scholarly journals Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39

Marine Drugs ◽  
2021 ◽  
Vol 19 (2) ◽  
pp. 51
Author(s):  
Vittoria Lopez ◽  
Laura Schäkel ◽  
H. J. Maximilian Schuh ◽  
Michael S. Schmidt ◽  
Salahuddin Mirza ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Extracellular Atp ◽  
Sulfated Polysaccharides ◽  
Nucleoside Triphosphate ◽  
Nucleotide Receptors ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Mechanism Of Inhibition ◽  
Immunotherapy Of Cancer ◽  
Dual Inhibitors

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

scholarly journals ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

2019 ◽  
Vol 476 (24) ◽  
pp. 3737-3750 ◽  
Author(s):  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Dependent Manner ◽  
Deficient Mutant ◽  
Concentration Dependent Manner ◽  
Glycoprotein P ◽  
Binding Domains ◽  
P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

scholarly journals Effect of ATP on preadipocyte migration and adipocyte differentiation by activating P2Y receptors in 3T3-L1 cells

2005 ◽  
Vol 393 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Mariko Omatsu-Kanbe ◽  
Kazuko Inoue ◽  
Yusuke Fujii ◽  
Takefumi Yamamoto ◽  
Takahiro Isono ◽  
...  
Keyword(s):  
Cell Line ◽  
Adipocyte Differentiation ◽  
P2y Receptors ◽  
Extracellular Atp ◽  
Dependent Manner ◽  
Fat Cell ◽  
Proliferating Cells ◽  
Concentration Dependent Manner ◽  
Membrane Ruffling

The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1–100 μM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density <50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.

Effects of extracellular ATP on ICa, [Ca2+]i, and contraction in isolated ferret ventricular myocytes

1993 ◽  
Vol 264 (3) ◽  
pp. C702-C708 ◽  
Author(s):  
Y. Qu ◽  
H. M. Himmel ◽  
D. L. Campbell ◽  
H. C. Strauss
Keyword(s):  
Action Potential ◽  
Ventricular Myocytes ◽  
Ventricular Myocardium ◽  
Inhibitory Effect ◽  
Extracellular Atp ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
The Right

The effects of extracellular ATP on the voltage-activated "L-type" Ca current (ICa), action potential, resting and transient intracellular Ca2+ levels, and cell contraction were examined in enzymatically isolated myocytes from the right ventricles of ferrets. With the use of the whole cell patch-clamp technique, extracellular ATP (10(-7) to 10(-3) M) inhibited ICa in a time- and concentration-dependent manner. ATP decreased the peak amplitude of ICa without altering the residual current at the end of 500-ms clamp steps. The concentration-response relationship for ATP inhibition of ICa was well described by a conventional Michaelis-Menten relationship with a half-maximal inhibitory concentration of 1 microM and a maximal effect of 50%. Consistent with its inhibitory effect on ICa, ATP hyperpolarized the plateau phase and shortened the action potential duration. In fura-2-loaded myocytes, extracellular ATP did not change the resting myoplasmic Ca2+ levels; however, when current was elicited under voltage-clamp conditions, ATP both decreased the myoplasmic intracellular Ca2+ transient and inhibited the degree of cell shortening. Our results suggest that ATP could be a genuine and potent extracellular modulator of cardiac function in ferret ventricular myocardium.

Low concentrations of A23187 increase calcium uptake by cardiac sarcoplasmic reticulum

1985 ◽  
Vol 249 (6) ◽  
pp. H1211-H1215
Author(s):  
J. J. Murray ◽  
A. V. Kuzmin ◽  
P. W. Reed ◽  
D. O. Levitsky
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Uptake ◽  
Atp Hydrolysis ◽  
Ionophore A23187 ◽  
Uptake System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Calcium Dependent ◽  
Uptake Measurement

