliculi, in the connective tissue of the portal triads and of the central veins, and in fat-storing cells probably corresponding to a special form of fibroblasts. In healthy animals the perisinusoidal ecto-5-nucleotidase activity was slightly higher in the pericentral than in the periportal area of the acinus whereas the inverse was observed for the staining of bile canaliculi. Anaemia provoked an increase of ecto-5-nucleotidase in fat-storing cells in the pericentral zone of the acinus and in fibroblasts around the central veins, resulting in steepended gradients along the sinusoids. The intralobular gradient of ecto-5-nucleotidase in perisinusoidal cells and the effect thereon of anaemia suggest that the expression of the ecto-5-nucleotidase might be directly or indirectly controlled by local oxygen tension.     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

Isolation and characterization of bovine brain myelin distribution of 5′-nucleotidase

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2,3-cyclic nucleotide 3-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH: cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.     

Electron microscopic localization of 5′-nucleotidase in rat salivary glands

Summary The localization of 5-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5-nucleotidase activity and compare it with the traditional lead-ion method.     

Lipid peroxidation as the mechanism of modification of brain 5′-nucleotidase activity in vitro

The effect of lipid peroxidation on the Mg²⁺-independent and Mg²⁺-dependent activity of brain cell membrane 5-nucleotidase was determined and the affinity of the active sites of Mg²⁺-dependent enzyme for 5-AMP (substrate) and Mg²⁺ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 M ascorbate and 25 M FeCl₂ (resultant) for 10 min. The activity of 5-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg²⁺-independent 5-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 mol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg²⁺, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg²⁺-dependent 5-nucleotidase for 5-AMP tripled, as indicated by a significant decrease inK _m (K _m=95±2 M AMP for control;K _m=32±2 MAMP for peroxidized).V _max was significantly reduced from 3.35±0.16 in control to 1.70±.09 moles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg²⁺ significantly increased (K _m=6.17±0.37 mM Mg²⁺ for control;K _m=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg²⁺-dependent 5-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5-nucleotidase activity and the loss of Mg²⁺-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5-nucleotidase during hypoxia.     

Ecto-5′-nucleotidase is expressed by pericytes and fibroblasts in the rat heart

Ecto-5-nucleotidase is anchored at the outer surface of cell membranes and thus its reaction product adenosine is released into the extracellular space. Extracellular adenosine displays via specific receptors a wide range of physiological effects in heart. There are discrepancies in the literature concerning the distribution of ecto-5-nucleotidase in heart. Since we suspected that these may be due to technical problems, in the present study on ecto-5-nucleotidase in rat heart we attempted to circumvent some technical pitfalls. Good preservation of the tissue with open capillary lumina, providing a clear identification of endothelium, was obtained by perfusion fixation. At the light microscopic level, the distribution of ecto-5-nucleotidase studied by enzyme histochemistry and immunohistochemistry using a monoclonal and a polyclonal antibody yielded congruent results. The enzyme was rather homogeneously distributed throughout the myocardium, with a slightly higher incidence of stained cells in the outer thirds than in the inner third of the wall. Consistently high levels of ecto-5-nucleotidase were seen only in interstitial cells. The walls of large vessels and heart muscle cells were constantly negative for ecto-5-nucleotidase. The endothelia of capillaries were mostly negative but a few profiles occasionally displayed a weak immunoreaction. The interstitial cells staining positive for ecto-5-nucleotidase could be identified as pericytes and as fibroblasts according to their shapes and localizations. The immunoreactivity of fibroblasts was confirmed by electron microscopy. These data indicate that adenosine may be formed extracellularly in the interstitium of the myocardium, where it would have direct access to important targets such as myocytes, arterioles and nerve endings.     

Diurnal variation in 5′-nucleotidase activity in rat liver

Summary The diurnal variation of 5-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Occurrence of adenosine 2′,5′-bisphosphate in rat liver

Adenosine 2′,5′-bisphosphate (pAp) is present in liver from 2-day-fasted rats, at a concentration of around 1 μM. pAp was obtained through perchloric acid extraction of the liver followed by two successive DEAE-cellulose chromatographies and an ion-pair high-pressure liquid chromatography. Both pAp extracted from liver and that obtained from a commercial source showed the same pattern of hydrolysis by alkaline phosphatase, i.e., more 5′-AMP than 2′-AMP was obtained as an intermediate of the reaction.     

Purification and some properties of cytosol 5′-nucleotidase from rat liver

A 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) was highly purified from rat liver. The preparation appeared homogeneous on the criteria of disc-gel electrophoresis.A pH optimum at about 6.5 was observed for all substrates tested. The activity of this enzyme was absolutely dependent on the presence of various bivalent metal salts. The highest V value was attained with MgCl₂ and the concentration at half-enzyme saturation was lowest with MnCl₂. The enzyme had markedly higher affinities for IMP, dIMP, GMP and dGMP than the other 5′-mononucleotides, although V values for all the substrates tested were in the same order of magnitude.The activity of this enzyme was stimulated by various alkali metal salts, some carboxylic acids and adenine nucleotides. When AMP was used as substrate, the substrate-velocity plot was sigmoidal and NaCl, Tris-maleate and ATP stimulated the enzyme by decreasing the sigmoidicity of the plot. When IMP was used as substrate, the substrate-velocity plot was hyperbolic and these three activators stimulated the enzyme by increasing the V and decreasing the K_m value.Some of these results provided consistent evidence for the identity of this enzyme and the cytosol 5′-nucleotidase, the presence of which had been reported in crude preparations from rat liver.     

Effect of ecto-5′-nucleotidase (eN) in astrocytes on adenosine and inosine formation

ATP is a gliotransmitter released from astrocytes. Extracellularly, ATP is metabolized by a series of enzymes, including ecto-5′-nucleotidase (eN; also known as CD73) which is encoded by the gene 5NTE and functions to form adenosine (ADO) from adenosine monophosphate (AMP). Under ischemic conditions, ADO levels in brain increase up to 100-fold. We used astrocytes cultured from 5NTE^+/+ or 5NTE^−/− mice to evaluate the role of eN expressed by astrocytes in the production of ADO and inosine (INO) in response to glucose deprivation (GD) or oxygen-glucose deprivation (OGD). We also used co-cultures of these astrocytes with wild-type neurons to evaluate the role of eN expressed by astrocytes in the production of ADO and INO in response to GD, OGD, or N-methyl-d-aspartate (NMDA) treatment. As expected, astrocytes from 5NTE^+/+ mice produced adenosine from AMP; the eN inhibitor α,β-methylene ADP (AOPCP) decreased ADO formation. In contrast, little ADO was formed by astrocytes from 5NTE^−/− mice and AOPCP had no significant effect. GD and OGD treatment of 5NTE^+/+ astrocytes and 5NTE^+/+ astrocyte-neuron co-cultures produced extracellular ADO levels that were inhibited by AOPCP. In contrast, these conditions did not evoke ADO production in cultures containing 5NTE^−/− astrocytes. NMDA treatment produced similar increases in ADO in both 5NTE^+/+ and 5NTE^−/− astrocyte-neuron co-cultures; dipyridamole (DPR) but not AOPCP inhibited ADO production. These results indicate that eN is prominent in the formation of ADO from astrocytes but in astrocyte-neuron co-cultures, other enzymes or pathways contribute to rising ADO levels in ischemia-like conditions.