Non-genomic inhibition of human P2X7 purinoceptor by 17beta-oestradiol

Abstract

1. Effects of oestrogen on the current evoked by ATP and benzoylbenzoyl ATP (BzATP) in CV-1 monkey kidney cells transformed by SV 40 (COS cells) expressing the human P2X7 (hP2X7) purinoceptor were studied using standard patch-clamp techniques. 2. 17beta-Oestradiol rapidly and reversibly inhibited the whole-cell hP2X7 receptor cation current. This inhibitory action resulted in a rightward shift of the dose-response curve to ATP and BzATP in the presence of physiological as well as low divalent cation concentrations. 3. The inhibitory effect of 17beta-oestradiol on the BzATP- or ATP-induced cation current was concentration dependent. The half-maximal inhibition was obtained with 3 microM 17beta-oestradiol. Progesterone and 17alpha-oestradiol had almost no effect on the hP2X7 receptor cation current. 4. The inhibition of the hP2X7 receptor cation current by 17beta-oestradiol did not depend on the membrane potential. 17beta-Oestradiol added to the extracellular side of outside-out patches inhibited BzATP-activated single-channel currents. 5. Activation of the hP2X7 receptor in both COS and U937 (human macrophage) cells did not induce the formation of large non-specific pores. 6. Since COS cells do not express endogenous nuclear oestrogen receptor, this study shows that, at pharmacological concentrations, 17beta-oestradiol inhibited the hP2X7 receptor cation channel in a non-genomic manner.

Figure 1. 17β-Oestradiol (β-oestradiol) inhibits BzATP-induced current in COS cells expressing hP2X₇ receptor

A, applications of 17β-oestradiol (5 μM) rapidly and reversibly inhibited the current induced by 10 μM BzATP. B, the inhibitory action of 5 μM 17β-oestradiol depended on the concentration of BzATP used (3, 10 or 30 μM applied to the same cell). C, concentration-response curves to BzATP and ATP under control conditions (filled symbols) and in the presence of 5 μM 17β-oestradiol (open symbols). ○, •, ▿ and ▾ represent curves obtained in the reference solution for BzATP and ATP, respectively. □ and ▪ represent the curves obtained for BzATP in the presence of low divalent cation concentrations (0.3 mM Ca²⁺, 0 mM Mg²⁺). Currents are normalized to their maximal amplitudes recorded in the absence of 17β-oestradiol. Each point represents the mean of 4–6 experiments. For all panels the holding potential was −40 mV.

Figure 2. Concentration dependence of the inhibitory effect of 17β-oestradiol (β-oestradiol) on the hP2X₇ receptor cation current

A, the inhibition of the BzATP- (10 μM) induced current was enhanced by increasing the concentration of 17β-oestradiol from 0.1 to 10 μM in the same cell. B, concentration-response curve for the inhibition of the BzATP- (10 μM) induced current by 17β-oestradiol. C, concentration-response curve for the inhibition of the ATP- (300 μM) induced current by 17β-oestradiol. Each point represents the mean of 4–8 experiments. D, ATP- (300 μM) induced current was not affected by 10 μM 17α-oestradiol (α-oestradiol) whereas it was inhibited by 5 μM 17β-oestradiol. E, 17α-oestradiol (α-Oest) or progesterone (Prog; 10 μM) did not mimic the inhibitory effect of 17β-oestradiol (β-Oest) on the ATP-induced current. Data displayed as means ±s.e.m. of 6–12 experiments. For all panels the holding potential was −40 mV.

Figure 3. The inhibitory effect of 17β-oestradiol (β-oestradiol) on the hP2X₇ receptor cation current did not depend on membrane potential

Current-voltage relationships of the current induced by 10 μM BzATP (A) or by 100 μM ATP (B). Voltage pulses of +80 (1), +40 (2), −40 (3) and -80 mV (4) were applied from the holding potential (-50 mV in A and −40 mV in B). The amplitude of the hP2X₇ receptor currents measured in the presence (open symbols) and in the absence of 17β-oestradiol (filled symbols) was subtracted from leakage and expressed as percentage of the current recorded at -130 mV (A) or -120 mV (B) in the absence of the inhibitor and plotted against membrane potential. Data displayed as means ±s.e.m. of 4–6 experiments. C, representative current traces from an outside-out patch maintained at -110 mV in the reference solution in the absence (left), in the presence of BzATP (10 μM) alone (middle), and after addition of 17β-oestradiol (5 μM, right). The open probability (NP_o) is indicated under the traces which are representative of results obtained in 3 different patches.

Figure 4. Ethidium uptake

A, trace representing a typical experiment recorded in COS cells expressing hP2X₇ receptors, showing that application of 100 μM BzATP in the presence of low divalent cation concentrations did not induce ethidium uptake, whereas permeabilization by β-escin led to ethidium uptake attested by fluorescence increase. B, summary of results obtained in COS and U937 cells. Control experiments were performed by stimulating COS cells expressing hP2X₁ receptors by 100 μM ATP. Data displayed as means ±s.e.m. of 10–15 cells.