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.     

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.     

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.     

Kinetics of the 5′-nucleotidase and the adenosine deaminase in subcellular fractions of rat brain

Suspensions of rat brain microsomes, synaptosomes, and synaptic vesicles were able to convert adenosine to inosine by means of adenosine deaminase. Isosbestic points of this transformation, at 222, 250 and 281 nm, remained unchanged with time-course. This fact suggests that adenosine deaminase (ADA, E.C. 3.5.4.4) is located on the surface of the vesicles whereas purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4) is located inside the vesicles. Kinetic parameters of the particulate 5-nucleotidase (5N, E.C. 3.1.3.5) and adenosine deaminase were analogous to those of the cytosolic enzymes. These results suggest that soluble and particulate enzymes represent different pools of the same molecular species.     

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.     

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.     

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.     

Localization of 5′-nucleotidase activity in the parotid acinar cells of a rat treated with isoproterenol

Summary Cytochemical localization of 5- nucleotidase (AMPase) has been investigated in the parotid acinar cells of rats at various stages of exocytic secretion induced by an administration of isoproterenol (IPR).In the resting stage, the acinar cells show AMPase activity located on the baso-lateral and luminal plasmalemma, and in the earliest secretory stage the luminal plasma membranes are devoid of the enzymatic activity. However, these particular regions exhibit AMPase activity during the advanced stages of secretion, and the AMPase positive membranes become absorbed into the cytoplasm by endocytic activity. The absorbed membrane components then seem to be degraded by the action of lysosomes.The intracellular fate of the endocytic vacuoles has been examined by the aid of ferritin particles introduced retrogradely through ductal lumina. Ferritin containing vacuoles are distributed in the cytoplasm, and these droplets change into secondary lysosomes. No tracer particles are recognized in the internal space of the Golgi lamella and its associated vesicles.The results suggested that in the exocytic secretion of parotid acinar cells, AMPase originating from plasma membrane intermingles with the membranes derived from secretion granules, and is translocated into cytoplasm by an endocytic mechanism. The internalized membrane components are, at least partly, degraded by lysosome action.     

Distribution of endogenous and exogenous 5′-nucleotidase on bovine spermatozoa

A polyclonal rabbit antibody against 5-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.     

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.     

Lymphocyte plasma membranes II. Cytochemical localization of 5′-nucleotidase in rat lymphocytes

Cytochemical studies of thymic and splenic lymphocytes from rats showed that 5′-nucleotidase was restricted to the plasma membranes. Isolated plasma membranes contained the highest specific activity of 5′-nucleotidase of any cellular fractions. The results indicate that this enzyme can be used as a plasma membrane marker for lymphocytes.     

Cytochemical investigations on the localization of 5′-nucleotidase in the rat hippocampus with special reference to synaptic regions

Summary The fine structural localization of the 5-nucleotidase was investigated in the CA₃ region of the rat hippocampus. The attention was focussed on the occurrence of the enzyme in the synaptic region. The 5-nucleotidase activity was demonstrated at the surface membranes of axons and dendrites. Prominent portions of enzyme activity were detectable in the nuclei and the nuclear envelope, whereas the cytoplasmic organelles were nearly devoid of reaction product. In synapses five types of 5-nucleotidase localization were revealed. A participation of the enzyme in the process of neurotransmission is discussed.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

5′-nucleotidase

Summary The distribution of 5-nucleotidase activity in rat liver shows a sexual dependence. In male liver the activity in the bile canalicular wall is most pronounced, whereas the activity at the sinusoidal border of the liver parenchymal cell is slightly more in the female rat. Castration and treatment with sex hormones change the distribution pattern. The greatest variations in enzyme activity are seen at the bile canalicular site of the liver cell. These changes are probably an expression of the altered functional state of the liver cell.     

Isolation and characterization of the ecto-5′-nucleotidase from a rat glioblastoma cell line

Summary 5-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5-AMP-Sepharose render the purified enzyme with a high specific activity (76.36 mol AMP-min^–1-mg^–1). The purified enzyme gives a single polypeptide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5-nucleotidase presents optimum activity at pH 7.8–8.1 either in the presence or in the absence of Me²⁺. A linear Arrhenius plot is observed in the 25–46° C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn²⁺, Mn²⁺, and Co²⁺. The hydrolysis of nucleosides 5-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins.     

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.     

A histochemical study of variations in the localization of 5′-nucleotidase activity in the acinar cell of the rat exocrine pancreas over the twenty-four hour period

The circadian variation of 5'-nucleotidase (AMPase) activity was studied in rat pancreatic exocrine cells. The localization of this enzyme, often associated with the plasmalemma, was studied by ultracytochemical methods at six time points over the 24-h period. The localization of AMPase activity exhibited a clear-cut circadian variation. During the light span strong activity was observed on the luminal plasmalemma, negative or weak activity on the baso-lateral plasmalemma and clearly visible activity on intracellular structures such as cytoplasmic vacuoles (fragmentation-like vesicles), dilated rims of the Golgi cisternae (or cisternal ends of the Golgi stacks), condensing vacuoles and lysosomal bodies. During the dark span the activity was detectable only on the baso-lateral plasmalemma. The fact that AMPase activity could not be found on the luminal plasmalemma during the dark span suggests that the luminal membranes may be replaced by the membranes of secretory granules, which do not display AMPase activity. The intracellular localization of AMPase activity during the light span, especially at 08.00 h, includes all cytoplasmic compartments which have hitherto been associated with the intracellular pathway for membrane retrieval from the plasmalemma. Moreover, the appearance of the activity in the dilated rims of the Golgi stack and condensing vacuoles indicates that these compartments may constitute a functional unit.     

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     

Immunolocalization of ecto-5′-nucleotidase in the kidney by a monoclonal antibody

Summary A monoclonal antibody IgG, has been raised against ecto-5-nucleotidase purified from rat kidney homogenate. The specificity of the antibody was verified by immunoprecipitation. The distribution of the corresponding antigen in the rat kidney was studied by immunocytochemistry (FITC and PAP technique) in 1 m thick cryostat sections. The antibody reacted with the brush border of proximal tubules, the apical cell membrane and the apical cytoplasm of intercalated cells in connecting tubules and collecting ducts and with interstitial cells of the cortex. Among the interstitial cells exclusively stellate shaped fibroblasts were reactive whereas rounded interstitial cells (type II interstitial cells) as well as pericytes and endothelial cells of peritubular capillaries were unreactive. Compared to the staining intensity of the fibroblasts in the cortical labyrinth the reactivity of the fibroblasts in the medullary rays of the cortex was weak or absent. Interstitial cells of the entire medulla were unreactive. Concerning the fibroblasts in the periarterial connective tissue, those surrounding the larger arteries (arcuate arteries, cortical radial arteries) were negative, those alongside afferent and efferent arterioles were positive. Endothelia of lymphatic capillaries travelling within the periarterial connective tissue were also positive. All components of the juxtaglomerular apparatus were negative.The findings are consistent with an interstitial production of adenosine, available extracellularly and thus being able to reach the major target sites of adenosine, the smooth muscles of glomerular arterioles, including the granular cells at the glomerular vascular pole.     

Absence of 5′-nucleotidase in porcine polymorphonuclear leucocyte membranes

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess $\text{p-}\text{nitrophenyl}$ phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5′-nucleotidase inhibitor α,β-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5′-nucleotidase. An antibody against mouse liver 5′-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5′-nucleotidase and the necessity for distinguishing between true 5′-nucleotidase and non-specific phosphatase activity is discussed.