Blockade by mefloquine of intercellular electrical coupling between vascular endothelial cells in the guinea-pig mesenteric arteries.

Mefloquine, an antimalarial drug, has been reported to block exogenously transfected gap junctions composed of either C x 36 or C x 50 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 C x 40 and C x 43 have been identified. Mefloquine (50 microM) 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 MOmega and 10.1 pF, respectively. While intercellular electrical coupling was well blocked by mefloquine (50 microM), 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.

Properties of acetylcholine-induced hyperpolarization in smooth muscle cells of the mouse mesenteric artery.

The properties of smooth muscle cell hyperpolarization produced by acetylcholine (ACh) were investigated in mesenteric arteries isolated from mice. The resting membrane potential of the smooth muscle cells was about -60 mV. When ACh (10 microM) was applied for 1 min, the membrane hyperpolarized with a peak amplitude of about 5 mV which was reached in about 1 min, following which the potential slowly reverted to the resting level over about 7 min following withdrawal of ACh from the superfusate (recovery component). Exposure of the artery to 0.5 mM Ba(2+), an inhibitor of inward rectifier K-channels, depolarized the membrane by about 13 mV, increased the amplitude of the ACh-induced hyperpolarization to about 10 mV, and facilitated the visualization of the recovery component. Indomethacine (10 microM), an inhibitor of cyclooxygenase, inhibited the recovery component and as a consequence reduced the duration of the hyperpolarization. The ACh-induced response was not markedly altered by either N(omega)-nitro-L-arginine (10 microM), an inhibitor of nitric oxide (NO) production, or catalase (130 U/ml), a super oxide scavenger. Exogenously applied hydrogen peroxide (H(2)O(2), 300 microM) hyperpolarized the membrane by about 5 mV, which was abolished by catalase. These results suggest that in the mouse mesenteric artery, the ACh-induced hyperpolarization has two components, both an indomethacin-sensitive and an indomethacin-insensitive component. The former component may be produced by prostanoids, while the latter may be produced by factors other than NO or H(2)O(2). The results also suggested that the inward rectifier K-channels may be important for producing the resting membrane potential, but they may not be the main contributor to the ACh-induced hyperpolarization of smooth muscle cell membranes in the mouse mesenteric artery.

Dependency of endothelial cell function on vascular smooth muscle cells in guinea-pig mesenteric arteries and arterioles.

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.

Effects of inhibitors of nonselective cation channels on the acetylcholine-induced depolarization of circular smooth muscle from the guinea-pig stomach antrum.

In circular smooth muscle bundles isolated from the guinea-pig stomach antrum, the effects of quinidine, Ni2+, flufenamic acid, niflumic acid, La3+, SKF-96365 and 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) on acetylcholine (ACh)-induced depolarization were investigated. Recording membrane potentials from smooth muscle cells with intracellular microelectrodes revealed that ACh (1 microM) depolarized the membrane by 5-8 mV and increased the amplitude and frequency of slow potentials. These effects were inhibited by atropine. Quinidine (10 microM) increased the amplitude of ACh-induced depolarization, with no alteration to the properties of slow potentials. Ni2+ (50 microM) transiently (5-10 min) depolarized the membrane by about 5 mV, with an associated increase in frequency and amplitude of slow potentials. In the stabilized condition with Ni2+, the amplitude of ACh-induced depolarization remained unchanged. Flufenamic acid (10 microM) inhibited the generation of slow potentials, with no change in either the amplitude of ACh-induced depolarization or of the amplitude and frequency of slow potentials generated during ACh stimulation. A high concentration of flufenamic acid (100 microM) depolarized the membrane and increased the amplitude of ACh-induced depolarization. Niflumic acid (10 microM) hyperpolarized the membrane and increased the amplitude and frequency of slow potentials and also the amplitude of ACh-induced depolarization. DIDS (100 microM) hyperpolarized the membrane and inhibited the amplitude and frequency of slow potentials, with no alteration to the amplitude of ACh-induced depolarization. SKF-96365 (3-50 microM) depolarized the membrane in a concentration-dependent manner, but did not change the level of ACh-induced depolarization. La3+ (50 microM) did not alter the properties of the slow potentials or the ACh-induced responses. These results provide evidence that ACh-induced depolarization is not inhibited by chemicals known to inhibit non-selective cation channels. We suggest that muscarinic receptor-mediated signal transduction may be different in smooth muscle and interstitial cells.

