Diadenosine pentaphosphate regulates dendrite growth and number in cultured hippocampal neurons.

During the establishment of neuronal circuits, axons and dendrites grow and branch to establish specific synaptic connections. This complex process is highly regulated by positive and negative extracellular cues guiding the axons and dendrites. Our group was pioneer in describing that one of these signals are the extracellular purines. We found that extracellular ATP, through its selective ionotropic P2X7 receptor (P2X7R), negatively regulates axonal growth and branching. Here, we evaluate if other purinergic compounds, such as the diadenosine pentaphosphate (Ap5A), may module the dynamics of dendritic or axonal growth and branching in cultured hippocampal neurons. Our results show that Ap5A negatively modulates the dendrite's growth and number by inducing transient intracellular calcium increases in the dendrites' growth cone. Interestingly, phenol red, commonly used as a pH indicator in culture media, also blocks the P2X1 receptors, avoided the negative modulation of Ap5A on dendrites. Subsequent pharmacological studies using a battery of selective P2X1R antagonists confirmed the involvement of this subunit. In agreement with pharmacological studies, P2X1R overexpression caused a similar reduction in dendritic length and number as that induced by Ap5A. This effect was reverted when neurons were co-transfected with the vector expressing the interference RNA for P2X1R. Despite small hairpin RNAs reverting the reduction in the number of dendrites caused by Ap5A, it did not avoid the dendritic length decrease induced by the polyphosphate, suggesting, therefore, the involvement of a heteromeric P2X receptor. Our results are indicating that Ap5A exerts a negative influence on dendritic growth.

Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells.

Extracellular tau promotes an increase in the level of intracellular calcium in cultured neuronal cells. We have found that such increase is impaired in the presence of antagonists of muscarinic receptors. In order to identify the nature of those receptors, we have tested the effect of different specific muscarinic receptor antagonists on tau promoted calcium increase. Our results indicate that the increase does not take place in the presence of antagonists of muscarinic (mainly M1 and M3) receptors. A similar increase in intracellular calcium was found in non-neuronal cells transfected with cDNA of M1 and M3 muscarinic receptors when tau was added. These results suggest that observed effect of tau protein on neuronal (neuroblastoma and primary cultures of hippocampal and cortical neurons) cells is through M1 and M3 muscarinic receptors. Therefore blocking M1 and for M3 receptors, by using specific receptor antagonists, can prevent that tau toxic effect that could take place in tauopathies.

Synaptic terminals from mice midbrain exhibit functional P2X7 receptor.

P2X(7) receptor has been recently localized in mice cerebellar granule neuron fibers. Here, the expression of this subunit has been detected in wild type mice midbrain, by quantitative real time-polymerase chain reaction, immunocytochemistry and Western blot assays. The functionality of this P2X(7) subunit has been confirmed using microfluorimetric experiments in isolated synaptic terminals from mice midbrain. 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) was 30-fold more potent than ATP and EC(50) values were 20 microM and 630 microM respectively. Brilliant Blue G (BBG) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) produced an inhibition in the responses induced by BzATP, with IC(50) values of 0.027 nM and 2.23 nM, respectively. In addition, P2X(7) inhibitors as ZnSO(4), BBG and suramin abolished partially or totally the responses induced by the physiological agonist ATP. According to immunochemical and PCR assays the presence of a "P2X(7)-like" protein in synaptosomes from validated P2X(7) knockout (KO) model have been detected. In KO animals, BzATP was sixfold more potent than ATP and the EC(50) values were 87 microM and 590 microM respectively. BBG and KN-62 also produced an inhibition in the responses induced by BzATP, with IC(50) value of 0.61 nM and 118 nM respectively, both of them higher than in wild type mice. Moreover, the calcium mobilization ability of native P2X(7) receptors was higher in control compared with KO mice. These biochemical and pharmacological experiments are consistent with the presence of a functional P2X(7) receptor in wild type mice midbrain, and the existence of a less efficient "P2X(7)-like" receptor in the KO model.

