M. Teresa Miras-Portugal

Presence of diadenosine polyphosphates— Ap4A and Ap5A— In rat brain synaptic terminals. Ca2+ dependent release evoked by 4-aminopyridine and veratridine

The study of the adenine nucleotides in middle brain synaptosomes from rat showed the presence of two diadenosine polyphosphates, Ap4A and Ap5A. HPLC techniques and phosphodiesterase digestion were employed in order to characterize and quantify the dinucleotides. The Ap4A content per mg of protein was 169 +/- 25 pmol and 159 +/- 22 pmol for Ap5A. The study of the exocytotic release of these compounds was carried out with 100 microM 4-aminopyridine or 10 microM veratridine in the presence and in the absence of calcium. 4-Aminopyridine released 14.5 +/- 3.0 pmol/mg protein of Ap4A and 11.6 +/- 2.4 pmol/mg protein of Ap5A in a calcium dependent process. Veratridine in the presence of calcium released 19.9 +/- 3.0 and 16.6 +/- 2.8 pmol/mg of protein of Ap4A and Ap5A respectively. The ratios of exocytosis were close to 7-9% and 10-12% of the total synaptosomal content in the presence of 4-aminopyridine and veratridine, respectively.

Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice

Prolonged seizures [status epilepticus (SE)] constitute a neurological emergency that can permanently damage the brain. SE results from a failure of the normal mechanisms to terminate seizures; in particular, γ‐amino butyric acid‐mediated inhibition, and benzodiazepine anticonvulsants are often incompletely effective. ATP acts as a fast neurotransmitter via ionotropic ligandgated P2X receptors. Here we report that SE induced by intra‐amygdala kainic acid in mice selectively increased hippocampal levels of P2X7 receptors relative to other P2X receptors. Using transgenic P2X7 reporter mice expressing enhanced green fluorescent protein, we identify dentate granule neurons as the major cell population transcribing the P2X7 receptor after SE. Pretreatment of mice with an intracerebroventricular microinjection of 1.75 nmol A438079, a P2X7 receptor antagonist, reduced seizure duration by 58% and reduced seizure‐induced neuronal death by 61%. Injection of brilliant blue G (1 pmol), another selective antagonist, reduced seizure duration by 48% and was also neuroprotective. A438079 was seizure‐suppressive when injected shortly after induction of SE, and coinjection of A438079 with lorazepam 60 min after triggering SE, when electrographic seizure‐responsiveness to lorazepam had decreased, also terminated SE. Our results suggest that P2X7 receptor antagonists may be a promising class of drug for seizure abrogation and neuroprotection in SE.—Engel, T., Gomez‐Villafuertes, R., Tanaka, K., Mesuret, G., Sanz‐Rodriguez, A., Garcia‐Huerta, P., Miras‐Portugal, M. T., Henshall, D. C., Diaz‐Hernandez, M. Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice. FASEB J. 26, 1616‐1628 (2012). www.fasebj.org

Characterization and quantification of diadenosine hexaphosphate in chromaffin cells: Granular storage and secretagogue-induced release

The presence of diadenosine hexaphosphate (Ap6A) in chromaffin cells is described. The characterization of Ap6A has been accomplished by HPLC techniques, using three different elution conditions, rechromatography, and coelution with standards. Treatment with phosphodiesterase from Crotalus durissus produced AMP and adenosine pentaphosphate. The HPLC techniques described allowed the quantification of Ap6A in the picomole range. Chromaffin granules store Ap6A in a quantity of 48.5 +/- 9.7 nmol/mg protein, with a molar ratio ATP/Ap6A of 27. In chromaffin cells the Ap6A value was 1.46 +/- 0.32 nmol/10(6) cells. Diadenosine hexaphosphate was released from chromaffin cells by the action of carbachol and a value of 64 +/- 15 pmol/10(6) cells was obtained, which represents 4-5% of the total cellular content.

A novel receptor for diadenosine polyphosphates coupled to calcium increase in rat midbrain synaptosomes

