RESUMO
Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-ß-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2',7'-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Receptores ErbB , Estradiol , Óxido Nítrico , Fosfatidilinositol 3-Quinases , Fitoestrógenos , Transdução de Sinais , Vasodilatação , Animais , Fitoestrógenos/farmacologia , Estradiol/farmacologia , Óxido Nítrico/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores ErbB/metabolismo , Masculino , Isoflavonas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Genisteína/farmacologia , Receptores de Estrogênio/metabolismo , Ratos WistarRESUMO
The vesicular nucleotide transporter (VNUT) is critical for sympathetic co-transmission and purinergic transmission maintenance. To examine this proposal, we assessed whether the bisphosphonate clodronate, claimed as a potent in vitro VNUT blocker, modified spontaneous and/or the electrically evoked overflow of ATP/metabolites and NA from mesentery sympathetic perivascular nerve terminals. Additionally, in primary endothelial cell cultures derived from this tissue, we also evaluated whether clodronate interfered with ATP/metabolite cell outflow and metabolism of N6-etheno adenosine 5'-triphosphate (eATP), N6-etheno adenosine (eADO), and adenosine deaminase enzyme activity. Rat mesenteries were perfused in the absence or presence of .01-1,000 nM clodronate, 1-1,000 nM Evans blue (EB), and 1-10 µM DIDS; tissue perfusates were collected to determine ATP/metabolites and NA before, during, and after perivascular electrical nerve terminal depolarization. An amount of 1-1,000 nM clodronate did not modify the time course of ATP or NA overflow elicited by nerve terminal depolarization, and only 10 nM clodronate significantly augmented perfusate adenosine. Electrical nerve terminal stimulation increased tissue perfusion pressure that was significantly reduced only by 10 nM clodronate [90.0 ± 18.6 (n = 8) to 35.0 ± 10.4 (n = 7), p = .0277]. As controls, EB, DIDS, or reserpine treatment reduced the overflow of ATP/metabolites and NA in a concentration-dependent manner elicited by nerve terminal depolarization. Moreover, mechanical stimulation of primary endothelial cell cultures from the rat mesentery added with 10 or 100 nM clodronate increased adenosine in the cell media. eATP was metabolized by endothelial cells to the same extent with and without 1-1,000 nM clodronate, suggesting the bisphosphonate did not interfere with nucleotide ectoenzyme metabolism. In contrast, extracellular eADO remained intact, indicating that this nucleoside is neither metabolized nor transported intracellularly. Furthermore, only 10 nM clodronate inhibited (15.5%) adenosine metabolism to inosine in endothelial cells as well as in a commercial crude adenosine deaminase enzyme preparation (12.7%), and both effects proved the significance (p < .05). Altogether, present data allow inferring that clodronate inhibits adenosine deaminase activity in isolated endothelial cells as in a crude extract preparation, a finding that may account for adenosine accumulation following clodronate mesentery perfusion.
RESUMO
P2 × 4R is allosterically modulated by Zn(II), and despite the efforts to understand the mechanism, there is not a consensus proposal; C132 is a critical amino acid for the Zn(II) modulation, and this residue is located in the receptor head domain, forming disulfide SS3. To ascertain the role of the SS2/SS3 microenvironment on the rP2 × 4R Zn(II)-induced allosteric modulation, we investigated the contribution of each individual SS2/SS3 cysteine plus carboxylic acid residues E118, E160, and D170, located in the immediate vicinity of the SS2/SS3 disulfide bonds. To this aim, we combined electrophysiological recordings with protein chemical alkylation using thiol reagents such as N-ethylmaleimide or iodoacetamide, and a mutation of key amino acid residues together with P2 × 4 receptor bioinformatics. P2 × 4R alkylation in the presence of the metal obliterated the allosteric modulation, a finding supported by the site-directed mutagenesis of C132 and C149 by a corresponding alanine. In addition, while E118Q was sensitive to Zn(II) modulation, the wild type receptor, mutants E160Q and D170N, were not, suggesting that these acid residues participate in the modulatory mechanism. Poisson-Boltzmann analysis indicated that the E160Q and D170N mutants showed a shift towards more positive electrostatic potential in the SS2/SS3 microenvironment. Present results highlight the role of C132 and C149 as putative Zn(II) ligands; in addition, we infer that acid residues E160 and D170 play a role attracting Zn(II) to the head receptor domain.
