RESUMO
Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Animais , Afidicolina/farmacologia , Células Cultivadas , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiadenina/farmacologia , Didesoxinucleotídeos/farmacologia , Feminino , Fase G2 , Indóis/farmacologia , Camundongos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oócitos , Cultura Primária de Células , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/metabolismo , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
2'-deoxy-ATP (dATP) is a naturally occurring small molecule that has shown promise as a therapeutic because it significantly increases cardiac myocyte force development even at low dATP/ATP ratios. To investigate mechanisms by which dATP alters myosin crossbridge dynamics, we used Brownian dynamics simulations to calculate association rates between actin and ADP- or dADP-bound myosin. These rates were then directly incorporated in a mechanistic Monte Carlo Markov Chain model of cooperative sarcomere contraction. A unique combination of increased powerstroke and detachment rates was required to match experimental steady-state and kinetic data for dATP force production in rat cardiac myocytes when the myosin attachment rate in the model was constrained by the results of a Brownian dynamics simulation. Nearest-neighbor cooperativity was seen to contribute to, but not fully explain, the steep relationship between dATP/ATP ratio and steady-state force-development observed at lower dATP concentrations. Dynamic twitch simulations performed using measured calcium transients as inputs showed that the effects of dATP on the crossbridge alone were not sufficient to explain experimentally observed enhancement of relaxation kinetics by dATP treatment. Hence, dATP may also affect calcium handling even at low concentrations. By enabling the effects of dATP on sarcomere mechanics to be predicted, this multi-scale modeling framework may elucidate the molecular mechanisms by which dATP can have therapeutic effects on cardiac contractile dysfunction.
Assuntos
Nucleotídeos de Desoxiadenina/farmacologia , Modelos Cardiovasculares , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Animais , Valor Preditivo dos Testes , RatosRESUMO
Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor α-haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y1 and P2Y12 receptors might ameliorate the septic response to HlyA-producing E. coli. The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y1 and P2Y12 antagonists. Our data show that the P2Y1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y1, by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y1 receptor antagonists.
Assuntos
Toxinas Bacterianas/toxicidade , Plaquetas/patologia , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Sepse/patologia , Infecções Urinárias/patologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Animais , Nucleotídeos de Desoxiadenina/farmacologia , Modelos Animais de Doenças , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Sepse/complicações , Sepse/tratamento farmacológico , Resultado do Tratamento , Infecções Urinárias/complicações , Infecções Urinárias/tratamento farmacológico , Escherichia coli Uropatogênica/efeitos dos fármacosRESUMO
4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) and 4'-ethynyl-2'-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4'-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.
Assuntos
Antivirais/síntese química , Nucleotídeos de Desoxiadenina/síntese química , Desoxiadenosinas/síntese química , Vírus da Hepatite B/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Nucleotídeos de Desoxiadenina/farmacologia , Desoxiadenosinas/farmacologia , Vírus da Hepatite B/fisiologia , HumanosRESUMO
Influenza A viruses leading to infectious respiratory diseases cause seasonal epidemics and sometimes periodic global pandemics. Viral polymerase is an attractive target in inhibiting viral replication, and 4'-ethynyladenosine, which has been reported as a highly potent anti-human immunodeficiency virus (HIV) nucleoside derivative, can work as an anti-influenza agent. Herein, we designed and synthesized a 4'-ethynyl-2'-deoxyadenosine 5'-monophosphate analog called EdAP (5). EdAP exhibited potent inhibition against influenza virus multiplication in Madin-Darby canine kidney (MDCK) cells transfected with human α2-6-sialyltransferase (SIAT1) cDNA and did not show any toxicity toward the cells. Surprisingly, this DNA-type nucleic acid analog (5) inhibited the multiplication of influenza A virus, although influenza virus is an RNA virus that does not generate DNA.