1 Diadenosine polyphosphates, AP4A and Ap5A, as well as ATP, α,β‐MeATP and ADP‐β‐S, were able to elicit variable intrasynaptosomal Ca2+ increases in rat midbrain synaptic terminals. The origin of the Ca2+ increment was the extrasynaptosomal space since the elimination of extracellular Ca2+ abolished the effect of all the agonists. 2 The P2‐purinoceptor antagonist, suramin, did not affect the Ca2+–increase evoked by diadenosine polyphosphates but dramatically blocked the Ca2+ entry induced by ATP and its synthetic analogues. 3 The actions of Ap5A and ATP on the intrasynaptosomal Ca2+ increase did not cross‐desensitize. 4 Concentration‐response studies for diadenosine polyphosphates showed pD2 values of 54.5 ±4.2 μm and 55.6 ±3.8 μm for AP4A and Ap5A, respectively. 5 The entry of calcium induced by diadenosine polyphosphates could be separated into two components. The first represented a selective voltage‐independent Ca2+ entry; the second, a sustained phase which was voltage‐dependent. 6 Studies on the voltage‐dependent Ca2+–channels involved in the effects of the diadenosine polyphosphates, demonstrated that Ω‐conotoxin G‐VI‐A inhibited the sustained Ca2+–entry, suggesting the participation of an N‐type Ca2+–channel. This toxin was unable to abolish the initial cation entry induced by AP4A or Ap5A. Ω‐Agatoxin IV‐A, tetrodotoxin, or nifedipine did not inhibit the effects of the diadenosine polyphosphates. 7 The effect of ATP on Ca2+–entry was abolished by nifedipine and Ω‐conotoxin G‐VI‐A, suggesting the participation of L‐ and N‐type Ca2+–channels in the response to ATP. 8 These data suggest that AP4A, Ap5A and ATP activate the same intracellular Ca2+ signal through different receptors and different mechanisms. AP4A and Ap5A induce a more selective Ca2+–entry in a voltage‐independent process. This is the first time that a selective action of diadenosine polyphosphate through receptors other than P1 and P2‐purinoceptors has been described.

Tissue-nonspecific Alkaline Phosphatase Promotes the Neurotoxicity Effect of Extracellular Tau

There is solid evidence indicating that hyperphosphorylated tau protein, the main component of intracellular neurofibrillary tangles present in the brain of Alzheimer disease patients, plays a key role in progression of this disease. However, it has been recently reported that extracellular unmodified tau protein may also induce a neurotoxic effect on hippocampal neurons by activation of M1 and M3 muscarinic receptors. In the present work we show an essential component that links both effects, which is tissue-nonspecific alkaline phosphatase (TNAP). This enzyme is abundant in the central nervous system and is mainly required to keep control of extracellular levels of phosphorylated compounds. TNAP dephosphorylates the hyperphosphorylated tau protein once it is released upon neuronal death. Only the dephosphorylated tau protein behaves as an agonist of muscarinic M1 and M3 receptors, provoking a robust and sustained intracellular calcium increase finally triggering neuronal death. Interestingly, activation of muscarinic receptors by dephosphorylated tau increases the expression of TNAP in SH-SY5Y neuroblastoma cells. An increase in TNAP activity together with increases in protein and transcript levels were detected in Alzheimer disease patients when they were compared with healthy controls.

Ap4A and ADP‐β‐S binding to P2 purinoceptors present on rat brain synaptic terminals

1 Diadenosine tetraphosphate (Ap4A) a dinucleotide stored and released from rat brain synaptic terminals presents two types of affinity binding sites in synaptosomes. When [3H]‐Ap4A was used for binding studies a Kd> value of 0.10 ± 0.014 nm and a Bmax value of 16.6 ± 1.2 fmol mg−1 protein were obtained for the high affinity binding site from the Scatchard analysis. The second binding site, obtained by displacement studies, showed a Ki value of 0.57 ± 0.09 μm. 2 Displacement of [3H]‐Ap4A by non‐labelled Ap4A and P2‐purinoceptor ligands showed a displacement order of Ap4A > adenosine 5′‐O‐(2‐thiodiphosphate) (ADP‐β‐S) > 5′‐adenylyl‐imidodiphosphate (AMP‐PNP) > α,β‐methylene adenosine 5′‐triphosphate (α,β‐MeATP) in both sites revealed by the Ki values of 0.017 nm, 0.030 nm, 0.058 nm and 0.147 nm respectively for the high affinity binding site and values of 0.57 μm, 0.87 μm, 2.20 μm and 4.28 μm respectively for the second binding site. 3 Studies of the P2‐purinoceptors present in synaptosomes were also performed with [35S]‐ADP‐β‐S. This radioligand showed two binding sites the first with Kd and Bmax values of 0.11 ± 0.022 nm and 3.9 ± 2.1 fmol mg−1 of protein respectively for the high affinity binding site obtained from the Scatchard plot. The second binding site showed a Ki of 0.018 ± 0.0035 μm obtained from displacement curves. 4 Competition studies with diadenosine polyphosphates of [35S]‐ADP‐β‐S binding showed a displacement order of Ap4A > Ap5A > Ap6A in the high affinity binding site and Ki values of 0.023 nm, 0.081 nm and 5.72 nm respectively. The second binding site potency order was Ap5A > Ap4A > Ap6A, with Ki values of 0.28 μm, 0.53 μm and 5.32 μm respectively. 5 Displacement studies of [35S]‐ADP‐β‐S with P2‐purinoceptor agonists showed the following potency pattern: ADP‐β‐S > AMP‐PNP > α,β‐MeATP with Ki values of 0.021 nm, 0.029 nm 0.215 nm respectively in the high affinity binding site. 2‐Methylthio‐adenosine 5′‐triphosphate (2MeSATP) was unable to displace [35S]‐ADP‐β‐S in this binding site. The second binding site showed a profile of ADP‐β‐S > α,β‐MeATP> AMP‐PNP > 2MeSATP and Ki values of 0.018 μm, 0.212 μm, 0.481 μm and 18.04 μm respectively. 6 These studies suggest the presence of a new P2‐purinoceptor in rat brain synaptosomes with high affinity for diadenosine polyphosphates which we tentatively designate as P2d.

