Jesús Pintor

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

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). 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. 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. 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. The potency order for agonists of P2X2 receptors was: ATP>2‐MeS‐ATPATPγS> ATPαS>>CTPBzATP, 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, ATPγS and ATPαS), without altering the potency order. The potency order for antagonists at P2X2 receptors was: Reactive blue‐2>trinitrophenol‐ATPPalatine fast blackCoomassie brilliant bluePPADS>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 μM, at pH 7.4; 78±5 nM, at pH 6.5; 30±6 nM, at pH 5.5). 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.

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.

Full sensitivity of P2times2 purinoceptor to ATP revealed by changing extracellular pH

A full pharmacological characterization was carried out on a recombinant ATP‐gated ion channel (P2times2 purinoceptor) expressed in Xenopus oocytes. This slowly‐desensitizing neuronal P2times2 purinoceptor, activated by ATP (EC50 = 4.6 ± 1 μm at pH 7.4; n 4), showed the agonist potency order: ATP ≥ 2‐ MeSATP = ATPγS ≥ ATPαS < < 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 P2times2 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 P2times2 purinoceptors.

Involvement of melatonin MT3 receptors in the regulation of intraocular pressure in rabbits

Melatonin, a neurohormone secreted by the pineal gland, can stimulate three subtypes of receptors, namely: mt(1), MT(2) and MT(3). We examined the ability of melatonin and the selective MT(3) receptor agonist, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), to modify intraocular pressure in rabbits. Both compounds significantly reduced intraocular pressure, maximally by 24% and 43%, respectively, with IC(50) values of 363+/-23.0 and 423+/-30.0 ng/10 microl (1.6+/-0.1 and 1.8+/-0.1 nmol, respectively). The non-specific melatonin receptor antagonist, luzindole, abolished the depressant effect of both compounds, thus confirming the involvement of melatonin receptors. Our results show, for the first time, a functional response that may be regulated by melatonin MT(3) receptors, and provide evidence that supports a role of melatonin in the circadian changes of intraocular pressure.

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.

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.

Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors

The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP‐activated inward currents at rat P2X1–4 receptors expressed in Xenopus oocytes and studied under voltage‐clamp conditions. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5′‐adenylic deaminase, and purified using reverse‐phase chromatography. Against ATP‐responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)>Ip4I (6.3)>Ip3I (>4.5). Ip5I (10–100 nM) caused a concentration‐dependent rightwards displacement of the ATP concentration‐response curve without reducing the maximum ATP effect. However, the Schild plot was non‐linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non‐surmountable antagonist. Against ATP‐responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6.0)>Ip5I (5.6)>Ip3I (>4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. Diinosine polyphosphates failed to inhibit ATP‐responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1–4 receptors. Thus, Ip5I acted as a potent and relatively‐selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high‐affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (100 nM) concentrations but has more complex actions at higher (>100 nM) concentrations.

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.

Ocular hypotensive effects of melatonin receptor agonists in the rabbit: further evidence for an MT3 receptor

Melatonin is involved in the control of intraocular pressure during the night and day photoperiod. We have investigated the receptor that regulates intraocular pressure in New Zealand white rabbits by means of agonists and antagonists of melatonin receptors. Melatonin and its analogues: 2‐Phe‐melatonin, 6‐Cl‐melatonin, 2‐I‐melatonin, 5‐ methoxycarbonylamino‐N‐acetyltryptamine (5‐MCA‐NAT) and N‐acetyltryptamine all produced a reduction in intraocular pressure. Dose‐response analysis for these compounds gave pD2 values of 9.3±0.24 for melatonin; 9.0±0.09 for 6‐Cl‐melatonin; 9.0±0.84 for 2‐I‐melatonin; 8.9±0.07 for 5‐MCA‐NAT; 8.7±0.18 for 2‐Phe‐melatonin and 9.4±0.30 for N‐acetyltryptamine (all n=8). At a dose of 0.5 nmol (in 10 μl) melatonin and the selective melatonin MT3 agonist 5‐MCA‐NAT, induced greater reductions of intraocular pressure (22.8±2.3% and 32.5±1.4%, respectively) than the other compounds. The melatonin‐receptor antagonists, prazosin, DH‐97 and 4‐P‐PDOT, reversed the effect of 5‐MCA‐NAT in a dose‐dependent manner, with pA2 values of 13.5±0.17 for prazosin, 10.6±0.16 for DH‐97 and 9.4±0.20 for 4‐P‐PDOT (n=8). Cholinoceptor antagonists (hexamethonium and atropine) and α2‐ and β2‐adrenoceptor antagonists (yohimbine and ICI 118,551) partially reversed the effects produced by melatonin and 5‐MCA‐NAT, suggesting the possible involvement of cholinergic and noradrenergic systems in the hypotensive actions mediated by melatonin agonists. The α1‐adrenoceptor antagonist, corynanthine, had no significant effect. The strong hypotensive effect of the MT3 agonist, 5‐MCA‐NAT, suggests that this compound may be a useful agent for treating those pathologies where intraocular pressure is abnormally elevated.

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.