Chelerythrine and other benzophenanthridine alkaloids block the human P2X₇ receptor

Abstract

1. Extracellular ATP can activate a cation-selective channel/pore on human B-lymphocytes, known as the P2X₇ receptor. Activation of this receptor is linked to PLD stimulation. We have used ATP-induced ⁸⁶Rb⁺ (K⁺) efflux to examine the effect of benzophenanthridine alkaloids on P2X₇ channel/pore function in human B-lymphocytes.

2. Both ATP and the nucleotide analogue 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) induced an ⁸⁶Rb⁺ efflux, which was completely inhibited by the isoquinoline derivative 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62), a potent P2X₇ receptor antagonist.

3. The benzophenanthridine alkaloid chelerythrine, a potent PKC inhibitor, inhibited the ATP-induced ⁸⁶Rb⁺ efflux by 73.4±3.5% and with an IC₅₀ of 5.6±2.3 μM. Similarly, other members of this family of compounds, sanguinarine and berberine, blocked the ATP-induced ⁸⁶Rb⁺ efflux by 58.8±4.8 and 61.1±8.0%, respectively.

4. Concentration–effect curves to ATP estimated an EC₅₀ value of 78 μM and in the presence of 5 and 10 μM chelerythrine this increased slightly to 110 and 150 μM, respectively, which fits a noncompetitive inhibitor profile for chelerythrine.

5. Chelerythrine at 10 μM was effective at inhibiting the ATP-induced PLD stimulation in B-lymphocytes by 94.2±21.9% and the phorbol 12-myristate 13-acetate-induced PLD stimulation by 68.2±7.4%.

6. This study demonstrates that chelerythrine in addition to PKC inhibition has a noncompetitive inhibitory action on the P2X₇ receptor itself.

Introduction

The P2X₇ receptor is an ATP-gated channel with intracellular N- and C-termini with two transmembrane domains, and is expressed in cells of haemopoietic origin (Dubyak, 2001). The large extracellular loop contains a number of glycosylation sites and putative ATP-binding domains (Worthington et al., 2002). The P2X₇ channel is selective to cations and its opening results in an influx of Ca²⁺ and efflux of K⁺ from the cell (Dubyak, 2001). ATP-mediated Ca²⁺ influx through the P2X₇ receptor is associated with the activation of PLD in B-lymphocytes (Gargett et al., 1996). However, it is not known what regulates PLD downstream of ATP-induced P2X₇ activation. PKC is well recognized as an important regulator of PLD (Exton, 2002). It has been recently shown that the PKC antagonist chelerythrine inhibits ATP-induced PLD activity in rat submandibular acinar gland and ductal cells (Perez-Andres et al., 2002; Pochet et al., 2003), suggesting a role for PKC in this process. These studies, however, did not determine if chelerythrine inhibited the P2X₇ receptor directly.

Chelerythrine is the only member of the benzophenanthridine alkaloids with potent and selective PKC inhibitory actions (IC₅₀ 0.66 μM in a permeabilized cell system) (Herbert et al., 1990). Structurally related compounds, sanguinarine and berberine, do not display such potent inhibition of PKC (Wang et al., 1997). However, chelerythrine also inhibits enzymes such as alanine aminotransferase and Na⁺/K⁺-ATPase independently of PKC (Cohen et al., 1978; Walterova et al., 1981), raising the possibility that chelerythrine may interact with other molecules such as the P2X₇ receptor. In this study, we demonstrate that chelerythrine blocks the ATP-induced cation fluxes mediated by the P2X₇ receptor, as well as the ATP-induced stimulation of PLD in human B-lymphocytes.

