Inhibitory Effect Of ATP Research Articles

MEDIATORS OF THE SYMPATHETIC NERVOUS SYSTEM ATP AND NORADRENALINE IN THE MODULATION OF ANTIBODY FORMATION DURING DEEP COOLING OF THE ORGANISM

In experiments on rats, the participation of co-mediators of the sympathetic nervous system in the suppressive effect of deep cooling on the antibody-forming function of the spleen was studied. Studied: 1) the effect of deep cooling (decrease in deep temperature by 3–4°С), 2) the introduction of the mediator of the sympathetic nervous system norepinephrine (NE, 1 mg/ml), 3) its co-transmitter ATP (0.01 mg/ml and 10 mg/ml) and 4) P2X-purinergic receptor blocker PPADS on the number of antibody-forming cells of the spleen in response to immunization with sheep erythrocytes. Deep cooling as well as ATP inhibited, while NE stimulated antibody formation in the spleen. Blockade of P2X-purinergic receptors by PPADS stimulated antibody formation in the norm. Against the background of blockade of P2X-purinergic receptors, the inhibitory effect of ATP and deep cooling on antibody formation was not manifested. The results obtained indicate the opposite effects of co-mediators of the sympathetic nervous system on antibody formation in the spleen and suggest that the inhibitory effect of cold on antibody formation in the spleen occurs with the participation of ATP through P2X-purinergic receptors.

Adenine-related compounds modulate UDP-glucuronosyltransferase (UGT) activity in mouse liver microsomes

ABSTRACTAdenine-related compounds are allosteric inhibitors of UDP-glucuronosyltransferase (UGT) in rat liver microsomes (RLM) and human UGT isoforms treated with detergent or pore-forming peptide, alamethicin.To clarify whether the same is true beyond species, the effects of adenine-related compounds on 4-methylumbelliferone (4-MU) glucuronidation were examined using detergent-treated mouse liver microsomes (MLM).Brij-58 treatment of MLM increased the Vmax and the Michaelis constant, Km, of 4-MU. This study was performed using Brij-58-treated MLM as an enzyme source. ATP- and ADP-inhibited 4-MU glucuronidation. In contrast, AMP caused a 1.5-fold increase in glucuronidation. Oxidised forms, NAD+ and NADP+, potently inhibited 4-MU glucuronidation, whereas the reduced forms, NADH and NADPH, did not. Furthermore, the IC50 values of ATP, ADP, NAD+, and NADP+ were approximately 15 μM.In our previous study, ATP was the strongest inhibitor of UGT activity in RLM. However, in this study, the above-mentioned compounds inhibited 4-MU UGT in a comparable and noncompetitive manner. Furthermore, AMP antagonised the inhibitory effects of ATP and ADP.These results suggest that ATP, ADP, NAD+, and NADP+ are common endogenous inhibitors of UGT beyond species.

Взаимодействие механизмов угнетения квантового выделения ацетилхолина при активации ваниллоидных (TRPV1) и пуриновых рецепторов в нервно-мышечном синапсе мыши

The main goal of the study was to analyze the relationship between the signaling pathways of the regulation of acetylcholine (ACh) quantal release in the peripheral synapse triggered by the activation of vanilloid (TRPV1) and purine receptors. In electrophysiological experiments carried out at the neuromuscular synapses of the m. Levator Auris Longus, it was found that the frequency of miniature endplate potentials (mEPPs) and the quantal content of endplate potentials (EPPs) decrease in the presence of TRPV1 agonist capsaicin. This effect was completely reversed by SB366791, a specific competitive antagonist of TRPV1 receptors. ATP, like capsaicin, decreased the frequency of mEPPs and the EPP quantal content. Against the background of the TRPV1 antagonist, the inhibitory effect of ATP on ACh secretion was realized in full. At the same time, against the background of TRPV1 channels activation by capsaicin, the effect of ATP on both spontaneous and evoked ACh release was absent. It was suggested that the mechanisms of action of ATP and capsaicin may be associated with a change in Ca2+ entry into the nerve ending. To test this hypothesis, experiments were carried out to assess the changes in the presynaptic calcium level (Ca2+ transient) using a fluorescent calcium dye upon nerve stimulation. The amplitude of the calcium transient did not change either with the application of ATP or with the addition of capsaicin. Thus, in the neuromuscular synapse of mammals, along with purinergic pathway of ACh secretion regulation, there is also a mechanism of neurosecretion modulation mediated by the activation of TRPV1 channels. The triggering of these mechanisms leads to the suppression of the processes of both spontaneous and evoked release of ACh quanta from the motor nerve endings. It was demonstrated that both pathways of regulation are not accompanied by a change in the Ca2+ transient, but have a common link of regulation of the quantal neurosecretion.

