A. D. Michel

Pharmacological characterization of ATP- and LPS-induced IL-1β release in human monocytes

We have utilized the human monocytic cell line, THP‐1, and freshly isolated adherent human monocytes with the compounds pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disuphonic acid (PPADS), oxidized ATP, and 1‐(N,O‐bis{5‐isoquinolinesufonyll}‐N‐methyl‐L‐tyrosyl)‐4‐phenylpiperazine (KN‐62) to pharmacologically characterize the P2 receptor involved in ATP‐induced release of interleukin 1β (IL‐1β). We have also investigated the involvement of P2 receptors in lipopolysaccharide (LPS)‐induced IL‐1β release from both cell types. ATP caused release of IL‐1β from LPS primed THP‐1 cells in both a time‐ and concentration‐dependent manner, with a minimal effective ATP concentration of 1 mM. Stimulation of cells with 5 mM ATP resulted in detectable concentrations of IL‐1β in cell supernatants within 30 min. The ATP analogue benzoylbenzoyl ATP (DBATP), a P2X7 receptor agonist, was approximately 10 fold more potent than ATP at eliciting IL‐1β release. KN‐62 (1 μM), PPADS (100 μM) or oxidized ATP (100 uM) significantly inhibited 5 mM ATP‐induced IL‐1β release by 81, 90 and 66% respectively, but failed to significantly inhibit LPS‐induced IL‐1β release in both THP‐1 cells and in freshly isolated human monocytes. In both THP‐1 cells and freshly isolated human monocytes, addition of the ATP degrading enzyme apyrase (0.4 U ml−1) to cell supernatants prior to LPS activation failed to significantly inhibit the LPS‐induced IL‐1β release. In addition there was no correlation between extracellular ATP concentrations and IL‐1β release in THP‐1 cells when studied over a 6 h time period. In conclusion our data confirm the involvement of P2X7 receptors in ATP‐induced IL‐1β release in human monocytes. However no evidence was obtained which would support the involvement of either endogenous ATP release or P2X7 receptor activation as the mechanism by which LPS‐induces IL‐1β release in either the THP‐1 cell line or in freshly isolated human monocytes.

Properties of the pore‐forming P2X7 purinoceptor in mouse NTW8 microglial cells

We have used whole‐cell patch clamping methods to study and characterize the cytolytic P2X7 (P2Z) receptor in the NTW8 mouse microglial cell line. At room temperature, in an extracellular solution containing 2 mM Ca2+ and 1 mM Mg2+, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐adenosine‐5′‐triphosphate (Bz‐ATP; 300 μM), or ATP (3 mM), evoked peak whole cell inward currents, at a holding potential of −90 mV, of 549±191 and 644±198 pA, respectively. Current‐voltage relationships generated with 3 mM ATP reversed at 4.6 mV and did not display strong rectification. In an extracellular solution containing zero Mg2+ and 500 μM Ca2+ (low divalent solution), brief (0.5 s) application of these agonists elicited larger maximal currents (909±138 and 1818±218 pA, Bz‐ATP and ATP, respectively). Longer application of ATP (1 mM for 30 s) produced larger, slowly developing, currents which reached a plateau after approximately 15–20 s and were reversible on washing. Under these conditions, in the presence of ATP, ethidium bromide uptake could be demonstrated. Further applictions of 1 mM ATP produced rapid currents of the same magnitude as those observed during the 30 s application. Subsequent determination of concentration‐effect curves to Bz‐ATP, ATP and 2‐methylthio‐ATP yielded EC50 values of 58.3, 298 and 505 μM, respectively. These affects of ATP were antagonized by pyridoxal‐phosphate‐6‐azophenyl‐ 2′, 4′‐disulphonic acid (PPADS; 30 μM) but not suramin (100 μM). In low divalent solution, repeated application of 1 mM ATP for 1 s produced successively larger currents which reached a plateau, after 8 applications, of 466% of the first application current. PPADS (30 μM) prevented this augmentation, while 5‐(N,N‐hexamethylene)‐amiloride (HMA) (100 μM) accelerated it such that maximal augmentation was observed after only one application of ATP in the presence of HMA. At a bath temperature of 32°C, current augmentation also occurred in normal divalent cation containing solution. These data demonstrate that mouse microglial NTW8 cells possess a purinoceptor with pharmacological characteristics resembling the P2X7 receptor. We suggest that the current augmentation phenomenon observed reflects formation of the large cytolytic pore characteristic of this receptor. We have demonstrated that pore formation can occur under normal physiological conditions and can be modulated pharmacologically, both positively and negatively.

