RESUMEN
Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.
Asunto(s)
Adenosina Trifosfato , Adenilil Ciclasas , Relajación Muscular , Músculo Liso , Testosterona , Tráquea , Uridina Trifosfato , Animales , Uridina Trifosfato/farmacología , Uridina Trifosfato/metabolismo , Cobayas , Relajación Muscular/efectos de los fármacos , Masculino , Adenosina Trifosfato/metabolismo , Tráquea/metabolismo , Tráquea/efectos de los fármacos , Testosterona/farmacología , Testosterona/metabolismo , Adenilil Ciclasas/metabolismo , Músculo Liso/metabolismo , Músculo Liso/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/metabolismo , Transducción de Señal/efectos de los fármacos , Receptores Purinérgicos P2/metabolismoRESUMEN
AIMS: Prostate cancer is the second most frequently malignancy in men worldwide. Most deaths are caused by metastasis, and tumor cell dissemination involves the interaction with endothelial cells. However, the endothelial cell signaling involved in such interaction is not entirely understood. The tumor microenvironment contains extracellular ATP, an endogenous agonist of the purinergic P2Y2 receptor (P2Y2R). P2Y2R signaling changes endothelial cell phenotype, which may be relevant to cancer pathophysiology. Therefore, we hypothesized that P2Y2R activation could favor the metastatic prostate cancer cells adhesion to endothelial cells. MAIN METHODS: For adhesion assays, confluent endothelial cells EA.hy926 were treated with P2Y2R agonists before adding and imaging stained DU-145 cells. Alternatively, fluorescent probes and antibodies were used to determine intracellular endothelial Ca2+, nitric oxide (NO), and flow cytometry assays. KEY FINDINGS: Endothelial P2Y2R activation with ATP, UTP, or the selective agonist 2-thio-UTP increased DU-145 cell adhesion to EA.hy926 cells. This effect required endothelial cell Ca2+ mobilization and relied on the endothelial expression of VCAM-1 and ICAM-1. Conversely, inhibiting this proadhesive endothelial phenotype could impair DU-145 cell adhesion. To evaluate this, we chose atorvastatin based on its notable improvement of endothelial cell dysfunction. Atorvastatin blocked UTP-induced DU-145 cell adhesion to endothelial cell monolayer in a NO-dependent manner, unveiling a P2Y2R and NO signaling crosstalk. SIGNIFICANCE: Endothelial P2Y2R signaling contributes to the adhesion of metastatic prostate cancer cells suggesting that the downstream signaling blockade by statins could be a putative mechanism to reduce prostate cancer metastasis.
Asunto(s)
Células Endoteliales , Neoplasias de la Próstata , Adenosina Trifosfato/metabolismo , Atorvastatina/metabolismo , Adhesión Celular , Células Endoteliales/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/patología , Microambiente Tumoral , Uridina Trifosfato/metabolismoRESUMEN
Extracellular nucleotides are signaling elements present in the tumor microenvironment; however, their role in tumor growth is not completely understood. In the present study, we asked whether nucleotides regulate cell migration in ovarian carcinoma-derived cells. We observed that 100 µM UTP induced migration in SKOV-3 cells (1.57 ± 0.08 fold over basal), and RT-PCR showed expression of transcripts for the P2RY2 and P2RY4 receptors. Knockdown of P2RY2 expression in SKOV-3 cells (P2RY2-KD) abolished the UTP-induced migration. The mechanism activated by UTP to induce migration involves transactivation of the epidermal growth factor receptor (EGFR) since we observed that the EGFR kinase inhibitor AG1478 and the PI3K inhibitor Wortmannin inhibit this response (to 0.76 ± 0.23 and 0.46 ± 0.14 relative to the control, respectively). In agreement with these observations, UTP was able to modify the phosphorylation state of the EGFR; likewise, the induction of ERK1/2 phosphorylation promoted by UTP was abolished by a 30-60 min treatment with AG1478. Our data also suggested that the enhanced cell migration involves the epithelium to mesenchymal transition (EMT) process, since a 12 h stimulation of SKOV-3 cells with 100 µM UTP showed an increase in vimentin and SNAIL protein levels (459.8 ± 132.4% over basal for SNAIL). Interestingly, treatment with apyrase (10 U/mL) reduces the migration of control cells and induces a considerable enrichment of E-cadherin in the cell-cell contacts, favoring an epithelial phenotype and strongly suggesting that the nucleotides released by tumor cells and acting through the P2RY2 receptor are potential regulators of invasiveness.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Receptor Cross-Talk/efectos de los fármacos , Receptores Purinérgicos P2Y2/genética , Uridina Trifosfato/farmacología , Androstadienos/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/metabolismo , Femenino , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tirfostinos/farmacología , Uridina Trifosfato/metabolismo , Vimentina/genética , Vimentina/metabolismo , WortmaninaRESUMEN
BACKGROUND: Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. MATERIALS AND METHODS: In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca(2+) levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. RESULTS: Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. CONCLUSION: Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms.