Assuntos
Receptores Purinérgicos P2X4/metabolismo , Zinco/metabolismo , Regulação Alostérica/fisiologia , Substituição de Aminoácidos , Animais , Humanos , Mutação de Sentido Incorreto , Domínios Proteicos , Receptores Purinérgicos P2X4/genética , Xenopus laevisRESUMO
Since the mechanism of human diabetic peripheral neuropathy and vascular disease in type 1 diabetes mellitus remains unknown, we assessed whether sympathetic transmitter overflow is altered by this disease and associated to vascular dysfunction. Diabetes was induced by streptozotocin (STZ)-treatment and compared to vehicle-treated rats. Aliquots of the ex vivo perfused rat arterial mesenteric preparation, denuded of the endothelial layer, were collected to quantify analytically sympathetic nerve co-transmitters overflow secreted by the isolated mesenteries of both groups of rats. Noradrenaline (NA), neuropeptide tyrosine (NPY), and ATP/metabolites were detected before, during, and after electrical field stimulation (EFS, 20 Hz) of the nerve terminals surrounding the mesenteric artery. NA overflow was comparable in both groups; however, basal or EFS-secreted ir-NPY was 26% reduced (p < 0.05) in diabetics. Basal and EFS-evoked ATP and adenosine (ADO) overflow to the arterial mesentery perfusate increased twofold and was longer lasting in diabetics; purine tissue content was 37.8% increased (p < 0.05) in the mesenteries from STZ-treated group of rats. Perfusion of the arterial mesentery vascular territory with 100 µM ATP, 100 nM 2-MeSADP, or 1 µM UTP elicited vasodilator responses of the same magnitude in controls or diabetics, but the increase in luminally accessible NO was 60-70% lower in diabetics (p < 0.05). Moreover, the concentration-response curve elicited by two NO donors was displaced downwards (p < 0.01) in diabetic rats. Parallel studies using primary cultures of endothelial cells from the arterial mesentery vasculature revealed that mechanical stimulation induced a rise in extracellular nucleotides, which in the cells from diabetic rats was larger and longer-lasting when comparing the extracellular release of ATP and ADO values to those of vehicle-treated controls. A 5 min challenge with purinergic agonists elicited a cell media NO rise, which was reduced in the endothelial cells from diabetic rats. Present findings provide neurochemical support for the diabetes-induced neuropathy and show that mesenteric endothelial cells alterations in response to mechanical stimulation are compatible with the endothelial dysfunction related to vascular disease progress.
RESUMO
The vas deferens is a simple bioassay widely used to study the physiology of sympathetic neurotransmission and the pharmacodynamics of adrenergic drugs. The role of ATP as a sympathetic co-transmitter has gained increasing attention and furthered our understanding of its role in sympathetic reflexes. In addition, new information has emerged on the mechanisms underlying the storage and release of ATP. Both noradrenaline and ATP concur to elicit the tissue smooth muscle contractions following sympathetic reflexes or electrical field stimulation of the sympathetic nerve terminals. ATP and adenosine (its metabolic byproduct) are powerful presynaptic regulators of co-transmitter actions. In addition, neuropeptide Y, the third member of the sympathetic triad, is an endogenous modulator. The peptide plus ATP and/or adenosine play a significant role as sympathetic modulators of transmitter's release. This review focuses on the physiological principles that govern sympathetic co-transmitter activity, with special interest in defining the motor role of ATP. In addition, we intended to review the recent structural biology findings related to the topology of the P2X1R based on the crystallized P2X4 receptor from Danio rerio, or the crystallized adenosine A2A receptor as a member of the G protein coupled family of receptors as prototype neuro modulators. This review also covers structural elements of ectonucleotidases, since some members are found in the vas deferens neuro-effector junction. The allosteric principles that apply to purinoceptors are also reviewed highlighting concepts derived from receptor theory at the light of the current available structural elements. Finally, we discuss clinical applications of these concepts.