Assuntos
Antivirais/farmacologia , Nucleotídeos de Desoxiadenina/farmacologia , Desoxiadenosinas/síntese química , Influenza Humana/tratamento farmacológico , Animais , Antivirais/síntese química , Antivirais/química , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxiadenina/química , Desoxiadenosinas/química , Desoxiadenosinas/farmacologia , Cães , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Replicação Viral/efeitos dos fármacosRESUMO
P2Y1 receptors play an essential role in inhibitory neuromuscular transmission in the gastrointestinal tract. The signalling pathway involves the opening of small conductance calcium activated potassium-channels (Kca2 family) that results in smooth muscle hyperpolarization and relaxation. Inorganic polyphosphates and dinucleotidic polyphosphates are putative neurotransmitters that potentially act on P2Y1 receptors. A pharmacological approach using both orthosteric (MRS2500) and allosteric (BPTU) blockers of the P2Y1 receptor and openers (CyPPA) and blockers (apamin) of Kca2 channels was used to pharmacologically characterise the effect of these neurotransmitters. Organ bath and microelectrodes were used to evaluate the effect of P1,P4-Di (adenosine-5') tetraphosphate ammonium salt (Ap4A), inorganic polyphosphates (PolyP) and CyPPA on spontaneous contractions and membrane potential of mouse colonic smooth muscle cells. PolyP neither modified contractions nor membrane potential. In contrast, Ap4A caused a concentration-dependent inhibition of spontaneous contractions reaching a maximum effect at 100⯵M Ap4A response was antagonised by MRS2500 (1⯵M), BPTU (3⯵M) and apamin (1⯵M). CyPPA (10⯵M) inhibited spontaneous contractions and this response was antagonised by apamin but it was not affected by MRS2500 or BPTU. Both CyPPA and Ap4A caused smooth muscle hyperpolarization that was blocked by apamin and MRS2500 respectively. We conclude that Ap4A but not PolyP activates P2Y1 receptors causing smooth muscle hyperpolarization and relaxation. Ap4A signalling causes activation of Kca2 channels through activation of P2Y1 receptors. In contrast, CyPPA acts directly on Kca2 channels. Further studies are needed to evaluate if dinucleotidic polyphosphates are released from inhibitory motor neurons.
Assuntos
Colo/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Receptores Purinérgicos P2Y1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Colo/fisiologia , Nucleotídeos de Desoxiadenina/farmacologia , Feminino , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Músculo Liso/citologia , Músculo Liso/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Transdução de Sinais/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismoRESUMO
Ribonucleotide reductases (RNRs) are essential enzymes producing de novo deoxynucleotide (dNTP) building blocks for DNA replication and repair and regulating dNTP pools important for fidelity of these processes. A new study reveals that the class Ia Escherichia coli RNR is regulated by dATP via stabilization of an inactive α4ß4 quaternary structure, slowing formation of the active α2ß2 structure. The results support the importance of the regulatory α4ß4 complex providing insight in design of experiments to understand RNR regulation in vivo.
Assuntos
Nucleotídeos de Desoxiadenina/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Domínio Catalítico , Escherichia coli/enzimologia , Modelos Moleculares , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismoRESUMO
Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α2 subunit that houses the active site and allosteric regulatory sites and a ß2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact α2ß2 state allowing for radical transfer from ß2 to α2 RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied Escherichia coli class Ia RNR, dATP binding to an allosteric site on α promotes formation of an α4ß4 ring-like state. Here, we investigate whether the α4ß4 formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-ß interface (S37D/S39A-α2, S39R-α2, S39F-α2, E42K-α2, or L43Q-α2) that disrupt the α4ß4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that α4ß4 formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the α-ß interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the α-ß interface whose substitution has previously been shown to cause a mutator phenotype in Escherichia coli (i.e. S39F-α2 or E42K-α2) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.