Dual effects of cyclopiazonic acid on excitation of circular smooth muscle isolated from the guinea-pig gastric antrum.

The effects of cyclopiazonic acid (CPA), a known Ca2+-pump inhibitor at internal stores, were investigated on electrical responses of the membrane of smooth muscle cells in small segments (0.3-0.5 mm long) of circular smooth muscle isolated from the guinea-pig gastric antrum. In most preparations, the membrane was spontaneously active with the generation of unitary potentials and regenerative slow potentials. Low concentrations (< 1 microM) of CPA did not alter either the membrane potential or the amplitude and frequency of slow potentials. CPA at a concentration of 1 microM initially increased the frequency of slow potentials, but this was followed by a decrease in the frequency as a result of sustained exposure to CPA, with no alteration of either the membrane potential or the amplitude of slow potentials. Higher concentrations of CPA (2-5 microM) depolarized the membrane and decreased the amplitude and frequency of slow potentials. CPA at higher than 10 microM abolished slow potentials with depolarization of the membrane. Intracellular electrical responses recorded simultaneously from paired cells were synchronized, indicating electrical coupling of the cells. Depolarization of the membrane with current stimuli through one electrode evoked regenerative slow potentials superimposed on the electrotonic potentials. The evoked slow potential had a refractory period of about 7 s. CPA (up to 10 microM) did not prevent the synchronization of paired cells. The refractory period for slow potentials was reduced by low concentrations of CPA (< 1 microM) and increased by higher concentrations of CPA (2-10 microM). These results suggest that lower concentrations of CPA produce excitatory actions on gastric smooth muscles due to a secondary effect of increased intracellular [Ca2+], while higher concentrations of CPA produce inhibitory actions as a result of reduced release of Ca2+ from depleted internal stores.

Effects of RHC-80267, an inhibitor of diacylglycerol lipase, on excitation of circular smooth muscle of the guinea-pig gastric antrum.

In small segments of circular smooth muscle isolated from the guinea-pig gastric antrum, the effects of RHC-80267, an inhibitor of diacylglycerol lipase, were investigated both on regenerative slow potentials (either occurring spontaneously or as the result of a depolarizing intracellular current injection) and on the actions of acetylcholine (ACh). As diacylglycerol is a known activator of protein kinase C (PKC), it would therefore be expected that RHC-80267 would activate PKC indirectly. In circular smooth muscle bundles, spontaneously generating slow potentials recorded simultaneously from two given cells were synchronized, indicating that these two cells were electrically coupled. RHC-80267 (0.3-1 microM) increased the frequency of slow potential generation, with no alteration to the amplitude of either the slow potentials or the resting membrane potential. Synchronous electrical activity in a given pair of cells was also unchanged by RHC-80267, indicating that intercellular electrical coupling was not altered. The input resistance of smooth muscle cells calculated from the amplitude of electrotonic potentials produced by injection of current was not significantly altered by RHC-80267. The refractory period for the generation of slow potentials evoked by depolarizing stimuli was about 8 s, and it was decreased to about 5 s by RHC-80267, with no significant alteration to the amplitude of spontaneous or evoked slow potentials. ACh (0.5 microM) depolarized the membrane by about 5 mV and increased the amplitude and frequency of slow potentials. The actions of ACh on the frequency of slow potentials were enhanced by RHC-80267, with no alteration to the amplitudes of both the ACh-induced depolarization and slow potentials. These results support the idea that PKC is involved in determining the frequency of slow potentials, by shortening the refractory period for excitation of gastric smooth muscle cells.

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

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 IK(Ca) 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 Ca(2+)-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.

Hypoxia differentially modulates the activity of pacemaker and smooth muscle cells in the guinea pig stomach antrum.