The divalent cation ionophore A23187 at a concentration of 1 nM produced an increased rate of oxalate-supported calcium uptake by isolated cardiac sarcoplasmic reticulum as determined by absorbance changes of the calcium-sensitive dye murexide. Addition of a higher concentration of A23187 (0.1 microM) produced a decreased rate of calcium uptake. Measurement of the time during which ATPase was activated by calcium addition also suggested an increased rate of calcium uptake in the presence of 1 nM A23187 and an inhibition of calcium uptake at a higher concentration of the ionophore (0.1 microM). Ca2+-stimulated ATPase activity and incorporation of 32Pi from [gamma-32P]ATP into sarcoplasmic reticular proteins were increased by A23187 at concentrations of 1 nM or greater. An increased coupling of calcium uptake to ATP hydrolysis was observed at 1 nM A23187, while concentrations of the ionophore greater than or equal to 10 nM produced a decreased coupling. Addition of an inhibitor of cyclic AMP-dependent protein kinase decreased the rate of calcium uptake, and this inhibition was reversed in a concentration-dependent manner by 0.01–1 nM A23187. These data suggest that A23187 can activate a mechanism involving the calcium-dependent phosphorylation of protein that may regulate the activity of the calcium uptake system of the sarcoplasmic reticulum. These observations appear to provide an explanation for some of the contractile effects of A23187 in intact cardiac muscle that suggest that treatment with the ionophore results in an increased sequestration of calcium from the cytoplasm.

scholarly journals Extracellular ATP inhibits transport in medullary thick ascending limbs: role of P2X receptors

2009 ◽  
Vol 297 (5) ◽  
pp. F1168-F1173 ◽  
Author(s):  
Guillermo B. Silva ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Receptor Antagonist ◽  
P2x Receptors ◽  
Extracellular Atp ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Nitric Oxide Synthase Inhibitor

Absorption of NaCl by the thick ascending limb (TAL) involves active transport and therefore depends on oxidative phosphorylation. Extracellular ATP has pleiotropic effects, including both stimulation and inhibition of transport and inhibition of oxidative phosphorylation. However, it is unclear whether ATP alters TAL transport and how this occurs. We hypothesized that ATP inhibits TAL Na absorption by reducing Na entry. We measured oxygen consumption in TAL suspensions. ATP reduced oxygen consumption in a concentration-dependent manner. The purinergic (P2) receptor antagonist suramin (300 μM) blocked the effect of ATP on TAL oxygen consumption (147 ± 15 vs. 146 ± 16 nmol O2·min−1·mg protein−1). In contrast, the adenosine receptor antagonist theophylline did not block the effect of ATP on oxygen consumption. When Na-K-2Cl cotransport and Na/H exchange were blocked with furosemide (100 μM) plus dimethyl amiloride (100 μM), ATP did not inhibit TAL oxygen consumption (from 78 ± 13 to 98 ± 5 nmol O2·min−1·mg protein−1). The Na ionophore nystatin (200 U/ml) increased TAL oxygen consumption to a similar extent in both ATP- and vehicle-treated samples (368 ± 41 vs. 397 ± 47 nmol O2·min−1·mg protein−1). The nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (3 mM) blocked the ATP effects on TAL oxygen consumption (157 ± 10 vs. 165 ± 15 nmol O2·min−1·mg protein−1). The P2X-selective receptor antagonist NF023 blocked the effect of ATP on oxygen consumption, whereas the P2X-selective agonist β-γ-Me-ATP reduced oxygen consumption in a concentration-dependent manner. We conclude that ATP inhibits Na transport-related oxygen consumption in TALs by reducing Na entry and P2X receptors and nitric oxide mediate this effect.

scholarly journals Carnosine Decreases PMA-Induced Oxidative Stress and Inflammation in Murine Macrophages

Antioxidants ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 281 ◽  
Author(s):  
Giuseppe Caruso ◽  
Claudia G. Fresta ◽  
Annamaria Fidilio ◽  
Fergal O’Donnell ◽  
Nicolò Musso ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Defense Mechanisms ◽  
Energy State ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Inhibitory Effect ◽  
Nucleoside Triphosphate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Beneficial Effects