Assuntos
Junção Neuroefetora/fisiologia , Ducto Deferente/fisiologia , Animais , Epitélio/fisiologia , Humanos , Masculino , Junção Neuroefetora/anatomia & histologia , Receptores Purinérgicos/metabolismo , Ducto Deferente/anatomia & histologiaRESUMO
Translation initiation from the human immunodeficiency virus type-1 (HIV-1) mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS)-1, a cis-acting regulatory element present within the gag open reading frame (ORF). In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1.
Assuntos
Genes gag/genética , HIV-1/genética , Fases de Leitura Aberta/genética , Células HeLa , Humanos , Immunoblotting , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genéticaRESUMO
Although the physiological function of the cellular prion protein (PrP(C)) remains unknown, several evidences support the notion of its role in copper homeostasis. PrP(C) binds Cu(2+) through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu(2+) of the adenosine triphosphate (ATP)-evoked currents in the P2X(4) receptor subtype, highlighting a modulatory role for PrP(C) in synaptic transmission through regulation of Cu(2+) levels. Here, we study the effect of full-length PrP(C) in Cu(2+) inhibition of P2X(4) receptor when both are coexpressed. PrP(C) expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X(4) receptors. However, the presence of PrP(C) reduces the inhibition by Cu(2+) of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu(2+) binding domain. Thus, our observations suggest a role for PrP(C) in modulating synaptic activity through binding of extracellular Cu(2+).
RESUMO
The development of sympathetic overactivity and hypertension in rats submitted to chronic intermittent hypoxia (CIH) involve alterations in the central mechanisms controlling respiratory and autonomic functions. Herein, we assessed whether CIH alters glutamatergic and/or purinergic signaling in the ventrolateral medulla (VLM), a region that encompasses the pre-sympathetic neurons and respiratory neurons of the ventral respiratory column. Groups of juvenile rats were exposed for 10 days to CIH (6% O(2) for 40s, every 9min, 8h/day) or normoxia (controls). Following treatment, in situ working heart-brainstem preparations were performed to record simultaneously respiratory and sympathetic motor outputs. In separate CIH and control groups, the VLM was dissected for western-blot analyses of ionotropic glutamatergic and P2 receptors. l-glutamate microinjections (1, 3 or 10mM) into VLM of control (n=6) and CIH groups (n=10) produced similar increases of sympathetic and abdominal activities associated with phrenic nerve inhibition; immunoreactive NMDAR1 and GluR2/3 densities at the VLM were also alike between groups (n=4). In contrast, VLM microinjections of ATP (1, 10 or 50mM) evoked larger sympatho-excitatory responses in CIH (n=8) than in control rats (n=7, P<0.05) whilst the abdominal increase and phrenic nerve inhibition were of comparable magnitudes. The immunoreactive densities of P2X3 and P2X4 receptors, but not P2X1 and P2Y2, were 20% higher in VLM of CIH (n=8; P<0.05) than controls (n=8). Altogether, our findings suggest that CIH augments purinergic signaling in the VLM, supporting the concept that nucleotides play a role in the dynamic central control of the sympathetic autonomic function.
Assuntos
Trifosfato de Adenosina/metabolismo , Ácido Glutâmico/metabolismo , Hipóxia/fisiopatologia , Bulbo/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Animais , Western Blotting , Hipóxia/metabolismo , Masculino , Bulbo/metabolismo , Ratos , Ratos Wistar , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X4/metabolismoRESUMO
Zn²(+) is an essential ion that is stored in and co-released from glutamatergic synapses and it modulates neurotransmitter receptors involved in long-term potentiation (LTP). However, the mechanism(s) underlying Zn²(+) -induced modulation of LTP remain(s) unclear. As the purinergic P2X receptors are relevant targets for Zn²(+) action, we have studied their role in LTP modulation by Zn²(+) in the CA1 region of rat hippocampal slices. Induction of LTP in the presence of Zn²(+) revealed a biphasic effect - 5-50 µm enhanced LTP induction, whereas 100-300 µm Zn²(+) inhibited LTP. The involvement of a purinergic mechanism is supported by the fact that application of the P2X receptor antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP) and periodate-oxidized ATP fully abolished the facilitatory effect of Zn²(+) . Notably, application of the P2X7 receptor-specific antagonist Brilliant Blue G did not modify the Zn²(+) -dependent facilitation of LTP. Exogenous ATP also produced a biphasic effect - 0.1-1 µm ATP facilitated LTP, whereas 5-10 µm inhibited LTP. The facilitatory effect of ATP was abolished by the application of TNP-ATP and was modified in the presence of 5 µm Zn²(+) , suggesting that P2X receptors are involved in LTP induction and that Zn²(+) leads to an increase in the affinity of P2X receptors for ATP. The latter confirms our previous results from heterologous expression systems. Collectively, our results indicate that Zn²(+) at low concentrations enhances LTP by modulating P2X receptors. Although it is not yet clear which purinergic receptor subtype(s) is responsible for these effects on LTP, the data presented here suggest that P2X4 but not P2X7 is involved.