Methods

Source and preparation of human peripheral blood B-lymphocytes

Blood was collected from patients with informed consent and with approval from the Wentworth Area Health Service Human Ethics Committee (Penrith, Australia). Peripheral blood B-lymphocytes from nine different patients with chronic lymphocytic leukaemia (CLL) were isolated by density centrifugation as described (Wiley et al., 1992). Cells were either resuspended in NaCl medium (145 mM NaCl, 5 mM KCl, 10 mM HEPES, 0.1% w v⁻¹ BSA, 5 mM D-glucose) for rubidium-86 (⁸⁶Rb⁺) loading or in supplemented RPMI-1640 medium containing 10% foetal calf serum, 2 mM L-glutamine and 5 μg ml⁻¹ gentamicin for [³H]oleic acid labelling.

⁸⁶Rb⁺ efflux measurements

ATP-induced ⁸⁶Rb⁺ efflux from B-lymphocytes (5 × 10⁶ cells ml⁻¹) was performed as described (Wiley et al., 2003). ⁸⁶Rb⁺-loaded cells were incubated for 5 min at 37°C in the presence or absence of inhibitor, before the addition of agonist for 4 min. ⁸⁶Rb⁺ efflux was expressed as (1−N_t/N_total), where N_t was the level of cell-associated radioactivity at time t (determined by Cerenkov counting) and N_total the amount of cell-associated radioactivity at time zero. The data were linearized by log transformation calculation of series time constants.

Phospholipase D assay

B-lymphocytes (1 × 10⁷ cells ml⁻¹) were cultured at 37°C/5% CO₂ overnight in supplemented RPMI-1640 medium containing [³H]oleic acid (2–5 μCi ml⁻¹). Labelled cells were resuspended in KCl medium (150 mM KCl, 10 mM HEPES, 5 mM D-glucose, 0.1% w v⁻¹ BSA, pH 7.5) containing 1 mM BaCl₂ and pre-incubated for 5 min in the presence of the primary alcohol, 1-butanol (30 mM), which yields a stable phosphatidylalcohol end product following PLD stimulation. The cells were then incubated in the presence or absence of chelerythrine (10 μM) for 15 min at 37°C before incubation in the presence or absence of ATP (500 μM) or phorbol 12-myristate 13-acetate (PMA; 0.1 μM) for a further 15 min. Membrane lipids were extracted and the level of phosphatidylbutanol (PBut) was determined as described (Gargett et al., 1996).

Statistics

Differences in ⁸⁶Rb⁺ efflux and PLD activity were compared using the two-tailed unpaired Student's t-test.

Materials

ATP, BzATP, BaCl₂, HEPES, D-glucose, BSA, L-glutamine, gentamicin, RPMI-1640 medium, PMA, iodine crystals, organic solvents and Hyperfilm MS were from Sigma Chemical Co., U.S.A. (St Louis, MO, U.S.A.). Foetal calf serum was from Life Technologies (Grand Island, NY, U.S.A.). Chelerythrine chloride and 1-[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) were from BIOMOL (Plymouth Meeting, PA, U.S.A.). Ficoll-Paque (d=1.077) was obtained from Pharmacia (Uppsala, Sweden). [9,10-³H]oleic acid (5 mCi ml⁻¹; specific radioactivity 10 Ci mmol⁻¹) and ⁸⁶RbCl (1.5 mCi ml⁻¹; specific radioactivity 3 Ci mmol⁻¹) were purchased from Amersham International (Little Chalfont, Buckinghamshire, U.K.). Di-n-butyl phthalate and di-isooctyl phthalate from BDH Chemicals (Poole, England) were blended 80 : 20 (v v⁻¹) to give a mixture of density 1.030 g ml⁻¹. LK6D thin-layer chromatography plates were from Whatman (Maidstone, Kent, U.K.). En³Hance autoradiography spray was from DUPONT (Boston, MA, U.S.A.).

