Subcellular distribution and early signalling events of P2X7 receptors from mouse cerebellar granule neurons.

The subcellular distribution and early signalling events of P2X7 receptors were studied in mouse cerebellar granule neurons. Whole-cell patch-clamp recordings evidenced inwardly directed non-desensitizing currents following adenosine 5'-triphosphate (ATP; 600 µM) or 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 µM) administration to cells bathed in a medium with no-added divalent cations (Ca(2+) and Mg(2+)). Nucleotide-activated currents were inhibited by superfusion of 2.5 mM Ca(2+), 1.2 mM Mg(2+) or 100 nM Brilliant Blue G (BBG), hence indicating the expression of ionotropic P2X7 receptors. Fura-2 calcium imaging showed [Ca(2+)]i elevations in response to ATP or BzATP at the somas and at a small number of axodendritic regions of granule neurons. Differential sensitivity of these [Ca(2+)]i increases to three different P2X7 receptor antagonists (100 nM BBG, 10 µM 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester, KN-62, and 1 µM 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine hydrochloride hydrate, A-438079) revealed that P2X7 receptors are co-expressed with different P2Y receptors along the plasmalemma of granule neurons. Finally, experiments with the fluorescent dye YO-PRO-1 indicated that prolonged stimulation of P2X7 receptors does not lead to the opening of a membrane pore permeable to large cations. Altogether, our results emphasise the expression of functional P2X7 receptors at both the axodendritic and somatic levels in mouse cerebellar granule neurons, and favour the notion that P2X7 receptors might function in a subcellular localisation-specific manner: presynaptically, by controlling glutamate release, and on the cell somas, by supporting granule neuron survival against glutamate excytotoxicity.

Brain-derived neurotrophic factor and the course of experimental cerebral malaria.

The role of neurotrophic factors on the integrity of the central nervous system (CNS) during cerebral malaria (CM) infection remains obscure, but the long-standing neurocognitive sequelae often observed in rescued children can be attributed in part to the modulation of neuronal survival and synaptic plasticity. To discriminate the contribution of key responses in the time-sequence of the pathogenic events that trigger the development of neurocognitive malaria syndrome we defined four stages (I-IV) of the neurological progression of CM in C57BL/6 mice infected with Plasmodium berghei ANKA. Upregulation of ICAM-1, VCAM-1, e-selectin and p-selectin expression was detected in all cerebral regions before parasitized red blood cells (pRBC) accumulation. As the severity of symptoms increased, BDNF mRNA progressively diminished in several brain regions, earliest in the thalamus-hypothalamus, cerebellum, brainstem and cortex, and correlated with a four-stage disease sequence. Immunohistochemical confocal microscopy revealed changes in the BDNF distribution pattern, suggesting altered axonal transport. During CM progression, molecular markers of neurological infection and inflammation in the parasite and the host, respectively, were accompanied by a switch in the brain constitutive proteasome to the immunoproteasome, which could impede normal protein turnover. In parallel with BDNF downregulation, NCAM expression also diminished with increased CM severity. Together, these data suggest that changes in BDNF availability could be involved in the pathogenesis of CM.

P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

Caracterización farmacológica y análisis funcional de los receptores nicotínicos α 9 α 10 en células romafines aisladas de la médula adrenal de rata / Pharmacological characterization and functional analysis of α 9 α 10 acetylcholine nicotinic receptors in isolated chromaffin cells from the rat adrenal medulla

El descubrimiento de receptores nicotínicos (nAChRs) formadospor las subunidades α9 y α10 en las células de los epitelios sensitivosdel sistema vestibular y auditivo ha motivado su búsqueda enestructuras del sistema nervioso autónomo, como las células cromafinesde la médula adrenal, en las que podrían inducir la hiperpolarizaciónde la membrana celular mediante la activación de canales deK+ dependientes de Ca2+ de baja conductancia iónica (canales SK). Elobjetivo fundamental de nuestro trabajo ha sido determinar la presenciay, en su caso, caracterizar funcionalmente el nAChR α9α10 enlas células cromafines de la médula adrenal de la rata. Con ese propósito,hemos empleado cultivos primarios de células cromafines obtenidasde la glándula adrenal de rata y recurrido a la técnica electrofisiológicade patch-clamp para registrar las corrientes iónicasgeneradas por la activación de los nAChRs del conjunto de la membranacelular. Asimismo, hemos empleado la α-conotoxina RgIA, unpéptido capaz de bloquear de forma selectiva los nAChRs formadospor las subunidades α9 y α10. Los resultados obtenidos aportan evidenciasfarmacológicas que permiten concluir que las células cromafinesde la rata expresan nAChRs α9α10 funcionales que, además, desempeñarían un papel modulador comportamiento eléctrico de dichascélulas(AU)