Existence of high and low affinity dinucleotides pentaphosphate-induced calcium responses in individual synaptic terminals and lack of correlation with the distribution of P2X1-7 subunits.

Individual analysis of synaptic terminals calcium responses, induced by dinucleotides pentaphosphate, Ap(5)A or Gp(5)G, demonstrates the presence of two main groups considering the concentration required for stimulation. The first group corresponds to those responding to Ap(5)A or Gp(5)G at nanomolar concentration, representing 16% and 12%, respectively, and the second one responds to micromolar concentration and represents, respectively, 17% and 14%, of the total functional synaptosomal population in rat midbrain. Dose-response curves in single terminals showed an Ap(5)A EC(50) values of 0.9+/-0.2 nM and 11.8+/-0.9 microM, being the maximal intrasynaptosomal calcium increase of 200+/-0.3 and 125+/-0.2 nM for the high and low affinity responding terminals, respectively. Combination of microfluorimetric and immunocytochemical studies showed lack of correlation between dinucleotides pentaphosphate responses and P2X receptor subunits expression, in spite of the abundance of P2X(2), P2X(3) and P2X(7) at the presynaptic level in rat midbrain synaptosomes. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a P2X receptors antagonist, showed no effect on low affinity dinucleotides receptors population, and partial inhibition on the high affinity one. On the other hand, diinosine pentaphosphate (Ip(5)I) completely abolished the low affinity dinucleotides responses, and 60% inhibition of the high affinity ones.

Role of CaCMKII in the cross talk between ionotropic nucleotide and nicotinic receptors in individual cholinergic terminals.

Ionotropic P2X receptors for ATP are formed, to date, by seven different subunits named P2X (Torres et al., 1999; Cunha and Ribeiro, 2000; North and Surprenant, 2000; Pintor et al., 2000; Hervás et al., 2003; Miras-Portugal et al., 2003; Illes and Ribeiro, 2004), which are cloned from various mammalian species (Illes and Ribeiro, 2004). These subunits can occur as homo- or hetero-oligomeric assemblies of more than one subunit (North and Surprenant, 2000), except P2X (Miras-Portugal et al., 2003) receptor, which has been described not to coassemble with other subunits (Torres et al., 1999). They are abundantly expressed in the peripheral and central nervous systems and exhibit high permeability to Ca2+ ions (Cunha and Ribeiro, 2000). The existence of presynaptic ionotropic receptors for nucleotides, either for ATP or dinucleotides, has been reported in isolated synaptic terminals from mammalian brain, and both exhibit good permeability to Ca2+ ions (Pintor et al., 2000; Hervás et al., 2003; Miras-Portugal et al., 2003). Studies on isolated single terminals have confirmed the existence of independent and specific responses to ATP and dinucleotides on the same or different terminals (Miras-Portugal et al., 1999; Díaz-Hernández et al., 2002; Hervás et al., 2005; Sánchez-Nogueiro et al., 2005). The activation of presynaptic ionotropic nucleotide receptors can induce the release of other neurotransmitters such as acetylcholine, glutamate, or GABA. In these specific terminals, ionotropic nucleotide receptors can be modulated by interaction with metabotropic receptors, such as GABAB and adenosine receptors (Khakh and Henderson, 1998; Gómez-Villafuertes et al., 2001), and ionotropic, such as nicotinic cholinergic receptors (Díaz-Hernández et al., 2004; Sánchez-Nogueiro et al., 2005). Here, we discuss a relevant finding on the interaction between ionotropic nucleotide and nicotinic receptors in cholinergic synaptic terminals and the role of CaCMKII in this interaction.

Effects of insulin on glucose transporters and metabolic patterns in Harding-Passey melanoma cells.