Asunto(s)
Tonsila Faríngea/citología , Tonsila Faríngea/metabolismo , Modelos Biológicos , Mucina 5AC/metabolismo , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Células Cultivadas , Niño , Preescolar , Cilios/metabolismo , Femenino , Humanos , Masculino , Transporte de Proteínas/fisiología , Uridina Trifosfato/metabolismoRESUMEN
Nucleotides are released into the extracellular milieu from infected cells and cells at inflammatory sites. The extracellular nucleotides bind to specific purinergic (P2) receptors and thereby induce a variety of cellular responses including anti-parasitic effects. Here we investigated whether extracellular nucleotides affect leishmanial infection in macrophages, and found that UTP reduces strongly the parasite load in peritoneal macrophages. Ultrastructural analysis of infected cells revealed that UTP induced morphological damage in the intracellular parasites. Uridine nucleotides also induced dose-dependent apoptosis of macrophages and production of ROI and RNI only in infected macrophages. The intracellular calcium measurements of infected cells showed that the response to UTP, but not UDP, increased the sensitivity and amplitude of cytosolic Ca(2+) changes. Infection of macrophages with Leishmania upregulated the expression of P2Y(2) and P2Y(4) receptor mRNA. The data suggest indirectly that Leishmania amazonensis infection induces modulation and heteromerization of P2Y receptors on macrophages. Thus UTP modulates the host response against L. amazonensis infection. UTP and UTP homologues should therefore be considered as novel components of therapeutic strategies against cutaneous leishmaniasis.
Asunto(s)
Leishmania/patogenicidad , Receptores Purinérgicos/metabolismo , Uridina Trifosfato/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Células Cultivadas , Etiquetado Corte-Fin in Situ , L-Lactato Deshidrogenasa , Leishmania/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Microscopía Electrónica , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno/metabolismo , Receptores Purinérgicos/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Superóxidos/metabolismoRESUMEN
The pyrH-encoded uridine 5'-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg²(+) as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents.
Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mycobacterium tuberculosis/enzimología , Pirimidinas/metabolismo , Transferasas/genética , Transferasas/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Genes Supresores , Guanosina Trifosfato/metabolismo , Cinética , Ligandos , Datos de Secuencia Molecular , Peso Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Espectrometría de Masa por Ionización de Electrospray , Transferasas/química , Transferasas/aislamiento & purificación , Uridina Trifosfato/metabolismoRESUMEN
Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y(2) receptor (P2Y(2)R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-beta-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1-10 mum UTP, a selective P2Y(2)R agonist, displaced the P2Y(2)R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y(2)R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y(2)R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y(2)R in intact human blood vessels.
Asunto(s)
Corion/irrigación sanguínea , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Receptores Purinérgicos P2/biosíntesis , Uridina Trifosfato/metabolismo , Actinas/química , Arterias/metabolismo , Femenino , Humanos , Ligandos , Microdominios de Membrana/metabolismo , Placenta/metabolismo , Embarazo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2Y2 , Transducción de Señal , Uridina Trifosfato/química , Sistema Vasomotor/fisiologíaRESUMEN
Purinergic receptors are expressed in the membrane of the follicular cell layer that communicates with the Xenopus oocyte. Adenosine (Ado) generates a cAMP-dependent K(+) current (I(K,cAMP)), whereas ATP activates a Cl(-) current (F(Cl)) and has a dual effect on I(K,cAMP), provoking both its activation and inhibition. Here, purinergic responses were studied electrophysiologically, first in the whole follicle (w.f.), and then in the same follicle after removal of its epithelium/theca layers (e.t.r. follicle). Responses were analyzed as the ratio of the current amplitudes (i(etr)/i(wf)) in the two preparations. For ATP activation of I(K,cAMP) and F(Cl), the ratios i(etr)/i(wf) were 0.053 and 22, respectively, whereas that for Ado was 0.75. Thus, epithelium/theca removal drastically altered the ATP response, suggesting a change in the signaling pathway that correlated with changes in the pharmacological characteristics: the half-maximal effective concentration for activation of the main current in w.f. (I(K,cAMP)) was 14 +/- 3.8 microM [Hill coefficient (nH) = 2.7 +/- 0.61], and that in e.t.r. follicles (F(Cl)) was 1.8 +/- 0.68 microM (nH = 0.76 +/- 0.09), whereas Ado-response parameters did not change. Responses to UTP and beta,gamma-methylene-ATP, specific agonists for I(K,cAMP) inhibition and activation, respectively, indicated that in e.t.r. follicles inhibition increased and activation decreased drastically. Thus, purinergic responses were not independent; instead, they were functionally linked. We hypothesize that this property was due to direct interactions between receptors for Ado (A2 subtype) and ATP (P2Y subtype) in the Xenopus follicle.
