Ma Teresa Miras-Portugal

Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production.

Sodium nitroprusside, S‐nitroso‐N‐acetyl‐D,L‐penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38±0.02 μM. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half‐life (t1/2) of 3.9±0.2 min. The t1/2 for SPER/NO was 37±3 min. SNAP decomposed more slowly (t1/2=37±4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 μM and maximal effective concentration between 2.5 and 5 μM. For SNAP, threshold activation was seen at 1 μM, whereas full activation required a higher concentration (500–750 μM). The dose‐response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide‐deficient SNAP analogue or other NO donors. Although NO‐donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.

Ca2+‐stores mobilization by diadenosine tetraphosphate, Ap4A, through a putative P2Y purinoceptor in adrenal chromaffin cells

1 Diadenosine tetraphosphate (Ap4A) evoked a concentration‐dependent increase in cytosolic [Ca2+] in resting chromaffin cells. The EC50 value for this action was 28.2 ± 6.6 μm. This effect was also produced by diadenosine pentaphosphate (Ap5A) with an EC50 of 50 ± 7 μm. 2 In contrast with this effect, pretreatment with Ap4A or Ap5A induced a 30% reduction in Ca2+ entry following 10 μm dimethylphenylpiperazinium. 3 The elevation in cytosolic [Ca2+] induced by AP4A was persistent in ≅ 100 nm external [Ca2+] and was sensitive to depletion of internal Ca2+ stores by a bradykinin prepulse or whole cell depletion in Ca2+. 4 The effect of Ap4A was mimicked and desensitized by the agonist adenosine 5′‐O‐(2‐thiodiphosphate), and blocked by the P2Y‐receptor antagonist, cibachrome blue. The P2X‐receptor agonist α,β‐methylene adenosine 5′‐triphosphate was inactive both by itself or in combination with Ap4A. This is compatible with a P2Y‐purinoceptor‐mediated action.

Coexpression of functional P2X and P2Y nucleotide receptors in single cerebellar granule cells

The present study describes the presence and expression of functional nucleotide receptors, both ionotropic and metabotropic, in highly purified cultures of cerebellar granule neurons. Microfluorimetric experiments have been carried out to record specific [Ca2+]i transients in individual granule neurons after challenge with diverse nucleotides. Although great heterogeneity was found in nucleotide responses in single cells, these responses all became modified during the course of granule cell differentiation, not only at the level of the number of responding cells, but also in the magnitude of the response to nucleotides. These in vitro developmental changes were more significant in metabotropic responses to pyrimidine nucleotides, UTP and UDP, which were down‐ and upregulated, respectively, during the time in culture. At least two types of ADP‐specific receptors seem expressed in different granule cell subpopulations responding to 2MeSADP, as the specific P2Y1 antagonist MRS‐2179 inhibited Ca2+ responses in only one of these populations. The great diversity of metabotropic responses observed was confirmed by the RT‐PCR expression of different types of P2Y receptors in granule cell cultures: P2Y1, P2Y4, P2Y6, and P2Y12. Similarly, ionotropic nucleotide responses were confirmed by the presence of specific messengers for different P2X subunits, and by immunolabeling studies (P2X1, P2X2, P2X3, P2X4 and P2X7). Immunolabeling reflected great variety in the P2X subunit distribution along the granule neuron cytoarchitecture, with P2X2, P2X3 and P2X4 present at somatodendritic locations, and P2X1, P2X7, and P2X3, located at the axodendritic prolongations. The punctuated labeling pattern obtained for P2X3 and P2X7 subunits is particularly notable, as it presents a high degree of colocalization with synaptophysin, a specific marker of synaptic vesicles, suggesting specialized localization and function in granule neurons.

P2X7 and P2Y13 purinergic receptors mediate intracellular calcium responses to BzATP in rat cerebellar astrocytes.