Dual regulatory effects of PI(4,5)P2 on TREK-2 K+ channel through antagonizing interaction between the alkaline residues (K330 and R355-357) in the cytosolic C-terminal helix.

TWIK-related two-pore domain K+ channel-2 (TREK-2) has voltage-independent activity and shows additional activation by acidic intracellular pH (pHi) via neutralizing the E332 in the cytoplasmic C terminal (Ct). We reported opposite regulations of TREK-2 by phosphatidylinositol 4,5-bisphosphate (PIP2) via the alkaline K330 and triple Arg residues (R355-357); inhibition and activation, respectively. The G334 between them appeared critical because its mutation (G334A) endowed hTREK-2 with tonic activity, similar to the mutation of the inhibitory K330 (K330A). To further elucidate the role of putative bent conformation at G334, we compared the dual mutation forms, K330A/G334A and G334A/R355-7A, showing higher and lower basal activity, respectively. The results suggested that the tonic activity of G334A owes to a dominant influence from R355-7. Since there are additional triple Arg residues (R377-9) distal to R355-7, we also examined the triple mutant (G334A/R355-7A/R377-9A) that showed tonic inhibition same with G334A/R355-7A. Despite the state of tonic inhibition, the activation by acidic pHi was preserved in both G334A/R355-7A and G334A/R355-7A/R377-9A, similar to the R355-7A. Also, the inhibitory effect of ATP could be commonly demonstrated under the activation by acidic pHi in R355-7A, G334A/R355-7A, and G334A/R355-7A/R377-9A. These results suggest that the putative bent conformation at G334 is important to set the tug-of-war between K330 and R355-7 in the PIP2-dependent regulation of TREK-2.

SO040ROLE OF INTRACELLULAR CA2+ IN THE REGULATION OF THE RENAL CHLORIDE CHANNEL CLC-KA

Abstract Background and Aims It has been recognized that the physiological role of ClC-Ka and its mouse homolog ClC-K1 in the nephron is to confer high Cl- permeability in the thin Ascending Limb of loop of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla in the countercurrent system. Accordingly ClC-K1 KO mice display a severe polyuria resembling a Nephrogenic Diabetes Insipidus (NDI) phenotype. Thus, ClC-Ka in human nephron could be an interesting target for diuretic and anti-hypertensive drugs. Unfortunately the regulation of ClC-Ka in and its electrophysiological properties are still not completely known. Method We analyzed, by whole-cell voltage-clamp, Cl- currents in HEK293 cells co-expressing GFP-ClC-Ka and the accessory m-Cherry-tagged barttin subunit, upon intracellular Ca2+ mobilization. The patch clamp protocol consists of a holding potential from 0 mV, which is stepped to various test potentials from -195 and +125 mV for 150 ms. Pulses ended with a tail pulse to -125 mV for 20 ms. Concomitantly we monitored by live confocal microscopy, the membrane expression of either GFP-ClC-Ka or barttin-mCherry. Results In control conditions we found a linear current/voltage relationship over a broad range of membrane voltages except of hyperpolarization below -100 mV, where the channel open probability was reduced, providing the well-known hook-shaped appearance of the I-V curve for ClC-Ka channel. Interestingly, both 100 µM ATP and 400 µg/ml of dandelion root extract (DRE), both increasing intracellular Ca2+ in HEK293, significantly inhibited Cl- currents in a time dependent way. Pre-treatment of cells with either the Ca2+ chelator BAPTA-AM or the PCK inhibitor Calphostin C, reverted the inhibitory effects of both ATP and DRE. Interestingly, 1µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of ATP and DRE on Cl- currents. We finally demonstrated that 30 µM Heclin, an inhibitor of E3 ubiquitin ligase, did not revert the DRE- and ATP- induced Cl- currents inhibition, thus suggesting that the ClC-Ka inhibition is independent of channel ubiquitination. Accordingly, live confocal analysis showed that all the Ca2+ mobilizing compounds tested did not induced neither ClC-Ka nor Barttin internalization. We hypothesized that phosphorylation in the putative PKC consensus sites present in ClC-Ka amino acidic sequence may be involved in the inhibition of channel activity. Conclusion In conclusion, we demonstrated for the first time that the activity of ClC-Ka in renal cells could by inhibited by either receptor agonists or natural molecules able to activate an intracellular Ca2+/PKC pathway, thus opening venues for the development of new diuretic drugs.

