Circadian-Related Heteromerization of Adrenergic and Dopamine D4 Receptors Modulates Melatonin Synthesis and Release in the Pineal Gland

Dopamine and adrenergic receptor complexes form under a circadian-regulated cycle and directly modulate melatonin synthesis and release from the pineal gland

Circadian-Related Heteromerization of Adrenergic and Dopamine D4 Receptors Modulates Melatonin Synthesis and Release in the Pineal Gland

Dopamine and adrenergic receptor complexes form under a circadian-regulated cycle and directly modulate melatonin synthesis and release from the pineal gland

Respuesta de las células gliales al daño neuronal "in vitro"

[spa] La mayoría de los estudios sobre la activación glial utilizan cultivos puros o mixtos de células gliales, y en general, los agentes que se emplean para inducirla ejercen directamente sobre las células gliales. En contraste, hay pocos trabajos donde se considere la muerte neuronal como el estímulo que se desencadena la activación glial. Se desconoce la señal que desencadena la activación glial en respuesta al daño neuronal, pero se sugiere que tanto alteraciones en los contactos neurona-glía como la presencia de determinados factores solubles secretados por las neuronas dañadas pueden jugar un papel importante en este proceso. En este trabajo se profundizó en el conocimiento del papel de la glía en respuesta al daño neuronal y del proceso de activación glial desencadenado por dos modelos de muerte neuronal: la muerte neuronal por excitotoxicidad mediante concentraciones elevadas de glutamato y la muerte neuronal por apoptosis mediante la deprivación de K+ del medio de cultivo. Se ha comprobado que las células gliales de cultivos neurona-glía de cerebelo responden al daño neuronal con cambios funcionales asociados con una activación glial, tal como ocurre in vivo. Sin embargo, aunque en los dos modelos experimentales de daño neuronal utilizados (excitotoxicidad y apoptosis) se produce la muerte de la mayoría de las neuronas a las 24 h, hay diferencias en la respuesta de los distintos parámetros gliales evaluados en función del tipo de muerte neuronal inducida. En respuesta a la muerte neuronal por excitotoxicidad, en las células gliales hay producción o activación de factores relacionados con una respuesta proinflamatoria, incrementan su proliferación y su actividad fagocítica. En respuesta a la muerte neuronal por apoptosis las células gliales no producen moléculas proinflamatorias ni proliferan, pero su actividad fagocítica se induce de manera más rápida que en el modelo de excitotoxicidad. En este caso, la activación glial queda restringida a la detección y eliminación de las neuronas dañadas sin que haya producción de factores que puedan amplificar la activación glial y transformarla en nociva. Los resultados obtenidos muestran que el estado en que se encuentran las células gliales puede tener un papel importante en la respuesta neuronal a estímulos nocivos. Esto podría ser de gran importancia para el diseño de estrategias que permitan potenciar aquellas propiedades protectoras de las células gliales activadas o inhibir aquellas que son nocivas para las neuronas, con la finalidad de favorecer los mecanismos de neuroprotección ante un determinado estímulo nocivo.[eng] In physiological conditions, glial cells play a key role in the normal function of the central nervous system (CNS) both during development and in the adult. Moreover, they are essential to the response of the CNS to pathological conditions. Astroglial and microglial cells respond to neuronal damage with morphological and functional changes and thus become reactive or activated glial cells. Given the complexity of studying glial activation in vivo, numerous authors have approached the subject using in vitro experimental models. However, most experimental approaches use glial cell cultures, and/or the stimulus used to induce glial activation is an in?ammatory agent or a mixture of cytokines that have a direct effect and cause signi?cant modi?cations of glial cells. In contrast, comparatively few studies have addressed glial activation in the presence of neurons employing neuronal damage as the inducing stimulus. The nature of the signal that induces glial activation in response to neuronal damage remains unknown, although direct contact between neuronal and glial cells as well as soluble factors delivered by the damaged neurons probably play an important role. In the present work, we have studied glial activation occurring in cerebellar neuronal-glial cell cultures in response to different forms of neuronal death. First, these neuronal-glial cultures were exposed to a high concentration of glutamate, which induces excitotoxic neuronal death. Second, we exposed cerebellar neuronal-glial cultures, which need to be cultured in a medium containing a high concentration of K+ (25 mM K+), to serum-free medium containing a normal concentration of K+ (5 mM K+), a well established and widely used model of apoptosis in cerebellar granule neurons. Our results show that glial cells in neuronal-glial cultures respond to neuronal damage with functional changes associated with glial activation, as occurs in vivo. However, the pattern of response differs depending on the kind of damage induced in the neurons. Although the two experimental models of neuronal damage used (excitotoxicity and apoptosis) resulted in the death of most neuronal cells after 24 h, differences were observed in the response of the various glial parameters evaluated. In the presence of excitotoxic neuronal death, glial cells increase their production of agents associated with an in?ammatory response, as well as proliferate and become phagocytic. This appears to be a drastic reactive response of glial cells that, on the one hand, clears the extracellular milieu of any neuronal debris or factors released by the dying neurons that may negatively affect the cellular homeostasis, and on the other, results in the production of factors that in turn can be deleterious for the remaining live cells. In contrast, glial cells do not produce pro-in?ammatory molecules in the presence of apoptotic neuronal death, but phagocytic activity is quickly induced. In this case, glial activation appears to constitute a preventative response resulting from a crosstalk between dying neuronal cells and glial cells, aimed at preventing alterations in the extracellular milieu through the release of injuring factors by dying neurons, as well as limiting the glial response as much as possible. Our results show that the response of neurons to a certain stimulus depends on the presence of glial cells. Our results also show that different aspects of glial activation are independently regulated in response to neuronal damage. It would be of interest to identify the different signal transduction pathways involved in each case. In this way, it may be possible to speci?cally interfere with individual aspects of glial activation in order to favour actions of reactive glial cells that could help neurons to overcome a negative stimulus and, likewise, to inhibit activities that enhance neuronal damage