Previous work has established the presence of functional P2X7 subunits in rat cerebellar astrocytes, which after stimulation with 3′‐O‐(4‐benzoyl)benzoyl ATP (BzATP) evoked morphological changes that were not reproduced by any other nucleotide. To further characterize the receptor(s) and signaling mechanisms involved in the action of BzATP, we have employed fura‐2 microfluorometry and the patch‐clamp technique. BzATP elicited intracellular calcium responses that typically exhibited two components: the first one was transient and metabotropic in nature – sensitive to phospholipase C inhibition and pertussis toxin treatment –, whereas the second one was sustained and depended on the presence of extracellular calcium. The ionotropic nature of this latter component was corroborated by measurements of Mn2+ entry and macroscopic non‐selective cation currents evoked by either BzATP (100 μM) or ATP (1 mM). The two components of the calcium response to BzATP differed in their pharmacological sensitivity. The metabotropic component was partially sensitive to pyridoxalphosphate‐5′‐phosphate‐6‐azo‐(‐2‐chloro‐5‐nitrophenyl)‐2,4‐disulfonate, a selective antagonist of P2Y13 receptors, while the ionotropic component was modulated by external magnesium and markedly reduced by brilliant blue G and 3‐(5‐(2,3‐dichlorophenyl)‐1H‐tetrazol‐1‐yl)methyl pyridine (A438079), thus implying the involvement of P2X7 purinergic receptors. It is concluded that P2Y13 and P2X7 purinergic receptors are functionally expressed in rat cerebellar astrocytes and mediate the increase in intracellular calcium elicited by BzATP in these cells.

Ca2+/calmodulin‐dependent kinase II signalling cascade mediates P2X7 receptor‐dependent inhibition of neuritogenesis in neuroblastoma cells

ATP, via purinergic P2X receptors, acts as a neurotransmitter and modulator in both the central and peripheral nervous systems, and is also involved in many biological processes, including cell proliferation, differentiation and apoptosis. Previously, we have reported that P2X7 receptor inhibition promotes axonal growth and branching in cultured hippocampal neurons. In this article, we demonstrate that the P2X7 receptor negatively regulates neurite formation in mouse Neuro‐2a neuroblastoma cells through a Ca2+/calmodulin‐dependent kinase II‐related mechanism. Using both molecular and immunocytochemical techniques, we characterized the presence of endogenous P2X1, P2X3, P2X4 and P2X7 subunits in these cells. Of these, the P2X7 receptor was the only functional receptor, as its activation induced intracellular calcium increments similar to those observed in primary neuronal cultures, exhibiting pharmacological properties characteristic of homomeric P2X7 receptors. Patch‐clamp experiments were also conducted to fully demonstrate that ionotropic P2X7 receptors mediate nonselective cation currents in this cell line. Pharmacological inhibition of the P2X7 receptor and its knockdown by small hairpin RNA interference resulted in increased neuritogenesis in cells cultured in low serum‐containing medium, whereas P2X7 overexpression significantly reduced the formation of neurites. Interestingly, P2X7 receptor inhibition also modified the phosphorylation state of focal adhesion kinase, Akt and glycogen synthase kinase 3, protein kinases that participate in the Ca2+/calmodulin‐dependent kinase II signalling cascade and that have been related to neuronal differentiation and axonal growth. Taken together, our results provide the first mechanistic insight into P2X7 receptor‐triggered signalling pathways that regulate neurite formation in neuroblastoma cells.

PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions

The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome.

Modulation of the dihydropyridine-insensitive Ca2+ influx by 8-bromo-guanosine-3′:5′-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 microM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca2+]i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca2+ influx pathways. Combined incubations with nifedipine 10 microM and 8-Br-cGMP reduced KCl-evoked intracellular Ca2+ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca2+]i transient induced by the L-type Ca2+ channel agonist Bay K 8644 (1 microM) under conditions of low depolarization. Neomycin (0.2 mM) and omega-AgaToxin-IVA (AgTx) (1 microM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca2+ influx pathway in the chromaffin cell.

