NPPase Activity Research Articles

Structure-activity relationship study of NPP1 inhibitors based on uracil-N1-(methoxy)ethyl-β-phosphate scaffold

Overexpression of ecto-nucleotide pyrophosphatase-1 (NPP1) is associated with diseases such as calcium pyrophosphate dihydrate deposition disease, calcific aortic valve disease, and type 2 diabetes. In this context, NPP1 inhibitors are potential drug candidates for the treatment of these diseases. The present study focuses on the analysis of the structure-activity relationship of NPP1 inhibitors based on acyclic uracil-nucleotides. For this purpose, we synthesized acyclic uridine-monophosphate analogs, 10-11, uridine-diphosphate analogs, 12–14, and uridine-Pα,α-dithio-triphosphate analogs, 15–17. We evaluated their inhibitory activity and selectivity towards NPP1, -3, NTPDase1, -2, -3, and -8, and P2Y2,4,6 receptors. Analogs 16 and 17 were the most selective and potent NPP1 inhibitors (Ki 0.94 and 0.73 μM, respectively) among the tested molecules. Analogs 10–17 had only minute effect on uracil-nucleotide responding P2Y2,4,6 receptors. Analog 17 (100 μM) displayed 96% inhibition of NPPase activity in osteoarthritic human chondrocytes. Analogs 14–17 displayed weak inhibitory effect on alkaline phosphatase activity at equimolar concentrations in human chondrocytes. All tested analogs showed no toxicity at human chondrocytes. We concluded that ribose-ring to chain transformation, as well as the type of the nucleobase, are parameters of minor significance to NPP1 inhibition, whereas the major parameter is Pα-dithio-substitution. In addition, the length of the phosphate chain also significantly affects inhibition. Overall, the experimental results were well reproduced by molecular docking. A correlation was observed between the activities of the compounds and the number of H-bonds and salt bridges formed between the inhibitors and NPP1 binding site residues. Uracil-N1-(methoxy)ethyl-β-Pα,α-dithio, Pβ,γ-methylene tri-phosphate, 17, was identified as the most potent, selective, and non-toxic NPP1 inhibitor among the tested analogs, and may be used as a lead structure for further drug development.

Identification of adenine-N9-(methoxy)ethyl-β-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes.

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate)dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the Pα,β-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-β-bisphosphonate, 10, was identified as an NPP1 inhibitor (Ki 16.3μM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60μM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (Ki 12.6μM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100μM.

Cytochemical localization of Na+/K+-ATPase activity in cochlear strial marginal cells after various catecholamine administrations.

Sodium/potassium-activated adenosine triphosphatase (Na+/K+-ATPase) activity in the kidney and brain is high, and is regulated by catecholamines. Na+/K+-ATPase activity is also high in the basolateral infoldings of the strial marginal cells, where it aids in maintaining the characteristic electrolyte composition of the endolymph. To clarify the involvement of humoral control in strial function, particularly the role of catecholamines, the K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of strial marginal cells was investigated in guinea pigs using a cerium-based cytochemical method. The effects of reserpine, serotonin (5-HT), norepinephrine (NE), epinephrine (EP), both alone and in combination, were studied. High doses of reserpine cause depletion of sympathetic substances. Strial K+-NPPase activity was decreased after reserpine or dopamine treatment, and was increased after 5-HT, NE, and EP treatment. After reserpinization, repeated treatment with 5-HT, NE, or EP led to detectable strial enzyme activity. Thus, exogenous 5-HT, NE, and EP were able to restore strial K+-NPPase activity in the reserpine-treated animals. These results suggested that biogenic amines regulate strial K+-NPPase activity. Thus, the function of the stria vascularis may be regulated by the opposing actions of these catecholamines, and 5-HT.

Characterization of a new plasma membrane-associated ecto-5′-phosphodiesterase/nucleotide-pyrophosphatase from rat hepatocarcinoma AS-30D cells

We have identified in plasma membrane fractions isolated from rat hepatocarcinoma AS-30D ascites cells three glycoproteins of 125 kDa, 115 kDa and 105 kDa (gp125, gp115 and gp105) which become adenylylated using ATP as substrate, most readily in the presence of EDTA. The gp115 becomes also phosphorylated. The adenylylation of these tumor glycoproteins was much lower than that of a group of analogous adenylylatable glycoproteins (gp130, gp120-gp110 dimer and gp100) present in normal rat liver plasma membrane. The tumor glycoproteins were reversibly O-adenylylated at threonine residues, as was the case for their normal rat liver counterparts. The tumor gp115, and the gp120-gp110 dimer from normal rat liver were both isolated using either ATP-affinity chromatography and/or AMP-affinity chromatography. The gp120-gp110 dimer from normal rat liver was identified as the plasma cell differentiation antigen-1 (PC-1 protein), an ecto-5' phosphodiesterase/nucleotide-pyrophosphatase (5'-PDE/NPPase). The gp115 from tumor cells also exhibited Zn2+ stimulated 5'-PDE and NPPase activities in alkaline conditions, although it appears to be distinct from the PC-1 protein. We have determined that the gp115 is an ecto-enzyme that catalyzes the hydrolysis of extracellular ATP, since its adenylylation and phosphorylation were detected in intact cells using extracellularly added [alpha-32P]ATP or [gamma-32P]ATP, respectively, in the absence of any permeabilizing agent.