Asunto(s)
Folículo Ovárico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos/metabolismo , Xenopus laevis/metabolismo , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , AMP Cíclico/metabolismo , Femenino , Cinética , Técnicas de Placa-Clamp , Uridina Trifosfato/metabolismoRESUMEN
Cryptococcus neoformans is the causative agent of pulmonary cryptococcosis and cryptococcal meningoencephalitis, which are major clinical manifestations in immunosuppressed patients. In the present study, a surface ATPase (ecto-ATPase) was identified in C. neoformans yeast cells. Intact yeasts hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 29.36+/-3.36nmol Pi/hx10(8) cells. In the presence of 5 mM MgCl(2), this activity was enhanced around 70 times, and an apparent K(m) for Mg-ATP corresponding to 0.61mM was determined. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases, V-ATPases, Na(+)-ATPases or P-ATPases had no effect on the cryptococcal ATPase, but extracellular impermeant compounds reduced enzyme activity in living cells. ATP was the best substrate for the cryptococcal ecto-enzyme, but it also efficiently hydrolyzed inosine 5'-triphosphate (ITP), cytidine 5'-triphosphate (CTP), guanosine 5'-triphosphate (GTP) and uridine-5'-triphosphate (UTP). In the presence of ATP, C. neoformans became less susceptible to the antifungal action of fluconazole. Our results are indicative of the occurrence of a C. neoformans ecto-ATPase that may have a role in fungal physiology.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cryptococcus neoformans/enzimología , Adenosina Trifosfato/metabolismo , Antifúngicos/farmacología , Pared Celular/metabolismo , Cryptococcus neoformans/efectos de los fármacos , Citidina Trifosfato/metabolismo , Relación Dosis-Respuesta a Droga , Fluconazol/farmacología , Guanosina Trifosfato/metabolismo , Inosina Trifosfato/metabolismo , Cloruro de Magnesio/farmacología , Uridina Trifosfato/metabolismoRESUMEN
1. Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X(7) subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2. P2Y and P2X receptor agonists ATP, uridine 5'-triphosphate (UTP) and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X(7) receptor. Activation was maximal at 5 - 10 min and was sustained for 60 min; the EC(50) for BzATP was approximately 50 microM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X(7) receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5. In conclusion, our data indicate that stimulation of astrocytic P2X(7) receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Astrocitos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Astrocitos/efectos de los fármacos , Calcio/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ácido Glutámico/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt , Ratas , Receptores Purinérgicos P2/clasificación , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X7 , Transducción de Señal/fisiología , Uridina Trifosfato/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Extracellular purines (adenosine triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine) and pyrimidines (uridine 5'-triphosphate (UTP) and UDP) are important signaling molecules that mediate diverse biological effects via P1 and P2 purinergic receptors. The human glioma cell lines U87 MG, U251 MG and U138 MG were treated with purines and pyrimidines for 24 or 48 h and proliferation was measured by [3H]-thymidine incorporation, flow cytometry and cell counting. The studies showed that extracellular nucleotides and nucleosides induce proliferation of the studied glioma cells. Incorporation of [3H]-thymidine followed the order of ATP approximately equal to guanosine approximately equal to inosine approximately equal to adenosine > UTP > ADP while ATPgammaS and 2MeSATP had no effect. The effect of ATP was partially inhibited by suramin and by reactive blue 2 (RB2). Co-treatment with the following antagonists of P1 purinoreceptors DPCPX, CPT or 8PT did not block the effect of adenosine while a specific antagonist of the A3 receptor, MRS1220, totally blocked the effect of adenosine. ATP and adenosine also increased the overall uptake of [3H]-thymidine into the cell, producing a positive effect on the [3H]-thymidine incorporation measurements. These data indicate that the uptake of thymidine and proliferation of gliomas can be induced by purines and pyrimidines via both P1 and P2 purinoceptors.