Assuntos
Substituição de Aminoácidos , Escherichia coli/enzimologia , Multimerização Proteica/genética , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Nucleotídeos de Desoxiadenina/farmacologia , Modelos Moleculares , Estrutura Quaternária de Proteína/genética , Ribonucleotídeo Redutases/químicaRESUMO
BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique that uses magnetic pulses over the cranium to induce electrical currents in underlying cortical tissue. Although rTMS has shown clinical utility for a number of neurological conditions, we have only limited understanding of how rTMS influences cellular function and cell-cell interactions. OBJECTIVE: In this study, we sought to investigate whether repeated magnetic stimulation (rMS) can influence astrocyte biology in vitro. METHOD: We tested four different rMS frequencies and measured the calcium response in primary neonatal astrocyte cultures. We also tested the effect of rMS on astrocyte migration and proliferation in vitro. We tested 3 to 4 culture replicates and 17 to 34 cells for each rMS frequency (sham, 1âHz, cTBS, 10âHz and biomemetic high frequency stimulation - BHFS). RESULTS: Of all frequencies tested, 1âHz stimulation resulted in a statistically significant rise in intracellular calcium in the cytoplasmic and nuclear compartments of the cultured astrocytes. This calcium rise did not affect migration or proliferation in the scratch assay, though astrocyte hypertrophy was reduced in response to 1âHz rMS, 24 hours post scratch injury. CONCLUSION: Our results provide preliminary evidence that rMS can influence astrocyte physiology, indicating the potential for a novel mechanism by which rTMS can influence brain activity.
Assuntos
Astrócitos/efeitos da radiação , Etanol , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Bromodesoxiuridina/metabolismo , Cafeína/farmacologia , Cálcio/metabolismo , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Células Cultivadas , Córtex Cerebral , Nucleotídeos de Desoxiadenina/farmacologia , Relação Dose-Resposta à Radiação , Edema/terapia , Campos Eletromagnéticos , Etanol/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo , Estimulação Magnética Transcraniana , Ferimentos e Lesões/terapiaRESUMO
BACKGROUND: Human gastric fundus relaxation is mediated by intrinsic inhibitory pathway. We investigated the roles of nitrergic and purinergic pathways, two known inhibitory factors in gastric motility, on spontaneous and nerve-evoked contractions in human gastric fundus muscles. METHODS: Gastric fundus muscle strips (12 circular and 13 longitudinal) were obtained from patients without previous gastrointestinal motility disorder who underwent gastrectomy for stomach cancer. Using these specimens, we examined basal tone, peak, amplitude, and frequency of spontaneous contractions, and peak and nadir values under electrical field stimulation (EFS, 150 V, 0.3 ms, 10 Hz, 20 s). To examine responses to purinergic and nitrergic inhibition without cholinergic innervation, atropine (muscarinic antagonist, 1 µM), MRS2500 (a purinergic P2Y1 receptor antagonist, 1 µM), and N-nitro-L-arginine (L-NNA, a nitric oxide synthase inhibitor, 100 µM) were added sequentially for spontaneous and electrically-stimulated contractions. Tetrodotoxin was used to confirm any neuronal involvement. RESULTS: In spontaneous contraction, L-NNA increased basal tone and peak in both muscle layers, while amplitude and frequency were unaffected. EFS (up to 10 Hz) uniformly induced initial contraction and subsequent relaxation in a frequency-dependent manner. Atropine abolished initial on-contraction and induced only relaxation during EFS. While MRS2500 showed no additional influence, L-NNA reversed relaxation (p = 0.012 in circular muscle, and p = 0.006 in longitudinal muscle). Tetrodotoxin abolished any EFS-induced motor response. CONCLUSIONS: The relaxation of human gastric fundus muscle is reduced by nitrergic inhibition. Hence, nitrergic pathway appears to be the main mechanism for the human gastric fundus relaxation.