Results

ATP-induced ⁸⁶Rb⁺ efflux from B-lymphocytes is mediated by the P2X₇ receptor

Chelerythrine and other benzophenanthridine alkaloids are fluorescent compounds, making them unsuitable for use in fluorescent-based assays of P2X₇ function, such as flow cytometry and fluorimetry. Therefore, P2X₇ channel/pore activity was assessed by measuring ATP-induced ⁸⁶Rb⁺ efflux from B-lymphocytes. This method has been previously used to measure P2X₇ function in lymphocytes, monocytes and macrophages (Steinberg & Silverstein, 1987; Wiley et al., 1992; 2003; Sluyter et al., 2004). Over a period of 4 min, ⁸⁶Rb⁺ efflux followed first-order kinetics with a rate constant of 0.02±0.01 min⁻¹ (n=9) in the absence of ATP and 0.20±0.03 min⁻¹ (n=9) in the presence of 100 μM ATP (Figure 1). Moreover, the potent P2X₇ agonist BzATP (20 μM), induced a similar rate of ⁸⁶Rb⁺ efflux from human B-lymphocytes at a rate of 0.23 min⁻¹ (n=2, Figure 1). To confirm that the ATP-induced ⁸⁶Rb⁺ efflux was mediated by P2X₇, B-lymphocytes were pre-incubated with the specific P2X₇ antagonist, KN-62 (Gargett & Wiley, 1997). Pre-incubation of B-lymphocytes for 5 min with 1 μM KN-62 had no effect on basal rates of ⁸⁶Rb⁺ efflux, but inhibited ATP-induced fluxes by 96.0% (n=2, Figure 1).

Figure 1.

P2X₇ activation mediates ⁸⁶Rb⁺ efflux from human B-lymphocytes. ⁸⁶Rb⁺-loaded CLL B-lymphocytes in KCl (for ATP) or NaCl (for BzATP) medium supplemented with 20 μM CaCl₂ were pre-incubated at 37°C for 5 min in the absence or presence of 1 μM KN-62, before the addition of 100 μM ATP or 20 μM BzATP for a further 4 min. The data are one representative of nine experiments for ATP and one representative of two experiments for BzATP and KN-62.

Chelerythrine and its structural analogues inhibit P2X₇ receptor-mediated ⁸⁶Rb⁺ efflux in B-lymphocytes

To examine the effect of chelerythrine, a PKC inhibitor, on P2X₇ receptor function, we measured ATP-induced ⁸⁶Rb⁺ efflux. Pre-incubation of B-lymphocytes with 10 μM chelerythrine inhibited the ⁸⁶Rb⁺ efflux induced by 100 μM ATP by 73.4±3.5% (n=13, P<0.001, Figure 2). At 100 μM ATP, chelerythrine inhibited ⁸⁶Rb⁺ efflux with an IC₅₀ of 5.6±2.3 μM (data not shown). The Hill coefficient for ATP was 2.1 in the presence of chelerythrine and 2.0 in its absence. Pre-incubation of B-lymphocytes with 10 μM chelerythrine also inhibited the ⁸⁶Rb⁺ efflux induced by 20 μM BzATP by 51% (data not shown).

Figure 2.

Chelerythrine and its structural analogues, sanguinarine and berberine, block ATP-induced ⁸⁶Rb⁺ efflux. ⁸⁶Rb⁺-loaded CLL B-lymphocytes in KCl medium containing 20 μM CaCl₂ were pre-incubated for 5 min at 37°C in the presence of 10 μM sanguinarine, 10 μM berberine or 10 μM chelerythrine, or in the absence of inhibitors, before the addition of 100 μM ATP for a further 4 min. The data are one representative of 13 experiments for chelerythrine and three experiments for sanguinarine and berberine.

To determine if the inhibitory effect of chelerythrine on the P2X₇ receptor was PKC dependent, we studied two structural analogues of chelerythrine, sanguinarine, which has poor PKC inhibitory activity with an IC₅₀ of 217 μM, and berberine, which has no known PKC inhibitory actions (Wang et al., 1997). Both sanguinarine and berberine at 10 μM inhibited ATP-induced ⁸⁶Rb⁺ efflux by 58.8±4.8% (n=3, P<0.02) and 61.1±8.0% (n=3, P<0.02), respectively (Figure 2). These analogues had no significant effect on the rate of ⁸⁶Rb⁺ efflux in the absence of ATP (data not shown).