Neuroprotection Mediated by P2Y13 Nucleotide Receptors in Neurons

ADP-specific P2Y13 receptor constitutes one of the most recently identified nucleotide receptor and the understanding of their physiological role is currently under investigation. Cerebellar astrocytes and granule neurons provide excellent models to study P2Y13 expression and function since the first identification of ADP-evoked calcium responses not attributable to the related P2Y1 receptor was performed in these cell populations. In this regard, all responses induced by ADP analogues in astrocytes resulted to be Gi-coupled activities mediated by P2Y13 instead of P2Y1 receptors. Similarly, both glycogen synthase kinase-3 (GSK3) and ERK1/2 signaling triggered by 2MeSADP in cerebellar granule neurons were also dependent on Gi-coupled receptors, and mediated by PI3K activity. In granule neurons, P2Y13 receptor was specifically coupled to the main neuronal survival PI3K/Akt-cascade targeting GSK3 phosphorylation. GSK3 inhibition led to nuclear translocation of transcriptional targets, including β-catenin and Nrf2. The activation of the Nrf2/heme oxygenase-1 (HO-1) axis was responsible for the prosurvival effect against oxidative stress. In addition, P2Y13-mediated ERK1/2 signaling in granule neurons also triggered activation of transcription factors, such as CREB, which underlined the antiapoptotic action against glutamate-induced excitotoxicity. Finally, a novel signaling mechanism has been recently described for a P2Y13 receptor in granule neurons that involved the expression of a dual protein phosphatase, DUSP2. This activity contributed to regulate MAPK activation after genotoxic stress. In conclusion, P2Y13 receptors harbored in cerebellar astrocytes and granule neurons exhibit specific signaling properties that link them to specialized functions at the level of neuroprotection and trophic activity in both cerebellar cell populations.

Neuroprotection elicited by P2Y13 receptors against genotoxic stress by inducing DUSP2 expression and MAPK signaling recovery.

Nucleotides activating P2Y13 receptors display neuroprotective actions against different apoptotic stimuli in cerebellar granule neurons. In the present study, P2Y13 neuroprotection was analyzed in conditions of genotoxic stress. Exposure to cisplatin and UV radiation induced caspase-3-dependent apoptotic cell death, and p38 MAPK signaling de-regulation. Pre-treatment with P2Y13 nucleotide agonist, 2methyl-thio-ADP (2MeSADP), restored granule neuron survival and prevented p38 long-lasting activation induced by cytotoxic treatments. Microarray gene expression analysis in 2MeSADP-stimulated cells revealed over-representation of genes related to protein phosphatase activity. Among them, dual-specificity phosphatase-2, DUSP2, was validated as a transcriptional target for P2Y13 receptors by QPCR. This effect could explain 2MeSADP ability to dephosphorylate a DUSP2 substrate, p38, reestablishing the inactive form. In addition, cisplatin-induced p38 sustained activation correlated perfectly with progressive reduction in DUSP2 expression. In conclusion, P2Y13 receptors regulate DUSP2 expression and contribute to p38 signaling homeostasis and survival in granule neurons.

Cross-talk among epidermal growth factor, Ap5A, and nucleotide receptors causing enhanced ATP Ca2+ signaling involves extracellular kinase activation in cerebellar astrocytes

In previous papers, we reported that ATP calcium responses in cerebellar astrocytes were strongly potentiated by preincubation with nanomolar concentrations of the diadenosine pentaphosphate Ap5A. However, the intracellular signaling pathway mediating this effect was not defined. We also showed that stimulation of astrocytes with the dinucleotide led to the activation of extracellular regulated kinases (ERKs). Here, we examined whether ERKs are involved in the potentiating mechanism and intracellular mechanism leading to their activation. Epidermal growth factor (EGF) exactly reproduced the potentiation displayed by the dinucleotide. Moreover, the potentiation of ATP responses by Ap5A and EGF was completely abolished by the MAP kinase (MEK) inhibitor U‐0126, indicating that ERK activation is a required step for the potentiation event. Our data also indicated that ERK activation and the potentiation of ATP calcium responses were sensitive to the src‐like kinase inhibitor herbimycin A, p21ras farnesyltransferase inhibitor peptide, and some PKC inhibitors. Taken together, our findings reveal that Ap5A triggers the potentiation of ATP calcium responses through an intracellular mechanism that is insensitive to pertussis toxin and that this potentiation requires src protein‐mediated ERK activation and the participation of an atypical protein kinase C isoform activated downstream from ERK.