Biomedical subjects

J Pintor

Publications and source records attributed to J Pintor.

At least 37 records · Page 2Linked to original sources

PubMed1999

Characterization of nucleotide transport into rat brain synaptic vesicles.

ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The Vmax values obtained were 13.5 +/- 1.4 pmol x min(-1) x mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol x min(-1) x mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.

Adenosine Triphosphate↗

PubMed1999

Studies of chromaffin granule functioning by flow cytometry: transport of fluorescent epsilon-ATP and granular size increase induced by ATP.

Flow cytometry techniques, usually employed to characterize cellular populations, are reported here to be a valuable tool to approach the study of subcellular organelle functioning. Chromaffin granules rendered fluorescent by using an antibody against their membrane protein, synaptophysin, are detectable by flow cytometry. Moreover, these storage granules are able to transport the fluorescent ATP analogue, epsilon-ATP (1,N6-ethenoadenosine 5'-triphosphate), and the resulting granular fluorescence increase can also be followed by this technique. The saturation studies show a non-hyperbolic kinetic behaviour, with a two step curve. The K0.5 values were 0.26 and 2.5 mM and Hill numbers 1 and 6 respectively. In addition, an unexpected granular size increase, which was dependent on the epsilon-ATP concentration, occurred together with the fluorescence increase. Other nucleotide triphosphate substrates of V-ATPase, such as ATP or GTP, but not the non-hydrolyzable analogue ATP gamma S (adenosine 5'-O-(3-thiotriphosphate), mimic this effect, which exhibited sigmoidal saturation curves with K0.5 values of 1.8 and 3.1 mM for ATP and epsilon-ATP respectively. The V-ATPase inhibitors, suramin, EGTA or EDTA significantly reduced the granular size increase in the presence of ATP. Extragranular addition of noradrenaline has no effect by itself on the granular size, but significantly reduced the granular size increase induced by ATP. This effect was reversed by the amine transport inhibitor reserpine. The granular size increase induced by ATP was more effective in the presence of Cl- than Br- or I-. Moreover, no increase occurred in the presence of F- or acetate. The Cl- channel blockers were poorly effective, and only 2-(phenylamino)-benzoic acid (DPC) exhibited an effect on the ATP-induced granular size increase.

Adrenal Glands↗

PubMed1998

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↗

PubMed1998

Intracellular diadenosine polyphosphates: a novel second messenger in stimulus-secretion coupling.

In pancreatic beta-cells, stimulatory glucose concentrations increase cytosolic diadenosine polyphosphates ([ApnA]i) to concentrations sufficient to block ATP-sensitive K+ (KATP) channels. High-performance liquid chromatography and patch clamp techniques were used to study the metabolic pathways by which pancreatic beta-cells synthesize ApnA and the mechanism through which ApnA inhibit KATP channels. ApnA show a glucose- and time-dependent cytosolic concentration increase parallel, though 30- to 50-fold higher, to changes observed in adenine nucleotides. Other fuel secretagogues, leucine and 2-ketoisocaproate, raise [ApnA]i as efficiently as 22 mM glucose. Blockade of glycolysis or Krebs cycle decreases glucose-induced [ApnA]i. No significant increase in cytosolic ApnA concentrations is induced by nonnutrient secretagogues or nonmetabolizable nutrient secretagogues. Inorganic pyrophosphatase inhibition with sodium fluoride blocks 22 mM glucose-induced [ApnA]i increase. ApnA inhibition of KATP channel resembles that of ATP in efficacy, but shows clear functional differences. Unlike ATP, Ap4A does not restore channel activity after rundown. Furthermore, these compounds do not compete with each other for the same site. These features suggest a prominent role for Ap4A in beta-cell function, comparable to ATP. We conclude that nutrient metabolism through pyrophosphatase activation is necessary to induce ApnA synthesis, which in turn constitutes a new, ATP-independent, metabolic regulator of KATP channel activity.

Adenosine↗

PubMed1997

Diadenosine polyphosphates evoke Ca2+ transients in guinea-pig brain via receptors distinct from those for ATP.