Asunto(s)
Neoplasias Encefálicas/metabolismo , ADN/biosíntesis , Glioma/metabolismo , Purinas/metabolismo , Uridina Difosfato/metabolismo , Uridina Trifosfato/metabolismo , Adenosina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , División Celular/fisiología , Espacio Extracelular/metabolismo , Humanos , Nucleósidos/metabolismo , Nucleótidos/metabolismo , Agonistas del Receptor Purinérgico P1 , Agonistas del Receptor Purinérgico P2 , Pirimidinas/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Transducción de Señal/fisiología , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
Both ATP and acetylcholine can induce the mobilization of intracellular calcium in the early developing chick embryo retina, a response that decreases during retinal development. In this study, the effects of these transmitters on the turnover of phosphoinositides and proliferation of developing retinal cells in culture were characterized. While ATP, UTP or carbachol were able to induce a >400% accumulation of phosphoinositides in retinal cell cultures, only ATP promoted a dose-dependent increase in [(3)H]-thymidine incorporation in cultured cells (EC(50)=8.6 microM), a response that was inhibited by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (0.1 or 0.25 mM). ADP, but not UTP or adenosine, also stimulated the proliferation of retinal cells (EC(50)=5.8 microM), indicating that activation of P2Y1 receptors mediates the proliferative response of retinal cells to ATP. The mitogenic effect of ATP was completely prevented by the PKC inhibitor chelerythrine chloride (0.5 microM) and the phospholipase C (PLC) inhibitor U73122 (0.5 microM). PD 98059 (25 or 50 microM), an inhibitor of the activation of extracellular signal-regulated kinases (ERKs) also blocked the increase in [(3)H]-thymidine incorporation induced by ATP. Moreover, the effect of ATP was pronounced in cultures obtained from retinas at embryonic days 6-8, but not at day 9. Since Müller and bipolar cells are the predominant cell types that proliferate at these embryonic stages, our data suggest that ATP, through activation of P2Y1 receptors coupled to phospholipase C, PKC and MAP kinases, affects DNA synthesis in one or both of these cell types in culture.
Asunto(s)
Adenosina Trifosfato/metabolismo , División Celular/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Fosfatidilinositoles/metabolismo , Proteína Quinasa C/metabolismo , Fosfato de Piridoxal/análogos & derivados , Retina/embriología , Fosfolipasas de Tipo C/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Carbacol/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Modelos Animales , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Proteína Quinasa C/efectos de los fármacos , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Fosfato de Piridoxal/farmacología , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1 , Retina/efectos de los fármacos , Retina/metabolismo , Timidina/metabolismo , Fosfolipasas de Tipo C/efectos de los fármacos , Uridina Trifosfato/metabolismo , Uridina Trifosfato/farmacologíaRESUMEN
We have recently reported the existence of ATPase activity capable of hydrolyzing extracellular ATP and localized at the external cell membrane of goldfish hepatocytes [Am. J. Physiol. (1998) 274 R1031]. In the present study, we investigated whether one or more enzymes of the ATP diphosphohydrolase family (called E-NTPDases) are responsible for the hydrolysis of extracellular ATP and other nucleotides. Using soluble extracts from goldfish liver, enzyme activity was detected in the presence of ATP (32.1+/-4.0 nmol Pi liberated mg protein(-1) min(-1)), ADP (20.7+/-3.3 nmol Pi liberated mg protein(-1) min(-1)) and UTP (20.7+/-1.2 nmol Pi liberated mg protein(-1) min(-1)). In line with the presence of this hydrolytic activity, liver samples separated by non-denaturing gel electrophoresis and subsequently exposed to either ATP, ADP or UTP yielded a single band with enzyme activity and similar electrophoretic mobility. Subsequent SDS-PAGE electrophoresis of the active bands resulted in the appearance of two protein bands with molecular masses of 70 and 64 kDa. Immunoblotting of soluble extracts and microsomes obtained from goldfish liver, using a monoclonal antibody against CD39 (a well-known E-NTPDase), detected a single 97-kDa protein. The enzyme activity measured in solution and in native gels, together with structural information from denaturing gels plus immunoblots, points to the existence, in goldfish liver, of at least two different E-NTPDases.