Assuntos
Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Receptores Purinérgicos P2X/metabolismo , Zinco/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Eletrofisiologia , Potenciação de Longa Duração/fisiologia , Masculino , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Receptores Purinérgicos P2X7/metabolismoRESUMO
In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.
Assuntos
Regiões 5' não Traduzidas , Vírus do Tumor Mamário do Camundongo/genética , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Animais , Linhagem Celular , Humanos , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oócitos/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas , Capuzes de RNA/antagonistas & inibidores , RNA Mensageiro/química , Coelhos , Xenopus laevis , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
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.
Assuntos
Córion/irrigação sanguínea , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Receptores Purinérgicos P2/biossíntese , Uridina Trifosfato/metabolismo , Actinas/química , Artérias/metabolismo , Feminino , Humanos , Ligantes , Microdomínios da Membrana/metabolismo , Placenta/metabolismo , Gravidez , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2Y2 , Transdução de Sinais , Uridina Trifosfato/química , Sistema Vasomotor/fisiologiaRESUMO
P2X receptor channels (P2XRs) are allosterically modulated by several compounds, mainly acting at the ectodomain of the receptor. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X(2)R and inhibits the activity of P2X(4)R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X(2) subunit, termed P2X(2a), and a splice variant lacking a 69 residue segment in the C terminal, termed P2X(2b), as the donors for intracellular and transmembrane segments and the P2X(4) subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X(4/2a) chimera but was absent in oocytes expressing P2X(4/2b) chimera. Site-directed mutagenesis experiments revealed that the Cys(430) residue mediates effects of mercury on the P2X(2a)R activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H(2)O(2)) and mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These experiments, done in both oocytes and human embryonic kidney HEK293 cells, revealed that these compounds potentiated the ATP-evoked P2X(2a)R and P2X(4/2a)R currents but not P2X(2b)R and P2X(2a)-C430A and P2X(2a)-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H(2)O(2) potentiation. Alkylation of Cys(430) residue with methylmethane-thiosulfonate also abolished the mercury and H(2)O(2) potentiation. Altogether, these results are consistent with the hypothesis that the Cys(430) residue is an intracellular P2X(2a)R redox sensor.
Assuntos
Cisteína/química , Cisteína/fisiologia , Líquido Intracelular/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/fisiologia , Animais , Linhagem Celular , Cisteína/genética , Feminino , Humanos , Líquido Intracelular/química , Oxirredução , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Xenopus laevis/metabolismoRESUMO
The human embryonic-lethal abnormal vision (ELAV)-like protein, HuR, has been recently found to be involved in the regulation of protein synthesis. In this study we show that HuR participates in the translational control of the HIV-1 and HCV IRES elements. HuR functions as a repressor of HIV-1 IRES activity and acts as an activator of the HCV IRES. The effect of HuR was evaluated in three independent experimental systems, rabbit reticulocyte lysate, HeLa cells, and Xenopus laevis oocytes, using both overexpression and knockdown approaches. Furthermore, results suggest that HuR mediated regulation of HIV-1 and HCV IRESes does not require direct binding of the protein to the RNA nor does it need the nuclear translocation of the IRES-containing RNAs. Finally, we show that HuR has a negative impact on post-integration steps of the HIV-1 replication cycle. Thus, our observations yield novel insights into the role of HuR in the post-transcriptional regulation of HCV and HIV-1 gene expression.