Effects of hypoxic solution (O(2) tension, 161 +/- 11 mmHg) on electrical responses of the membrane (slow waves), intracellular Ca(2+)-responses measured by Fura-2 fluorescence (Ca-transients) and isometric mechanical responses (phasic contraction) were observed in circular smooth muscles isolated from the guinea-pig stomach antrum. In normoxic solution (O(2) tension, 362 +/- 28 mmHg), muscle cells generated slow waves spontaneously, and switching to hypoxic solution caused an increase in frequency and decrease in duration of slow waves, with no significant change in the resting membrane potential. Hypoxia also reduced the amplitude and duration and increased the frequency of Ca-transients. The increase in frequency of slow waves by hypoxia was prevented by cyclopiazonic acid (CPA) but not by carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), potassium cyanide (KCN) or low-Ca solution. The reduction by hypoxia of the duration of slow waves was prevented by CCCP or KCN but not by CPA or low-Ca solution. Hypoxia resulted in an increase in frequency and decrease in amplitude of phasic contractions, and the changes were prevented by CPA but not by CCCP. These results suggested that in antrum smooth muscle tissues, the increase in frequency of spontaneous activity by hypoxia is related to the enhanced function of the CPA-sensitive internal Ca-stores in pacemaker cells, while the inhibition in amplitude of phasic contractions by hypoxia may be mainly related to the decrease in Ca(2+) release from the CPA-sensitive internal stores in smooth muscle cells. It is concluded that in hypoxic solution, the function of internal Ca(2+) stores is enhanced in ICC-MY and is inhibited in smooth muscle cells in the guinea-pig stomach antrum.

Effects of increased intracellular Cl- concentration on membrane responses to acetylcholine in the isolated endothelium of guinea pig mesenteric arteries.

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.

Excitation of smooth muscles isolated from the guinea-pig gastric antrum in response to depolarization.

In small segments of circular smooth muscle bundle isolated from the guinea-pig gastric antrum, depolarization of the tissue with intracellular current stimuli evoked regenerative slow potentials after a refractory period of 5-10 s. The refractory period changed inversely with the amplitude and duration of the stimulating depolarization. Thapsigargin (an inhibitor of calcium-ATPase at internal stores), 2-aminoethoxydiphenyl borate (2-APB, an inhibitor of inositol 1,4,5-trisphosphate (IP3)-receptor-mediated Ca2+ release), and carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial protonophore) reduced the amplitude of slow potentials, with no significant alteration of the refractory period. Bisindolylmaleimide I or chelerythrine (inhibitors of protein kinase C, PKC) increased the refractory period and inhibited the amplitude of slow potentials. These results indicate that the refractory period and amplitude of slow potentials are related to the activation of PKC and the amount of Ca2+ released from the internal stores through activation of IP3 receptors, respectively. Acetylcholine (ACh) reduced the refractory period and increased the amplitude of slow potentials: the former was antagonized by chelerythrine and the latter by 2-APB. The results suggest that ACh has dual actions; stimulation of the metabolism of inositol phosphate and activation of PKC. Phorbol-12-myristate-13-acetate, a selective stimulant of PKC, at low concentrations (< 10 nM) mimicked the actions of ACh and at high concentrations reduced the frequency of slow potentials and increased the refractory period. The possible involvement of the concentration-dependent differences in the actions of phorbol ester on the translocation of PKC was considered.

[Cellular mechanism of the generation of spontaneous activity in gastric muscle].

In gastric smooth muscles, interstitial cells of Cajal (ICC) might be the pacemaker cells of spontaneous activities since ICC are rich in mitochondria and are connected with smooth muscle cells via gap junctions. Several types of ICC are distributed widely in the stomach wall. A group of ICC distributed in the myenteric layer (ICC-MY) were the pacemaker cells of gastrointestinal smooth muscles. Pacemaker potentials were generated in ICC-MY, and the potentials were conducted to circular smooth muscles to trigger slow waves and also conducted to longitudinal muscles to form follower potentials. In circular muscle preparations, interstitial cells distributed within muscle bundles (ICC-IM) produced unitary potentials, which were conducted to circular muscles to form slow potentials by summation. In mutant mice lacking inositol trisphosphate (IP(3)) receptor, slow waves were absent in gastric smooth muscles. The generation of spontaneous activity was impaired by the inhibition of Ca(2+)-release from internal stores through IP(3) receptors, inhibition of mitochondrial Ca(2+)-handling with proton pump inhibitors, and inhibition of ATP-sensitive K(+)-channels at the mitochondrial inner membrane. These results suggested that mitochondrial Ca(2+)-handling causes the generation of spontaneous activity in pacemaker cells. Possible involvement of protein kinase C (PKC) in the Ca(2+) signaling system was also suggested.