The identification of acetylcholine nicotinic receptors (nAChRs)formed by α9 and α10 subunits in the sensory cells of the vestibularand auditory systems, prompted us to investigate their presence inadrenomedullary chromaffin cells, in which they could mediate membranehyperpolarization through the activation of small-conductanceCa2+-dependent K+ channels (SK channels). The aim of the currentstudy has been to pharmacologically identify α9α10 nAChRs and initiatetheir functional characterization in isolated chromaffin cellsfrom the rat adrenal medulla. We have employed the patch clamptechnique to record either the ionic currents generated by the activationof nAChRs or the associated changes in membrane potential. Wetook advantage of the specificity of α-conotoxin RgIA for the nAChRsformed by α9 and α10 subunits. Our pharmacological results suggestthat the rat chromaffin cells express functional α9α10 nAChRs thatwould influence the electrical behaviour of these cells(AU)

Interacción entre receptores de glutamato y receptores de nucleótidos en neuronas granulares de cerebelo en cultivo / Cross-talk between glutamate and nucleotide receptors in cerebellar granule neurons in culture

ATP elicits Ca2+ transients in cultured cerebellar granule neuronsacting through specific ionotropic (P2X) and metabotropic (P2Y)purinergic receptors. In these neurons, application of L-Glutamate(L-Glu) immediately before ATP induced a prolonged reductionof ATP-mediated responses that remains at least 5 minutes afterL-Glu wash out. alpha-amino-3-hydro-5-methyl-4-isoxazolpropionicacid (AMPA), N-methyl-D-aspartate (NMDA) and 3,5-dihydroxyphenyl-glycine (DHPG), selective agonists of ionotropic non-NMDA,NMDA and Group I metabotropic glutamate receptors respectively,mimicked Glu-induced attenuating effects. The activity of calciumcalmodulindependent protein kinase II (CaMKII) seems to beinvolved, at least at long term, because inhibitors of CaMKII, 1-[N,Obis(5-isoquinolinesulfonyl)-N-methyl-L-(KN-62) and N-[2-[[[3-(4'-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-methoxybenzenesulfonamide(KN-93), abolished the inhibitory effect of L-Glu on ATP-mediatedresponses. However, it is likely that other protein kinases could beinvolved in the cross-talk process between both groups of receptorsat short term. Therefore, these results demonstrate that the activationof glutamate receptors is able to modulate nucleotide responses incerebellar granule neurons(AU)

Interacción entre receptores de glutamato y receptores denucleótidos en neuronas granulares de cerebelo en cultivoEl ATP induce un incremento de Ca2+ en neuronas granulares decerebelo en cultivo actuando a través de receptores purinérgicos específicosionótropicos (P2X) y metabotrópicos (P2Y). En estas neuronas,la aplicación de L-Glutamato (L-Glu) inmediatamente antes del ATPinduce una prolongada disminución de las respuestas mediadas porATP que se mantiene al menos durante cinco minutos tras el lavadodel L-Glu. Los agonistas selectivos de los receptores ionotrópicosde glutamato no-NMDA, NMDA y del Grupo I, el ácido alfa-amino-3-hidro-5-metil-4-isoxazolpropiónico (AMPA), el N-metil-D-aspartato(NMDA) y el 3,5-dihidroxifenil-glicina (DHPG), respectivamente, mimetizanlos efectos atenuantes inducidos por el glutamato. La actividadde la proteína calcio-calmodulina quinasa II (CaMKII) pareceestar implicada en este proceso, al menos a largo plazo, puesto quelos inhibidores de la CaMKII, 1-[N,O-bis(5-isoquinolinesulfonil)-Nmetil-L-tirosil]-4fenilpiperazine (KN-62) y N-[2-[[[3-(4'-clorofenil)-2-propenil]metilamino]metil]fenil]-N-(2-hidroxietil)-4'-metoxibenzenosulfonamida(KN-93), revierten el efecto inhibitorio del L-Glu sobrelas respuestas mediadas por ATP. Sin embargo, es probable que puedanestar implicadas otras proteín quinasas en los procesos de interacciónentre ambos grupos de receptores a corto plazo. Por lo tanto,estos resultados demuestran que la activación de los receptores deglutamato son capaces de modular las respuestas a nucleótidos enneuronas granulares de cerebelo(AU)