Abstract 5021: LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment

Abstract The ectonucleotidases CD39 and CD73 have emerged as promising therapeutic targets to enhance antitumor immune responses, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on cancer cells and support the tumor in escaping immune recognition and destruction. Accordingly, in order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to reduce adenosine concentrations in the tumor microenvironment. As an alternative approach to small molecule inhibitors or antibodies, we developed antisense oligonucleotides (ASOs) using the OligofyerTM tool to suppress the expression of CD39 and CD73. ASOs were synthesized as Gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP. The impact of ASO-mediated target knockdown in vivo on anti-tumor immune responses was analyzed in a syngeneic mouse tumor model. Unformulated CD39- and CD73-specific ASOs achieved potent target knockdown on mRNA and protein level in tumor cell lines and primary human T cells in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability if T cells expressed CD39 or CD73. Notably, ASO-mediated knockdown of CD39 or CD73 reversed this inhibitory effect of ATP. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Treatment of tumor-bearing mice with CD39-specific ASO led to dose dependent reduction of CD39 protein expression e.g. in Tregs, tumor-associated macrophages and myeloid-derived suppressor cells. Strikingly, the frequency of intratumoral Tregs was considerably diminished in CD39 ASO-treated animals. Moreover, tumor growth was strongly reduced by CD39-specific ASO. Anti-tumor efficacy was improved when combining CD39 ASO with anti-PD-1 treatment. Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors. Citation Format: Julia Festag, Tamara Thelemann, Abhishek S. Kashyap, Richard Klar, Sandra Kallert, Melanie Buchi, Lisa Hinterwimmer, Monika Schell, Stefanie Raith, Sven Michel, Alfred Zippelius, Frank Jaschinski. LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5021.

Abstract 5909: Inhibition of Cd39 and Cd73 by 3rd-generation antisense oligonucleotides to improve immunity against tumors

Abstract During the last decades it became obvious that the immune system can be utilized to evoke effective antitumor responses. However, cancer cells develop mechanisms to circumvent this. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on a range of cancer cells and the latter are recognized to co-opt both ectonucelotidases for circumventing antitumor immune responses. In order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to simultaneously reduce adenosine concentrations in the tumor microenvironment. We designed antisense oligonucleotides (ASOs) with specificity for human and mouse CD39 and CD73. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP and adenosine. The impact of ASO-mediated target knockdown in vivo on antitumor immune responses was analyzed in a syngeneic mouse tumor model. CD39- and CD73-specific ASOs suppressed expression of CD39 and CD73 mRNA and protein in different cancer cell lines and in primary human T cells without the need of any transfection reagent in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability of CD39- expressing CD8+ T cells. Strikingly, CD39-knockdown by ASO reversed the inhibitory effects of ATP on cell proliferation and viability. Extracellular AMP strongly inhibited proliferation and viability of CD73-expressing CD4+ T cells. Notably, AMP-mediated inhibition of proliferation was considerably less pronounced in cells treated with CD73-specific ASO. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Moreover, CD39-ASO treatment of tumor-bearing mice significantly reduced CD39-protein expression on tumor-associated macrophages, as well as intratumoral CD8+ T cells and Tregs. Remarkably, frequency of intratumoral Tregs was potently reduced by ASO-mediated CD39 knockdown. Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors. Citation Format: Tamara Hilmenyuk, Sandra Kallert, Richard Klar, Lisa Hinterwimmer, Monika Schell, Marina Van Ark, Alfred Zippelius, Frank Jaschinski. Inhibition of Cd39 and Cd73 by 3rd-generation antisense oligonucleotides to improve immunity against tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5909.

Inhibitory Effects of ATP and Adenosine on Cholangiocarcinoma Cell Proliferation and Motility.