Asunto(s)
Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/aislamiento & purificación , Hígado/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Carpa Dorada , Hepatocitos/metabolismo , Immunoblotting , Nucleósido-Trifosfatasa , Especificidad por Sustrato , Uridina Trifosfato/metabolismoRESUMEN
A rat brain extract, able to synthesize from UDP-Glc an alpha-1,4-glucan covalently bound to a protein in the absence of added primer is described. The compound formed is precipitable by dilute trichloroacetic acid (TCA). In the presence of glycogen, added as primer, this molecule is enlarged and is not precipitable by TCA. Unprimed and primed activities differ in several aspects, such as the behavior in the presence of some effectors, and the optimum pH. Umprimed and primed activities presented two pHs optima, both sharing only one. The proteoglucans synthesized under the different pHs gave different patterns after analysis under denaturing PAGE and the oligosaccharides synthesized on the protein backbone differ in the glucosyl length. It is concluded that also in rat brain, the initiation process of glycogen biosynthesis is mediated through the formation of a glycoprotein. Our present results showed that the step of the putative "Glycogen Initiator" proposed by use before, requires two enzymes UDPGlc-transglucosylating activities, Glycogen Initiator 1 and Glycogen Initiator 2, before Glycogen Synthase in the alpha-1,4-glucosidic linkages formation.
Asunto(s)
Encéfalo/enzimología , Glucógeno Sintasa/metabolismo , Glucógeno/biosíntesis , Animales , Cromatografía Líquida de Alta Presión , Glucosa/metabolismo , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Manganeso/metabolismo , Proteoglicanos/metabolismo , Ratas , Ratas Endogámicas , Uridina Trifosfato/metabolismoRESUMEN
Purified sarcoplasmic reticulum ATPase was phosphorylated by either ATP or UTP under otherwise identical conditions. Calcium, pH, and nucleotide concentrations were adjusted to permit maximal steady-state accumulation of phosphoenzyme (EP). Either 4 or 8.5 nmol of EP/mg of protein were obtained with ATP or UTP, respectively. Tryptic digestion of phosphorylated ATPase followed by acid gel electrophoresis showed that EP from UTP was on fragment A1, similar to the report in the literature for EP from ATP. Phosphorylation with Pi in the absence of calcium gave EP levels similar to those obtained from UTP. Thus, comparison of EP levels from different substrates measured in parallel in the same preparation reveal that with ATP half of the sites are phosphorylated. Illumination of the ATPase with UV light in the presence of [3H]UTP caused photolabeling of the ATPase at a maximal level of 1 nmol of [3H]UTP incorporated/mg of ATPase. The UTP concentration dependence for photolabeling was the same as that for promoting catalysis. ATP when present in the illumination protected with a competitive pattern against photolabeling with UTP. Tryptic digestion and autoradiography of photolabeled ATPase revealed that UTP was covalently attached to tryptic fragment A2. The data indicate that a peptide sequence of fragment A2 is involved in the binding of the nucleoside moiety of UTP and possibly belongs to the nucleotide domain of the ATPase in addition to the sequence of fragment A1 which contains the phosphorylation residue.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Retículo Sarcoplasmático/enzimología , Nucleótidos de Uracilo/metabolismo , Uridina Trifosfato/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Marcadores de Afinidad , Calcio/farmacología , Electroforesis en Gel de Poliacrilamida , Fluoresceína-5-Isotiocianato , Fluoresceínas/farmacología , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Cinética , Fosfoproteínas/metabolismo , Fosforilación , Fotoquímica , Temperatura , Tiocianatos/farmacología , Tripsina/metabolismoRESUMEN
Hormones play a role in the regulation of gene expression by inducing changes in enzyme patterns in target cells mediated by the synthesis of specific RNA molecules. Erythropoiesis has been used as a system for studying the molecular mechanism of regulation of gene action by means of two hormones: erythropoietin and testosterone. Experiments designed to correlate the biochemical action of both hormones on rat marrow cells are herein reported. Both factors seems to act at different biochemical and citological levels. Erythropoietin triggers the erythropoietic process acting on the erythropoietin sensitive cells (ESC), in which the hormone induces the synthesis of a high molecular weight RNA, which is the precursor of a functional 9 S messenger RNA. Testosterone seems to act on polychromatophilic erythroblasts, in which the synthesis of ribosomal RNA or its precursor is stimulated. The steroid enhances the nuclear ribonuclease activity, which could represent a control mechanism for the processing (maturation) of high molecular weight RNAs. The incorporation of 3H-GTP and 3H-UTP into RNA by isolated rat bone marrow nuclei is stimulated by erythropoietin and testosterone. Using alpha-amanitine and different ionic strength conditions it was found that erythropoietin enhances preferentially RNA polymerase II activity while testosterone increases RNA polymerase I activity. It is postulated that erythropoietin and testosterone act synergically to create the biochemical machinery for hemoglobin synthesis, the macromolecule that characterizes the erythropoietic process.