In vitro dose dependent inverse effect of nantenine on synaptosomal membrane K+-p-NPPase activity.

The effect of nantenine, an aporphine alkaloid, on ATPase K+-dependent dephosphorylation was evaluated using p-nitrophenylphosphate (p-NPP) as substrate. Basal K+-p-NPPase activity was significantly increased with 3 x 10(-4) M, remained unchanged with 3 x 10(-6) M, 3 x 10(-5) M but was reduced with 7.5 x 10(-4) M and 1 x 10(-3) M nantenine, whereas Mg2+-p-NPPase activity was not modified. Kinetic studies showed that K+-p-NPPase inhibition by nantenine is competitive to KCl but non-competitive to substrate p-NPP, whereas K+-p-NPPase stimulation by nantenine is non-competitive to KCl but competitive to p-NPP. These data suggest that there may be two acceptor sites for nantenine in p-NPPase, one eliciting stimulation and the other inhibition of K+-dependent p-NPP hydrolysis. Considering the biphasic action of nantenine on seizures and the correlation between decreased ATPase activity and seizure development, alkaloid anticonvulsant effect observed at low nantenine doses is attributable to the stimulation of phosphatase activity whereas the convulsant effect at high alkaloid doses seems related to Na+, K+-ATPase inhibition.

Effects of Epinephrine on Ouabain-Sensitive, K+-DependentP-Nitrophenylphosphatase Activity in Strial Marginal Cells of Guinea Pigs

In strial marginal cells, Na+/K(+)-ATPase activity is abundant, and contributes to maintain the characteristic electrolyte composition of the cochlear endolymph. In the present study, to clarify the relationship between epinephrine and strial Na+/K(+)-ATPase activity, the ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of strial marginal cells was investigated with a cerium-based method in normal guinea pigs and guinea pigs treated with reserpine, epinephrine, and reserpine plus epinephrine. In our previous study, K(+)-NPPase activity had almost completely decreased 3 to 20 days after reserpine administration. In the present study, at 10 days after reserpinization and following repeated epinephrine treatment, enzyme activity was detectable. These results suggest that exogenous epinephrine was able to restore strial K(+)-NPPase activity in the reserpine-treated animals, and that epinephrine might increase strial Na+/K(+)-ATPase activity.

Gill morphology and ATPase activity in the goldfishcarassius carassiusvar.auratusexposed to experimental lead intoxication

Abstract The effect of acute lead intoxication (5 mg 1‐1) on the structure and function of the gills in Carassius carassius var. auratus was studied. The gills absorbed Pb very quickly and the storage increased progressively in the longest treated specimens. Light and electron microscopy observations on gill filaments from unexposed fish showed an organization similar to that described for other teleosteans. Gill histological and cytological alterations induced by lead were detected after 24 h of treatment and became more evident after 48 h. The salient changes were the following: 1) free parts of secondary lamellae increased in length and had irregular outlines; 2) an increased number of mucocytes, prevalently distributed over the superficial layer of the primary epithelium; 3) chloride and epithelial cell ultrastructure were particularly damaged; the degenerating chloride cells were more numerous; 4) K+‐NPPase activity was not particularly affected; on the contrary, Ca2+‐ATPase increased particularly in...

Extralysosomal localization of acid paranitrophenylphosphatase activity in the developing rat cerebellar cortex.

Using para-nitrophenyl phosphate as substrate, extralysosomal localization of acid phosphatase activity was investigated in the developing and adult rat cerebellar cortex, particularly in the Purkinje (P) cells. Light microscopic observations revealed that acid para-nitrophenylphosphatase (acid NPPase) activity appeared increasingly in the P cell and molecular layers throughout their postnatal development. Under an electron microscope, the acid NPPase activity was found in the cisterns of nuclear envelope and endoplasmic reticulum of the P cells, in addition to the lysosomal activity. In the synapses on the P cells, the acid NPPase activity was predominantly localized in the presynaptic membrane of such parallel fibers. The activity of nonlysosomal acid NPPase was inhibited by fluoride (0.1mM) and CuSO4 (1.0mM), but not by L(+)-tartrate (10mM). The present results are discussed in a possible relationship to an activity of phosphotyrosine protein phosphatase in the developing rat cerebellar cortex.

Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H(+)-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na(+)-K(+)-ATPase (Mayahara et al. 1980) and gastric H(+)-K(+)-ATPase (Fujimoto et al. 1986). K(+)-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na(+)-K(+)-ATPase and Ca2(+)-ATPase. The K(+)-independent NPPase activity was diminished by the inhibitors of H(+)-ATPase such as N-ethylmaleimide (NEM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H(+)-ATPase which plays a role in acidification.

Cytochemical localization of K+-NPPASE in the heminode of Ranvier in the rat optic nerve.

The localization of ouabain sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase), a component of the transport adenosine triphosphatase (Na+, K+-ATPase) system, was ultracytochemically investigated in the heminode of Ranvier in the rat optic nerve. K+-NPPase activities were noted on the nodal and paranodal axolemma, and on the paranodal myelin lateral loops. In the nodal region, the axolemma, which lies above the dense cytoplasmic undercoating, showed remarkably strong K+-NPPase activity.

Localization of K+-NPPase Activity in the Developing Tegmentum Vasculosum of the Chick Cochle

Localization of K+-NPPase Activity in the Developing Tegmentum Vasculosum of the Chick Cochle

Prefeeding of aldose reductase inhibitor and galactose cataractogenesis

Our recent investigations have shown that the Eisai compound, E-0722, (2R-4S-6-fluoro-1-2-methylspirochroman 4,4'-imidazolidine 2,5'-dione) is a more potent aldose reductase inhibitor than Sorbinil (D-6-fluorospirochroman 4,4'-imidazolidine 2,5'-dione). In the previous studies these aldose reductase inhibitors were added to the 50% galactose diet fed to rats to determine their effect on galactose-induced alterations in the lens and the development of cataract. In this report we present our results on the effect of prefeeding the aldose reductase inhibitor, E-0722, on the alterations in rat lens following subsequent feeding of galactose. For this study, young Sprague Dawley rats were prefed either rat chow or rat chow plus 50% galactose containing 1mg/day/Kg body weight of E-0722 for 1 or 2 weeks. After this dietary regimen, the animals were transferred to diets containing 50% galactose for different periods. For controls, rats were fed either rat chow or 50% galactose without the prefeeding of E-0722. Our results obtained through gross observation of the lenses, light microscopic studies of lens sections and assay of Na+-K+-ATPase (NPPase) activity show that the prefeeding of E-0722 prior to galactose feeding delays galactose-induced alterations and the development of mature cataract.

Elevated nucleotide pyrophosphatase activity in cultured skin fibroblasts from patients with Lowe's syndrome

In previous papers we reported that nucleotide pyrophosphatase (NPPase) activities are markedly elevated in skin fibroblasts from patients with Lowe's syndrome. To reveal the molecular basis for this elevation, we investigated to see whether or not the activities are proportional to the amounts of enzyme protein. Determination of the amounts of NPPase proteins by a radioimmunoassay using monoclonal antibodies revealed that the amounts of NPPase proteins were proportional to the enzyme activities. The NPPase proteins from patients' and normal cells were indistinguishable, after SDS-polyacrylamide gel electrophoresis, on Western blot analysis with staining with polyclonal antibodies raised against denatured NPPase. These data suggested that the elevated NPPase activities in fibroblasts in this disorder resulted from increased amounts of the enzyme proteins.

Ultracytochemical Study of Ouabain-sensitive, Potassium-dependent p-Nitrophenylphosphatase Activity in the Inner Ear of the Squirrel Monkey

The localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of the Na(+),K(+)-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K(+)-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K(+)-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.

Ultracytochemical Study of Ouabain-sensitive, Potassium-dependent p-Nitrophenylphosphatase Activity in the Inner Ear of the Squirrel Monkey

The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of the Na+, K+-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K+-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K+-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.

Cytochemical study of K+-p-nitrophenyl phosphatase (K+-NPPase) in planum semilunatum and dark cells of the squirrel monkey.

We examined the ultrastructure of the planum semilunatum, and also cytochemically demonstrated K+-dependent p-nitrophenyl phosphatase (K+-NPPase) activity in planum semilunatum and ampullary dark cells of the squirrel monkey. K+-NPPase activity was found in the basal part of the planum semilunatum. The reaction products were limited to the cytoplasmic side of the plasma membranes. A more intense reaction was found in the basal infoldings and the lateral interdigitations of the dark cells. On the other hand, the apical cell surface of both cell types showed no K+-NPPase activity. The present results show that both the planum semilunatum and the dark cells may both be involved in maintaining endolymph homeostasis.

Ultracytochemical studies of adenosine nucleotidases in aortic endothelial and smooth muscle cells - Ca++-ATPase and Na+, K+-ATPase.