Inhibition of the P2X₇ receptor by chelerythrine is noncompetitive

To establish whether the inhibition of chelerythrine was competitive or noncompetitive, the ⁸⁶Rb⁺ efflux was measured over a range of ATP concentrations in the absence or presence of 5 or 10 μM chelerythrine (Figure 3). The addition of ATP (25–1000 μM) increased ⁸⁶Rb⁺ efflux in a concentration-dependent manner (Figure 3). Maximum rates of ⁸⁶Rb⁺ efflux were observed at ATP concentrations above 200 μM. Chelerythrine was an effective inhibitor at all ATP concentrations tested. The EC₅₀ for ATP was 78 μM in the absence of inhibitor. In the presence of 5 and 10 μM chelerythrine, the EC₅₀ for ATP rose modestly to 110 and 150 μM, respectively. However, the maximum response to ATP was reduced by 35.1% in the presence of 5 μM chelerythrine and further reduced by 64.9% in the presence of 10 μM chelerythrine. The data are consistent with the idea that the inhibitory action of chelerythrine is predominantly noncompetitive. Similar results were also obtained with berberine, which has no known PKC inhibitory action (data not shown).

Figure 3.

Allosteric noncompetitive inhibition of ATP-induced ⁸⁶Rb⁺ efflux by chelerythrine. ⁸⁶Rb⁺-loaded CLL B-lymphocytes were incubated for 5 min at 37°C in the presence of either 5 or 10 μM chelerythrine or in the absence of chelerythrine before addition of ATP, as indicated for a further 4 min. The data are presented as means ±s.e.m. from three separate experiments.

Chelerythrine inhibits the ATP- and PMA-induced PLD activation in B-lymphocytes

To determine if chelerythrine also impairs ATP-induced PLD stimulation, in B-lymphocytes, cells were labelled with [³H]oleic acid by overnight incubation and PLD activity was measured by the transphosphatidylation reaction in the presence of 1-butanol. As previously observed (Gargett et al., 1996; Gargett & Wiley, 1997), but with a different cohort of B-CLL patients, we found that both ATP and PMA stimulated PLD activity in B-lymphocytes (Figure 4). Pre-incubation of B-lymphocytes with chelerythrine at 10 μM abolished the ATP-induced PLD activity by 94.2±21.9% (n=5, P<0.03) and reduced the PMA-induced PLD activity by 68.2±7.4% (n=5, P< 0.05; Figure 4). Chelerythrine did not inhibit the basal activity of PLD.

Figure 4.

Chelerythrine inhibits both ATP- and PMA-induced PLD activity. [³H]oleic acid-labelled CLL B-lymphocytes were incubated in KCl medium containing 1 μM BaCl₂ in the absence or presence of 10 μM chelerythrine for 15 min prior to the addition of 500 μM ATP or 0.1 μM PMA for a further 15 min at 37°C. The data are presented as means ±s.e.m. from five separate experiments (^*P<0.03, ^**P<0.05, chelerythrine versus control).

Discussion

In this study, we used ATP-induced ⁸⁶Rb⁺ efflux from B-lymphocytes to assess the effect of chelerythrine and other benzophenanthridine alkaloids on P2X₇ channel/pore activity. In a series of preliminary experiments, other commonly used techniques such as flow cytometry and fluorimetry (Wiley et al., 2001) were found to be unsuitable due to the high fluorescence of chelerythrine (data not shown). The ATP-induced ⁸⁶Rb⁺ efflux from B-lymphocytes was mediated via the P2X₇ receptor, as this process was also stimulated by the potent P2X₇ agonist BzATP. In addition, the P2X₇ antagonist KN-62 inhibited ATP-induced ⁸⁶Rb⁺ efflux. Moreover, we have recently shown that ATP-induced ⁸⁶Rb⁺ efflux is impaired in B-lymphocytes from subjects heterozygous for a loss-of-function mutation at amino-acid 568 of the P2X₇ receptor (Wiley et al., 2003).

