RESUMO
ATP-gated P2X(2) receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X(2) receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X(2)-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X(2)-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X(2)-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X(2) receptor distribution on the seconds time scale.
Assuntos
Dendritos/metabolismo , Hipocampo/citologia , Neurônios/citologia , Neurônios/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Substituição de Aminoácidos/genética , Animais , Tamanho Celular/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Condutividade Elétrica , Ácido Glutâmico/farmacologia , Hipocampo/embriologia , Neurônios/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Óptica e Fotônica , Pseudópodes/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Proteínas Recombinantes de Fusão/metabolismo , Sindbis virus/genética , Xenopus laevisRESUMO
ATP is found in every cell, where it is a major source of energy. But in the nervous system, ATP also has additional actions, which include its role in fast synaptic transmission and modulation. Here I discuss the 'fast' actions of ATP at synapses, the properties of the receptors that are activated by ATP and the physiology of ATP signalling, with emphasis on its role in pain processing.
Assuntos
Trifosfato de Adenosina/metabolismo , Neurônios/fisiologia , Receptores Purinérgicos P2/metabolismo , Sinapses/metabolismo , Animais , Eletrofisiologia , Modelos Moleculares , Dor/metabolismo , Transdução de Sinais/fisiologiaRESUMO
ATP acts as a humoral mediator to control cell function extracellularly. The receptors that mediate the actions of ATP belong to two classes, the metabotropic P2Y receptors and the transmitter-gated, ion channel P2X receptors. This review describes the structure, distribution, function, and ligand recognition characteristics of P2X receptors, which comprise seven distinct subunits that can function as both homo- and hetero- polymers. The pharmacology of P2X receptors is complicated by marked differences between species orthologues. The current nomenclature is based largely on recombinant receptor studies and detailed knowledge of endogenous P2X receptors in native tissues is limited because of lack of good selective agonists and antagonists for each receptor type.
Assuntos
Receptores Purinérgicos P2/classificação , Terminologia como Assunto , Animais , Eletrofisiologia , Humanos , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiologiaRESUMO
Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.
Assuntos
Ansiedade/genética , Dopamina/metabolismo , Mutação Puntual , Receptores Nicotínicos/metabolismo , Animais , Feminino , Heterozigoto , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Gravidez , Ratos , Receptores Nicotínicos/genéticaRESUMO
Trigeminal mesencephalic nucleus (MNV) neurones express functional P2X receptors. In order to determine the molecular identity of the P2X receptors in this nucleus we have used whole cell patch clamp recording of P2X receptor-mediated currents to determine the pharmacological properties of the receptors, and have compared them with those of cloned P2X receptor subunits. The purine nucleotides ATP (300 microM), ATP-gamma-S (30 microM) and alphabetameATP (300 microM) evoked inward currents in all MNV neurones whereas alphabetameADP (300 microM) did not. betagammame-L-ATP (300 microM) evoked only a small ( approximately 20 pA) current in 3 out of 6 MNV neurones. The P2X receptor antagonist TNP-ATP (10 nM-10 microM) and raised extracellular Ca(2+) (8 and 30 mM) reduced, but did not abolish, the current evoked by ATP-gamma-S. The current remaining in TNP-ATP was insensitive to blockade by raised Ca(2+). These properties suggest that MNV neurones do not express homomeric P2X(3), P2X(4) or P2X(6) receptors. Whilst the TNP-ATP-insensitive ATP-gamma-S-evoked current has many characteristics similar to both homomeric P2X(2) and P2X(5) receptors, its insensitivity to blockade by raised Ca(2+) is difficult to reconcile with the receptor being a P2X(2) or P2X(5) homomeric channel. More likely, the receptor is a heteromer that comprises either or both of these subunits. The TNP-ATP-sensitive component of the ATP-gamma-S-evoked current is dissimilar to known cloned homomeric or heteromeric P2X receptors.
Assuntos
Mesencéfalo/metabolismo , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Núcleos do Trigêmeo/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Eletrofisiologia , Técnicas In Vitro , Masculino , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Nucleotídeos de Purina/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Ratos Wistar , Receptores Purinérgicos P2X , Núcleos do Trigêmeo/citologia , Núcleos do Trigêmeo/efeitos dos fármacosRESUMO
Transmitter-gated cation channels are detectors of excitatory chemical signals at synapses in the nervous system. Here we show that structurally distinct alpha3beta4 nicotinic and P2X2 channels influence each other when co-activated. The activation of one channel type affects distinct kinetic and conductance states of the other, and co-activation results in non-additive responses owing to inhibition of both channel types. State-dependent inhibition of nicotinic channels is revealed most clearly with mutant P2X2 channels, and inhibition is decreased at lower densities of channel expression. In synaptically coupled myenteric neurons, nicotinic fast excitatory postsynaptic currents are occluded during activation of endogenously co-expressed P2X channels. Our data provide a molecular basis and a synaptic context for cross-inhibition between transmitter-gated channels.
