Raquel P. Sen

P2X7 Receptors in Rat Brain: Presence in Synaptic Terminals and Granule Cells

ATP stimulates [Ca2+]i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X3 subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg2+-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X7 receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca2+]i measurements in single synaptic terminals. Additionally, the P2X7 receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining.

Effect of P2Y agonists on adenosine transport in cultured chromaffin cells.

Abstract: Adenosine transport in cultured chromaffin cells was inhibited by purinergic P2y‐receptor agonists without significant changes in the affinity constant, the values being between 1 ± 0.4 and 1.6 ± 0.6 μM. The Vmax parameter was modified significantly, being 40 ± 1.0, 26 ± 5.0, 32 ± 3.0, and 22 ± 4.7 pmol/106 cells/min for control, adenosine‐5′‐O‐(2‐thiodiphosphate), 5′‐adenylylimidodiphosphate, and P1,P4‐di(adenosine‐5′‐) tetraphosphate (Ap4A) (100 μM for every effector), respectively. Ap4A, a physiological ligand for P2y receptors in chromaffin cells, showed the highest inhibitory effect (45%). This transport inhibition is explained by an increase in the cytosolic Ca2+ concentration ([Ca2+]i) and the activation of protein kinase C (PKC). Experiments of [Ca2+]i measurement with the fura‐2 technique showed that P2y agonists, as well as bradykinin, were able to increase [Ca2+]i, this effect being independent of the presence of extracellular Ca2+. The peptide bradykinin, determined to be coupled to phosphatidylinositol hydrolysis and internal Ca2+ mobilization in chromaffin cells, exhibited a behavior similar to that of P2y agonists in adenosine transport inhibition (39%). P2y agonists and bradykinin increased PKC activity associated with the membrane fraction (about 50% increase in particulate PKC activity with respect to controls). The present studies suggest that adenosine transport is regulated by P2y‐purinergic receptors mediated via Ca2+ mobilization and PKC activation.

Effect of 5′-(N-Ethylcarboxamido)adenosine on Adenosine Transport in Cultured Chromaffin Cells

Abstract: Extracellular adenosine is transported into chromaffin cells by a high‐affinity transport system. The action of adenosine receptor ligands was studied in this cellular model 5‐(N‐Ethylcarboxamido)adenosine (NECA), an agonist receptors, activated adenosine transport. Km values for adenosine were 4.6 ± 1.0 (n = 5) and 10.2 ± 3.0 μM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 ± 23.5 and 170.2 ± 30 pmol/106 cells/min for control and 100 nM NECA, respectively. The A1 agonist N6‐cyclohexyladenosine, the A1 antagonist 8‐cyclopentyl‐1, 3‐dipropylxanthine, and the A1‐A2 antagonist 1,3‐dipropyl‐8‐{4‐[(2‐aminoethyl)amino]‐carbonylmethyloxyphenyl}‐xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]‐nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 ± 800 and 23,200 ± 700 transporters/cell for controls and 100 nM NECA, repectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin.

Effect of forskolin and cyclic AMP analog on adenosine transport in cultured chromaffin cells

The adenosine transport in cultured chromaffin cells was inhibited by the presence of the adenylate cyclase activator, forskolin, and a cAMP analog. The V(max) values of this transport obtained for control and in the presence of 8-(-4-chlorophenylthio)adenosine-3?:5?-monophosphate cyclic (ClPhcAMP, 100 ?M) or forskolin (0.5 ?M) were 85 +/- 5; 45 +/- 1.5 and 38 +/- 3 pmol/10(6) cells/min, respectively. The K(m) values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by nitrobenzylthioinosine (NBTI) binding, were decreased by the above mentioned effectors. The values of binding sites per cell were 30,000 +/- 3200; 12,000 +/- 1000 and 21,300 +/- 2000 for control, ClPhcAMP and forskolin, respectively; without changing the dissociation constant. When the binding studies were conducted with cellular homogenates, a significant decrease in the maximal binding capacity for nitrobenzylthioinosine was obtained. The values were as follows: 0.087 +/- 0.01 pmol/mg protein for control, 0.044 +/- 0.02 pmol/mg protein for ClPhcAMP; and 0.032 +/- 0.01 pmol/mg protein for forskolin. In this neural tissue, the adenosine transport system seems to be inhibited by stimulation of the adenylate cyclase or by the cyclic AMP analogue that enters the cells. These results suggest that this inhibition could be mediated by a molecular modification of adenosine transporters, the binding with NBTI is therefore a possible parameter of this modification.