Assuntos
Antígenos de Superfície/metabolismo , HIV-1/metabolismo , Hepacivirus/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Animais , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação Viral da Expressão Gênica , HIV-1/fisiologia , Células HeLa , Hepacivirus/fisiologia , Humanos , Oócitos , RNA Viral/metabolismo , Coelhos , Ribossomos/metabolismo , Xenopus laevisRESUMO
Epinephrine plays a key role in the control of vasomotor tone; however, the participation of the NO/cGMP pathway in response to beta-adrenoceptor activation remains controversial. To evaluate the involvement of the endothelium in the vascular response to epinephrine, we assessed NO production, endothelial NO synthase phosphorylation, and tissue accumulation of cGMP in the perfused arterial mesenteric bed of rat. Epinephrine elicited a concentration-dependent increase in NO (EC(50) of 45.7 pM), which was coupled to cGMP tissue accumulation. Both NO and cGMP production were blocked by either endothelium removal (saponin) or NO synthase inhibition (N(omega)-nitro-L-arginine). Blockade of beta(1)- and beta(2)-adrenoceptors with 1 microM propranolol or beta(3)-adrenoceptor with 10 nM SR 59230A displaced rightward the concentration-NO production curve evoked by epinephrine. Selective stimulation of beta(1)-, beta(2)-, or beta(3)-adrenoceptors also resulted in NO and cGMP production. Propranolol (1 microM) inhibited the rise in NO induced by isoproterenol or the beta(2)-adrenoceptor agonists salbutamol, terbutaline, or fenoterol. Likewise, 10 nM SR 59230A reduced the effects of the beta(3)-adrenoceptor agonists BRL 37344, CGP 12177, SR 595611A, or pindolol. The NO production induced by epinephrine and BRL 37344 was associated with the activation of the phosphatidylinositol 3-kinase/Akt pathway and phosphorylation of eNOS in serine 1177. In addition, in anaesthetized rats, bolus administration of isoproterenol, salbutamol, or BRL 37344 produced NO-dependent reductions in systolic blood pressure. These findings indicate that beta(1)-, beta(2)-, and beta(3)-adrenoceptors are coupled to the NO/cGMP pathway, highlighting the role of the endothelium in the vasomotor action elicited by epinephrine and related beta-adrenoceptor agonists.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Epinefrina/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/biossíntese , Receptores Adrenérgicos beta/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , GMP Cíclico/biossíntese , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Luminescência , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Contração Muscular/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Circulação Esplâncnica/efeitos dos fármacos , Circulação Esplâncnica/fisiologia , Resistência Vascular/efeitos dos fármacosRESUMO
The nucleotide P2Y(1) receptor (P2Y(1)R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y(1)R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y(1)R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl beta-cyclodextrin reduced the raft partitioning of the P2Y(1)R and obliterated the P2Y(1)R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y(1)R agonist, not only displaced within 4 min the P2Y(1)R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N(6)-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y(1)R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y(1)R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y(1)R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y(1)R to membrane rafts, highlighting the role of this microdomain in P2Y(1)R signaling.
Assuntos
Vasos Sanguíneos/metabolismo , Microdomínios da Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Agonistas do Receptor Purinérgico P2 , Vasos Sanguíneos/fisiologia , Feminino , Humanos , Técnicas In Vitro , Contração Muscular , Músculo Liso Vascular/fisiologia , Placenta/irrigação sanguínea , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2Y1 , Transdução de SinaisRESUMO
Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.
Assuntos
Sítio Alostérico/fisiologia , Encéfalo/metabolismo , Cobre/metabolismo , Receptores Purinérgicos P2/metabolismo , Oligoelementos/metabolismo , Zinco/metabolismo , Regulação Alostérica/fisiologia , Animais , Barreira Hematoencefálica/metabolismo , Química Encefálica/fisiologia , Cobre/química , Homeostase , Humanos , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Potenciais da Membrana , Neurônios/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X , Oligoelementos/química , Zinco/químicaRESUMO
Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X4 receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His140, a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr123 and Thr146 for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys132, but not Cys126, proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp138, but not Asp131 was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp129, Asp131, and Thr133 had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability.