Altered nucleotide receptor expression in a murine model of cerebral malaria.

In cerebral malaria, the most severe complication of malaria, both neurotransmission mechanisms and energy metabolism are affected. To understand how metabolic changes modify neurotransmission, we examined P2 receptor expression in a murine model of cerebral malaria. Quantitative polymerase chain reaction experiments revealed that parasite deposition was greatest in the cerebellum, compared with other areas of the brain, suggesting a correlation between brain parasitemia and loss of control of movement. Infected mice showed modified patterns of expression of P2 receptor subtype messenger RNA (mRNA), depending on both the specific purinergic receptor and the cerebral region analyzed. Immunohistochemical studies indicated altered levels of protein expression by these receptors in infected brains and, in some cases, a pattern of expression different from that noted in control mice. These differences in both the amount of mRNA and the protein distribution of P2 receptors observed in the different brain sites in infected mice suggest an important role for P2 receptors in either provoking cerebral damage or conferring neuroprotection.

Axodendritic fibres of mouse cerebellar granule neurons exhibit a diversity of functional P2X receptors.

Distribution and functional expression of P2X receptors were analyzed in mouse cerebellum axodendritic fibres, using different experimental approaches such as RT-PCR, western blot, immunochemistry, microfluorimetric experiments and exocytotic studies. RT-PCR and western blot demonstrated the presence of P2X1-4,7 subunits in both whole cerebellum and mouse cerebellar granule cultured neurons. Immunochemistry analysis of tissular and cellular location of P2X1-4,7 receptors confirmed their presence and unequal distribution between somas and axodendritic prolongations. Microfluorimetric experiments using a variety of modulators of the P2X subunits revealed the presence of different functional P2X receptors in the axodendritic fibres. The use of the synthetic agonist alpha,beta-meATP and the antagonist Ip(5)I revealed the activation of functional P2X1 and P2X3 receptors. Responses mediated by P2X1 subunits were also confirmed by using ZnSO(4). Activation of functional P2X4 receptors is observed when stimulated in the presence of ivermectin. Exocytotic studies confirmed the role of most P2X subunits in the activation of neurotransmitter release in axodendritic fibres from mouse cerebellar granule neurons.

Altered P2X7-receptor level and function in mouse models of Huntington's disease and therapeutic efficacy of antagonist administration.

The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntington's disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP-gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7-mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7-receptor level and altered P2X7-mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7-receptor stimulation. Finally, in vivo administration of the P2X7-antagonist Brilliant Blue-G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor-coordination deficits. These in vivo data strongly suggest that altered P2X7-receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.

Una aproximación farmacológica al tratamiento de la acondroplasia / A pharmacological appoach to achondroplasia treatment

