Yoshimichi Yamamoto

Acetylcholine (ACh)-induced membrane currents were investigated using freshly isolated endothelial layers prepared from the guinea-pig mesenteric artery. Gap junctions were blocked by mefloquine and the whole-cell patch clamp method was applied to individual endothelial cells within each multicellular preparation. While mefloquine effectively blocked the gap junctions, it hyperpolarized the membrane by some 10 mV. As this hyperpolarization was absent when the intracellular Cl– concentration was increased, mefloquine may increase the membrane conductance for Cl–. Besides this minor hyperpolarizing effect, mefloquine did not have serious side effects and ACh could activate a sustained outward current producing a membrane hyperpolarization at concentrations as low as 100 nM. At the beginning of ACh application, the reversal potential of the ACh-induced current was around the equilibrium potential for K+ indicating that this was a K+ current. The reversal potential then gradually became less negative suggesting that other ionic conductances with less negative equilibrium potentials were involved. As the ACh-induced outward current was completely blocked by charybdotoxin (CTX, 100 nM), this current seemed to be due to CTX-sensitive K+ channels, possibly IKCa channels. After the K+ current had been blocked, ACh gradually activated the membrane current which reversed the polarity at around –10 mV, which was most likely due to Ca2+-activated non-selective cation channels. These ionic conductances may be responsible for the variety in agonist-induced membrane responses observed in different types of vascular preparations.

Mefloquine, an antimalarial drug, has been reported to block exogenously transfected gap junctions composed of either Cx36 or Cx50 more potently than those composed of other connexins. Using the conventional whole-cell clamp technique, we investigated the effects of mefloquine on intercellular electrical coupling in vascular endothelial cells of guinea-pig mesenteric arteries, where expressions of Cx40 and Cx43 have been identified. Mefloquine (50 μM) almost abolished the current required to impose a 10 mV command step, leaving only rapid capacitive currents and some sustained currents after about 3 min. The relaxation of the capacitive current could be well fitted with a single exponential function. The effect of mefloquine was reversible and the time course of the current induced by the voltage step gradually changed back after mefloquine was removed. The mean input resistance and capacitance in the presence of mefloquine were 323 MΩ and 10.1 pF, respectively. While intercellular electrical coupling was well blocked by mefloquine (50 μM), the membrane hyperpolarized from -24.0 to -32.5 mV. The results indicate that mefloquine effectively blocks gap junctions without producing major side effects in vascular endothelial cells and that this compound is a useful tool in the investigation of gap junctions.

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.

Using guinea-pig mesenteric arteries and arterioles, we investigated the membrane potential of endothelial cells at rest and during application of acetylcholine (ACh) with and without the smooth muscle layers attached. When smooth muscle and endothelial layers were in close apposition, the resting membrane potentials of the two types of cells were closely related and were slightly more negative in the smooth muscle cells than in the endothelial cells. Once the endothelial layer was separated from the smooth muscle layer, the endothelial cells depolarized (the average, -4.2 mV). In the isolated endothelial layer, ACh did not induce a membrane hyperpolarization as expected, but did induce a quick depolarization soon after conventional whole-cell recording was started. However, as the pipette solution (high K+) gradually diffused into the endothelial layer, the membrane response to ACh gradually changed toward hyperpolarization. ACh-induced hyperpolarization was also observed after incubating preparations in a high-potassium bath solution. Our results indicate that vascular smooth muscle cells and endothelial cells are influencing each other as a functional unit and that the endothelial cells rely on the smooth muscle cells for their intracellular ionic composition and resting membrane potential.