Carnosine is an endogenous dipeptide composed of β-alanine and L-histidine. This naturally occurring molecule is present at high concentrations in several mammalian excitable tissues such as muscles and brain, while it can be found at low concentrations in a few invertebrates. Carnosine has been shown to be involved in different cellular defense mechanisms including the inhibition of protein cross-linking, reactive oxygen and nitrogen species detoxification as well as the counteraction of inflammation. As a part of the immune response, macrophages are the primary cell type that is activated. These cells play a crucial role in many diseases associated with oxidative stress and inflammation, including atherosclerosis, diabetes, and neurodegenerative diseases. In the present study, carnosine was first tested for its ability to counteract oxidative stress. In our experimental model, represented by RAW 264.7 macrophages challenged with phorbol 12-myristate 13-acetate (PMA) and superoxide dismutase (SOD) inhibitors, carnosine was able to decrease the intracellular concentration of superoxide anions (O2−•) as well as the expression of Nox1 and Nox2 enzyme genes. This carnosine antioxidant activity was accompanied by the attenuation of the PMA-induced Akt phosphorylation, the down-regulation of TNF-α and IL-6 mRNAs, and the up-regulation of the expression of the anti-inflammatory mediators IL-4, IL-10, and TGF-β1. Additionally, when carnosine was used at the highest dose (20 mM), there was a generalized amelioration of the macrophage energy state, evaluated through the increase both in the total nucleoside triphosphate concentrations and the sum of the pool of intracellular nicotinic coenzymes. Finally, carnosine was able to decrease the oxidized (NADP+)/reduced (NADPH) ratio of nicotinamide adenine dinucleotide phosphate in a concentration dependent manner, indicating a strong inhibitory effect of this molecule towards the main source of reactive oxygen species in macrophages. Our data suggest a multimodal mechanism of action of carnosine underlying its beneficial effects on macrophage cells under oxidative stress and inflammation conditions.

scholarly journals Purinergic signaling in the pancreas and the therapeutic potential of ecto-nucleotidases in diabetes.

2014 ◽  
Vol 61 (4) ◽  
Author(s):  
Marek Cieślak ◽  
Katarzyna Roszek
Keyword(s):  
Therapeutic Potential ◽  
Current Knowledge ◽  
Purinergic Signaling ◽  
Receptor Activation ◽  
Extracellular Nucleotides ◽  
P2x Receptor ◽  
Nucleotide Receptors ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Adenosine Concentration

It is widely accepted that purinergic signaling is involved in the regulation of functions of all known tissues and organs. Extracellular purines activate two classes of receptors, P1-adenosine receptors and P2-nucleotide receptors, in a concentration-dependent manner. Ecto-enzymes metabolizing nucleotides outside the cell are involved in the termination of the nucleotide signaling pathway through the release of ligands from their receptors. The pancreas is a central organ in nutrient and energy homeostasis with endocrine, exocrine and immunoreactive functions. The disturbances in cellular metabolism in diabetes mellitus lead also to changes in concentrations of intra- and extracellular nucleotides. Purinergic receptors P1 and P2 are present on the pancreatic islet cells as well as on hepatocytes, adipocytes, pancreatic blood vessels and nerves. The ATP-dependent P2X receptor activation on pancreatic β-cells results in a positive autocrine signal and subsequent insulin secretion. Ecto-NTPDases play the key role in regulation of extracellular ATP concentration. These enzymes, in cooperation with 5'-nucleotidase can significantly increase ecto-adenosine concentration. It has been demonstrated that adenosine, through activation of P1 receptors present on adipocytes and pancreatic islets cells, inhibits the release of insulin. Even though we know for 50 years about the regulatory role of nucleotides in the secretion of insulin, an integrated understanding of the involvement of purinergic signaling in pancreas function is still required. This comprehensive review presents our current knowledge about purinergic signaling in physiology and pathology of the pancreas as well as its potential therapeutic relevance in diabetes.

scholarly journals The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase.

1987 ◽  
Vol 105 (1) ◽  
pp. 313-324 ◽  
Author(s):  
K A Conzelman ◽  
M S Mooseker
Keyword(s):  
Enzymatic Activity ◽  
Nucleoside Triphosphate ◽  
Dependent Manner ◽  
Binding Interaction ◽  
Ph Optimum ◽  
Concentration Dependent Manner ◽  
Nitrophenyl Phosphate ◽  
Ph Range ◽  
Mgatpase Activity ◽  
Actin Concentration