Ca++-activated adenosine triphosphatase (Ca++-ATPase) and ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase), a component of Na+, K+-ATPase system, were studied in rat aortic endothelium and smooth muscle cell using the recent improved cytochemical methods introduced by Ando et al. (1981) and Mayahara et al. (1980) respectively.In the endothelium, Ca++-ATPase activity was localized on the external side of luminal, lateral and abluminal membrane, on the inside of caveolae and vesicles of both luminal and abluminal surfaces and on the matrix of mitochondria. Furthermore, Ca++-ATPase activity on only the luminal surface was affected by the concentrations of Ca++ in reaction medium. K+-NPPase activity was recognized on the cytoplasmic side of or exactly on the membrane covering abluminal caveolae, abluminal vesicles and Golgi apparatus. These findings indicate the existence of a cytochemical difference on the aortic endothelial cell-surface corresponding to the function.On the other hand, in the smooth muscle cell, reaction products indicating Ca++-ATPase activity were localized on the plasma membrane, the caveolae, the sarcoplasmic reticulum, the Golgi apparatus and the matrix of mitochondria. K+-NPPase activity was observed on the cytoplasmic side of or just on the caveolae and the Golgi apparatus in the smooth muscle cell. These findings confirm the previously reported biochemical studies and support the mechanism of the smooth muscle relaxation in relation to the sequestration or extrusion of Ca++ and the function of adenine nucleotides and nucleosides.

Redistribution of gastric K+-NPPase in vertebrate oxyntic cells in relation to hydrochloric acid secretion: a cytochemical study.

Gastric K+-NPPase represents a partial reaction of the (K+-H+)ATPase system, which is considered to be the proton pump in mammalian parietal cells. In the present paper, K+-NPPase activity was cytochemically studied by the method of Mayahara et al. (1980) in gastric glands of birds, amphibia, and mammals, either in the resting state induced by cimetidine or after stimulation of HCl secretion by histamine. The gastric K+-NPPase cytochemical reaction was localized only in oxyntic cells of the gastric mucosa in the three species tested. The subcellular distribution of the K+-NPPase reaction product drastically changes with the secretory state of HCl. In resting cells, the K+-NPPase staining is associated with the membranes of the endocellular tubular system while in HCl-secreting cells, it is associated with the plasma membrane of the elaborate secretory surface characteristic of this functional state. The above results demonstrate that the same enzymatic activity, which is associated with the gastric proton pump, is present in both membranous systems of the oxyntic cell secretory pole. This fact supports the proposal that the tubular system represents a membrane reserve that inserts the proton pump into the luminal plasma membrane in vertebrate oxyntic cells under the action of HCl secretagogues.

Two Ca2+-requiring p-nitrophenylphosphatase activities of the highly purified Ca2+-pumping adenosinetriphosphatase of human erythrocyte membranes, one requiring calmodulin and the other ATP.

The highly purified Ca2+-pumping ATPase from human erythrocyte membranes displays two p-nitrophenylphosphatase (NPPase) activities: one of these requires calmodulin and low concentrations of Ca2+, while the other requires ATP and higher Ca2+ concentrations. The free Ca2+ concentrations required for the expression of the two NPPase activities differed very substantially. Both activities required high free Mg2+ concentrations and displayed simple hyperbolic kinetics toward p-nitrophenyl phosphate (NPP) with a Km in the range of 5-20 mM. Study of the dependence of the calmodulin-stimulated NPPase on Mg2+ and NPP indicated that the Mg-NPP complex is not the substrate of the enzyme. Under conditions optimal for ATP-requiring NPPase (1 mM free Ca2+), the Ca2+-ATPase displayed simple hyperbolic kinetics with a low Km for ATP. NPP competitively inhibited this activity, and the apparent Ki for NPP was less than 1 mM, much lower than the Km for NPP as a substrate. If NPP were inhibiting the ATPase by binding at the same site at which NPP is hydrolyzed, the apparent Ki for NPP as inhibitor would be the same as the Km for NPP as substrate. (Under these circumstances, the apparent Ki and the Km can be directly compared, since NPP was being hydrolyzed under both circumstances.) Since Ki was much lower than Km, NPP must have been inhibiting at another site; thus, these data show the existence of two types of NPP sites on the enzyme, one at which NPP is hydrolyzed and the other at which it inhibits ATP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig.

Ca++-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity, which represents the second dephosphorylative step of the Na+-K+-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca++-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca++- and substrate-dependent and showed sensitivity to oligomycin. When Mg++-ions were used instead of Ca++-ions, a distinct reaction was also found on mitochondrial inner membranes. In contrast to the localization of the Ca++-ATPase activity, the K+-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K+ from the incubation medium.