Inhibitory effects of extracellular nucleotides have been investigated in many types of cancers. Herein, we aimed to determine the effects of ATP and adenosine and their receptor profile on cholangiocarcinoma (CCA) cells. Two CCA and one immortalized cholangiocyte cell line were used. The effects of ATP and adenosine on cell proliferation and motility were examined by MTT and wound-healing/trans-well invasion assays, respectively. Purinergic receptor profiling was carried out by reverse transcription-polymerase chain reaction (RT-PCR). ATP and adenosine induced proliferation-inhibitory and motility-inhibitory effects in all cell lines tested. However, immortalized cholangiocytes showed resistance in proliferation inhibition. Several P2 receptors were commonly expressed in all cells, whereas no adenosine receptor was expressed. Furthermore, no synergistic effects of ATP and adenosine were observed in CCA cells. ATP and adenosine had anti-proliferative and anti-motility effects in CCA cells, while there was a smaller effect on normal cholangiocytes. These data indicate the potential use of ATP and odenosine as a novel therapy for CCA.

Mitochondrial cytochrome c oxidase is inhibited by ATP only at very high ATP/ADP ratios.

In the past, divergent results have been reported based on different methods and conditions used for enzymatic activity measurements of cytochrome c oxidase (CytOx). Here, we analyze in detail and show comparable and reproducible polarographic activity measurements of ATP-dependent inhibition of CytOx kinetics in intact and non-intact rat heart mitochondria and mitoplasts. We found that this mechanism is always present in isolated rat heart mitochondria and mitoplasts; however, it is measurable only at high ATP/ADP ratios using optimal protein concentrations. In the kinetics assay, measurement of this mechanism is independent of presence or absence of Tween-20 and the composition of measuring buffer. Furthermore, the effect of atractyloside on intact rat heart mitochondria confirms that (i) ATP inhibition occurs under uncoupled conditions [in the presence of carbonly cyanide m-chlorophenyl hydrazone (CCCP)] when the classical respiratory control is absent and (ii) high ATP/ADP ratios in the matrix as well as in the cytosolic space are required for full ATP inhibition of CytOx. Additionally, ATP inhibition measured in intact mitochondria extends in the presence of oligomycin, thus indicating further that the problem to measure the inhibitory effect of ATP on CytOx is apparently due to the lack of very high ATP/ADP ratios in isolated mitochondria.

Action of ATP on Ca2+-Transient in Different Parts of the Frog Motor Nerve Ending

Electrophysiological evidence indicates a difference in neurotransmitter secretion along the motor nerve terminals of the frog neuromuscular junction. This includes a decrease of the minimal synaptic delay value and a reduction in the quantal content of the evoked endplate currents from the proximal to distal portion of the motor nerve ending. Besides, various physiologically active compounds may have different effects on the acetylcholine secretion in the proximal and distal parts of the nerve terminal. Here, we explored the effects of ATP on Ca2+-transient using optical detection methods with high-speed camera in different parts of the frog nerve terminal. There was shown a significant inhibitory effect of ATP on Ca2+-transient in both the proximal and distal regions of nerve terminals. However, in different parts of nerve endings, any significant differences in ATP effects were not found. Thus, ATP decreases the Ca2+-transient along the entire presynaptic terminal.

Nucleotides metabolizing ectoenzymes as possible markers of mesenchymal stem cell osteogenic differentiation

Growing murine mesenchymal stem cells (mMSCs) from mouse bone marrow decreased their rate of proliferation in the presence of benzoylbenzoyl-ATP persistently, but the inhibitory effect of ATP was strong only in a concentration of 50 μmol·L(-1) and lasted for 48 h in culture. These results hinted at ATP hydrolysis by the cell surface enzymes at the lower concentrations and thus it may be not able to inhibit MSCs. By using ATP, ADP, or AMP as substrates, we tested the ectonucleotidase activity on the surface of undifferentiated MSCs and MSC-derived osteoblasts. Here, we report that although nucleoside triphosphate diphosphohydrolase (NTPDase)1 and NTPDase8 are engaged in the metabolism of ATP in MSC-derived osteoblasts, NTPDase3 is responsible for its metabolism in undifferentiated MSCs. In this study, we also realized that osteoblasts effectively metabolize ADP to ATP and AMP. The enzymatic activity of adenylate kinase (AK) is consistent with the high expression level of the AK gene. Therefore, it was tempting to suggest that this enzyme, together with NTPDase1 and NTPDase8, assume the role of specific markers that allowed distinction between differentiated osteoblasts and early undifferentiated MSCs. Additionally, unlike osteoblasts, undifferentiated MSCs demonstrated the activity of 5'-nucleotidase (CD73). However, the expression analysis of CD73 mRNA did not show any differences; CD73 mRNA was expressed in both kinds of cells to the same extent.

