Sodium-calcium exchanger contributes to membrane hyperpolarization of intact endothelial cells from rat aorta during acetylcholine stimulation

Abstract

1. The role of sodium-calcium exchanger in acetylcholine (Ach)-induced hyperpolarization of intact endothelial cells was studied in excised rat aorta. The membrane potential was recorded using perforated patch-clamp technique. 2. The mean resting potential of endothelial cells was -44.1+/-1.4 mV. A selective inhibitor of sodium-calcium exchanger benzamil (100 microm) had no significant effect on resting membrane potential, but reversibly decreased the amplitude of sustained Ach-induced endothelial hyperpolarization from 20.9+/-1.4 to 5.7+/-1.1 mV when applied during the plateau phase. 3. The blocker of reversed mode of the exchanger KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate, 20 microm) reversibly decreased the amplitude of sustained Ach-induced hyperpolarization from 20.5+/-2.9 to 7.5+/-1.8 mV. 4. Introduction of tetraethylammonium (10 mm) in the continuous presence of Ach decreased the sustained phase of hyperpolarization from 17.9+/-1.5 by 12.9+/-0.9 mV. Subsequent addition of 20 microm KB-R7943 further depolarized endothelial cells by 4.8+/-1.1 mV. 5. Substituting external sodium with N-methyl d-glucamine during the plateau phase of Ach-evoked hyperpolarization reversibly decreased the hyperpolarization from -61.8+/-2.7 to -54.2+/-1.9 mV. In the majority of preparations, the initial response to removal of external sodium was a transient further rise in the membrane potential of several mV. Sodium ionophore monensin hyperpolarized endothelium by 10.3+/-0.7 mV. 6. The inhibitory effect of benzamil on Ach-induced endothelial sustained hyperpolarization was observed in endothelium mechanically isolated from smooth muscle. 7. These results suggest that the sodium-calcium exchanger of intact endothelial cells is able to operate in reverse following stimulation by Ach, contributing to sustained hyperpolarization. Myoendothelial electrical communications do not mediate the effect of blockers of sodium-calcium exchanger.

Figure 1

Sustained endothelial hyperpolarization in response to Ach requires extracellular Ca²⁺ entry. (a) A typical response of endothelial membrane potential to Ach in the presense of 2.4 mM extracellular Ca²⁺. (b, c) Transient hyperpolarization in response to Ach (b) in Ca²⁺-free solution and (c) in the presence of 2 mM Ni²⁺.

Figure 2

Effect of benzamil on Ach-induced sustained hyperpolarization of endothelial cells. (a) Transient hyperpolarization to application of 2 μM Ach in the presence of 100 μM benzamil. (b) Inhibition of sustained hyperpolarization to Ach by benzamil application during the plateau phase of hyperpolarization. (c) Effect of benzamil on Ach-induced sustained endothelial hyperpolarization.

Figure 3

Effect of KB-R7943 on sustained Ach-induced hyperpolarization of endothelial cells. (a) Inhibition of sustained Ach-induced hyperpolarization of endothelial cells by 20 μM KB-R7943. (b) Inhibitory effect of KB-R7943 on Ach-induced hyperpolarization of endothelial cells in the presence of 10 mM TEA. Addition of 20 μM KB-R7943 further depolarized endothelium. (c, d) Effect of KB-R7943 on the membrane potential fluctuations caused by Ca²⁺ addition after the strip was exposed to Ach in Ca²⁺-free solution, (c) Ca²⁺ was added in the absence of KB-R7943 and (d) Ca²⁺ was added in the presence of 20 μM KB-R7943. Note that KB-R7943 does not affect the amplitude of the hyperpolarization caused by Ca²⁺ influx, but accelerates the decay of the hyperpolarization. (e) Effect of KB-R7943 (20 μM) on Ach-induced sustained endothelial hyperpolarization.

Figure 4

Dependence of endothelial hyperpolarization on Na⁺ gradient. (a) Effect of external Na⁺ withdrawal during Ach exposure on endothelial membrane potential fluctuations. (b) Effect of external Na⁺ withdrawal before Ach exposure on endothelial membrane potential fluctuations. (c) Effect of Na⁺ loading by monensin on endothelial membrane potential.

Figure 5

Evaluation of the role of gap junctions in inhibitory effect of benzamil on endothelial sustained hyperpolarization to Ach. (a) 18α-GA (20 μM) attenuates the effect of benzamil on Ach-induced sustained hyperpolarization. (b) 18α-GA (20 μM) does not inhibit the phenylephrine-induced oscillations in endothelial membrane potential. (c) Effect of 100 μM benzamil on Ach-induced hyperpolarization of mechanically isolated endothelium.