La acondroplasia es una patología caracterizada por una mutación en el receptorpara el factor de crecimiento de fibroblastos de tipo 3 (FGFR3). Esta alteracióncausa la patología que conocemos habitualmente con el nombre de enanismo congénitoy que se manifiesta con individuos de talla baja con diversos problemasmúsculo-esqueléticos. La aplicación de nucleótidos y dinucleótidos ha permitidoobservar que las células acondroplásicas pueden fenomenológicamente comportarsecomo células normales, en especial cuando son tratadas con el dinucleótidoAp4A. Este compuesto reestablece los niveles de calcio en los condrocitos acondroplásicos,permitiendo que se comporten como células absolutamente normales enlo que respecta a este ion. Por otro lado, también este dinucleótido permite que elreceptor de FGFR3, que no se internaliza y degrada con normalidad, pueda pasara ser degradado por las vías proteosomales y lisosomales, como sucede en lascélulas normales, haciendo que el receptor motivo de la patología desaparezca de las membranas de los condrocitos acondroplásicos. Por último, hemos podidocomprobar cómo el derivado del piridoxalfosfato, el PPADS, presenta propiedadesextraordinarias al reducir prácticamente a cero los niveles de fosforilación de lasproteínas ERK, que son anormalmente elevadas por el receptor FGFR3 acondroplásicoy que originan la patología. En resumen, se plantean una serie de nuevasestrategias encaminadas al tratamiento de la acondroplasia por medio de estrategiasde tipo farmacológico en claro contrate con las estrategias actuales de tipoquirúrgico

Achondroplasia is a pathology due to a mutation in the receptor for thefibroblast growth factor type 3 (FGFR3). This alteration produces problems inindividuals’ stature as well as other muscle-skeletal problems. The application ofnucleotides and dinucleotides permit achondroplasic cells (chondrocytes) torecover, aparently, from this pathology. In particular, the application of thedinucleotide Ap4A, permits to restore the correct calcium levels in achondroplasiccell. Moreover, this dinucleotide permits the right degradation of the FGFR3receptor, which does not downregulate properly in achondropasic chondrocytes,by facilitating the proteosomal and lysosomal pathways alter the dinucleotideapplication. Also we have discovered that the pyridoxal phosphate derivative PPADScan dramatically reduce the activation of ERK casacade which is abnormalyelevated by the achondroplasic FGFR3 receptor raising the pathology. In summary,we wish to introduce a series of new pharmacological strategies for the treatmentof achondroplasia in clear contrast with the current surgery ones

Glutamate release and synapsin-I phosphorylation induced by P2X7 receptors activation in cerebellar granule neurons.

The present work reports that activation of P2X7 receptor induces synaptic vesicle release in granule neurons and phosphorylation of synapsin-I by calcium-calmodulin-dependent protein kinase II (CaMKII), which in turn modulates secretory event. ATP, in absence of magnesium, induced a concentration-dependent glutamate release with an EC50 value of 1.95 microM. The involvement of P2X7 receptor was suggested when maximal secretory response was significantly reduced by the selective P2X7 antagonist Brilliant Blue G (BBG; 100 nM) and abolished by removing extracellular Ca2+. The involvement of P2X7 receptor on synaptic vesicle release was confirmed by measuring the release of FM 1-43 dye. In this case, pharmacological activation of P2X7 was achieved with the more selective agonist 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 microM) showing a significant FM 1-43 release that was blocked by BBG (100 nM), by Zn2+ ions (100 microM), both P2X7 blockers, but not by suramin (100 microM), antagonist of P2X1, P2X2, P2X3 and P2X5. In addition, BzATP, through P2X7 receptor activation, significantly increased the phosphorylation of synapsin-I, the main presynaptic target of CaMKII. Both effects mediated by BzATP were inhibited by the CaMKII inhibitors KN-62 (10 microM) and KN-93 (10 microM). These results suggest, therefore, that Ca2+ entrance mediated by P2X7 receptor induces glutamate release and in parallel synapsin-I phosphorylation.

Physiological role of extracellular nucleotides at the central nervous system: signalling through P2X and P2Y receptors