Assuntos
Ativação do Canal Iônico , Lidocaína/análogos & derivados , Receptores Nicotínicos/metabolismo , Receptores Purinérgicos P2/metabolismo , Acetilcolina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Lidocaína/farmacologia , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Antagonistas Nicotínicos/farmacologia , Receptor Cross-Talk , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Proteínas Recombinantes/metabolismo , Sinapses , XenopusRESUMO
There is now considerable evidence demonstrating that ligand-gated cation channels (i.e., P2X, nicotinic, kainate, NMDA, AMPA and 5-HT(3) receptors), in addition to mediating fast excitatory neurotransmission, may be located presynaptically on nerve terminals in the peripheral and central nervous systems where they function to modulate neurotransmitter release. This modulation can be facilitation, inhibition or both. In this article, we first outline the multiple mechanisms by which activation of presynaptic ligand-gated cation channels can modulate spontaneous and evoked neurotransmitter release, before reviewing in detail published electrophysiological studies of presynaptic P2X, nicotinic, kainate, NMDA, AMPA and 5-HT(3) receptors.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Receptores Pré-Sinápticos/fisiologia , Transmissão Sináptica/fisiologia , Animais , Humanos , Ligantes , Receptores de Glutamato/fisiologia , Receptores Purinérgicos P2/fisiologiaRESUMO
The CNS abundantly expresses P2X receptor channels for ATP; of these the most widespread in the brain is the P2X(4) channel. We show that ivermectin (IVM) is a specific positive allosteric effector of heterologously expressed P2X(4) and possibly of heteromeric P2X(4)/P2X(6) channels, but not of P2X(2), P2X(3), P2X(2)/P2X(3,) or P2X(7) channels. In the submicromolar range (EC(50,) approximately 250 nM) the action of IVM was rapid and reversible, resulting in increased amplitude and slowed deactivation of P2X(4) channel currents evoked by ATP. IVM also markedly increased the potency of ATP and that of the normally low-potency agonist alpha, beta-methylene-ATP in a use- and voltage-independent manner without changing the ion selectivity of P2X(4) channels. Therefore, IVM evokes a potent pharmacological gain-of-function phenotype that is specific for P2X(4) channels. We also tested whether IVM could modulate endogenously expressed P2X channels in the adult trigeminal mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM produced no significant effect on the fast ATP-evoked inward currents in either type of neuron, despite the fact that IVM modulated P2X(4) channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric P2X(4) channels are not the primary subtype of P2X receptor in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons.
Assuntos
Ivermectina/farmacologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/fisiologia , Regulação Alostérica , Animais , Linhagem Celular , Clonagem Molecular , Feminino , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Cinética , Mamíferos , Potenciais da Membrana/efeitos dos fármacos , Neuropeptídeos/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Fenótipo , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Receptores Purinérgicos P2X4 , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Suramina/farmacologia , Transfecção , Xenopus laevisRESUMO
Fast synaptic transmission depends on the selective ionic permeability of transmitter-gated ion channels. Here we show changes in the ion selectivity of neuronal P2X transmitter-gated cation channels as a function of time (on the order of seconds) and previous ATP exposure. Heterologously expressed P2X2, P2X2/P2X3 and P2X4 channels as well as native neuronal P2X channels possess various combinations of mono- or biphasic responses and permeability changes, measured by NMDG+ and fluorescent dye. Furthermore, in P2X4 receptors, this ability to alter ion selectivity can be increased or decreased by altering an amino-acid residue thought to line the ion permeation pathway, identifying a region that governs this activity-dependent change.