1. The ability of diadenosine polyphosphates, namely P1,P2-di(adenosine) pyrophosphate (Ap2A), P1,P3-di(adenosine) triphosphate (Ap3A), P1,P4-di(adenosine) tetraphosphate (Ap4A), P1,P5-di(adenosine) pentaphosphate (Ap5A) and P1,P6-di(adenosine) hexaphosphate (Ap6A) to evoke Ca2+ signals in synaptosomes prepared from three different regions of the guinea-pig brain was examined. 2. In synaptosomal preparations from the paleocortex (cortex), diencephalon/brainstem (midbrain) and cerebellum all the dinucleotides evoked Ca2+ signals that were concentration dependent over the range 1-300 microM. ATP and its synthetic analogues, alpha,beta-methylene ATP, 2-methylthio ATP and adenosine 5'-O-(2-thio)diphosphate (all 100 microM) also evoked Ca2+ signals in these preparations. 3. In the midbrain and cerebellum preparations, responses to ATP and its analogues were attenuated or abolished by the P2 receptor antagonist suramin (100 microM) but responses to the dinucleotides were not. Also, desensitization by a dinucleotide blocked responses to dinucleotides but not mononucleotides, and desensitization by a mononucleotide blocked responses to mononucleotides but not dinucleotides. 4. In cortical preparations, suramin (100 microM) blocked responses to both classes of nucleotides. Furthermore, there was mutual cross-desensitization between the mono- and dinucleotides. 5. The adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, did not affect responses evoked by the dinucleotides, nor did the pyrimidine UTP. 6. It is concluded that there are specific dinucleotide receptors, activated by diadenosine polyphosphates, but not ATP or UTP, on synaptic terminals in guinea-pig diencephalon/ brainstem and cerebellum. These receptors bear a similarity to the dinucleotide receptor (P4 receptor) in rat brain. In guinea-pig cerebral cortex synaptosomes, diadenosine polyphosphates appear to act via the same receptor as ATP.

Adenosine Triphosphate↗

PubMed1997

Antagonism of P2X receptors in guinea-pig vas deferens by diinosine pentaphosphate.

Diinosine pentaphosphate (Ip5I) antagonized contractions, mediated via P2X receptors, evoked by diadenosine pentaphosphate (Ap5A) and ATP in the guinea-pig isolated vas deferens with pA2 values of 6.4 +/- 0.17 (10 d.f.) and 6.5 +/- 0.10 (10 d.f.), respectively. Ip5I (30 microM) did not affect contractile responses evoked by noradrenaline. Ip5I (up to 100 microM) did not antagonize P2Y receptors in the guinea-pig taenia coli, nor P1 or P2 receptors in the guinea-pig left atrium.

Animals↗

PubMed1997

Effects of extracellular pH on agonism and antagonism at a recombinant P2X2 receptor.

1. Under voltage-clamp conditions, the activity of agonists and antagonists at a recombinant P2X2 receptor expressed in Xenopus oocytes was examined at different levels of extracellular pH (pHe). 2. In normal Ringer (Mg2+ ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pHe (8.0-5.5). ATP-responses reached a maximum at pH 6.5 with a 5 fold increase in ATP-affinity; the apparent pKa was 7.05 +/- 0.05. 3. Receptor affinity for ATP was lowered when extracellular Ca2+ ions were replaced with equimolar Mg2+ ions. However, the amplitude of the ATP-responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP-affinity; the apparent pKa was 7.35 +/- 0.05. 4. ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X2 receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important. 5. The potency order for agonists of P2X2 receptors was: ATP> 2-MeS-ATP ATPgammaS> ATPalphaS> >CTP >BzATP, while other nucleotides were inactive. EC50 and nH values for full agonists were determined at pH 7.4 and re-examined at pH 6.5. Extracellular acidification increased the affinity by approximately 5 fold for full agonists (ATP, 2-MeSATP, ATPyS and ATP alpha S), without altering the potency order. 6. The potency order for antagonists at P2X2 receptors was: Reactive blue-2 >trinitrophenol-ATP > or = Palatine fast black > or = Coomassie brilliant blue > or = PPADS>suramin (at pH 7.4). IC50 values and slopes of the inhibition curves were re-examined at different pH levels. Only blockade by suramin was affected significantly by extracellular acidification (IC50 values: 10.4 +/- 2 microM, at pH 7.4; 78 +/- 5 nM, at pH 6.5; 30 +/-6 nM, at pH 5.5). 7 In summary, a lowered pHe enhanced the activity of all agonists at P2X2 receptors but, with the exception of suramin, not antagonists. Since a lowered pHe is also known to enhance agonist activity at P2x receptors on sensory neurones containing P2X2 transcripts, the sensitization by metabolic acidosis of native P2x receptors containing P2X2 subunits may have a significant effect on purinergic cell-to-cell signalling.