The effect of insulin on glucose metabolism through different pathways and the glucose transporters in Harding-Passey melanoma cells have been studied. Glucose was utilized at a rate of 6.9 +/- 2.3 (SD) mumol X g-1 X h-1 with 86% transformed into lactate and pyruvate and only 0.43 and 3% metabolized through the tricarboxylic acid cycle and the pentose phosphate pathway, respectively. Of the total glucose consumed 2% was used in protein synthesis and 2% was used for lipid synthesis. Hexokinase isoenzyme was type I and enolase was present mainly in the alpha gamma hybrid form. The glucose transporters were cytochalasin B sensitive. The number of high affinity cytochalasin B binding sites was 175,000 receptors/cell (about 0.6 pmol/mg protein) and Kd = 1 X 10(-7) M. Insulin increased glucose utilization and lactate production by about 70% and caused a 56% increase in transport without alterations in the Kd of the site. Insulin receptors were quantified by binding assay using 125I-insulin. Kd was 11 X 10(-9) M with the number of receptors calculated as 11,500/cell. Harding-Passey melanoma cells could thus be a useful model to study basic metabolic events and their modulation by hormones or other effectors.

Adenosine transporters in chromaffin cells: subcellular distribution and characterization.

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.

Bovine adrenal medullary chromogranin A: studies on the structure and further evidence for identity with dopamine-beta-hydroxylase subunit.

1. Chromogranin A was purified by the use of polyacrylamide gel electrophoresis. The amino acid composition of chromogranin A appeared to be nearly identical to that reported by other investigators and, moreover, was confirmed to be similar to that of dopamine beta-hydroxylase. 2. Dansyl-end group analysis revealed the presence of leucine as the only amino-terminal residue and quantitative estimations showed the presence of two leucine residues per molecule of 77 000 molecular weight. 3. Tryptic and CNBr patterns were obtained. Data are in good agreement with the concept of two nearly identical polypeptide chains per chromogranin A molecule of mol. wt 77 000. Patterns were compared with those obtained in parallel dopamine beta-hydroxylase and support the idea that chromogranin A and the dopamine beta-hydroxylase subunit are identical. Digestion with leucine amino peptidase gave further additional evidence for this suggestion. 4. Chromogranin A appeared to be free of carbohydrates. No cross-reaction was detected between chromogranin A and rabbit antibody against bovine adrenal dopamine beta-hydroxylase.

Down-regulation and recycling of the nitrobenzylthioinosine-sensitive nucleoside transporter in cultured chromaffin cells.

The dynamics of the nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporter were studied in cultured chromaffin cells. Photolabelling of transporters with [3H]NBTI induced a down-regulation of this protein from the plasma membrane with a half-life value of 2.31 +/- 0.61 h, measured by specific isolation of plasma membrane on polycationic beads. In this internalization step 50-60% of transporters were destroyed. The remaining labelled protein reappeared in plasma membranes and underwent a new disappearance cycle with a longer half-life period (34.65 +/- 3.9 h). A similar pattern of internalization and reappearance of nucleoside transporters was observed in cells cross-linked with non-labelled NBTI, with a half value of reappearance of 33 h. Chromaffin cells cultured in the presence of the protein synthesis inhibitor, cycloheximide, had a component of disappearance for NBTI binding sites with a half-life value of 24.6 +/- 1.4 h.

Diadenosine polyphosphates. A novel class of glucose-induced intracellular messengers in the pancreatic beta-cell.

Diadenosine polyphosphates are a group of low-weight compounds that increase after exposure to a wide variety of oxidants and have been suggested to act as "alarmones," alerting the cell to the onset of metabolic stress. We demonstrate here that glucose at concentrations that induce insulin release produce a 30- to 70-fold increase in the concentration of diadenosine triphosphate (Ap3A) and tetraphosphate (Ap4A) in beta-cells. Furthermore, Ap3A and Ap4A, at the concentrations found in glucose-stimulated cells, are effective inhibitors of the ATP-regulated K+ channels when applied to the intracellular side of excised membrane patches from cultured beta-cells. We suggest that Ap3A and Ap4A act as second messengers mediating a glucose-induced blockade of the pancreatic beta-cell ATP-regulated potassium channel.

Effect of diabetic hyperglycemia and other sugars on plasma dopamine-beta-hydroxylase activity.

