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 microm. 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 microm and in the presence of 5 and 10 microm chelerythrine this increased slightly to 110 and 150 microm, respectively, which fits a noncompetitive inhibitor profile for chelerythrine. 5 Chelerythrine at 10 microm 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.

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.

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.

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.

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).