Inhibition of Hippocampal Synaptic Activity by ATP, Hypoxia or Oxygen-Glucose Deprivation Does Not Require CD73

Adenosine, through activation of its A1 receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A1 receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5′-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73+/+ and CD73−/− mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC50 values of approximately 25 µM. In contrast, ATP was a less potent inhibitor (IC50 = 100 µM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73+/+ and CD73−/− slices were blocked by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73−/− and CD73+/+ slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73−/− mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.

Alanine substitution scanning of pannexin1 reveals amino acid residues mediating ATP sensitivity

Pannexin1 is a prime candidate to represent an ATP release channel. The pannexin1 channel can be activated by extracellular ATP through purinergic receptors P2X7 or P2Y. Recent studies have shown that the Pannexin1 channel is inhibited by its own permeant ion, ATP, and also by P2X7 receptor agonists and antagonists. However, the dose dependence of this inhibition indicated that significant inhibition was prominent at ATP concentrations higher than required for activation of purinergic receptors, including P2X7 and P2Y2. The inhibitory effect of ATP is largely decreased when R75 in the first extracellular loop of Pannexin1 is mutated to alanine, indicating that ATP regulates this channel presumably through binding. To further investigate the structural property of the putative ATP binding site, we performed alanine-scanning mutagenesis of the extracellular loops of pannexin1. Mutations on W74, S237, S240, I247 and L266 in the extracellular loops 1 and 2 severely impaired the inhibitory effect of BzATP, indicating that they might be the essential amino acids in the putative binding site. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 moderately (≥50%) decreased BzATP sensitivity, suggesting their supporting roles in the binding. Mutations of other residues did not change the BzATP potency compared to wild-type except for some nonfunctional mutants. These data demonstrate that several amino acid residues on the extracellular loops of Pannexin1 mediate ATP sensitivity. Cells expressing mutant Panx1W74A exhibited an enhanced release of ATP, consistent with the removal of a negative feedback loop controlling ATP release.

Involvement of P2X Receptors in the Regulation of Insulin Secretion, Proliferation and Survival in Mouse Pancreatic β-Cells

In order to clarify the functional role of ionotropic purinergic (P2X) receptors in pancreatic β-cells, we examined the effect of several P2 receptor agonists and antagonists on insulin secretion by mouse pancreatic islets, mouse Beta-TC6 cell proliferation and survival of dispersed islet cells in culture. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the expression of mRNAs of P2X₄ receptor in mouse islets and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅ and P2X₇ receptors in Beta-TC6 cells. The presence of P2X₄ receptor proteins in islets and Beta-TC6 cells was confirmed by immunofluorescent staining and Western blot analysis. We have previously found that the functional P2Y₁ receptor but not P2Y₂ and P2Y₄ receptors was present in islets. In this study we found that a nonspecific P2 receptor agonist, ATP (1 µM) stimulated insulin secretion by islets in the presence of high glucose (20 mM) in culture. The effect of ATP was partially inhibited by a P2 receptor antagonist PPADS as well as a P2Y₁ receptor antagonist MRS2179. In addition, a P2X₄ receptor potentiator ivermectin per se augmented glucose-induced insulin secretion and slightly potentiated the effect of ATP. These results suggested the involvement of P2Y₁and P2X receptors. We also found that ATP inhibited proliferation of Beta-TC6 cells in a concentration-dependent manner during 72 h culture. The inhibitory effect of ATP was completely reversed by PPADS and partially by treating cells with small interfering RNA targeted for P2X₄ receptor mRNA. Furthermore, we found that the phosphorylation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) was suppressed by treatment with ATP in Beta-TC6 cells. In addition, we found that ATP reduced the cell viability and DNA synthesis of islet cells in culture. The effect of ATP on the cell viability was blocked by PPADS or MRS2179. These results suggested that P2X receptors as well as the P2Y₁ receptor played a role in the modulation of insulin secretion, proliferation and cell viability in mouse pancreatic β-cells.