Chelerythrine inhibited the efflux of ⁸⁶Rb⁺ in a dose-dependent manner and acted noncompetitively with respect to ATP at the P2X₇ receptor. Moreover, the structural analogues of chelerythrine, sanguinarine and berberine, which have limited or no known PKC-inhibitory activity (Wang et al., 1997), also inhibited ATP-induced ⁸⁶Rb⁺ efflux. These results suggest that the inhibitory action of these compounds on the P2X₇ receptor is structurally related and may be a result of chelerythrine acting directly with the receptor. Chelerythrine, like KN-62, may be binding to a portion of the extracellular loop (Chen et al., 2002). However, it seems unlikely that chelerythrine binds to the ATP-binding site of the P2X₇ receptor as its locus of action on PKC is independent of the ATP active site (Herbert et al., 1990), consistent with chelerythrine acting as a noncompetitive inhibitor of P2X₇ receptor. However, given that a number of potential PKC phosphorylation sites are present in the P2X₇ receptor (Watters et al., 2001), and that phosphorylation of a conserved threonine residue in the N-terminus of P2X₂ and P2X₃ receptors is necessary for their normal function (Boue-Grabot et al., 2000; Paukert et al., 2001), we cannot exclude the possibility that a component of the inhibitory action of chelerythrine on P2X₇ function may involve PKC. Chelerythrine has been reported to inhibit other channels such as acetylcholine-induced K⁺ channel in mouse atrial myocytes (Shi & Wang, 1999) and acetylcholine-induced currents in PC12 cells (Cho et al., 2001). Nevertheless, this study confirms that the P2X₇ receptor can be added to the list of ion channels directly blocked by chelerythrine.

In addition to ATP-induced ⁸⁶Rb⁺ efflux, chelerythrine also inhibited ATP-induced PLD stimulation. Chelerythrine blocked 500 μM ATP-induced PLD activity with greater efficacy than 100 μM ATP-induced ⁸⁶Rb⁺ efflux in human B-lymphocytes (94.2±21.9 versus 73.4±3.5%, respectively) suggesting that chelerythrine not only blocks P2X₇ but may also act on signalling events, such as PKC, downstream of this receptor. Consistent with this idea, chelerythrine inhibited PLD activity induced by PMA, a known activator of PKC. Moreover, recent studies have indicated that ATP via P2X₇ receptor activation stimulates other kinases such as stress-activated protein kinase and Rho-effector kinase (Humphreys et al., 2000; Verhoef et al., 2003). The ATP- and PMA-induced PLD activity could not be investigated in the presence of sanguinarine or berberine, as these compounds are only soluble in alcohol and this alcohol interferes with the transphosphatidylation assay used to determine PLD activity (data not shown). Collectively, these results suggest that further studies are needed to confirm if PKC is activated by P2X₇ and if PKC is involved in the regulation of ATP-induced PLD stimulation via the P2X₇ receptor.

Studies using P2X₇-deficient mice have shown that these animals have reduced inflammatory responses (Labasi et al., 2002) possibly due to reduced P2X₇-mediated secretion of interleukin-1β from cells of the monocyte–macrophage lineage (Solle et al., 2001), which is due, in part, to K⁺ efflux (of which Rb⁺ is a surrogate) (Perregaux & Gabel, 1994). Interestingly, chelerythrine and sanguinarine have potent anti-inflammatory effects in vivo (Lenfeld et al., 1981). Thus, it is tempting to speculate that the previously described anti-inflammatory effects of these benzophenanthridine alkaloids may arise from inhibition of the P2X₇ receptor.

In conclusion, our data highlight the importance of ensuring that kinase inhibitors employed in studying signalling events downstream of P2X₇ do not affect the receptor directly.

Abbreviations

BzATP

2′-3′-O-(4-benzoylbenzoyl)-ATP

CLL

chronic lymphocytic leukaemia

KN-62, 1-(N

O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine

PBut

phosphatidylbutanol

PMA

phorbol 12-myristate 13-acetate

⁸⁶Rb⁺

rubidium-86