Ionic effects on human recombinant P2X7 receptor function.

The actions of monovalent and divalent ions on the P2X7 receptor have been assessed by measuring their effect on responses to the P2 receptor agonist, 2’- and 3’-O-(4-benzoyl-benzoyl)-ATP (DbATP), in HEK293 cells expressing the human recombinant P2X7 receptor. In these cells, DbATP increased the cellular accumulation of the DNA binding, fluorescent dye, YO-PRO-1. The potency of DbATP to elicit this effect was decreased by both calcium and magnesium ions. In addition, when the pH was increased above 8 or reduced below 6.5, the potency of DbATP was less than obtained at pH 7.5. Monovalent ions also affected the P2X7 receptor such that the potency of DbATP was 19-fold higher in NaCl-free buffer containing 280 mM sucrose (pEC50=6.48) than in 140 mM NaCl containing buffer (pEC50=5.19). Monovalent cations differentially affected the potency of DbATP. Thus, when the chloride concentration was maintained at 140 mM, pEC50 values for DbATP were 6.14, 5.87 and 5.19 when the counter cation was 140 mM choline, potassium or sodium, respectively. Monovalent anions also differentially affected the potency of DbATP and in the presence of 140 mM sodium ions, pEC50 values for DbATP were 6.14, 6.07, 5.19 and 4.53, respectively, when the counter anion was 140 mM aspartate, glutamate, chloride or iodide. The inhibitory effect of monovalent anions on P2X7 receptor function was also observed in electrophysiological studies. Thus in sodium glutamate containing buffer the potency of DbATP (pEC50=5.55) was approximately 22-fold higher than in NaCl containing buffer (pEC50=4.20). This study has demonstrated that P2X7 receptor function can be markedly affected by a wide range of ions and that physiological concentrations of sodium and chloride ions, as well as divalent cations, contribute to the low potency of ATP as an agonist at this receptor.

Effects of antagonists at the human recombinant P2X7 receptor

1 We have used whole‐cell patch clamping methods to examine the properties of the recombinant human P2X7 (P2Z) receptor stably expressed in HEK‐293 cells. 2 In an extracellular solution with lowered concentrations of divalent cations (zero Mg2+ and 0.5 mm Ca2+), both ATP and the nucleotide analogue, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐adenosine 5′‐triphosphate (Bz‐ATP) evoked concentration‐dependent whole‐cell inward currents with maxima of 4658±671 and 5385±990 pA, respectively, at a holding potential of −90 mV. Current‐voltage relationships determined using 100 μm Bz‐ATP reversed at −2.7±3.1 mV, and did not display significant rectification. 3 Repeated applications of 300 μm Bz‐ATP produced inward currents with similar rise‐times (approx. 450 ms, 5–95% current development) but with progressively slower 95–5% decay times, with the eighth application of this agonist yielding a decay time of 197% of the first application. 4 Concentration‐effect curves to ATP and Bz‐ATP produced estimated EC50 values of 780 and 52.4 μm, respectively. Consecutive concentration‐effect curves to Bz‐ATP produced curves with similar maxima and EC50 values. 5 The non‐selective P2 antagonists, pyridoxal‐phosphate‐6‐azophenyl‐, 2′,4′‐disulphonic acid (PPADS) and suramin, both produced concentration‐dependent increases in maximal inward currents to Bz‐ATP, with IC50 concentrations of approximately 1 μm and 70 μm, respectively. The profile of antagonism produced by PPADS was not that of a competitive antagonist. 6 The isoquinolene derivatives 1‐(N,O‐bis[5‐isoquinolinesulphonyl]‐N‐methyl‐l‐tyrosyl)‐4‐phenylpiperazine (KN‐62) and calmidazolium both produced antagonism which was not competitive, with IC50 concentrations of approximately 15 and 100 nm, respectively. HMA (5‐(N,N‐hexamethylene)‐ amiloride) was also an effective antagonist at a concentration of 10 μm. The group IIb metal, copper, also displayed antagonist properties at the human P2X7 receptor, reducing the maximum response to Bz‐ATP by about 50% at a concentration of 1 μm. 7 These data demonstrate that the human recombinant P2X7 receptor displays functional behaviour which is similar to the recombinant rat P2X7 receptor, but has a distinct pharmacological profile with respect to agonist and antagonist sensitivity.