Adenosine Triphosphate↗

PubMed1997

Dinucleotide receptor modulation by protein kinases (protein kinases A and C) and protein phosphatases in rat brain synaptic terminals.

The diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate (Ap5A), can activate an ionotropic dinucleotide receptor that induces Ca2+ transients into synaptosomes prepared from rat brain. This receptor, also termed the P4 purinoceptor, is sensitive only to adenine dinucleotides and is insensitive to ATP. Studies on the modulatory role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases on the response of diadenosine polyphosphate receptors were performed by measuring the changes in the intracellular Ca2+ levels with fura-2. Activation and inhibition of PKA were carried out by means of forskolin and the PKA inhibitory peptide (PKA-IP), respectively. The Ap5A response was inhibited by forksolin to 35% of control values, but PKA-IP induced an increase of 37%. The effect of PKC activation was similar to that observed for PKA. PKC stimulation with phorbol 12,13-dibutyrate produced an inhibition of 67%, whereas the PKC inhibitors staurosporine and PKC inhibitory peptide enhanced the responses elicited by Ap5A to 40% in both cases. Protein phosphatase inhibitors diminished the responses elicited by Ap5A to 17% in the case of okadaic acid, to 50% for microcystin, and to 45% in the case of cyclosporin A. Thus, the activity of dinucleotide receptors in rat brain synaptosomes appears to be modulated by phosphorylation/dephosphorylation. These processes could be of physiological significance in the control of transmitter release from neurons that are postsynaptic to nerves that release diadenosine polyphosphates.

Adenosine Triphosphate↗

PubMed1997

Characterization of diadenosine polyphosphate transport into chromaffin granules from adrenal medulla.

The transport of diadenosine polyphosphates into chromaffin granules from bovine adrenal medulla has been studied by using the radiolabeled substrate [3H]Ap5A and the fluorescent substrate analog di(1,N6-ethenoadenosine)polyphosphate, epsilon-(Ap(n)A) (n=3-5). The vesicular concentration increase was time dependent and the substrates were not metabolized to any extent during the transport experiments. The saturation curve indicates the existence of kinetic and allosteric cooperativity during Ap(n)A (diadenosine polyphosphates) transport and could be the result of the presence of various affinity states of the transporter with K values of 16 +/- 1 microM and 75 +/- 6 microM, and corresponding Hill numbers of 2 and 4, when epsilon-(Ap4A) was the substrate. The saturation studies for [3H]Ap5A were performed in a broader concentration range; in this case a three-step curve was obtained with K values of 16 +/- 2 microM, 125 +/- 9 microM, and 545 +/- 11 microM; the corresponding Hill numbers were 2, 4, and 6. This kinetic behavior can be explained on the basis of a mnemonic model, as already demonstrated for the vesicular transport of ATP. The nonhydrolyzable adenine nucleotide analogs, ATPgammaS and ADPbetaS, inhibited the diadenosine polyphosphate transport at concentrations in the millimolar range. Ap(n)A transport was also inhibited by the P2 receptor antagonist suramin, the mitochondrial ATP/ADP exchange inhibitor atractyloside, the proton translocator FCCP, and N-ethylmaleimide.

Adrenal Medulla↗

PubMed1997

Diinosine polyphosphates, a group of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor.