Assuntos
Trifosfato de Adenosina/farmacologia , Cátions/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Receptores Purinérgicos P2/fisiologia , Substituição de Aminoácidos , Animais , Benzoxazóis , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , DNA Complementar/genética , Corantes Fluorescentes/metabolismo , Transporte de Íons , Meglumina/metabolismo , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/efeitos dos fármacos , Técnicas de Patch-Clamp , Compostos de Quinolínio , Ratos , Receptores Nicotínicos/fisiologia , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Receptores Purinérgicos P2X4 , Proteínas Recombinantes de Fusão/fisiologia , Fatores de TempoRESUMO
ATP-gated cation channels (P2X receptors) exist on the soma of proprioceptive neurons in the trigeminal mesencephalic nucleus (MNV) in the brain stem. However, these pseudomonopolar neurons seem to receive no synaptic input to their soma; we therefore hypothesized that in MNV neurons, the P2X receptors of importance may be those located on their central terminal projections. Here, we show in trigeminal mesencephalic motor nucleus neurons, which receive their major input from the MNV, that both exogenous ATP (1 mM) and high frequency focal stimulation to evoke endogenous ATP release enhanced the frequency of spontaneous fast excitatory postsynaptic currents (EPSCs) with no change in their amplitude. The enhancement was reduced by the antagonists suramin (300 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (30 microM) and persisted when action potential conduction was blocked with tetrodotoxin (1 microM). Thus, functional P2X receptors are expressed on nerve terminals in the brain stem, where they increase the spontaneous release of glutamate onto trigeminal mesencephalic motor nucleus neurons.
Assuntos
Trifosfato de Adenosina/metabolismo , Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Estimulação Elétrica , Neurônios/metabolismo , RatosRESUMO
1. We studied the voltage-dependent current activated by membrane hyperpolarization in sensory proprioceptive trigeminal mesencephalic nucleus (MNV) neurones. 2. Membrane hyperpolarization (from -62 to -132 mV in 10 mV steps) activated slowly activating and non-inactivating inward currents. The hyperpolarization-activated currents could be described by activation curves with a half-maximal activation potential (V ) of -93 mV, slope (k) of 8.4 mV, and maximally activated currents (Imax) of around 1 nA. The reversal potential of the hyperpolarization-activated currents was -57 mV. 3. Extracellular Cs+ blocked hyperpolarization-activated currents rapidly and reversibly in a concentration-dependent manner with an IC50 of 100 microM and Hill slope of 0.8. ZD7288 (1 microM; 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride), the compound developed as an inhibitor of the cardiac hyperpolarization-activated current (If), also blocked the hyperpolarization-activated currents in MNV neurones. Extracellular Ba2+ (1 mM) did not affect hyperpolarization-activated currents. We tested whether the hyperpolarization-activated currents contribute to the somatic membrane properties of MNV neurones by performing some experiments using current-clamp recording. In such experiments application of Cs+ (1 mM) produced no effect on neuronal resting membrane potentials. 4. During the course of our experiments we noticed that activating ATP-gated non-selective cation channels (P2X receptors) caused an inhibition of Ih associated with a V shift of 10 mV in the hyperpolarizing direction. This P2X receptor-mediated inhibition of Ih was blocked in recordings made with the rapid calcium chelator BAPTA (11 mM) in the pipette solution. 5. We conclude that the current activated by membrane hyperpolarization in MNV neurones is Ih on the basis of its similarity to Ih observed in other neuronal preparations. Activation of Ih can account for the anomalous time-dependent inward rectification that has previously been described in MNV neurones.
Assuntos
Canais Iônicos/metabolismo , Mesencéfalo/metabolismo , Neurônios/metabolismo , Núcleos do Trigêmeo/metabolismo , Animais , Estimulação Elétrica , Eletrofisiologia , Feminino , Técnicas In Vitro , Canais Iônicos/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mesencéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Ratos , Ratos Wistar , Receptores Purinérgicos P2/metabolismo , Núcleos do Trigêmeo/efeitos dos fármacosAssuntos
Purinas/metabolismo , Receptores Purinérgicos P1/fisiologia , Receptores Purinérgicos P2/fisiologia , Adenosina/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Linhagem Celular , Receptores Purinérgicos P1/ultraestrutura , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/ultraestrutura , Receptores Purinérgicos P2X2RESUMO
1. We have investigated whether receptors for ATP exist on neurones of the trigeminal mesencephalic nucleus (MNV) of the rat using whole-cell and outside-out patch-clamp recording in coronal brainstem slices. 2. With whole-cell recording, the batch application of ATP, adenosine 5'-O-[3-thiotriphosphate] (ATP gamma S) and alpha,beta methylene adenosine triphosphate (alpha beta meATP) caused concentration-dependent inward currents in all cells tested (holding potential, -62 mV), with EC50 values of 437, 15 and 254 microM, respectively. All three agonist-evoked currents developed rapidly (rise time, approximately 10-25 s), desensitized slowly (over approximately 20-50 s), cross-desensitized with each other, were associated with an increase in membrane conductance and were attenuated by the application of suramin (30 microM). 3. The inward current evoked by ATP decreased as the membrane potential was made less negative and had a zero current potential of +1.0 +/- 3.7 mV. The current-voltage relationship showed marked inward rectification. 4. Brief flickery single-channel openings could be resolved in response to ATP (3 microM) in outside-out membrane patches. Unitary current at -82 mV was -1.81 +/- 0.2 pA, which corresponds to a unitary conductance of 22 pS. 5. We conclude that proprioceptive MNV neurones contain ATP-gated cation channels. Such P2X purinoceptors may be involved in the processing of proprioceptive information, thus suggesting a potentially important physiological role of ATP.