The microvillus 110-kD protein-calmodulin complex (designated 110K-CM) shares several properties with all myosins. In addition to its well-defined ATP-dependent binding interaction with F-actin, 110K-CM is an ATPase with diagnostically myosin-like divalent cation sensitivity. It exhibits maximum enzymatic activity in the presence of K+ and EDTA (0.24 mumol P1/mg per min) or in the presence of Ca++ (0.40 mumol P1/mg per min) and significantly less activity in physiological ionic conditions of salt and Mg++ (0.04 mumol P1/mg per min). This MgATPase is activated by F-actin in an actin concentration-dependent manner (up to 2.5-3.5-fold). The specific MgATPase activity of 110K-CM is also enhanced by the addition of 5-10 microM Ca++, but in the isolated complex, there is often also a decrease in the extent of actin activation in this range of free Ca++. Actin activation is maintained, however, in samples with exogenously added calmodulin; under these conditions, there is an approximately sevenfold stimulation of 110K-CM's enzymatic activity in the presence of 5-10 microM Ca++ and actin. 110K-CM is relatively indiscriminant in its nucleoside triphosphate specificity; in addition to ATP, GTP, CTP, UTP, and ITP are all hydrolyzed by the complex in the presence of either Mg++ or Ca++. Neither AMP nor the phosphatase substrate p-nitrophenyl phosphate are substrates for the enzymatic activity. The pH optimum for CaATPase activity is 6.0-7.5; maximum actin activation of MgATPase occurs over a broad pH range of 6.5-8.5. Finally, like myosins, purified 110K-CM crosslinks actin filaments into loosely ordered aggregates in the absence of ATP. Collectively these data support the proposal of Collins and Borysenko (1984, J. Biol. Chem., 259:14128-14135) that the 110K-CM complex is functionally analogous to the mechanoenzyme myosin.

A tryptophan residue involved in the inhibition of plant vacuolar H+-ATPase by 2-hydroxy-5-nitrobenzyl bromide

1998 ◽  
Vol 25 (6) ◽  
pp. 679
Author(s):  
Soong Yu Kuo ◽  
May Whei Lin ◽  
Shih Sheng Jiang ◽  
Shu Hsien Hung ◽  
Chi Meng Tzeng ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Conformational Change ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Order Rate Constant ◽  
Tryptophan Residue ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Significant Conformational Change ◽  
State Dissociation

Treatment of the vacuolar H+ -ATPase from mung bean seedlings (Vigna radiata L.) with the tryptophan modifying agent 2-hydroxyl-5-nitrobenzyl bromide (HNBB), caused a progressive decline of the ATP hydrolysis activity and proton translocation in a time- and concentration-dependent manner. Dithiothreitol could not restore the inhibition of H+ -ATPase by HNBB, indicating possible involvement tryptophan, and not cysteine residues. Protection studies suggested that modified sites might not locate in the active domain. Kinetic analysis shows that Vmax but not Km of H+ -ATPase was changed by HNBB. The reaction order of inactivation by HNBB was calculated as 0.98, implying that at least one tryptophan was labelled. The steady-state dissociation constant (Ki) and the pesudo-first-order rate constant of inhibition (k2) were determined as 1.61 mM and 0.22 min-1, respectively. Furthermore, stoichiometry experiments indicated that 8 mol tryptophan/mol ATPase were modified, when the enzyme activity was completely inhibited. However, a Tsou analysis showed that only one out of these modified tryptophans was crucial to the enzymatic activity. In addition, modification of the vacuolar H+ -ATPase by HNBB led to a decrease in intrinsic fluorescence, suggesting a possible conformational change of the enzyme. Taken together, our data indicate that the tryptophan residue is indispensable to vacuolar H+ -ATPase and the modification of this residue may induce a significant conformational change, consequently resulting in the loss of enzymatic activity.

scholarly journals Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin

2011 ◽  
Vol 301 (3) ◽  
pp. C619-C629 ◽  
Author(s):  
John J. Enyeart ◽  
Haiyan Liu ◽  
Judith A. Enyeart
Keyword(s):  
Atp Hydrolysis ◽  
Water Soluble ◽  
Zona Fasciculata ◽  
Dependent Manner ◽  
Ang Ii ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Whole Cell ◽  
Dependent Inhibition

Bovine adrenocortical cells express bTREK-1 K+ (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca2+ and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca2+-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca2+ ionophore ionomycin (1 μM) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5–10 μM) produced a sustained inhibition of bTREK-1 that was reversible upon washing, even in the absence of hydrolyzable [ATP]i. BAPTA was much more effective than EGTA at suppressing bTREK-1 inhibition by ANG II. When intracellular Ca2+ concentration ([Ca2+]i) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 ± 4.5% ( n=11) and 59.3 ± 8.4% ( n=4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP2) analog DiC8PI(4,5)P2 in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability ( Po) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca2+ (10–35 μM) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K+ channels by a Ca2+-dependent mechanism that does not require the depletion of membrane-associated PIP2. They further indicate that the Ca2+ source is located in close proximity within a “Ca2+ nanodomain” of bTREK-1 channels, where [Ca2+]i may reach concentrations of >10 μM. bTREK-1 is the first two-pore K+ channel shown to be inhibited by Ca2+ through activation of a G protein-coupled receptor.

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