A new family of dinucleotide derivatives, diinosine polyphosphates, has been synthesized through the use of the enzyme 5' adenylic acid deaminase from Aspergillus sp., starting from the corresponding diadenosine polyphosphates. Functional studies were performed on rat brain synaptic terminals in which a dinucleotide receptor has been described that is specific for adenine dinucleotides. The results demonstrated that diinosine polyphosphates did not behave as agonists on the diadenosine polyphosphate receptor (also know as P4 purinoceptor), but they were very efficient as antagonists in abolishing the Ca2+ responses elicited by diadenosine pentaphosphate. The IC50 values for diinosine triphosphate, diinosine tetraphosphate, and diinosine pentaphosphate were 4.90 +/- 0.10 microM, 8.33 +/- 0.22 microM, and 4.23 +/- 0.12 nM, respectively. The diinosine polyphosphates also antagonized the ATP receptors present in synaptic terminals, showing IC50 values of 100.08 +/- 5.72 microM for diinosine triphosphate, 29.51 +/- 1.40 microM for diinosine tetraphosphate and 27.75 +/- 1.65 microM for diinosine pentaphosphate. The antagonistic ability of these diinosine nucleotides was studied in comparison with other P1 and P2 purinoceptor antagonists, such as suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, and 8-cyclopentyl-1,3-dipropylxanthine. These purinergic antagonists did not inhibit the response of the P4 purinoceptor; only the diinosine polyphosphates were able to act as antagonists on the dinucleotide receptor. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid attenuated the responses elicited by ATP, as did the diinosine polyphosphate compounds. The most antagonistic diinosine polyphosphate for the dinucleotide and ATP receptors was diinosine pentaphosphate, which was 6000 times more selective for the P4 purinoceptor than it was for the ATP receptor.

Animals↗

PubMed1996

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.

Adenine Nucleotides↗

PubMed1996

Effects of P2-purinoceptor antagonists on degradation of adenine nucleotides by ecto-nucleotidases in folliculated oocytes of Xenopus laevis.

The aim of the present study was to examine the effects of a number of P2-purinoceptor antagonists on degradation of adenine nucleotides by Xenopus laevis oocyte ecto-nucleotidase. Folliculated oocytes readily metabolize all three naturally-occurring nucleotides, the order of preferential substrates being ATP >ADP > AMP. The degradation of ATP and ADP was decreased significantly in the presence of several P2X- and P2Y-purinoceptor antagonists, including suramin, PPADS, Cibacron blue, Coomassie Brilliant blue, Evans blue, Trypan blue, Congo red, and PIT (each compound was used at 100 microM). All these compounds inhibited the degradation of ATP by up to 60%, whereas the hydrolysis of ADP was inhibited by Congo red and PIT by 75-80%. In addition, DIDS (100 microM) and TNP-ATP (100 microM) selectively inhibited the breakdown of ATP, and sodium azide (10 mM) selectively inhibited the breakdown of ADP. The enzymatic breakdown of either ATP or ADP was unaffected by 8-pSPT (100 microM), an antagonist of P1-purinoceptors, or by oxidized ATP (100 microM), an antagonist of P2Z-purinoceptors. The degradation of AMP was prevented completely by PIT (100 microM) and ingibited significantly by Congo red (100 microM). In conclusion, the present study shows that most of currently available antagonists of P2-purinoceptors inhibit the enzymatic breakdown of extracellular ATP and ADP. The inhibitory effect on ecto-nucleotidase activity should be taken into account when these antagonists are used in pharmacological experiments.

Adenine Nucleotides↗

PubMed1996

Nucleotide vesicular transporter of bovine chromaffin granules. Evidence for a mnemonic regulation.

The nucleotide vesicular transport has been studied with the fluorescent substrate analogues, the (1,N6-ethenoadenosine) nucleotides. The transport experiments were carried out with granular preparations from bovine adrenal medulla, and epsilon-ATP, epsilon-ADP, and epsilon-AMP were quantified after separation by high performance liquid chromatography. The granular concentration increase of all three nucleotides was time-dependent. The concentration dependence of epsilon-nucleotide transport to chromaffin granules did not follow the Michaelis-Menten kinetics and presented a similar three-step curve with cooperativity. This shape can be considered to be the result of the addition of three sigmoidal curves with their corresponding kinetic parameters. epsilon-ATP exhibited K values of 0.25, 1, and 3 mM and Vmax values of 0.02, 0.04 and 0.19 nmol.min-1.mg of protein-1, for the first, second, and third curves for each step, respectively. epsilon-ADP exhibited K values of 0.15, 0.9, and 3.6 mM and Vmax values of 0.025, 0.035, and 0.3 nmol.min-1.mg of protein-1, respectively for the first, second, and third curves. epsilon-AMP exhibited K values of 0.2, 1.2, and 3.2 mM, and Vmax values of 0.01, 0.04, and 0.055 nmol.min-1.mg of protein-1, also for the first to third steps. The Hill numbers for epsilon-ATP, epsilon-ADP, and epsilon-AMP were not constant but a function of the transport saturation. The nonhydrolyzable ATP analogues AMPPNP, ATP gamma S, and ADP beta S were activators of epsilon-nucleotide transport at concentrations under 1 mM and inhibitors at higher concentrations. Atractyloside and N-ethylmaleimide partially inhibited the nucleotide granular transport. High extragranular ATP concentrations specifically induced the exit of the previously transporter granular epsilon-ATP.