Assuntos
Trifosfato de Adenosina/fisiologia , Ativação do Canal Iônico/fisiologia , Mesencéfalo/fisiologia , Neurônios/fisiologia , Receptores Purinérgicos P2/fisiologia , Núcleos do Trigêmeo/fisiologia , Animais , Eletrofisiologia , Hibridização In Situ , Técnicas In Vitro , Masculino , Potenciais da Membrana/fisiologia , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ponte/citologia , Ponte/metabolismo , Ponte/fisiologia , Propriocepção/fisiologia , Ratos , Ratos Wistar , Receptores Purinérgicos P2/metabolismo , Núcleos do Trigêmeo/citologia , Núcleos do Trigêmeo/metabolismoRESUMO
Significant advances in understanding of P2X purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becoming available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for P2X receptor heterogeneity, from data with agonists, has recently been reported. A number of new antagonists at P2X purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P2X purinoceptor. Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P2X purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P2X purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P2X purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P2X purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better understanding of the physiological and pathological importance of ATP and its activation of P2X purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.
Assuntos
Trifosfato de Adenosina/farmacologia , Receptores Purinérgicos P2/fisiologia , Animais , DNA Complementar/genética , DNA Complementar/metabolismo , Eletrofisiologia , Humanos , Canais Iônicos/fisiologia , Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Ensaio Radioligante , Receptores Purinérgicos P2/genética , Relação Estrutura-AtividadeRESUMO
The effects of the putative selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate (beta gamma me-L-ATP), were determined at rat neuronal and smooth muscle P2X purinoceptors. beta gamma Me-L-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 microM. In contrast, the archetypal P2X purinoceptor agonist, alpha,beta-methylene ATP (alpha beta meATP; 1-100 microM), produced concentration-related depolarisation responses with a mean EC50 value of 10.8 microM. The depolarising effects of alpha beta meATP were not attenuated by beta gamma me-L-ATP (100 microM). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 microM), but not beta gamma me-L-ATP (1-300 microM), evoked rapid (< 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, beta gamma me-D-ATP and alpha beta meATP competed with high affinity for [3H]alpha beta meATP binding sites, with mean pIC50 values of 7.7 and 8.3, respectively. However, beta gamma me-L-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 microM (mean pIC50 value 4.1). In prostatic segments of the rat vas deferens, beta gamma me-L-ATP (1-100 microM) and alpha beta meATP (0.3-100 microM) each produced concentration-related contractile responses with mean EC50 values of 17.1 and 3.6 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Trifosfato de Adenosina/análogos & derivados , Músculo Liso/metabolismo , Neurônios/metabolismo , Agonistas do Receptor Purinérgico P2 , Trifosfato de Adenosina/metabolismo , Animais , Condutividade Elétrica , Eletrofisiologia , Técnicas In Vitro , Masculino , Ensaio Radioligante , Ratos , Ratos Wistar , Receptores Purinérgicos P2/metabolismo , Nervo Vago/metabolismo , Ducto Deferente/metabolismoRESUMO
1. We have studied both the electrophysiological and contractile effects of the purine nucleotide, adenosine-5'-triphosphate (ATP), as well as a number of its structural analogues as agonists at P2X purinoceptors in the rat vas deferens in vitro. 2. Electrophysiological effects were investigated by a whole cell voltage clamp technique (holding potential-70 mV) with fast flow concentration-clamp applications of agonists in single isolated smooth muscle cells. ATP, 2-methylthio adenosine-5'-triphosphate (2-MeSATP) and alpha,beta methylene adenosine-5'-triphosphate (alpha,beta-meATP) all evoked inward currents over a similar concentration range (0.3-10 microM), being approximately equipotent with similar concentrations for threshold effects (0.3 microM). ADP (10 microM) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 microM). 3. alpha,beta-meATP was the most potent agonist in producing concentrations of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 microM). However, ATP and 2-MeSATP were at least ten times less potent in studies measuring contraction than in the electrophysiological studies. Furthermore, their concentration-effect curves were shallow with smaller maximal responses than could be achieved with alpha,beta-meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was alpha,beta-meATP >> ATP = 2-MeSATP. 4. Measurement of inorganic phosphate (iP), as a marker of purine nucleotide metabolism in the vasdeferens whole tissue preparation, indicated that ATP and 2-MeSATP were rapidly metabolized,whereas alpha,beta-meATP was stable for up to 2 h. Removal of divalent cations prevented breakdown of ATP and 2-MeSATP, suggesting that metabolism involved a Ca2+/Mg2+-dependent enzyme.5. It appears that in isolated preparations of rat vas deferens, the low potency of ATP and 2-MeSATP can be explained by rapid agonist breakdown by ectonucleotidases. However, this is not the case in the single cell studies where the use of rapid concentration-clamp applications revealed the true potency of the agonists. Under such conditions the three agonists were all equal in potency indicating that the rank order of agonist potencies of alpha,beta-meATP>> ATP = 2-MeSATP is not in fact characteristic of smooth muscle P2x-purinoceptors as commonly believed.