Respuesta de las células gliales al daño neuronal in vitro

Tesis doctoral realizada en el Instituo de Investigaciones Biomédicas de Barcelona, CSIC-IDIBAPS.La mayoría de los estudios sobre la activación glial utilizan cultivos puros o mixtos de células gliales, y en general, los agentes que se emplean para inducirla ejercen directamente sobre las células gliales. En contraste, hay pocos trabajos donde se considere la muerte neuronal como el estímulo que se desencadena la activación glial. Se desconoce la señal que desencadena la activación glial en respuesta al daño neuronal, pero se sugiere que tanto alteraciones en los contactos neurona-glía como la presencia de determinados factores solubles secretados por las neuronas dañadas pueden jugar un papel importante en este proceso. En este trabajo se profundizó en el conocimiento del papel de la glía en respuesta al daño neuronal y del proceso de activación glial desencadenado por dos modelos de muerte neuronal: la muerte neuronal por excitotoxicidad mediante concentraciones elevadas de glutamato y la muerte neuronal por apoptosis mediante la deprivación de K+ del medio de cultivo. Se ha comprobado que las células gliales de cultivos neurona-glía de cerebelo responden al daño neuronal con cambios funcionales asociados con una activación glial, tal como ocurre in vivo. Sin embargo, aunque en los dos modelos experimentales de daño neuronal utilizados (excitotoxicidad y apoptosis) se produce la muerte de la mayoría de las neuronas a las 24 h, hay diferencias en la respuesta de los distintos parámetros gliales evaluados en función del tipo de muerte neuronal inducida. En respuesta a la muerte neuronal por excitotoxicidad, en las células gliales hay producción o activación de factores relacionados con una respuesta proinflamatoria, incrementan su proliferación y su actividad fagocítica. En respuesta a la muerte neuronal por apoptosis las células gliales no producen moléculas proinflamatorias ni proliferan, pero su actividad fagocítica se induce de manera más rápida que en el modelo de excitotoxicidad. En este caso, la activación glial queda restringida a la detección y eliminación de las neuronas dañadas sin que haya producción de factores que puedan amplificar la activación glial y transformarla en nociva. Los resultados obtenidos muestran que el estado en que se encuentran las células gliales puede tener un papel importante en la respuesta neuronal a estímulos nocivos. Esto podría ser de gran importancia para el diseño de estrategias que permitan potenciar aquellas propiedades protectoras de las células gliales activadas o inhibir aquellas que son nocivas para las neuronas, con la finalidad de favorecer los mecanismos de neuroprotección ante un determinado estímulo nocivo.Beca predoctoral del IDIBAPS (2002-2005). Proyectos de la Red Cien, del FIS y el SAF2001-2240 del MCYT.Peer Reviewe