Assuntos
Fundo Gástrico/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Atropina/farmacologia , Nucleotídeos de Desoxiadenina/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Feminino , Fundo Gástrico/fisiologia , Motilidade Gastrointestinal/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Antagonistas Muscarínicos/farmacologia , Contração Muscular/fisiologia , Relaxamento Muscular/fisiologia , Músculo Liso/fisiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , Antagonistas Purinérgicos/farmacologiaRESUMO
P2Y1 receptors mediate nerve mediated purinergic inhibitory junction potentials (IJP) and relaxations in the gastrointestinal (GI) tract in a wide range of species including rodents and humans. A new P2Y1 antagonist, with a non-nucleotide structure, BPTU, has recently been described using X-ray crystallography as the first allosteric G-protein-coupled receptor antagonist located entirely outside of the helical bundle. In this study, we tested its effect on purinergic responses in the gastrointestinal tract of rodents using electrophysiological and myographic techniques. BPTU concentration dependently inhibited purinergic inhibitory junction potentials and inhibition of spontaneous motility induced by electrical field stimulation in the colon of rats (EC50 = 0.3 µM) and mice (EC50 = 0.06 µM). Mechanical inhibitory responses were also concentration-dependently blocked in the stomach of both species. Compared to MRS2500, BPTU displays a lower potency. In the rat colon nicotine induced relaxation was also blocked by BPTU. BPTU also blocked the cessation of spontaneous contractility elicited by ADPßS and the P2Y1 agonist MRS2365. We conclude that BPTU is a novel antagonist with different structural and functional properties than nucleotidic antagonists that is able to block the P2Y1 receptor located at the neuromuscular junction of the GI tract.
Assuntos
Colo/efeitos dos fármacos , Fármacos Gastrointestinais/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Colo/fisiologia , Nucleotídeos de Desoxiadenina/farmacologia , Feminino , Motilidade Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/fisiologia , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Nicotina/farmacologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Ratos Sprague-Dawley , Receptores Purinérgicos P2Y1/metabolismo , Tionucleotídeos/farmacologia , Técnicas de Cultura de TecidosRESUMO
In the equine large intestine, the knowledge of the basic mechanisms underlying motility function is crucial to properly treat motility disorders. P2Y1 receptors are responsible for mediating purinergic colonic relaxation in several species. In vitro experimental studies of the circular muscle from the equine pelvic flexure (n = 6) were performed to characterize inhibitory and excitatory neuromuscular transmission. Electrophysiological studies showed that electrical field stimulation (EFS) evoked biphasic inhibitory junction potentials (IJPs) in smooth muscle cells: a fast IJP (IJPf) followed by a sustained IJP (IJPs). IJPs was sensitive to L-NNA 1 mM (a nitric oxide synthase inhibitor) (P <0.01), while IJPf was abolished by MRS2500 1 µM (a P2Y1 receptor antagonist) (P <0.001). EFS (5 Hz for 2 min) in the organ bath inhibited rhythmic contractions to 3.0 ± 2.5% of basal area under the curve (P <0.0001). EFS under MRS2500 1 µM or L-NNA 1 mM incubation inhibited contractions to 6.0 ± 2.8% (P <0.05) and 24.4 ± 11.3% respectively (P <0.05). Combination of MRS2500 1 µM and L-NNA 1 mM completely reversed the EFS-induced inhibition of colonic motility. Non-nitrergic, non-purinergic conditions were used to reveal voltage-dependent EFS-induced contractions sensitive to atropine 1 µM (P <0.001) and, therefore, cholinergic. In conclusion, nerve-mediated relaxation and contraction in the equine pelvic flexure involve the same mechanisms as those observed in the human colon. P2Y1 receptors mediate purinergic relaxations and are potential targets for the treatment of equine colonic motor disorders.