Adenine Nucleotides↗

PubMed1996

The diadenosine polyphosphate receptors: P2D purinoceptors.

Diadenosine polyphosphates-Ap4A, Ap5A and Ap6A-are co-stored in neurosecretory vesicles together with ATP and aminergic compounds. They are released from neural cells and synaptic terminals in a Ca(2+)-dependent process. Ligand binding and displacement experiments carried out with [3H]Ap4A on isolated chromaffin cells and synaptosomal preparations result in curvilinear Scatchard plots with Kd values close to 0.1 nM for the high-affinity binding sites. Displacement curves with two steps are obtained for homologous and heterologous nucleotide ligands; the lowest-affinity step exhibits Ki values in the micromolar range for ApnA compounds. The high-affinity binding sites were named P2D purinoceptors on the basis of their binding characteristics. Single-cell studies in neurochromaffin cells indicate the presence of P2X purinoceptors in noradrenergic cells that do not respond to Ap4A and in which noradrenaline secretion can be induced by influx of extracellular Ca2+. P2Y receptors that respond to ATP analogues and ApnAs are present in endothelial cells from adrenal medulla. Those cells that express P2U purinoceptors are unresponsive to ApnAs. Ectodiadenosine polyphosphate hydrolases with Km values of 0.3 to 2 microM are present in both neural and endothelial cells from adrenal medulla. In midbrain synaptic terminals diadenosine polyphosphates induce Ca2+ entry from the extracellular medium. The fact that the synaptic response is not cross-desensitized by ATP and its non-hydrolysable analogues, the non-blocking effect of suramin, and the differential effect of Ca2+ channel blockers, together suggest that there are different receptors for nucleotides and dinucleotides in rat brain synaptosomes, which we have called P4 purinoceptors on the basis of functional studies.

Adrenal Medulla↗

PubMed1996

Diadenosine polyphosphate-activated inward and outward currents in follicular oocytes of Xenopus laevis.

Ionic currents evoked by alpha, omega-adenine dinucleotides (ApXA; X = 2-6) in follicular oocytes of Xenopus laevis were studied under voltage-clamp conditions. Dinucleotides evoked inward and outward currents in Xenopus oocytes by activating native P1 and P2 purinoceptors known to be present on the follicle cell monolayer enveloping oocytes. Inward currents were mediated by a suramin-sensitive P2 purinoceptor which showed an agonist potency order (at 10 microM): Ap4A > ATP > Ap3A > > Ap5A, while Ap2A and Ap6A were inactive. Outward currents were mediated by a novel theophylline-sensitive P1 purinoceptor which showed an agonist potency order (at 10 microM): Ap2A > ATP > > Ap4A = Ap5A = Ap6A > Ap3A. Chromatographic analysis confirmed ectonucleotidase activity at the follicle cell layer of oocytes but at a very low rate of dinucleotide cleavage, indicating that currents evoked by dinucleotides resulted from a direct activation of oocyte P1 and P2 purinoceptors and not through their breakdown to ATP, ADP and AMP. There was no evidence for specific receptors (i.e., P4 purinoceptors) for diadenosine polyphosphates in Xenopus oocytes.

Animals↗

PubMed1996

Diadenosine polyphosphates selectively potentiate N-type Ca2+ channels in rat central neurons.