NEW INSIGHTS ON P2X PURINOCEPTORS

Significant advances in understanding of P2X purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becoming available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for P2X receptor heterogeneity, from data with agonists, has recently been reported.A number of new antagonists at P2X purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P2X purinoceptor.Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P2X purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P2X purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P2X purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P2X purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better understanding of the physiological and pathological importance of ATP and its activation of P2X purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.

Apparent species differences in the kinetic properties of P2X7 receptors

Apparent species differences in the responses of recombinant P2X7 receptors to repeated application of 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP) have been investigated. Repeated application of 100 μM BzATP resulted in a progressive increase in current magnitude (current growth) at mouse and human, but not rat P2X7 receptors. Current growth was thought to reflect progressive dilation of the P2X7 ion‐channel to a pore permeable to large molecules (MW<900), suggesting that channel dilation was not occurring at the rat P2X7 receptor. However, 100 μM BzATP produced a rapid influx of YO‐PRO‐1 (MW375) in cells expressing rat or human P2X7 receptors. There were, however, species differences in agonist potency such that 100 μM BzATP was a supra‐maximal concentration at rat, but not human or mouse, P2X7 receptors. Importantly, when sub‐maximal concentrations of BzATP or ATP were examined, current growth occurred at rat P2X7 receptors. The rate of current growth and YO‐PRO‐1 accumulation increased with agonist concentration and appeared more rapid at rat and human, than at mouse P2X7 receptors. The potency of BzATP and ATP was 1.5–10 fold lower in naïve cells than in cells repeatedly exposed to ATP. This study demonstrates that current growth occurs at mouse, rat and human P2X7 receptors but only when using sub‐maximal concentrations of agonist. Previously, current growth was thought to reflect the progressive increase in pore diameter of the P2X7 receptor ion channel, however, the results of this study suggest a progressive increase in agonist potency may also contribute.

Functional characterization of the P2X4 receptor orthologues

The aim of this study was to functionally characterize the recombinant mouse P2X4 receptor and to compare its pharmacological properties with those of the human and rat orthologues. Whole cell recordings were made from rafts of HEK‐293 cells stably expressing recombinant mouse, rat or human P2X4 receptors, using Cs‐aspartate containing electrodes (3–8 MΩ) in a HEPES‐buffered extracellular medium. The agonist potency of ATP at the three species orthologues was similar, with mean EC50 values of 2.3 μM, 1.4 μM and 5.5 μM, respectively. Adenosine‐5′‐tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X4 receptors (EC50=2.6 and 3.0 μM), but was significantly less potent at the rat orthologue (EC50=20.0 μM). α,β‐methylene adenosine‐5′‐triphosphate (α,β‐meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X4 and 24% at the human P2X4 receptor. In contrast to the other species orthologues, α,β‐meATP failed to elicit a significant agonist response at rat P2X4 receptors, and was found to act as an antagonist, with an IC50 of 4.6 μM, against 10 μM ATP. Mouse P2X4 receptors were found to be sensitive to the antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (IC50=10.5 μM), as were human P2X4 receptors (IC50=9.6 μM). The rat receptor however, showed a low sensitivity to PPADS (IC50>100 μM). All three orthologues were relatively suramin‐insensitive (IC50>100 μM) and insensitive to 1‐[N,O‐Bis(5‐isoquinoline sulphonyl)benzyl]‐2‐(4‐phenylpiperazine)ethyl]‐5‐isoquinoline sulphonamide (KN‐62; IC50>3 μM). Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X4 receptor, and differ markedly from the rat receptor.