Assuntos
Colo/efeitos dos fármacos , Nucleotídeos de Desoxiadenina/farmacologia , Inibidores Enzimáticos/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Cavalos/metabolismo , Nitroarginina/farmacologia , Antagonistas do Receptor Purinérgico P2Y/metabolismo , Animais , Colo/fisiologia , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacosRESUMO
Adenosine diphosphate directly induces platelet aggregation via the G-protein coupled P2Y1 and P2Y12 receptors. P2Y12, but not P2Y1, receptor antagonists are available in the clinic. The relevance of the P2Y1 receptor as an antiplatelet target has been studied in rodents, but not in higher species. We therefore examined effects of the pharmacological blockade of the P2Y1 receptor with its selective antagonist MRS2500 in monkey models of electrolytic-mediated arterial thrombosis (ECAT) and kidney bleeding time (KBT). Abciximab, a GPIIb-IIIa antagonist, and cangrelor, a P2Y12 antagonist, were utilized to validate these monkey models. Compounds were given IV at 15-60 min before thrombosis initiation in anesthetized monkeys. Scanning electron microscopy showed the luminal surface of thrombotic artery covered with platelet aggregates and fibrin network. Administration of abciximab at 0.25 and 0.7 mg/kg IV significantly reduced thrombus weight by 71 ± 1 and 100 ± 0 %, and increased KBT by 10.0 ± 0.1- and 10.1 ± 0-fold, respectively (n = 3/dose). Likewise, cangrelor at 0.6 and 2 mg/kg/h IV significantly reduced thrombus weight significantly by 72 ± 9 % and 100 ± 0 % and increased KBT by 2.1 ± 0.1- and 9.8 ± 0.2-fold, respectively (n = 3/dose). MRS2500 [mg/kg + mg/kg/h IV] at 0.09 + 0.14 and 0.45 + 0.68 significantly reduced thrombus weight by 57 ± 1 % and 88 ± 1 % and increased KBT by 2.1 ± 0.3- and 4.9 ± 0.6-fold, respectively (n = 4/dose). In summary, MRS2500 prevented occlusive arterial thrombosis at a dose that moderately prolonged KBT, indicating a role of P2Y1 receptors in arterial thrombosis and hemostasis in monkeys. Thus P2Y1 receptor antagonism provides a suitable target for drug discovery.
Assuntos
Artérias Carótidas , Nucleotídeos de Desoxiadenina/farmacologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Trombose/prevenção & controle , Animais , Avaliação Pré-Clínica de Medicamentos , Macaca fascicularisRESUMO
Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca(2+) sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM.
Assuntos
Cardiomiopatia Dilatada/tratamento farmacológico , Cardiotônicos/farmacologia , Nucleotídeos de Desoxiadenina/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Miosinas/metabolismo , Actomiosina/metabolismo , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Modelos Animais de Doenças , Cães , Metabolismo Energético/efeitos dos fármacos , Feminino , Cinética , Masculino , Miofibrilas/metabolismo , Fosforilação , Recuperação de Função FisiológicaRESUMO
In response to adenosine 5'-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.
Assuntos
Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiadenina/metabolismo , Antagonistas do Receptor Purinérgico P2Y/química , Receptores Purinérgicos P2Y1/química , Receptores Purinérgicos P2Y1/metabolismo , Uracila/análogos & derivados , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Nucleotídeos de Desoxiadenina/farmacologia , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Antagonistas do Receptor Purinérgico P2Y/metabolismo , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Tionucleotídeos/química , Tionucleotídeos/metabolismo , Uracila/química , Uracila/metabolismo , Uracila/farmacologiaRESUMO
We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction. We measured the contractile properties of chemically-demembranated multicellular ventricular wall preparations and isolated myofibrils from human subjects with end-stage heart failure. Isometric force was increased at both saturating and physiologic Ca(2+) concentrations with dATP compared to ATP. This resulted in an increase in the Ca(2+) sensitivity of force (pCa50) by 0.06 pCa units. The rate of force redevelopment (ktr) in demembranated wall muscle was also increased, as was the rate of contractile activation (kACT) in isolated myofibrils, indicating increased cross-bridge binding and cycling compared with ATP in failing human myocardium. These data suggest that dATP could increase dP/dT and end systolic pressure in failing human myocardium. Importantly, even though the magnitude and rate of force development were increased, there was no increase in the time to 50% and 90% myofibril relaxation. These data, along with our previous studies in rodent models, show the promise of elevating myocardial dATP to enhance contraction and restore cardiac pump function. These data also support further pre-clinical evaluation of this new approach for treating heart failure.
Assuntos
Nucleotídeos de Desoxiadenina/farmacologia , Insuficiência Cardíaca/fisiopatologia , Contração Miocárdica/efeitos dos fármacos , Adulto , Demografia , Feminino , Humanos , Contração Isométrica/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Miofibrilas/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD(+) and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca(2+)-activated small-conductance K(+) (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1(-/-) mice. Up4A induced P2Y1R-SK-channel-mediated hyperpolarization in isolated PDGFRα(+) cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca(2+) transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD(+), or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon.