En los últimos años los receptores de nucleótidos, receptores ionotrópicosP2X1-7 y metabotrópicos P2Y1, 2, 4, 6, 11, 12, 13, 14, han adquirido una importancia excepcionaldebido a su localización estratégica en órganos y tejidos, a su gran variedadjunto con la complejidad de vías de señalización a las que están asociados y a lasprimeras evidencias de importantes alteraciones debidas a su mal funcionamiento.Nuestro grupo ha sido pionero en la caracterización estos receptores en el sistemanervioso, donde definimos su localización y su funcionalidad. La abundante presencia,a nivel presináptico, de las subunidades P2X3 y P2X7 debe ser resaltada,donde gracias a la entrada de calcio inducen la exocitosis de varios neurotransmisores,como glutamato, GABA, catecolaminas y acetilcolina entre otros, como hasido descrito por nuestro grupo en trabajos previos. Además, estos receptores inducenuna profunda remodelación del citoesqueleto de las terminales nerviosas yde los mecanismos exocitóticos a través de la CaMKII y pueden interactuar conotros receptores ionotrópicos y metabotrópicos co-existentes en sus cercanías. Lamayoría de los receptores P2Y también están presentes en las células nerviosas,activando vías de señalización a través de una gran variedad de cascadas intracelulares.Recientemente hemos demostrado que los receptores metabotrópicos P2Ypertenecientes a la sub-familia de receptores activados por ADP, especialmente elP2Y13, están conectados con la señalización hacia GSK3 y β-catenina, lo que abrenuevas vías para la comprensión de la función de los nucleótidos en la supervivenciay el mantenimiento de las células nerviosas. Además, tanto los receptores P2Xcomo los P2Y juegan un papel en los estadíos iniciales del desarrollo y en lamaduración neuronal donde su función aún ha de ser plenamente comprendida.Los receptores de nucleótidos son también muy abundantes en las células gliales, y nuestro grupo ha demostrado que la mayoría de los receptores P2Y están presentesy son plenamente funcionales en astrocitos en cultivo, donde, dependiendo delsubtipo de receptor, activan una gran variedad de cascadas de señalización

In the last few years nucleotide receptors, the ionotropic P2X1-7 subunits andthe metabotropic P2Y1, 2, 4, 6, 11, 12, 13, 14, have acquired an excepcional importance dueto their strategic location in organs and tissues, their great variety along with thecomplexity of the associated signalling pathways and the first evidence of theserious alterations entailed in their dysfunctions. Our group has been pioneer inthe characterization of these receptors in the nervous system, where we definedtheir location and functionality. The abundant presence, at a presynaptic level, ofP2X3 and P2X7 should be emphasized, where by means of calcium intake theyinduce neurotransmitter exocytosis, such as glutamate, GABA, catecholamines andacetylcholine among others, as described in previous works by our group. In addition, they induce an extensive remodeling of the terminal’s cytoskeleton and exocytoticmechanisms through CaMKII and they can interact widely with other ionotropicand metabotropic receptors co-existing in nearby areas. Neural cells alsoexhibit the presence of most P2Y receptors signalling through a large variety ofintracellular cascades. Recently we have demostrated that P2Y metabotropic receptorsof the sub-family activated by ADP, especially P2Y13, are connected withthe signalling towards GSK3 and Beta-catenin, opening new ways of understading thenucleotide function in survival and maintenance of neural cells. In addition bothP2X and P2Y receptors play a role in early developmental stages and neural maturationwhere their function has to be fully understanded. Nucleotide receptorsare also very abundant in glial cells, and our group has shown that most P2Yreceptors are present and fully functional in cultured astrocytes, where, dependingon the subtype receptor they activate a large variety of signalling cascades

Glutamate determinations using Amplex Red Glutamic Acid Assay are affected by P2X agonist BzATP

Amplex Red (AR) reagent is the most stable and sensitive fluorogenic substrate known for horseradish peroxidase (HRP). Thus, in the presence of hydrogen peroxide (H2O2), AR (a nonfluorescent compound) is converted to resorufin (a strongly fluorescing product) by the action of HRP. In the last years, multiple assays have been developed using this reagent to quantify a diverse assortment of analyses and also to detect the activity of many different enzymes. We recently showed that BzATP, agonist of several ionotropic nucleotide receptors (P2X), interfered with Amplex Red oxidation catalyzed by HRP. In the present work we reported that glutamate determinations using Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit are also strongly interfered by BzATP. These data are really interesting because demonstrate that resorufin fluorescence, in the presence of BzATP, is not proportional to glutamate concentration and, therefore, it must not be used as method to measure glutamate concentration