Evidence that Nitric Oxide Causes Calcium‐Independent Release of [3H]Dopamine from Rat Striatum In Vitro

Abstract: Nitric oxide (NO), liberated from the photoactive donor Roussins black salt (RBS), was investigated for its ability to release tritium from [3H]dopamine‐loaded rat striatal slices. Our results show that illumination of RBS‐pretreated striatal slices caused an increase in basal dopamine release, which was reduced by ∼73% in the presence of oxyhaemoglobin (10 µM), indicating that it was mediated by liberation of NO. The release was insensitive to removal of extracellular calcium yet was not due to gross cellular damage of the tissue, as there was no detectable increase in lactate dehydrogenase release. Chelation of intracellular calcium with 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetra(acetoxymethyl) ester (BAPTA‐AM; 10 µM) had no effect on the dopamine release stimulated by illumination of RBS‐pretreated slices. The concentration of BAPTA‐AM was adequate to chelate intracellular calcium because it inhibited release evoked by the calcium ionophore ionomycin (10 µM). Superfusion with zaprinast (10 µM) had no effect on RBS‐induced dopamine release, suggesting that a mechanism independent of cyclic GMP is involved. This study indicates that NO has a stimulatory effect on striatal dopamine release in vitro that is independent of calcium.

Functional characterization of the P2X(4) receptor orthologues.

The aim of this study was to functionally characterize the recombinant mouse P2X4 receptor and to compare its pharmacological properties with those of the human and rat orthologues. Whole cell recordings were made from rafts of HEK‐293 cells stably expressing recombinant mouse, rat or human P2X4 receptors, using Cs‐aspartate containing electrodes (3–8 MΩ) in a HEPES‐buffered extracellular medium. The agonist potency of ATP at the three species orthologues was similar, with mean EC50 values of 2.3 μM, 1.4 μM and 5.5 μM, respectively. Adenosine‐5′‐tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X4 receptors (EC50=2.6 and 3.0 μM), but was significantly less potent at the rat orthologue (EC50=20.0 μM). α,β‐methylene adenosine‐5′‐triphosphate (α,β‐meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X4 and 24% at the human P2X4 receptor. In contrast to the other species orthologues, α,β‐meATP failed to elicit a significant agonist response at rat P2X4 receptors, and was found to act as an antagonist, with an IC50 of 4.6 μM, against 10 μM ATP. Mouse P2X4 receptors were found to be sensitive to the antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (IC50=10.5 μM), as were human P2X4 receptors (IC50=9.6 μM). The rat receptor however, showed a low sensitivity to PPADS (IC50>100 μM). All three orthologues were relatively suramin‐insensitive (IC50>100 μM) and insensitive to 1‐[N,O‐Bis(5‐isoquinoline sulphonyl)benzyl]‐2‐(4‐phenylpiperazine)ethyl]‐5‐isoquinoline sulphonamide (KN‐62; IC50>3 μM). Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X4 receptor, and differ markedly from the rat receptor.

Antagonist effects on human P2X7 receptor-mediated cellular accumulation of YO-PRO-1

We have examined the interaction of P2 antagonists with the human P2X7 receptor by studying their effect on 2′ and 3′‐O‐benzoyl‐benzoyl‐ATP (DbATP) stimulated cellular accumulation of the fluorescent, DNA binding dye, YO‐PRO‐1 (MW=375Da). In suspensions of HEK293 cells expressing human recombinant P2X7 receptors, DbATP produced time and concentration‐dependent increases in YO‐PRO‐1 fluorescence. This response presumably reflects YO‐PRO‐1 entry through P2X7 receptor channels and binding to nucleic acids. When studies were performed in a NaCl‐free, sucrose‐containing buffer, full concentration‐effect curves to DbATP could be constructed. The P2 antagonists, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) and periodate oxidized ATP (oATP), reduced the potency of DbATP and decreased its maximum response. 1‐[N,O‐bis(1,5‐isoquinolinesulphonyl)‐N‐methyl‐L‐tyrosyl]‐4‐phenylpiperazine (KN62) and its analogue, KN04, reduced the potency of DbATP. Schild slopes for KN62 and KN04 were shallow and exhibited a plateau at concentrations of compound greater than 1 μM, indicating that these compounds were not competitive antagonists. Calmidazolium and a monoclonal antibody to human P2X7 receptors attenuated DbATP‐stimulated YO‐PRO‐1 accumulation but they were not competitive antagonists and only produced 2–3 fold decreases in the potency of DbATP. The effects of PPADS and KN62 were partially reversible whereas those of oATP were not. PPADS protected cells against the irreversible antagonist effects of oATP suggesting a common site of action. In contrast KN62 was not effective suggesting that it may bind at a different site to oATP and PPADS. This study has demonstrated that P2X7 receptor function can be quantified by measuring DbATP stimulated YO‐PRO‐1 accumulation and has provided additional information about the interaction of P2 receptor antagonists with the human P2X7 receptor.