Enrique Castro

A purinergic component of the excitatory postsynaptic current mediated by P2X receptors in the CA1 neurons of the rat hippocampus

The pyramidal neurons in the CA1 area of hippocampal slices from 17‐ to 19‐day‐old rats have been investigated by means of patch clamp. Excitatory postsynaptic currents (EPSCs) were elicited by stimulating the Schaffer collateral at a frequency below 0.2 Hz. It was found that inhibition of glutamatergic transmission by 20 μm 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 100 μm 2‐amino‐5‐phosphonovaleric acid (D‐APV) left a small component of the EPSC uninhibited. The amplitude of this residual EPSC (rEPSC) comprised 25 ± 11% of the total EPSC when measured at a holding potential of −50 mV. The rEPSC was blocked by selective P2 blocker pyridoxal phosphate‐6‐azophenyl‐2′‐4′‐disulphonic acid (PPADS) 10 μm and bath incubation with non‐hydrolysable ATP analogues, ATP‐γ‐S and α,β‐methylene‐ATP at 50 and 20 μm, respectively. The rEPSC was dramatically potentiated by external Zn2+ (10 μm). In another series of experiments exogenous ATP was applied to the CA1 neurons in situ. An inward current evoked by ATP was inhibited by PPADS to the same extent as the rEPSC. It is concluded that, depending on membrane voltage, about one‐fifth to one‐quarter of the EPSC generated by the excitatory synaptic input to the hippocampal CA1 neurons of rat is due to the activity of P2X receptors.

Potentiation of ATP calcium responses by A2B receptor stimulation and other signals coupled to Gs proteins in type‐1 cerebellar astrocytes

We have studied the interaction between P1 and P2 purinoceptors in purified type‐1 astrocyte cultures from postnatal days 7–8 rat cerebella using single cell microfluorimetry with fura‐2. The stimulation of astrocytes with ATP elicits rapid [Ca2+]i transients showing an EC50 value of 7.9 ± 0.3 μM. Costimulation of type‐1 astrocytes with adenosine and ineffective ATP concentrations (0.1 or 1 μM) evoked [Ca2+]i transients that correspond to 60% of the maximal ATP response. NECA (5′‐N‐ethylcarboxamidoadenosine) was the only agonist that mimicked the adenosine effect and showed an EC50 value of 0.17 ± 0.01 μM. This value was identical to that obtained for the cAMP production stimulation, indicating that A2B receptors coupled to adenylate cyclase activation were involved. The presence of A2B adenosine receptors was also confirmed by immocytochemistry experiments. When astrocytes were costimulated with isoproterenol and ineffective ATP concentrations similar [Ca2+]i transients were observed. The treatment of astrocytes with cholera toxin potentiated ATP calcium signals, lowering the EC50 value for ATP to 1.5 ± 0.2 μM. However, the pretreatment of cells with forskolin or a permeable cAMP analogue had no effect on ATP calcium responses. These results indicated that the potentiation mechanism was elicited before the adenylate cyclase activation. We could conclude that in type‐1 astrocytes, the activation of A2B adenosine receptors or other signals positively coupled to adenylate cyclase stimulation strongly potentiate metabotropic calcium responses to ATP. The potentiation was parallel but independent on cAMP accumulation suggesting the involvement of βγ subunits released after Gs stimulation. GLIA26:119–128, 1999.

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.

Effect of diadenosine polyphosphates on catecholamine secretion from isolated chromaffin cells

1 The action of several diadenosine polyphosphates (AP3A, AP4A and AP5A) on basal, and on nicotine‐and high K+‐evoked, catecholamine (CA) release has been investigated. Each of the three diadenosine polyphosphates weakly but significantly increased basal CA secretion. This enhancement represented about 10% of the response evoked by 2 μm nicotine. 2 The evoked secretory response to diadenosine polyphosphates had an absolute requirement for extracellular Ca2+. 3 In contrast, these compounds had an inhibitory action on nicotine‐evoked release. This response was concentration‐dependent, EC50 values being 3.2 ± 0.4 μm, 4.0 ± 1.6 μm and 19.3 ± 4.0 μm for AP3A, AP4A, and AP5A, respectively. The lower the concentration of nicotine used to evoke secretion, the higher the inhibitory power of these compounds. 4 The CA secretion evoked by K+‐rich solutions was further enhanced by AP3A and AP5A, whereas AP4A inhibited it. The possible physiological role of these dual actions is discussed.

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.

Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

1 Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 ± 0.65 × 10−11 m and Bmax value of 5420 ± 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 ± 0.53 × 10−9 m and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2 The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]‐Ap4A from the two binding sites, the Ki values being 1.0 nm, 0.013 nm, 0.013 nm and 0.013 nm for the very high affinity binding site and 0.5 μm, 0.13 μm, 0.062 μm and 0.75 μm for the second binding site. 3 The ATP analogues displaced [3H]‐Ap4A with the potency order of the P2y receptors, adenosine 5′‐O‐(2 thiodiphosphate) (ADP‐β‐S) > 5′‐adenylyl imidodiphosphate (AMP‐PNP) > α,β‐methylene ATP (α,β‐MeATP), in both binding sites. The Ki, values were respectively 0.075 nm, 0.2 nm and 0.75 nm for the very high affinity binding site and 0.125 μm, 0.5 μm and 0.9 μm for the second binding site.

Diadenosine polyphosphates, extracellular function and catabolism.

Publisher Summary Diadenosine polyphosphates (Ap n A n = 2-6) are natural compounds that play an important role inside the cell, in both the nucleus and at the cytosol. The transport of Ap n A to cytoplasmatic storage granules provides both the end of their intracellular actions and at the same time the way to be released by controlled exocytosis. Diadenosine polyphosphates are co-stored and co-released from a large variety of synaptic terminals. Their physiological extracellular concentration range can be expected to be between the nM and low μM. Binding studies have detected the presence of high affinity sites, correlating with the physiological concentrations . From a functional point of view, specific receptors, called “dinucleotide receptors” or “P4 receptors,” have been described in presynaptic terminals and elicit calcium entrance through ionotropic receptors that are not susceptible to inhibition by voltage dependent calcium channel toxins. The extracellular actions of diadenosine polyphosphates are terminated by the enzymatic hydrolysis by an ecto-diadenosine polyphosphate hydrolase.

Chapter 19 ATP receptor-mediated component of the excitatory synaptic transmission in the hippocampus

Publisher Summary This chapter presents the pharmacological identification of a purinergic component in the excitatory synaptic input to CA1 neurons of the rat hippocampus. The investigation is initiated by the observation that the specific P2-purinoceptor antagonist pyridoxal phosphate-6-azophenyI-2’-4’-disulfonic acid (PPADS) had a small, but consistent and reversible, inhibitory effect on the excitatory post-synaptic currents (EPSC) recorded in CA1 pyramidal neurons in response to stimulation of Schaffer collateral at especially low stimulation frequencies (0.1–0.05 Hz). The current paradigm establishes that the excitatory synaptic transmission in hippocampal CAl/CA3 synapses is mediated solely by excitatory amino acid receptors. The chapter demonstrates the presence of a clear non-glutamatergic component in the EPSC measured in CA1 pyramidal neurons. Based on the selectivity of PPADS and the sensitivity to Zn2 + and ATP analogs the chapter concludes that the transmitter underlying this component is most likely ATP acting on ionotropic P2X receptors. The chapter reveals that about one fifth of the excitatory input to CA1 neurons is purinergic.

Single-cell fura-2 microfluorometry reveals different purinoceptor subtypes coupled to Ca2+ influx and intracellular Ca2+ release in bovine adrenal chromaffin and endothelial cells.

ATP and adenosine(5′)tetraphospho(5′)adenosine (Ap4A), released from adrenal chromaffin cells, are potent stimulators of endothelial cell function. Using single-cell fura-2 fluorescence recording techniques to measure free cytosolic Ca2+ concentration ([Ca2+]i), we have investigated the role of purinoceptor subtypes in the activation of cocultured chromaffin and endothelial cells. ATP evoked concentration-dependent [Ca2+]i rises (EC50=3.8 μM) in a subpopulation of chromaffin cells. Both ATP-sensitive and -insensitive cells were potently activated by nicotine, bradykinin and muscarine. Reducing extracellular free Ca2+ concentration to around 100 nM suppressed the [Ca2+]i transient evoked by ATP but not the [Ca2+]i response to bradykinin. ATP-sensitive chromaffin cells were also potently stimulated by 2-methylthioadenosine triphosphate (2MeSATP; EC50= 12.5 μM) and UTP, but did not respond to either adenosine 5′-[β-thio]diphosphate (ADP[βS]), a P2Y receptor agonist, adenosine 5′-[α,β-methylene]triphosphate (pp[CH2]pA), a P2X agonist or AMP. Adrenal endothelial cells displayed concentration-dependent [Ca2+]i responses when stimulated with ATP (EC50=0.86 μM), UTP (EC50=1.6 μM) and 2MeSATP (EC50= 0.38 μM). 2MeSATP behaved as a partial agonist. Ap4A and ADP[βS] also raised the [Ca2+]i in endothelial cells, whereas AMP and pp[CH2]pA were ineffective. Lowering extracellular free Ca2+ to around 100 nM did not affect the peak ATP-evoked [Ca2+]i rise in these cells. It is concluded that different purinoceptor subtypes are heterogeneously distributed among the major cell types of the adrenal medulla. An intracellular Ca2+-releasing P2U-type purinoceptor is specifically localized to adrenal endothelial cells, while a subpopulation of chromaffin cells expresses a non-P2X, non-P2Y subtype exclusively coupled to Ca2+ influx.

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.