El compuesto Amplex Red (AR) es el sustrato fluorogénico para la peroxidasa de rábano (HRP) más estable y sensible. Así, en presencia de peróxido de hidrógeno (H2O2), el AR (un compuesto no fluorescente) es convertido en resorufina (un producto fuertemente fluorescente) por la acción de la HRP. En los últimos años se han realizado muchos ensayos utilizando este compuesto para cuantificar un variado surtido de análisis, así como para detectar la actividad de muchas y variadas enzimas. Recientemente hemos demostrado que el BzATP, un agonista de varios receptores ionotrópicos de nucleótidos (P2X), interfiere con la oxidación del Amplex Red catalizada por la HRP. En el presente trabajo mostramos que las determinaciones de glutamato utilizando el kit Amplex Red Glutamic Acid/Glutamate Oxidase Assay están fuertemente interferidas por el BzATP. Estos datos son realmente interesantes porque demuestran que la fluorescencia de la resorufina, en presencia del BzATP, no es proporcional a la concentración de glutamato y, por lo tanto, no debe ser utilizada como un método para medir la concentración de glutamato

Characterization of a functional P2X(7)-like receptor in cerebellar granule neurons from P2X(7) knockout mice.

The presence of ionotropic P2X(7) receptor has been studied in mice brain from wild type and P2X(7) receptor knockout animals. Western blot and immunocytochemical assays show the presence of a protein containing the P2X(7) immunogenic epitopes in the brain of knockout model. Reverse transcriptase polymerase chain reaction experiments demonstrate the absence of the disrupted sequence, but other sequences of P2X(7) specific mRNA expression have been detected. Functional calcium imaging experiments in cultured granule neurons from P2X(7) knockout cerebella show the existence of a functional P2X(7)-like receptor that keeps some of the properties of the genuine receptor.

Interaction between dinucleotide and nicotinic receptors in individual cholinergic terminals.

Functional ionotropic nucleotidic receptors responding to diadenosine pentaphospate and nicotinic receptors responding to epibatidine coexpress in 19% of the total rat midbrain cholinergic terminals, as determined by the combination of immunological and microfluorimetric techniques. Activation of each independent receptor induces the intrasynaptosomal [Ca2+]i and acetylcholine (ACh) release in a dose-dependent way. The responses are inhibited by antagonists of the dinucleotide receptor and nicotinic receptors, thus confirming the involvement of specific receptors in both functions. Stimulation of single cholinergic terminal with both agonists altogether results in a significant decrease of the [Ca2+]i signaling compared with responses of each independent agonist. Inhibitory interaction between both receptors is reverted when one of them is blocked by specific antagonists, both in [Ca2+]i, and subsequent ACh release. The receptor's inhibitory cross talk confirm the involvement of calcium/calmodulin-dependent protein kinase II, CaMKII, as the inhibitory effects are reverted in the presence of the specific inhibitors KN-62 (2-[N-(4'-methoxybenzenesulfonyl)]-amino-N-(4'-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate) and KN-93 (N-(2-[N-[4-chlorocinnamyl]-N-methylaminomethyl]phenyl)-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide). These results demonstrate the existence of an efficient interaction between these two channel populations, opening a new understanding of the functioning of the cholinergic synaptic terminals or terminals containing other neurotransmitters but exhibiting these receptor types or ones that are similar.

Immunolocalisation of P2Y receptors in the rat eye.

Nucleotides present an important role in ocular physiology which has been demonstrated by recent works that indicate their involvement in many ocular processes. P2Y are important among P2 receptors since they can control tear production, corneal wound healing, aqueous humour dynamics and retinal physiology. Commercial antibodies have allowed us to investigate the distribution of P2Y receptors in the cornea, anterior and posterior chamber of the eye and retina. The P2Y(1) receptor was present mainly in cornea, ciliary processes, and trabecular meshwork. The P2Y(2) receptors were present in cornea, ciliary processes and retinal pigmented epithelium. P2Y(4) was present in cornea, ciliary processes, photoreceptors, outer plexiform layer and ganglion cell layer. The P2Y(6) presented almost an identical distribution as the P2Y(4) receptor. The P2Y(11) was also detectable in the retinal pigmented epithelium. The detailed distribution of the receptors clearly supports the recent findings indicating the relevant role of nucleotides in the ocular function.