Biotin ergopeptide probes for dopamine receptors

The incorporation of chemical modifications into the structure of bioactive compounds is often difficult because the biological properties of the new molecules must be retained with respect to the native ligand. Ergopeptides, with their high affinities at D and D dopamine receptors, are particularly complex examples. Here, we report the systematic derivatization of two ergopeptides with different peptide-based spacers and their evaluation by radioligand binding assays. Selected spacer-containing ergopeptides with minimal biological alteration and a proper anchoring point were further derivatized with a biotin reporter. Detailed characterization studies identified 13 as a biotin ergopeptide maintaining high affinity and agonist behavior at dopamine receptors, being a useful tool for the study of heteromers involving D R, D R, or D R

A1R–A2AR heteromers coupled to Gs and Gi/0 proteins modulate GABA transport into astrocytes

Astrocytes play a key role in modulating synaptic transmission by controlling extracellular gamma-aminobutyric acid (GABA) levels via GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that a further level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A(1) (A(1)R) and A(2A) (A(2A)R) receptors. This regulation occurs through A(1)R–A(2A)R heteromers that signal via two different G proteins, G(s) and G(i/0), and either enhances (A(2A)R) or inhibits (A(1)R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron–glia–neuron) synapse

Circadian-related heteromerization of adrenergic and dopamine D4 receptors modulates melatonin synthesis and release in the pineal gland

The role of the pineal gland is to translate the rhythmic cycles of night and day encoded by the retina into hormonal signals that are transmitted to the rest of the neuronal system in the form of serotonin and melatonin synthesis and release. Here we describe that the production of both melatonin and serotonin by the pineal gland is regulated by a circadian-related heteromerization of adrenergic and dopamine D4 receptors. Through α1B-D4 and β1-D4 receptor heteromers dopamine inhibits adrenergic receptor signaling and blocks the synthesis of melatonin induced by adrenergic receptor ligands. This inhibition was not observed at hours of the day when D4 was not expressed. These data provide a new perspective on dopamine function and constitute the first example of a circadian-controlled receptor heteromer. The unanticipated heteromerization between adrenergic and dopamine D4 receptors provides a feedback mechanism for the neuronal hormone system in the form of dopamine to control circadian inputs

Modulation of GABA transport by adenosine A 1R-A 2AR heteromers, which are coupled to both G s- and G i/o-Proteins

Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A(1) (A(1)R) and A(2A) (A(2A)R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A(1)R-A(2A)R that signal via two different G-proteins, G(s) and G(i/o), and either enhances (A(2A)R) or inhibits (A(1)R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse

Circadian-related heteromerization of adrenergic and dopamine D4 receptors modulates melatonin synthesis and release in the pineal gland

The role of the pineal gland is to translate the rhythmic cycles of night and day encoded by the retina into hormonal signals that are transmitted to the rest of the neuronal system in the form of serotonin and melatonin synthesis and release. Here we describe that the production of both melatonin and serotonin by the pineal gland is regulated by a circadian-related heteromerization of adrenergic and dopamine D4 receptors. Through alpha18-D4 and ß1-D4 receptor heteromers dopamine inhibits adrenergic receptor signaling and blocks the synthesis of melatonin induced by adrenergic receptor ligands. This inhibition was not observed at hours of the day when D4 was not expressed. These data provide a new perspective on dopamine function and constitute the first example of a circadian-controlled receptor heteromer. The unanticipated heteromerization between adrenergic and dopamine D4 receptors provides a feedback mechanism for the neuronal hormone system in the form of dopamine to control circadian inputs

Functionality of dopamine D₄ receptors in pineal gland and pinealocytes.

<p>Pineal glands extracted at 9:00 h were treated for 10 min with increasing amounts of dopamine or with 1 µM of RO 10-5824 (RO). The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) phosphorylation (A) and Akt (Ser⁴⁷³) phosphorylation (B), of two separate experiments performed in duplicate were quantified and values represent the mean ± S.D. of the fold increase relative to basal levels found in untreated cells. Significant differences with respect to basal levels were determined by one-way ANOVA followed by a Dunnett's multiple comparison post hoc test (*<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001). A representative Western blot is shown at the top (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>). (C) Pinealocytes were isolated from pineal glands extracted at 9:00 h and were treated with medium (Control), 1 µM of RO 10-5824 (RO), 1 µM phenylephrine (Phenyl), or 1 µM isoproterenol (Iso) for 10 min before labeling with anti-S-arrestin (green) and anti-phospho-ERK1/2 (red), as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Cell nuclei were stained with DAPI (blue). Scale bar, 5 µm.</p