Assuntos
Trifosfato de Adenosina/farmacologia , Músculo Liso/fisiologia , Receptores Purinérgicos P2/metabolismo , Ducto Deferente/fisiologia , Trifosfato de Adenosina/análogos & derivados , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Condutividade Elétrica , Eletrofisiologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Fosfatos/metabolismo , Agonistas do Receptor Purinérgico P2 , Ratos , Ratos Sprague-Dawley , Suramina/farmacologia , Ducto Deferente/efeitos dos fármacosRESUMO
1. Whole-cell recordings were made from guinea-pig coeliac, rat nodose and rat superior cervical (SCG) neurones in culture, and currents in response to fast-flow (concentration clamp) application of ATP and other ATP analogues were measured. 2. At a holding potential of -70 mV, ATP evoked inward currents in all neurons. ATP-induced currents reversed at approximately 0 mV and showed strong inward rectification. Halving the external sodium concentration shifted the reversal potential by -15 to -17 mV, while increasing external potassium from 2 to 20 mM produced a 6-10 mV shift in reversal potential. Latency to onset of ATP current was < 1 ms; rise time was concentration dependent with maximum time to peak of 5-20 ms in nodose and coeliac neurones but 80 ms in superior cervical neurones. 3. Threshold concentrations of ATP were 0.1 microM for nodose and coeliac neurones but 10 microM for superior cervical neurones; EC50 values were approximately 3 microM for both nodose and coeliac neurones and 43 microM for superior cervical ganglia. Hill slopes for ATP concentration-response curves were not significantly different from unity in nodose and coeliac neurones whereas the Hill slope in superior cervical neurones was two. 4. 2-MethylthioATP (2-MeSATP) acted as a full agonist in all three neuronal preparations; EC50 values were 0.4, 2.8 and 46 microM for nodose, coeliac and superior cervical neurones, respectively. alpha,beta-Methylene ATP (alpha,beta-MeATP) was a full agonist in nodose and coeliac neurones with EC50 values of 9 and 13 microM, respectively. 5. In superior cervical neurones alpha,beta-MeATP had little or no agonist action but produced a concentration-dependent attenuation of the ATP current. Thus, alpha, beta-MeATP appears to behave as a partial agonist at P2X-purinoceptors in superior cervical neurones. 6. The non-selective purinoceptor antagonists suramin (1-100 microM) and pyridoxal-5'-phosphate (30 microM), as well as the putative P2Y-selective antagonist, Cibacron Blue (30 microM), inhibited all agonist-evoked responses to a similar degree in all three neuronal populations. 7. This study demonstrates that an agonist potency profile of 2-MeSATP > or = ATP > or = alpha,beta-MeATP is characteristic of ligand-gated P2X-purinoceptors in isolated peripheral neurones. We also suggest that the P2X-receptor in superior cervical neurones may represent a distinct subtype of P2X-purinoceptor from that present in nodose and coeliac neurones.