Differential modulation of nucleoside transport types in neuroblastoma cells by protein kinase activation

Nucleoside transport regulation in undifferentiated Neuro-2A cells has been studied and found to include Na+-dependent adenosine transport and facilitated diffusion adenosine transport. The latter corresponded to nitrobenzylthioinosine-sensitive nucleoside transport. Short-term treatment of Neuro-2A cells with physiologically relevant signals only modulated the facilitated diffusion component. The stimulation of undifferentiated cells with forskolin or other activators of the protein kinase A pathway, decreased NBTI-sensitive adenosine transport. Treatment of cells with an inactive analogue of forskolin, 1,9-dideoxi-forskolin, had no effect on NBTI-sensitive nucleoside transport. Therefore, the inhibition of protein kinase A activity by pre-incubation with H-89 or the cAMP antagonist, Rp-8-Br-cAMPS, completely prevented the inhibitory effect of forskolin. Similarly, the activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore A-23187 decreased NBTI-sensitive adenosine transport. The effect of PDBu was reversed by pre-incubation of cells with staurosporine. Maximal transport inhibition was obtained by the simultaneous stimulation of cells with a phorbol ester and A-23187 or a phorbol ester and forskolin. The modulation of NBTI-sensitive nucleoside transport corresponded to changes in specific [3H]NBTI binding to Neuro-2A cells. Maximal inhibition correlated well with a maximal enhancement of cAMP production. However, the Na+-dependent adenosine transport in Neuro-2A cells was not modulated by any of these signals.

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 K i 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.

Nucleoside Transport in Neurons. Regulation by Secretagogues and Effectors of Protein Kinases A and C

Abstract In chromaffin cells, secretagogues and direct activators of protein kinase C and protein kinase A inhibited the nucleoside transport with a parallel decrease in the high affinity binding sites.

Control of Nucleoside Transport in Neural Cells Effect of Protein Kinase C Activation

Although nucleoside transport has been extensively characterized in synaptosomal preparations, they are not suitable for regulatory studies because the cellular integrity is necessary1,2. Adenosine transport is a highly regulated process in cultured neural cells. Long term effects of nerve growth factor (NGF) have been described in cultured chromaffin cells, with a significant increase in the nucleoside transport3. Short term regulation by the action of protein kinases has also been reported. Activation of protein kinase A, directly by cAMP analogs or through membrane receptors, results in a quick inhibition of adenosine transport capacity and in a parallel decrease in the transporters number at the plasma membrane, measured by [3H]nitrobenzylthioinosine binding4,5. There is also recent evidence of the interaction between adenosine receptors and transporters in this cellular model, where the A2 agonist, NECA, activates the adenosine transport6.

Extracellular Signals and Transduction Mechanisms Controlling the Adenosine Transport in Neural Cells

The adenosine transport in bovine chromaffin cells is highly regulated by different extracellular signals, both at short and long-term. The short-term regulation by extracellular signals such as secretagogues or direct activation of protein kinases A and C inhibit the adenosine transport capacity. Binding studies with nitrobenzylthioinosine show that the inhibition of the transport runs in parallel with a decrease in the number of binding sites for this ligand. The adenosine transport inhibition is mediated by a phosphorylation-dephosphorylation process. Extracellular signals that bind o nuclear receptors are responsible for the long-term regulation. Thyroid hormones increase the adenosine transport by increasing the number of adenosine transporters at the plasma membrane level. This transport activation requires the protein-synthesizing mchanism. On the contrary, dexamethasone and retinoic acid inhibit the adenosine transpor capacity and revert the action of thyroid hormones.

Allosteric Modulation of Nucleoside Transport by Adenosine and ATP

Adenosine levels in extracellular fluids depend on a large set of physiologic or pathologic events, and there is broad evidence for a cardiovascular and neuroprotective role of this nucleoside in hypoxic or ischemic situations through its cell surface receptors [1]. Moreover, at the purinergic synapses adenosine can be considered as the last extracellular product that still maintains signaling capability.