Assuntos
Colo/metabolismo , Fosfatos de Dinucleosídeos/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y1/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Antineoplásicos/farmacologia , Colo/inervação , Nucleotídeos de Desoxiadenina/farmacologia , Humanos , Camundongos , Camundongos Knockout , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/metabolismo , Receptores Purinérgicos P2Y1/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Suramina/farmacologia , Difosfato de Uridina/farmacologiaRESUMO
Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.
Assuntos
Nucleotídeos de Desoxiadenina/farmacologia , Fluorometria , Multimerização Proteica/efeitos dos fármacos , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Humanos , CinéticaRESUMO
The purine receptor involved in inhibitory responses in the gastrointestinal tract has been recently identified. P2Y1 receptor activation mediates the fast component of the inhibitory junction potential (IJPf) and the non-nitrergic relaxation. The aim of the present work has been to investigate which purinergic agonist better mimics endogenous responses. We used different agonist and antagonist of P2 receptors. Contractility and microelectrode experiments were used to compare the effects of exogenously added purines and electrical field stimulation (EFS)-induced nerve mediated effects in rat and human colonic strips. In rat colon, the IJPf and EFS-induced inhibition of contractions were concentration-dependently inhibited by the P2Y1 antagonist MRS2500 but not by iso-PPADS or NF023 (P2X antagonists) up to 1 µM. In samples from human colon, EFS-induced inhibition of contractions was inhibited by either MRS2500 or apamin (1 µM) but not by iso-PPADS. In both species, α,ß-meATP, a stable analog of ATP, caused inhibition of spontaneous contractions. α,ß-meATP effect was concentration-dependent (EC50: 2.7 µM rat, 4.4 µM human) and was antagonized by either MRS2500 or apamin but unaffected by P2X antagonists. ATP, ADP, ß-NAD and ADP-ribose inhibited spontaneous contractions but did not show the same sensitivity profile to purine receptor antagonists as EFS-induced inhibition of contractions. The effect of α,ß-meATP is due to P2Y1 receptor activation leading the opening of sKca channels. Accordingly, α,ß-meATP mimics the endogenous purinergic mediator. In contrast, exogenously added putative neurotransmitters do not exactly mimic the endogenous mediator. Quick degradation by ecto-nuclease or different distribution of receptors (junctionally vs extrajunctionally) might explain these results.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Colo/efeitos dos fármacos , Agonistas Purinérgicos/farmacologia , Antagonistas do Receptor Purinérgico P2/farmacologia , Receptores Purinérgicos P2Y1/fisiologia , Trifosfato de Adenosina/farmacologia , Idoso , Animais , Apamina/farmacologia , Colo/fisiologia , Nucleotídeos de Desoxiadenina/farmacologia , Estimulação Elétrica , Feminino , Humanos , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos Sprague-Dawley , Suramina/análogos & derivados , Suramina/farmacologiaRESUMO
Signaling within and between animal cells is controlled by the many receptor proteins in their membrane. They variously operate as trans-membrane monomers and homo- or hetero-dimers, and may assemble with ion-channels: analyses thereof are needed in studies of receptor actions in tissue physiology and pathology. Interactions between membrane proteins are detectable when pre-labeled with fluorophores, but a much fuller analysis is achievable via advanced optical techniques on living cells. In this context, the measurement of polarization anisotropy in the emitted fluorescence has been the least exploited. Here we demonstrate its methodology and particular advantages in the study of receptor protein assembly. Through excitation in both TIRF and EPI fluorescence illumination modes we are able to quantify and suppress contributions to the signal from extraneous intra-cellular fluorescence, and we show that the loss of fluorescence-polarization measured in membrane proteins reports on receptor protein assembly in real time. Receptor monomers and homo-dimers in the cell membrane can be analyzed quantitatively and for homo-dimers only a single fluorescent marker is needed, thus suppressing ambiguities that arise in alternative assays, which require multiple label moieties and which are thus subject to stoichiometric uncertainty.