Assuntos
Plexo Celíaco/fisiologia , Plexo Cervical/fisiologia , Gânglio Nodoso/fisiologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , Eletrofisiologia , Cobaias , Ratos , Receptores Purinérgicos P2/efeitos dos fármacos , Tionucleotídeos/farmacologia , Triazinas/farmacologiaRESUMO
In functional studies pyridoxalphosphate-6-azophenyl-2',5'-disulphonic acid (iso-PPADS), suramin, GR200282 (4,4'-[carbonyl-bis(imino-3- benzoylimino)]-bis[5-hydroxy-naphthalene-2,7-disulfonic acid] tetrapotassium salt), cibacron blue, trypan blue and congo red, each produced specific antagonism of the contractile responses of isolated rat vas deferens, induced by alpha,beta-methylene ATP (alpha,beta-meATP), with antagonist pKB estimates of 6.6 +/- 0.3, 5.5 +/- 0.2, 5.1 +/- 0.3, 5.8 +/- 0.2, 4.7 +/- 0.2 and 4.6 +/- 0.2, respectively. In radioligand binding studies, iso-PPADS, suramin, cibacron blue, GR200282, trypan blue and congo red competed for the high affinity [3H]alpha,beta-meATP binding sites in rat vas deferens membranes with pKi estimates of 5.6 +/- 0.04, 5.5 +/- 0.08, 5.6 +/- 0.15, 5.6 +/- 0.04, 4.3 +/- 0.06 and 4.9 +/- 0.10, respectively. Comparison of pKB and pKi estimates revealed a good agreement between the two approaches for estimating measures of affinity for the putative antagonists, except in the case of iso-PPADS. However, we found that two populations of [3H]alpha,beta-meATP binding sites can be identified by iso-PPADS, 26.4% of these having low affinity (pKi of 4.4 +/- 0.2), and 73.6% having high affinity (pKi of 6.5 +/- 0.02) for iso-PPADS. The pKi of 6.5 obtained at the high affinity sites identified by iso-PPADS was close to the equivalent pKB value of 6.6 from functional studies. These studies therefore show a good agreement between pKB and pKi estimates for several antagonists, and suggest that the high affinity binding sites labelled with [3H]alpha,beta-meATP in rat vas deferens represents binding to functional P2X purinoceptors.
Assuntos
Antagonistas Purinérgicos , Ducto Deferente/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ligação Competitiva , Vermelho Congo/metabolismo , Vermelho Congo/farmacologia , Glutationa Transferase/antagonistas & inibidores , Masculino , Contração Muscular/efeitos dos fármacos , Naftalenossulfonatos/metabolismo , Naftalenossulfonatos/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos/metabolismo , Suramina/análogos & derivados , Suramina/metabolismo , Suramina/farmacologia , Triazinas/metabolismo , Triazinas/farmacologia , Azul Tripano/metabolismo , Azul Tripano/farmacologia , Ducto Deferente/efeitos dos fármacosRESUMO
The antagonist properties of pyridoxal-5-phosphate, a synthesis precursor of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, were investigated on P2 purinoceptor-mediated responses of the rat isolated vagus nerve and vas deferens. In addition, the effect of this agent was studied on high affinity tritiated alpha,beta-methylene adenosine triphosphate (alpha,beta-meATP) binding to rat vas deferens membranes, thought to represent binding to functional P2x purinoceptors. In the rat vagus nerve, pyridoxal-5-phosphate (10(-5)-10(-4) M) produced concentration-related antagonism of depolarisation responses induced by alpha,beta-meATP, measured using an extracellular recording technique. In contrast, depolarisation responses to 5-hydroxytryptamine (5-HT) were unaffected by pyridoxal-5-phosphate. In the rat vas deferens, pyridoxal-5-phosphate (10(-5)-10(-4) M) antagonised contractile responses produced by alpha,beta-meATP while contractions to phenylephrine were unaffected. However, responses of the vagus nerve and the vas deferens to alpha,beta-meATP were not antagonised by pyridoxal hydrochloride (10(-4) M). Pyridoxal-5-phosphate competed for high affinity binding of [3H]alpha,beta-meATP to homogenised membranes of the rat vas deferens with a pKi estimate of 4.91 +/- 0.12 and a Hill slope of 0.80 +/- 0.03. Pyridoxal hydrochloride only competed for binding at concentrations in excess of 10(-4) M, yielding a pKi estimate of 3.21 +/- 0.04 and a Hill slope of 1.82 +/- 0.12. These findings indicate that pyridoxal-5-phosphate acts as a specific antagonist of P2 purinoceptors in the vagus nerve and vas deferens of the rat and that the phosphate moiety is required for activity.(ABSTRACT TRUNCATED AT 250 WORDS)