The plasma glycoprotein, dopamine-beta-hydroxylase (DBH), is present in markedly increased amounts in experimental, streptozocin (STZ)-diabetic rats, reaching a maximum at about the first week and maintaining a plateau for several months afterward. High glycemia values are observed simultaneously. Insulin treatment is observed to keep the glycemia and plasma DBH activity values at levels seen in control rats. The heterologous half-life of DBH in STZ-diabetic rats is significantly increased compared with that of control animals. The glucose analogue, 2-deoxy-D-glucose, has a similar effect on plasma DBH activity levels, eliciting high glycemia values. In STZ-diabetic animals, this increase is more significant, as if it were the additive effect of the two sugars. Other sugars that can compete for glycoprotein catabolic receptors can also modulate the plasma DBH activity levels. The lack of effect of galactose on DBH levels, together with the induced increase of DBH by alpha-methyl-D-mannoside and, to a lesser extent, by inulin, suggest an important rate for the mannose/glucose/N-acetyl glucosamine/fructose receptor in the catabolic clearance of DBH from plasma and explain the abnormal values seen for DBH in diabetes mellitus.

Diadenosine polyphosphate receptors. from rat and guinea-pig brain to human nervous system.

Diadenosine polyphosphates are a family of naturally occurring nucleotidic compounds present in secretory vesicles together with other chemical messengers. The exocytotic release of these compounds permits them to stimulate receptors termed "purinoceptors" or "ATP receptors." Purinoceptors for nucleotides are named P2 in contrast with those sensitive to nucleosides (P1). P2 receptors are further subdivided into metabotropic P2Y receptors, further divided into 5 subtypes, and ionotropic P2X receptors, with 7 different subtypes. Diadenosine polyphosphates can activate recombinant P2Y(1), P2Y(2), and P2Y(4) and recombinant homomeric P2X(1), P2X(2), P2X(3), P2X(4), and P2X(6). Heteromeric P2X receptors change their sensitivity to diadenosine polyphosphates when co-assembly between different subunits occurs. Diadenosine polyphosphates can activate specific receptors termed dinucleotide receptors or P4 receptors, which are insensitive to other nucleosides or nucleotides. The P4 receptor is a receptor-operated Ca(2)+ channel present in rat brain synaptic terminals, stimulated by diadenosine pentaphosphate and diadenosine tetraphosphate. This receptor is strongly modulated by protein kinases A and C and protein phosphatases. The dinucleotide receptor is present in different brain areas, such as midbrain (in rat and guinea-pig), cerebellum (in guinea-pig), and cortex (in human).

The neurotransmitter role of diadenosine polyphosphates.

Diadenosine polyphosphates present at the cytosol can be transported to secretory granules allowing their exocytotic release. Extracellularly, they can act through specific metabotropic or ionotropic receptors, or as analogues of P2X and P2Y nucleotide receptors. The specific ionotropic receptor P4 is present in synaptic terminals, and modulated by protein kinases (PK) A and C and protein phosphatases. Activation of PKA or PKC, directly or through membrane receptors, results in a decrease of affinity or in reduction of the Ca2+ transient respectively. Adenosine and ATP, both products of the extracellular destruction of diadenosine polyphosphates, acting through A1 or P2Y receptors respectively, are important physiological modulators at the P4 receptor.

Adenosine transporters in chromaffin cells. Quantification by dipyridamol monoacetate.

Chromaffin cells from bovine adrenal medulla are a useful model to approach adenosine transport and metabolism in neural cells. Dipyridamol has been shown to be an adenosine transport inhibitor with high affinity. To quantify the adenosine transporters a labelled dipyridamol analogue, [14C]dipyridamol acetate, was synthesized. This compound had a Ki = 5.3 +/- 0.43 nM according to the Dixon method, and 4.58 +/- 0.46 nM when the receptor number molarity was taken into account showing, like dipyridamol, a non-competitive mechanism. The high-affinity receptors present in chromaffin cells showed a Kd = 6.8 +/- 0.8 nM and the receptor number was 630 000 +/- 40 000 per cell.

Glucose transporters in chromaffin cells: subcellular distribution and characterization.