Carbachol induced release of diadenosine polyphosphates -Ap4A and Ap5A- from perfused bovine adrenal medulla and isolated chromaffin cells

The diadenosine polyphosphates--Ap4A and Ap5A--were released from perfused bovine adrenal glands and recently isolated chromaffin cells by the action of carbachol. The H.P.L.C. technique reported here allowed the quantification of pmol amounts of these compounds present in biological samples from the perfusion media after stimulation. Both compounds (Ap4A and Ap5A) were identified by the retention time in H.P.L.C. chromatography, co-elution with standards, re-chromatography and destruction by the phosphodiesterase action. Bovine adrenal glands stimulated with 100 microM carbachol released 0.47 +/- 0.12 nmol/gland of Ap4A and 1.11 +/- 0.26 nmol/gland of Ap5A. Isolated bovine chromaffin cells after 100 microM carbachol, as secretagogue, released 11.1 +/- 0.8 pmol/10(6) cells of Ap4A and 15.8 +/- 1.1 pmol/10(6) cells of Ap5A. The ratio of these compounds with respect to the exocytotically released ATP and catecholamines was in the same order as that found in isolated chromaffin granules.

P2 purinergic receptors for diadenosine polyphosphates in the nervous system.

1. The actions of diadenosine polyphosphates, diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap5A) and diadenosine hexaphosphate (Ap6A) in the nervous system have been reviewed. 2. In the peripheral nervous system, diadenosine polyphosphates bind to P2-purinergic receptors such as the P2Y in chromaffin cells and Torpedo synaptosomes, P2X in vas deferens and urinary bladder and also Torpedo synaptosomes and P2U in endothelial chromaffin cells. 3. In the central nervous system ApnA compounds can act through P2X-purinoceptors opening cation channels in nodose ganglion neurones. Diadenosine polyphosphates bind to a P2d-purinergic receptor in rat brain synaptic terminals and hippocampus, linked to protein kinase C (PKC) activation. 4. P4-purinoceptors are specific receptors for diadenosine polyphosphates, coupled to the Ca2+ influx, in the central synapses. This purinoceptor is not activated by ATP and synthetic analogs. The P4-purinoceptor could act as a positive modulator of the synaptic transmission, giving even more importance to diadenosine polyphosphates as neurotransmitters.

Characterization of a functional P2X7-like receptor in cerebellar granule neurons from P2X7 knockout mice

The presence of ionotropic P2X7 receptor has been studied in mice brain from wild type and P2X7 receptor knockout animals. Western blot and immunocytochemical assays show the presence of a protein containing the P2X7 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 P2X7 specific mRNA expression have been detected. Functional calcium imaging experiments in cultured granule neurons from P2X7 knockout cerebella show the existence of a functional P2X7‐like receptor that keeps some of the properties of the genuine receptor.

Coexpression of Several Types of Metabotropic Nucleotide Receptors in Single Cerebellar Astrocytes

Abstract: We have examined the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes could be responsible for ATP metabotropic calcium responses in single type 1 astrocytes. To identify these subtypes we have studied the pharmacological profile of ATP calcium responses using fura‐2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astrocytes also responded to 2‐methylthioATP and ADP challenges. The agonist potency order was 2‐methylthioATP > ADP > ATP = UTP. Cross‐desensitization experiments carried out with ATP, UTP, and 2‐methylthioATP showed that 2‐methylthioATP and UTP interact with different receptors, P2Y1 and P2Y2 or P2Y4. In a subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca2+ concentration responses at very low concentrations. 2‐MethylthioATP and UTP calcium responses exhibited different sensitivity to pertussis toxin and different inhibition patterns in response to P2 antagonists. The P2Y1‐specific antagonist N6‐methyl‐2′‐deoxyadenosine 3′,5′‐bisphosphate (MRS 2179) specifically blocked the 2‐methylthio‐ATP responses. We can conclude that all single astrocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y1 and either P2Y2 or P2Y4 receptors. Moreover, 30‐40% of astrocytes also coexpressed specific pyrimidine receptors of the P2Y6 subtype, highly selective for UDP coupled to pertussis‐toxin insensitive G protein.