Current clamp studies using two patch electrodes and morphological observations have been performed in guinea-pig mesenteric arterioles to evaluate intercellular electrical couplings. In electron micrographs, preparations were found to have a single layer of smooth muscle cells. Typical gap junctions were readily observed between endothelial cells only. While immunoreactivity to connexin 40 was strongly expressed on the membranes of endothelial cells only, that to connexin 43 was expressed on both smooth muscle and endothelial cell membranes. Neurobiotin injected into a smooth muscle cell diffused into several neighbouring smooth muscle cells while that injected into an endothelial cell diffused into many endothelial cells. Acetylcholine-induced hyperpolarizations were conducted from endothelial cells to smooth muscle cells with a relative amplitude of 80.1 %. Ba2+-induced action potentials were conducted in the opposite direction with a relative amplitude of 92.4 %. An electrotonic potential produced in a smooth muscle cell by current injection diminished steeply with distance as it spread along the muscle layer, plateauing at distances beyond 25 μm. An electrotonic potential produced in an endothelial cell spread within the intima with virtually no reduction. Electrotonic potentials could conduct through myoendothelial couplings, which seemed to behave as ohmic resistors without rectification. The coupling resistance between adjacent smooth muscle cells was estimated to be at least 90 MΩ and that between a smooth muscle cell and the whole endothelial layer to be 0.9 GΩ. The results indicate that although the resistance of myoendothelial couplings is appreciable, the endothelium may be important as a low resistance path connecting many smooth muscle cells.

1 Using the conventional whole-cell clamp method, the electrical responses of individual smooth muscle and endothelial cells to acetylcholine (ACh) were observed in multicellular preparations where the two types of cells remained in close apposition. 2 In both types of cells, ACh induced similar hyperpolarizing responses which, when recorded in current clamp mode, had two phases (an initial fast and a second slower phase). 3 After blocking gap junctions, including myoendothelial junctions, with 18β-glycyrrhetinic acid, ACh induced an outward current with two phases in voltage-clamped endothelial cells. The outward current appeared around −90 mV and increased linearly with the membrane depolarization. 4 In smooth muscle cells, ACh failed to induce a membrane current after gap junctions had been blocked with 18β-glycyrrhetinic acid. The inhibition of ACh-induced response by 18β-glycyrrhetinic acid was observed using either sharp or patch electrodes. 5 Nominally Ca²⁺-free solution reduced the initial phase and abolished the second phase of ACh-induced responses of endothelial cells. Both phases were also reduced by charybdotoxin (CTX). 6 Our results indicate that in guinea-pig mesenteric arterioles, ACh hyperpolarizes endothelial cells by activating Ca²⁺-activated K⁺ channels which are sensitive to CTX. On the other hand, hyperpolarizing responses detected in smooth muscle cells seem to originate in endothelial cells and conduct to the muscle layer via myoendothelial gap junctions.

Intercellular electrical communication between smooth muscle and endothelial cells was examined in guinea‐pig mesenteric arterioles using the whole‐cell patch‐clamp method. The time course of the current required to impose a 10 mV voltage clamp step was used to determine the extent of electrical coupling between them. Currents recorded from both smooth muscle and endothelial cells relaxed in a multi‐exponential manner, indicating the existence of electrical coupling between cells. 18β‐Glycyrrhetinic acid, a gap junction blocker, quickly blocked electrical communication at 40 μM, while neither heptanol nor octanol did so at concentrations of up to 1 mM. In the current clamp mode, repetitive spikes, induced by 10 mM Ba²⁺ solutions, could be recorded from both kinds of cells. After blocking gap junctions, spikes could only be recorded from the smooth muscle cell layer, indicating that they had been conducted through myoendothelial junctions. In endothelial cells, acetylcholine (ACh, 3 μM) induced hyperpolarizing responses, which had two phases (an initial fast and a second slower phase) in the current clamp condition. This ACh response persisted in the presence of 18β‐glycyrrhetinic acid, although this compound seemed to make the membrane slightly leaky. After blocking gap junctions, the membrane potential of a single cell in a multicellular preparation could be well clamped. Thus, 18β‐glycyrrhetinic acid may be useful in studying the function of both arteriolar smooth muscle and endothelial cells while they remain located within a multicellular preparation.