Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve

Abstract

1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization.

Figure 1

Effects of ACh on membrane potential in RAVEC. (Aa) Concentration-dependent effects of ACh on membrane potential. ACh (0.01–3 μM) was applied for 3 min. Recordings were all from the same preparation. (Ab) Hyperpolarization was maintained throughout a 10-min application of 3 μM ACh. (Ba) and (Bb) Summary of the effects of ACh on membrane potential. Bb shows the maximum hyperpolarization and depolarization elicited by each concentration of ACh (mean of data from three preparations, with s.e. shown by vertical lines). Ba indicates how these maxima were obtained.

Figure 2

Effects of ChTX and apamin on ACh-induced membrane potential changes. Actual tracings of the effects of ChTX and apamin. (a) Control; (b) in the presence of ChTX (50 nM); (c) in the presence of both ChTX (50 nM) and apamin (0.1 μM). Recordings were all obtained from the same cell.

Figure 3

Effects of Ca²⁺-free solution (a) and BAPTA-AM (b) on ACh-induced membrane potential changes. (a) ACh (3 μM) was first applied in normal Krebs solution (as control). The preparation was then exposed to Ca²⁺-free solution for 10 min, and ACh was subsequently applied at 225 min intervals. The traces were obtained from the same cell. Similar observations were made in two other cells. (b) BAPTA-AM (30 μM) was pretreated for 40 min followed by a 10 min washout, then ACh (3 μM) was applied. Similar observations were made in two other cells.

Figure 4

Effects of 2 mM Co²⁺ (a) and 5 mM Ni²⁺ (b and c) on ACh-induced membrane potential changes. ACh (3 μM) was applied for 3 min as indicated by bars (a and b). Co²⁺ and Ni²⁺ were added 10 min before ACh and were present throughout the application of ACh. (c) Ni²⁺ (5 mM) was applied during the sustained component of the hyperpolarization induced by 3 μM Ach.

Figure 5

Effect of CPA (3 μM) on ACh-induced membrane potential changes. (a) In the presence of CPA (3 μM), the recovery from the ACh-induced hyperpolarization that occurred after washout of ACh was largely prevented. When ACh was again applied after an interval of 25 min (in the continued presence of CPA), ACh evoked only a small hyperpolarization. The hyperpolarization was maintained for as long as CPA was present. (b) In the presence of CPA, ACh produced a hyperpolarization that was maintained after the removal of ACh. When Co²⁺ (2 mM) was applied during this maintained hyperpolarization, the membrane response was converted to a depolarization.

Figure 6

Effect of A23187 (0.1 μM) on ACh-induced membrane potential changes. The transient component of the ACh-induced hyperpolarization was attenuated in the presence of A23187.

Figure 7

Effects of NPPB, bumetanide and mannitol on ACh-induced membrane potential changes. ACh-induced responses obtained before and after application of inhibitors [NPPB (20 nM, a), bumetanide (10 μM, b) and mannitol (20 mM, c)] in the same cell.