Cardiac P2X purinergic receptors as a new pathway for increasing Na⁺ entry in cardiac myocytes

Abstract

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na(+). Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na(+) entry and modulate Na(+) handling. We further determined whether P2X receptor-induced stimulation of the Na(+)/Ca(2+) exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na(+)-K(+)-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca(2+) entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from -14 ± 2.3 to -25 ± 4.1 mV, causing an estimated intracellular Na(+) concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca(2+) entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca(2+) entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na(+) entry pathway through ligand-gated P2X4Rs in cardiomyocytes.

Keywords: Na+-K+-ATPase; Na+/Ca2+ exchanger; contraction; myocytes; purinergic receptors.

Fig. 1.

Peak Na⁺-K⁺-ATPase current (I_p) increases after 2-methylthio-ATP (2-meSATP) application in P2X4 receptor-overexpressing transgenic (P2X4R TG) cardiac myocytes. A, top: protocol of the rapid solution changes with 5.4 mM extracellular K⁺ concentration ([K⁺]_o) to elicit K⁺-activated I_p. Bottom, membrane voltage. I_p was recorded at −20 mV, and 2-meSATP was applied at −80 mV. CTR, control; WO, washout. B, top: individual traces in a typical myocyte showing the increase of [K⁺]_o-activated I_p after 2-meSATP application. Bottom, superimposed traces of I_p under CTR conditions and with 2-meSATP. C: peak I_p, normalized by cell capacitance, is presented as means ± SE. Average CTR peak I_p differed from that with 2-meSATP (n = 11 myocytes from 9 TG mice, P < 0.05). D: relationship between peak I_p and pipette Na⁺ concentration ([Na⁺]). Peak I_p was elicited by switching [K⁺]_o from 0 to 5.4 mM at −20 mV; pipette [Na⁺] was varied as shown. Normalized peak I_p is presented as means ± SE. Numbers of cells are indicated in the parentheses (from 18 TG mice).

Fig. 2.

2-meSATP increases Na⁺/Ca²⁺ exchanger (NCX) current (I_NCX). I_NCX, represented by Ni²⁺-sensitive current, in P2X4R TG ventricular myocytes was determined. A: representive trace showing membrane current when 10 mM Ni²⁺ was applied (time period bracketed by downward and upward arrows) under CTR conditions, during 2-meSATP application, and after WO. B, top: descending voltage ramps from 80 to −100 mV were used to contruct the current-voltage (I–V) relationship. Bottom, typical traces of membrane currents under CTR conditions and in the presence of Ni²⁺. C: mean I–V relationships of Ni²⁺-sensitive current under CTR conditions and in the presence of 2-meSATP. n = 10 myocytes from 8 TG mice. *P < 0.05. D: 2-meSATP increased the Ca²⁺ entry mode of I_NCX. Representative traces are shown for Ni²⁺ -sensitive currents at +30 mV before and after 2-meSATP application in a TG myocyte. E: plot of Ni²⁺-sensitive current at 30 mV under CTR conditions and during 2-meSATP application. n = 7 cells from 5 TG mice. P < 0.05.

Fig. 3.

Simulated I–V relationships with 10 and 11.2 mM intracellular [Na⁺] ([Na⁺]_i). A: simulation of I_p with [Na⁺]_i taken as cellular (pipette) [Na⁺] of 10 and 11.2 mM. B: simulation of I_NCX with intracellular Ca²⁺ concentration kept constant at 100 nM for cellular [Na⁺] of 10 and 11.2 mM. The actual experimental data under CTR conditions and after 2-meSATP application were also plotted for comparison with the simulation.

Fig. 4.

KB-R7943 (KBR) or YM-244769 (YM) inhibits the 2-meSATP-induced increase of cell shortening (CS). A: representative traces of CS in TG myocytes paced at 0.5 Hz for cells exposed to 2-meSATP and then to 2-meSATP plus KBR followed by WO. B: basal CS did not change after application of KBR (n = 12) or YM (n = 7). C: percent increases of CS above basal during application of 2-meSATP alone (percent above basal, n = 16 cells from 10 Tg mice) and during the addition of KBR plus 2-meSATP (percent above basal with both KBR and 2-meSATP vs. that with 2-meSATP alone, P < 0.05). D: percent increases of CS above basal during application of 2-meSATP alone (percent above basal, n = 10 cells from 6 Tg mice) and during exposure of YM plus 2-meSATP (percent above basal with both YM and 2-meSATP vs. that with 2-meSATP alone, P < 0.05).

Fig. 5.

Link between P2X receptors and NCX in wild-type (WT) myocytes. A: 2-meSATP-induced increase of I_NCX in WT myocytes. Ni²⁺-sensitive currents before and after 2-meSATP application are shown at membrane potentials of −100, −80, 50, and 70 mV. I_NCX at +50 and +70 mV was significantly larger in the presence of 2-meSATP than under CTR conditions (n = 8 cells from 7 WT mice). *P < 0.05. B: percent increases of CS above basal during application of 2-meSATP plus ivermectin (Ive; percent above basal, n = 8 cells from 7 WT mice) and during the subsequent exposure of KBR plus 2-meSATP and Ive (percent above basal with KBR plus 2-meSATP and Ive vs. that with 2-meSATP plus Ive, P < 0.05).