I P Chessell

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.

P2X receptor-mediated excitation of nociceptive afferents in the normal and arthritic rat knee joint

1 We tested the hypothesis that functional P2X receptors are present on peripheral terminals of primary afferent articular nociceptors in the rat knee joint. Neural activity was recorded extracellularly from the medial articular nerve innervating the knee joint in rats anaesthetized with pentobarbitone. 2 The selective P2X receptor agonist, αβ methylene ATP (αβmeATP), and the endogenous ligand, ATP, caused a rapid short‐lasting excitation of a sub‐population of C and Aδ nociceptive afferent nerves innervating normal knee joints when injected intra‐arterially or intra‐articularly, and this effect was antagonized by the non‐selective P2 receptor antagonist PPADS. 3 Induction of a chronic (14–21 days) unilateral inflammatory arthritis of the knee joint using locally injected Freunds adjuvant neither increased or decreased responsiveness of joint nociceptors to αβmeATP or ATP. 4 Our results support the hypothesis that αβmeATP‐sensitive P2X receptors are expressed on peripheral nociceptive afferents in the rat knee joint suggesting that they may be involved in the initiation of nociception and pain.

Cloning and functional characterisation of the mouse P2X7 receptor

We have isolated a 1785‐bp complementary DNA (cDNA) encoding the murine P2X7 receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80% and 85% homology to the human and rat P2X7 subunits, respectively. Functional properties of the heterologously expressed murine P2X7 homomeric receptor broadly resembled those of the P2X7 receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X7 receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.

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.

Excitatory effect of P2X receptor activation on mesenteric afferent nerves in the anaesthetised rat

1 We examined the effects of P2X purinoceptor agonists and P2 purinoceptor antagonists on mesenteric afferent nerves supplying the jejunum in the pentobarbitone sodium‐anaesthetised rat. 2 ATP (0.01–10 mg kg−1, i.a.) and α,β‐methylene‐ATP (1–30 μg kg−1, i.a.) each induced dose‐dependent increases in afferent nerve discharge and intrajejunal pressure. The effect on afferent nerves comprised an early (< 2 s after administration) intense burst of activity followed by a later increase (> 2 s after administration), less pronounced in comparison, which coincided with elevated intrajejunal pressure. 3 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (20 mg kg−1, i.v.) and suramin (80 mg kg−1, i.v.) each antagonised both the early and later increases in afferent nerve discharge elicited by α,β‐methylene‐ATP (30 μg kg−1, i.a.). 4 Co‐administration of ω‐conotoxin MVIIA and ω‐conotoxin SVIB (each at 25 μg kg−1, i.v.), or treatment with the selective 5‐HT3 receptor antagonist alosetron (30 μg kg−1, i.v.), did not affect the rapid burst of afferent nerve activity elicited by α,β‐methylene‐ATP (30 μg kg−1, i.a.). However, toxin treatment did attenuate the elevations in intrajejunal pressure and the corresponding later phases of evoked afferent discharge, while alosetron inhibited basal afferent nerve activity. 5 In summary, ATP and α,β‐methylene‐ATP each evoke excitation of mesenteric afferent nerves in the anaesthetised rat. We propose that the early increase in mesenteric afferent nerve activity represents a direct effect on the nerve ending, mediated by P2X receptors, whereas the later increase reflects activation of mechanosensitive fibres secondary to elevated intrajejunal pressure.

Activation of P2X receptors for adenosine triphosphate evokes cardiorespiratory reflexes in anaesthetized rats

1 We tested the hypothesis that activation of P2X receptors associated with vagal afferent nerves can evoke a Bezold‐Jarisch (B‐J) depressor reflex in anaesthetized rats. 2 Injection of αβ‐methylene ATP (αβ‐MeATP; 0.6‐600 nmol i.v.) evoked a dose‐dependent B‐J reflex comprising bradycardia, hypotension and apnoea in rats anaesthetized with pentobarbitone. Apnoea was commonly preceded by hyperventilation. Bilateral vagotomy significantly reduced the bradycardia and most of the apnoeic response without affecting hyperventilation, and unmasked a vasopressor response. Hypotension and apnoea were subject to desensitization, and ATP was about 100 times less potent than αβ‐MeATP in evoking the B‐J reflex. 3 ED50 values for responses to αβ‐MeATP were: bradycardia 14.6 ± 3.8 nmol; apnoea 47.1 ± 8.5 nmol; hyperventilation 23.3 ± 6.0 nmol, n= 14. The ED50 for apnoea was significantly greater than that for bradycardia or hyperventilation (P < 0.05). Atropine (2.8 μmol (kg body wt)−1 i.v.) antagonized the reflex bradycardia and hypotension. 4 The P2 antagonists suramin (14 μmol (kg body wt)−1 i.v.) and PPADS (17 μmol (kg body wt)−1 i.v.) antagonized the bradycardic and apnoeic components of the reflex response to αβ‐MeATP, without reducing the vasopressor or hyperventilatory responses to the agonist. 5 Recordings from vagal afferents showed that pulmonary inflation receptors were activated by αβ‐MeATP in 62 % of units recorded (ED50 22 ± 5 nmol) and this was blocked by PPADS (17 μmol (kg body wt)−1 i.v.); unidentified vagal afferents were also activated. 6 αβ‐MeATP activated carotid chemoreceptor afferents (ED50 23 ± 9 nmol), an action that was unaffected by PPADS or suramin. 7 The results support the hypothesis that P2X receptor subtypes for ATP are associated with specific sensory nerves that form part of the homeostatic mechanism for cardiovascular and respiratory regulation and these receptors therefore have physiological, pathological and therapeutic significance.

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.