The glucose transporter was identified and characterized by cytochalasin B binding in subcellular membrane fractions of chromaffin tissue. The binding was saturable with Kd of about 0.3 microM for each subcellular fraction. The Bmax capacity was 12-16 pmol/mg protein for enriched plasma membrane fractions, 6.3 pmol/mg protein for microsomal membrane preparations and 5.4 pmol/mg protein for chromaffin granule membranes. Irreversible photoaffinity labelling of the glucose-protectable binding sites with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis from enriched plasma membrane preparations demonstrated the presence of three molecular species: 97 +/- 10, 51.5 +/- 6 and 30 +/- 4 kDa. The chromaffin granule membranes showed only a molecular species of 80 +/- 10 kDa.

Requirement of nitric oxide and calcium mobilization for the induction of apoptosis in adrenal vascular endothelial cells.

Exposure of adrenal vascular endothelial cells (AVEC) to pharmacological nitric oxide (NO) donors, proinflammatory cytokines or lipopolysaccharide was unable to induce apoptosis as occurred when macrophages were treated under identical experimental conditions. However, when the intracellular Ca2+ concentration increased, AVEC displayed apoptotic features upon exposure to NO. This apoptosis was confirmed by the release of oligonucleosomes to the cytosol and by the characteristic DNA laddering observed after electrophoresis in agarose gels. Ca2+-mobilizing agents and interleukin-1beta (IL-1beta) also elicited an apoptotic response in these cells through a mechanism that required NO synthesis. The ability of NO and intracellular Ca2+ to promote apoptosis was dependent on the number of passages of the cells in culture, suggesting the loss of protective factors in the course of ex vivo cell culture. Because AVEC exhibit an important capacity to increase the intracellular Ca2+ concentration in response to a wide array of agonists, this condition might affect the integrity of the vascular system under pathological circumstances such as those prevailing in the course of septic shock.

Presence of epsilon-adenosine tetraphosphate in chromaffin granules after transport of epsilon-ATP.

Adenosine 5'-tetraphosphate (Ap4) is a natural constituent of chromaffin granules with concentration values of 2.2 +/- 0.1 nmol/mg of protein and a ratio 245 +/- 40 times lower with respect to ATP (n = 4). The granular transport of epsilon-ATP resulted in a time- and concentration-dependent production of epsilon-adenosine tetraphosphate (epsilon-Ap4) at the intragranular level. The epsilon-Ap4 formation followed a hyperbolic saturation kinetic at low epsilon-ATP concentrations with K(m) value of 0.4 microM epsilon-ATP intragranular (1.15 pmol/mg of granular protein). Intragranular concentrations of epsilon-ATP higher than 500 pmol/mg of protein (approximately to 175 microM intragranular) resulted in a non-saturable production of epsilon-Ap4.

PKC-independent inhibition of glutamate exocytosis by arachidonic acid in rat cerebrocortical synaptosomes.

In rat cerebrocortical synaptosomes, the addition of 4 beta-phorbol dibutyrate (4 beta-PDBu) and arachidonic acid enhances and decreases, respectively, the glutamate release evoked by 4-aminopyridine. Pretreatment of synaptosomes with 12-O-tetradecanoylphorbol 13-acetate (TPA) or pre-incubation with staurosporine, prevent the stimulatory effect of 4 beta-PDBu, but are without effect on the inhibitory action of arachidonic acid. Moreover, methyl arachidonate, which is not effective as a PKC activator, also strongly inhibits glutamate exocytosis. These results suggest that PKC is not involved in the inhibition of glutamate release by arachidonic acid.

Di(1,N6-ethenoadenosine)5', 5'''-P1,P4-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A.

Di(1,N6-ethenoadenosine)5',5'''-P1,P4-tetraphosphate, epsilon-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristics maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (lambda exc 307 nm, lambda em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split epsilon(Ap4A) and catabolize the resulting epsilon-nucleotide moieties up to epsilon-Ado.

Flow cytometric studies of nucleoside transport regulation in single chromaffin cells.

The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [3H]NBTI binding with a Ki of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (K[D] = 6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.