Effect of hypoxen on bioenergetic processes in mitochondria and activity of ATP-sensitive potassium channel

The effect of hypoxenum on bioenergetic processes in heart and liver mitochondria of rats, connected with respiration, the generation of hydrogen peroxide, and the activity of ATP-sensitive K-channel ((mitoK)ATP) has been studied. It was shown that hypoxenum in the concentration range of 0.05-10 microg/ml stimulates respiration, increases the coupling in the respiratory chain, and enhances the formation of H2O2 and energy-dependent swelling associated with potassium transport in mitochondria. Hypoxenum removes the inhibitory effect of ATP on the energy-dependent swelling of mitochondria and partially reduces the accumulation of H2O2 in the presence of ATP. The role of antihypoxic and antioxidant action of hypoxenum associated with the activation of (mitoK)ATP is discussed.

Presynaptic inhibition of neural vasodilator pathways to submucosal arterioles by release of purines from sympathetic nerves

Capsaicin-sensitive extrinsic sensory nerves and submucosal vasodilator neurons provide important vasodilator input to submucosal arterioles, but relatively little is known about the signaling between these populations and the sympathetic vasoconstrictor innervation. This study examined whether release of sympathetic purines can modulate dilator nerves. In vitro submucosal preparations from guinea pig ileum were modified to leave the parent mesenteric artery intact so that perivascular sympathetic and extrinsic afferent nerves could be activated by a bipolar stimulating electrode placed on the parent artery, and submucosal vasodilator neurons were activated using focal electrodes placed on submucosal ganglia. The outside diameter of submucosal arterioles was monitored using videomicroscopy, and dilator responses were examined after preconstricting vessels 80-95% with prostaglandin F(2alpha) (400 nM). Mesenteric nerve stimulation evoked a frequency-dependent dilation, with suramin (100 microM) present throughout to inhibit P(2X) receptor-mediated vasoconstrictions. In the presence of guanethidine (10 microM) to inhibit sympathetic purine release, superfusion of ATP (200 nM-6 microM) caused a concentration-dependent inhibition of nerve-evoked dilations. Vasodilations to substance P (10 nM) were not inhibited by ATP in the presence of guanethidine, implicating a presynaptic effect of ATP on neurotransmitter release. The inhibitory effect of ATP was blocked by the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 10 microM). In addition, 8-PT increased the amplitude of nerve-evoked dilations, suggesting a tonic inhibitory effect of adenosine receptors on vasodilator release. Dilations evoked by electrical stimulation of submucosal ganglia were also inhibited almost 50% by ATP (2 microM) and its nonhydrolyzable analog, alpha,beta-methylene-ATP (10 microM). These data suggest that sympathetic varicosities release ATP or a related purine that can act at presynaptic adenosine receptors on extrinsic sensory and submucosal vasodilator neurons to inhibit neurotransmitter release.

Interaction of hydrocortisone with ATP and adenosine on nerve-mediated contractions of frog skeletal muscle

The inhibitory effects of ATP and adenosine on the nerve-mediated contractile responses of isolated sartorius muscle of the frog, Rana ridibunda, evoked by electrical field stimulation (EFS) were studied using pharmacological organ-bath technique. The effects of hydrocortisone applied in vitro and in vivo on contractility of sartorius muscle were also examined. ATP (100 μM) significantly reduced the amplitude of contraction to EFS of sartorius muscle, while pyridoxalphosphate-6-azonphenyl-2′,4′-disulfonic acid (PPADS; 10 μM), a P2 receptor antagonist, abolished inhibitory effect of ATP. A similar inhibitory effect of adenosine (100 μM) was fully antagonized by 8-( p-sulfophenyl)-theophylline (8-SPT, 100 μM), a P1 receptor antagonist. Incubation of the tissue with hydrocortisone (10 μM) caused a slight, but significant, decrease of muscle contractions. After incubation of muscle preparations with both hydrocortisone and ATP, no inhibition of muscle contractility was registered. A single injection of hydrocortisone (100 mg/kg) 12 h prior to experiments to frogs did not significantly change the nerve-mediated contractility of isolated sartorius muscle; however, it abolished the inhibitory action of ATP without changing inhibitory activity of adenosine. After treatment of frogs with hydrocortisone for 14 days (100 mg/kg/day), both ATP and adenosine retained their inhibitory action on EFS-induced contractions of the muscle, and their effects were antagonized by PPADS and 8-SPT, respectively. It is concluded that hydrocortisone has antagonistic actions against the inhibitory effects of ATP at the frog neuromuscular junction, although this effect is lost following long-term treatment with hydrocortisone.

