Effects of Increased Intracellular Cl− Concentration on Membrane Responses to Acetylcholine in the Isolated Endothelium of Guinea Pig Mesenteric Arteries

Abstract

ACh-induced membrane responses in vascular endothelial cells that have been reported vary between preparations from a sustained hyperpolarization to a transient hyperpolarization followed by a depolarization; the reason for this variation is unknown. Using the perforated whole-cell clamp technique, we investigated ACh-induced membrane currents in freshly isolated endothelial layers having a resting membrane potential of less negative than -10 mV. A group of cells was electrically isolated using a wide-bore micropipette, and their membrane potential was well controlled. ACh activated K + and Cl -currents simultaneously. The K + current was blocked by a combination of charybdotoxin and apamin and appears to result from the opening of IK Ca and SK Ca channels. The Cl -current was partially blocked by tamoxifen, niflumic acid, or DIDS and appears to be produced by Ca 2+ -activated Cl -channels. When the pipettes contained 20 mM Cl -, the ACh-induced K + conductance started decreasing during a 1-min application of ACh while the Cl -conductance continued, making the ACh-induced hyperpolarization sustained. When the pipettes contained 150 mM Cl -, both conductances started decreasing during a 1-min application of ACh, making the ACh-induced hyperpolarization small and transient. [Cl -] i is very likely modified by experimental procedures such as the cell isolation and the intracellular dialysis with the pipette solution. Such a variability in [Cl -] i may be one of the reasons for the variations in the ACh-induced membrane response.Key words: endothelial cell, chloride channel, potassium channel, whole-cell voltage clamp, membrane potential.In vascular endothelial cells, vasoactive substances such as ACh increase the intracellular concentration of calcium ions, [Ca 2+ ] i , and hyperpolarize the membrane through the opening of charybdotoxin-sensitive intermediate-conductance Ca 2+ -activated K + channels (IK Ca ) and apamin-sensitive small-conductance Ca 2+ -activated K + channels (SK Ca ) [1][2][3][4][5][6][7][8]. The hyperpolarization conducts electrotonically to the smooth muscle cells [4], and the vessel dilates [9]. This is one of the explanations for the phenomenon caused by an endothelium-derived hyperpolarizing factor (EDHF) over which controversy still remains (see [10,11] for reviews).Agonist-induced membrane responses in vascular endothelial cells are complex and vary greatly between preparations. Bradykinin or ATP induces a sustained hyperpolarization in the cultured bovine aortic endothelium when the resting potential is less negative and a transient hyperpolarization followed by a depolarization when it is more negative [12]. In the intact endothelium of rat aorta with the smooth muscle layers attached, ACh-induced membrane responses vary between preparations, but in most preparations a biphasic response, a transient hyperpolarization followed by a depolarization, is detected [13]. ACh produces a sustained hyperpolarization followed by a transient depolarization during the washout process in th...

Analysis of acetylcholine-induced membrane responses in vascular endothelial cells of the guinea-pig mesenteric artery using mefloquine as a gap junction blocker

“…We have previously measured the input resistance to be around 2-3 MΩ when a patch electrode was applied to one of the endothelial cells within such a preparation (Yamamoto and Suzuki, 2007). To control the membrane potential of the patched cell accurately, mefloquine, an antimalarial drug, was used as a gap junction blocker (Yamamoto and Suzuki, 2008).…”

“…In mechanically-isolated population of endothelial cells where the input resistance is less than 100 MΩ, the membrane potential can be controlled with a patch electrode (Yamamoto and Suzuki, 2007). In the presence of mefloquine the average input resistance was more than 200 MΩ and the membrane potential of a patched cell should be properly controlled.…”

“…The other conductance was activated more slowly and gradually. In our previous paper using a small number of mechanically-isolated cells instead of chemically-isolated cells with a gap junction blocker, we concluded that the second conductance was a Cl -one because it had a reversal potential near ECl and was blocked by so called Cl -channel blockers (Yamamoto and Suzuki, 2007). However, the calculated ECl might not be correct because the intracellular ionic conditions were difficult to control in these coupled cells.…”

“…Preparations were made as described previously (Yamamoto and Suzuki, 2007). Briefly, a segment of artery was incubated in collagenase solution (nominally Ca 2+ -free physiological saline containing 0.5 mg/mL collagenase; Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 30-35 min at 37ºC.…”

“…As the arterial intima consists of thousands of electrically coupled endothelial cells having numerous gap junctions, the number of cells to be controlled needs to be reduced to get proper space-clamp conditions. To achieve such conditions, we have previously used a wide-bore micropipette to destroy a ring of cells, leaving an isolated population of intact cells inside (Yamamoto and Suzuki, 2007). Although the membrane potential of these mechanically-isolated cells could be easily controlled, it was not clear whether the intracellular solution of these cells could be quickly replaced with the pipette solution.…”