David León

P2Y1 and P2X7 receptors induce calcium/calmodulin-dependent protein kinase II phosphorylation in cerebellar granule neurons

The activation of nucleotide receptors − both ionotropic, P2X, and most of metabotropic, P2Y − increases intracellular calcium concentration, resulting in calcium/calmodulin‐dependent protein kinase II (CaMKII) activation. Stimulation of cerebellar granule neurons in culture − with different P2X and P2Y agonists and their effect on CaMKII phosphorylation − was studied using immunocytochemical and microfluorimetrical techniques. P2X agonist: 2′‐3′‐o‐(4‐benzoylbenzoyl)‐adenosine 5′‐triphosphate (BzATP), α,β‐methylene adenosine 5′‐triphosphate (α,β‐meATP) and diadenosine pentaphosphate (Ap5A); and P2Y agonists: 2‐(methylthyo)‐adenosine diphosphate (2MeSADP) and uridine 5′‐bisphosphate (UDP); tested induced a CaMKII phosphorylation but with a different immunostaining pattern in each group. Stimulation with 2MeSADP induced a Ca2+ release from intracellular stores and a significant CaMKII phosphorylation in cell somas and neurites. This agrees with the subcellular distribution of P2Y1. MRS 2179, a specific P2Y1 inhibitor, antagonized the 2MeSADP effect. On the other hand, cerebellar granule neuron stimulation with BzATP, in Mg2+‐free conditions, produced extracellular calcium entrance and, as a result, a significant increase in CaMKII phosphorylation mostly in fibres, which correspond with P2X7 subdistribution. Immunocytochemical and microfluorimetrical experiments, using Zn2+ and Brilliant Blue G (BBG), as a specific P2X7 antagonist, confirmed that BzATP was acting through the P2X7 receptor. These results indicate that P2Y1 and P2X7 produce a significant increase in CaMKII phosphorylation, but show important differences in subcellular distribution and in effect duration. P2X7 activation in granule neurons is not associated with pore formation, according to the absence of YO‐PRO‐1 fluorescence. The abundant presence of P2X7 at the synaptic structures suggests a relevant role played by this receptor in synaptic plasticity.

Dinucleoside polyphosphates and their interaction with other nucleotide signaling pathways

Dinucleoside polyphosphates or ApnA are a family of dinucleotides formed by two adenosines joined by a variable number of phosphates. Ap4A, Ap5A, and Ap6A are stored together with other neurotransmitters into secretory vesicles and are co-released to the extracellular medium upon stimulation. These compounds can interact extracellularly with some ATP receptors, both metabotropic (P2Y) and ionotropic (P2X). However, specific receptors for these substances, other than ATP receptors, have been described in presynaptic terminals form rat midbrain. These specific dinucleotide receptors are of ionotropic nature and their activation induces calcium entry into the terminals and the subsequent neurotransmitter release. Calcium signals that cannot be attributable to the interaction of ApnA with ATP receptors have also been described in cerebellar synaptosomes and granule cell neurons in culture, where Ap5A induces CaMKII activation. In addition, cerebellar astrocytes express a specific Ap5A receptor coupled to ERK activation. Ap5A engaged to MAPK cascade by a mechanism that was insensitive to pertussis toxin and required the involvement of src and ras proteins. Diadenosine polyphosphates, acting on their specific receptors and/or ATP receptors, can also interact with other neurotransmitter systems. This broad range of actions and interactions open a promising perspective for some relevant physiological roles for the dinucleotides. However, the physiological significance of these compounds in the CNS is still to be determined.

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.

Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways

Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways.

Angiopoietin-1 enhances neutrophil chemotaxis in vitro and migration in vivo through interaction with CD18 and release of CCL4

Angiopoietins are a family of growth factors that are ligands for the tyrosine kinase receptor, Tie2. Angiopoietin 1 (Ang-1) is agonistic for Tie2, plays a key role in blood vessel maturation and stability and has been shown to possess anti-inflammatory properties. However, Tie2 expression has been demonstrated on human neutrophils and the observation that neutrophils migrate in response to Ang-1 in vitro has confounded research into its exact role in inflammation as well as its potential use as a therapeutic agent. We used a mouse model of peritoneal neutrophilic inflammation to determine if Ang-1 could stimulate neutrophil migration in vivo. Tie2 expression was demonstrated on mouse neutrophils. In addition, recombinant human Ang-1 induced significant chemotaxis of isolated mouse neutrophils in a Tie2- and CD18-dependent manner. Subsequently, co-immunoprecipitation of Ang-1 and CD18 demonstrated their interaction. Intraperitoneal injection of an engineered angiopoietin-1, MAT.Ang-1, induced significant neutrophil migration into the peritoneum and a significant increase in the levels of CCL4 in peritoneal lavage fluid. Depletion of resident peritoneal macrophages prior to, or concomitant injections of an anti-CCL4 antibody with MAT.Ang-1 resulted in a significant reduction in neutrophil recruitment. These data indicate a pro-inflammatory role for Ang-1 with respect to neutrophil recruitment.