P2 Receptor-mediated Inhibition of Vasopressin-stimulated Fluid Transport and cAMP Responses in AQP2-transfected MDCK Cells

We cultured canine kidney (MDCK) cells stably expressing aquaporin-2 (AQP2) on collagen-coated permeable membrane filters and examined the effect of extracellular ATP on arginine vasopressin (AVP)-stimulated fluid transport and cAMP production. Exposure of cell monolayers to basolateral AVP resulted in stimulation of apical to basolateral net fluid transport driven by osmotic gradient which was formed by addition of 500 mM mannitol to basolateral bathing solution. Pre-exposure of the basolateral surface of cell monolayers to ATP (100 microM) for 30 min significantly inhibited the AVP-stimulated net fluid transport. In these cells, AVP-stimulated cAMP production was suppressed as well. Profile of the effects of different nucleotides suggested that the P2Y(2) receptor is involved in the action of ATP. ATP inhibited the effect of isoproterenol as well, but not that of forskolin to stimulate cAMP production. The inhibitory effect of ATP on AVP-stimulated fluid movement was attenuated by a protein kinase C inhibitor, calphostin C or pertussis toxin. These results suggest that prolonged activation of the P2 receptors inhibits AVP-stimulated fluid transport and cAMP responses in AQP2 transfected MDCK cells. Depressed responsiveness of the adenylyl cyclase by PKC-mediated modification of the pertussis-toxin sensitive G(i) protein seems to be the underlyihng mechanism.

Influential factor contributing to the isoform‐specific inhibition by ATP of human mitochondrial NAD(P)+‐dependent malic enzyme

Human mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in malic enzyme isoforms has not yet been identified. Sequence alignment of nucleotide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. In other ME isoforms, this residue is serine. As the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms, we have examined the possible role of Lys346 by replacing it to alanine, serine or arginine. Our kinetic data indicate that the K346S mutant enzyme displays a shift in its cofactor preference from NAD(+) to NADP(+) upon increasing k(cat,NADP) and decreasing K(m,NADP). Furthermore, the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wild-type is close to 2, but those of the K346 mutants are approximately 1.5. The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate, suggesting that the allosteric property is retained in these mutants. Our data strongly suggest that Lys346 in human m-NAD-ME is required for ATP inhibition. Mutation of Lys346 to Ser or Ala causes the enzyme to be much less sensitive to ATP, similar to cytosolic NADP-dependent malic enzyme. Substitution of Lys to Arg did not change the isoform-specific inhibition of the enzyme by ATP. The inhibition constants of ATP are increased for K346S and K346A, but are similar to those of the wild-type for K346R, suggesting that the positive charge rather than group specificity is required for binding affinity of ATP. Thus, ATP inhibition is proposed to be determined by the electrostatic potential involving the positive charge on the side chain of Lys346.

Dual Effect of ATP on Glucose-Induced Insulin Secretion in HIT-T15 Cells

: The study investigated the dual effect of purinergic nucleotides on the secretion of insulin from pancreatic beta cells. : The level of insulin secretion in HIT-T15 cells of static incubation was measured using a radioimmunoassay. : The adenine nucleotides reduced the level of glucose-induced insulin secretion in a concentration-dependent manner, and the relative potency order (IC50; muM) was BzATP (6.9) > ATP (20.4) >/= alpha, beta-methylene ATP (23.3) >/= 2-methylthio-ATP (24.9). Suramin and PPADS (200 muM), which are blockers of the purinergic receptors, had a little influence on the activity of ATP. However, the inhibitory effect of ATP was reversed by preincubation with oxidized ATP (200 muM), which is a P2X7 antagonist. The level of insulin secretion in these preincubated cells exposed to the purinergic nucleotides increased in the following order: ATP > alpha, beta-methylene ATP >/= 2-methylthio-ATP. A pretreatment with foskolin and PDBu (100 nM) potentiated the increasing effect of ATP on insulin secretion. The Western blotting showed the expression of P2X7 and P2Y11 receptors. : Purinergic stimulation has inhibitory activity on glucose-dependent insulin secretion through the activation of the P2X7 receptor, whereas it has enhancing effect through the activation of the P2Y11 receptor in HIT-T15 cells.