The action of diadenosine polyphosphates on Ca2+ channels was studied in two preparations: isolated hippocampal neurons and synaptosomes, both from the rat brain. High-voltage-activated Ca2+ channels were recorded in freshly isolated CA3 neurons using a whole-cell patch-clamp technique. Current-voltage relationships were measured in the control and after incubation in 5 microM diadenosine pentaphosphate. In the majority of tested pyramidal neurons, the latter procedure led to a reversible increase in the high-voltage-activated current through Ca2+ channels when measured at the holding potential of -100 mV but not at -40 mV. In experiments on synaptosomes from the whole brain, diadenosine pentaphosphate taken at a concentration of 100 microM increased the intrasynaptosomal calcium level measured by means of spectrofluorimetry for 26 +/- 1.8 nM (by 24 +/- 2%). Nifedipine failed to block this effect both in synaptosomes and hippocampal neurons. Potentiation of the current through Ca2+ channels in hippocampal neurons as well as the increase in intrasynaptosomal Ca2+ were irreversibly blocked by 5 microM omega-conotoxin, but not by 200 nM omega-Agatoxin-IVA. These data indicate that diadenosine polyphosphates enhance the activity of N-type Ca2+ channels in many central neurons of the rat brain.

Adenosine Triphosphate↗

PubMed1996

Full sensitivity of P2X2 purinoceptor to ATP revealed by changing extracellular pH.

A full pharmacological characterization was carried out on a recombinant ATP-gated ion channel (P2X2 purinoceptor) expressed in Xenopus oocytes. This slowly-desensitizing neuronal P2X2 purinoceptor, activated by ATP (EC50 = 4.6 +/- 1 microM at pH 7.4; n = 4), showed the agonist potency order: ATP > or = 2-MeSATP = ATP gamma S > or = ATP alpha S > > Bz-ATP. The receptor affinity for ATP was enhanced 5-10 fold by acidifying the bathing solution (to pH 6.5) but was diminished 4-5 fold in an alkaline solution (pH 8.0). The maximum activity of P2X2 purinoceptors and the activity order of a series of nucleotides were unaltered by changing extracellular pH. Interestingly, ATP sensitivity at a recombinant P2Y1 purinoceptor remained unaltered with changing extracellular pH. These results indicate that acidotic conditions in the synaptic cleft could strengthen purinergic transmission at neuronal P2X2 purinoceptors.

Adenosine Triphosphate↗

PubMed1996

The activation of P1- and P2-purinoceptors in the guinea-pig left atrium by diadenosine polyphosphates.

1. The effects of P1, P2-di(adenosine) pyrophosphate (AP2A), P1, P3-di(adenosine) triphosphate (AP3A), P1,P4-di(adenosine) tetraphosphate (AP4A), P1,P5-di(adenosine) pentaphosphate (AP5A), ATP, alpha, beta-methylene ADP and 2-chloroadenosine (2-ClAd) were examined in the guinea-pig driven left atrium. 2. All these purine compounds except alpha, beta-methylene ADP produced a negative inotropic response with a rank order of potency of: 2-ClAd > > AP2A > or = ATP > or = AP4A = AP3A = AP5A. The EC50 value for 2-ClAd was approximately 1 microM, while those for the remaining compounds were in the range 10 microM-100 microM, alpha, beta-Methylene ADP (10-300 microM), a selective P2Y-purinoceptor agonist, produced a small positive inotropism. 3. The P1-purinoceptor antagonist, 8-para-sulphophenyltheophylline (8-pSPT, 20 microM) caused a right-ward shift in the concentration-response curves for 2-ClAd, ATP and AP2A, but converted the responses of AP3A, AP4A, and AP5A into positive inotropisms. 4. The non-selective P2-purinoceptor antagonist, suramin (300 microM), had no significant effect on the concentration-response curves for 2-ClAd, ATP or AP2A, but significantly antagonized inhibitory responses to AP3A, AP4A and AP5A, and excitatory responses to alpha, beta-methylene ADP. 5. In the presence of 8-pSPT (20 microM), suramin (300 microM) abolished the positive inotropic responses evoked by the dinucleotides. 6. ATP was degraded far more rapidly than any of the dinucleotides, and AP3A was the least stable of the diadenosine compounds. The relative order of stability was AP2A > AP4A = AP5A > AP3A > > ATP. Suramin (300 microM) reduced the rate of degradation of ATP and AP3A by approximately 30%. Suramin had no significant effect on the degradation of AP2A, AP4A or AP5A. 7. It is concluded that the diadenosine polyphosphates cause negative inotropic responses via P1-purinoceptors and a hitherto undefined suramin-sensitive P2-purinoceptor, and that they appear to have positive inotropic effects mediated via another suramin-sensitive P2-purinoceptor.

Analysis of Variance↗