Assuntos
Substituição de Aminoácidos , Cobre/farmacologia , Mutação de Sentido Incorreto , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/metabolismo , Zinco/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sítio Alostérico/genética , Motivos de Aminoácidos/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Cobre/metabolismo , Relação Dose-Resposta a Droga , Feminino , Ativação do Canal Iônico/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismo , Oócitos/citologia , Estrutura Terciária de Proteína/genética , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X4 , Xenopus laevis , Zinco/metabolismoRESUMO
The P2X7 receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X7 receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 +/- 1.0 micromol/L. The inhibition was non-competitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219A were less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 +/- 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X7 receptor allosteric modulation.
Assuntos
Membrana Celular/metabolismo , Líquido Extracelular/metabolismo , Histidina/metabolismo , Metais/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Cátions Bivalentes/metabolismo , Cátions Bivalentes/farmacologia , Membrana Celular/efeitos dos fármacos , Cobre/metabolismo , Cobre/farmacologia , Feminino , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Magnésio/metabolismo , Magnésio/farmacologia , Metais/farmacologia , Mutação/genética , Oócitos , Prótons , Ratos , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Xenopus laevis , Zinco/metabolismo , Zinco/farmacologiaRESUMO
Successful trials with 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol, CQ) for Alzheimer's disease treatment prompted renewed interest in assessing whether its therapeutic action is related to the coordination of neurotoxic trace metals, such as Cu(II) and Zn(II). We now report conditional stability constants (K(C')) for CQ Cu(II) and Zn(II) complexes measured in a biological buffer containing Ca(II) and Mg(II) ions. UV-vis spectroscopy and polarography evidenced a 1:2 stoichiometry of Cu(II) and Zn(II) CQ complexes; the K(C')s calculated were: Cu(CQ)(2) 1.2x10(10), and Zn(CQ)(2) 7.0x10(8)M(-2); the CQ affinity for Cu(II) is at least an order of magnitude higher than for Zn(II). To test the possible functional relevance of the Cu(II) CQ complexes in the brain, we bioassayed free Cu(II) concentration by the metal-induced inhibition of ATP-gated currents of the P2X(4) receptor, a predominant brain P2X receptor. CQ reduced concentration-dependently the Cu(II) inhibition of the ATP-gated currents. In view that the stability constant of CQ for Zn(II) is similar to that of Abeta-amyloid for Zn(II), and the fact that CQ may form complexes with Cu(II), even in the presence of competing ions, the present results highlight that the formation of Cu(II) CQ complexes in the brain may act by diminishing free Cu(II) concentrations modifying thereby brain excitability, or favoring the degradation of beta-amyloid plaques or huntingtin, rather than through a specific effect of CQ itself.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Clioquinol/química , Clioquinol/uso terapêutico , Cobre/química , Doença de Huntington/tratamento farmacológico , Zinco/química , Trifosfato de Adenosina/fisiologia , Algoritmos , Animais , Fenômenos Químicos , Físico-Química , Cobre/metabolismo , Eletrofisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Microinjeções , Oócitos/fisiologia , Polarografia , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X4 , Soluções , Espectrofotometria Ultravioleta , Xenopus laevisRESUMO
Vasomotion was characterized using human placentae vessel rings; force displacement transducers recorded isometric contractions. Umbilical vein rings display rhythmic contractions occurring with a frequency of 1.47+/-0.01 min(-1) and 274+/-2.2 mg (n=211) of amplitude, which corresponds to 11.1+/-0.4% of the maximal KCl contracture. Vasomotion waves were recorded for up to 8 h; their amplitude and duration was larger in umbilical veins than arteries or chorionic vessels (p<0.001), vasomotion frequency was indistinguishable among these vessels. Segments of the umbilical vein closer to the fetus showed larger amplitudes and longer-lasting waves. Gap junction blockers, including peptide Gap 27, 18alpha-glycyrrhetinic acid, hexanol, heptanol or octanol, reduced the amplitude but not the frequency of vasomotion; all these drugs, in addition, decreased tissue basal tension. The role of intracellular calcium stores was evidenced using calcium-free buffer, which reduced oscillation amplitude and tissue basal tension. Cyclopiazonic acid increased wave amplitude and tissue basal tension, reducing oscillatory frequency. We propose that biological oscillators localized in the smooth muscle layer of the umbilical cord, trigger vasomotion waves, which are synchronized and propagated via gap junctions; internal calcium reservoirs are essential for their maintenance. These myogenic oscillations may be relevant for maternal-fetus blood flow and contribute to fetal nutrition and development.