Hiroyasu Fukuta

Blockade by 18β‐glycyrrhetinic acid of intercellular electrical coupling in guinea‐pig arterioles

1 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. 2 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. 3 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. 4 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. 5 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.

Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder.

1 The origin and propagation of waves of spontaneous excitation in bundles of smooth muscle of the guinea‐pig bladder were examined using intracellular recording techniques and visualization of the changes in the intracellular calcium concentration ([Ca2+]i). 2 Bladder smooth muscle cells exhibited spontaneous transient increases in [Ca2+]i which originated along a boundary of each smooth muscle bundle and then spread to the other boundary with a conduction velocity of 2.0 mm s−1. 3 Spontaneous increases in [Ca2+]i were always preceded by action potentials. Nifedipine (10 μM) abolished increases in both [Ca2+]i and action potentials. Caffeine (10 mM), ryanodine (50 μM) and cyclopiazonic acid (10 μM) reduced the amplitude of the associated increases in [Ca2+]i without preventing the generation of action potentials. 4 Spontaneous action potentials had conduction velocities of 40 mm s−1 in the axial direction and 1.3 mm s−1 in the transverse direction. The electrical length constants of the bundles of muscle were 425 μm in the axial direction and 12.5 μm in the transverse direction. 5 Neurobiotin, injected into an impaled smooth muscle cell, spread more readily to neighbouring cells located in the axial direction than those located in the transverse direction. The spread of neurobiotin was inhibited by 18β‐glycyrrhetinic acid (18β‐GA, 40 μM), a gap junction blocker. 6 Immunohistochemistry for Connexin 43 showed abundant punctate staining on the smooth muscle cell membranes. 7 These results suggested that spontaneous action potentials and associated calcium waves occur almost simultaneously along the boundary of bladder smooth muscle bundles and then propagate to the other boundary probably through gap junctions.

Spontaneous electrical activity and associated changes in calcium concentration in guinea-pig gastric smooth muscle

Spontaneous electrical activity and internal Ca2+ concentration ([Ca2+]i) were measured simultaneously using conventional microelectrodes and fura‐2 fluorescence, respectively, in isolated circular smooth muscle bundles of the guinea‐pig gastric antrum. The smooth muscle bundles generated periodic slow potentials with accompanying spike potentials and associated transient increases in [Ca2+]i (Ca2+‐transients). Nifedipine abolished the spike potentials but not the slow potentials, and reduced the amplitude of associated Ca2+‐transients. Caffeine, in the absence or presence of ryanodine, reduced resting [Ca2+]i levels and abolished the slow potentials and associated Ca2+‐transients. Depolarization elevated and hyperpolarization reduced resting [Ca2+]i levels with associated changes in the frequency of slow potentials. The amplitude of Ca2+‐transients changed in a bell‐shaped manner with the membrane potential change. Slow potentials and associated Ca2+‐transients were abolished if [Ca2+]i levels were reduced by BAPTA‐AM or if the internal Ca2+ pump was inhibited by cyclopiazonic acid. 2‐Aminoethoxy‐diphenylborate (2‐APB), a known inhibitor of inositol trisphosphate (IP3)‐mediated Ca2+ release, also blocked slow potentials and Ca2+‐transients. Carbonyl cyanide m‐chlorophenyl hydrazone (CCCP), a mitochondrial protonophore, depolarized the membrane, elevated [Ca2+]i levels and abolished slow potentials and Ca2+‐transients. Inhibition of mitochondrial ATP‐sensitive K+ channels by glybenclamide and 5‐hydroxydecanoic acid (5‐HAD) abolished slow potentials and Ca2+‐transients, without altering the smooth muscle [Ca2+]i. It is concluded that in antrum circular muscles, the frequency of slow potentials is correlated with the level of [Ca2+]i. The slow potential is coupled to release of Ca2+ from an internal store, possibly through the activation of IP3 receptors; this may be initiated by the activation of ATP‐sensitive K+ channels in mitochondria following Ca2+ handling by mitochondria.

Components of pacemaker potentials recorded from the guinea-pig stomach antrum

Abstract. Pacemaker potentials recorded intracellularly from the guinea pig stomach consisted of initial primary and following plateau components. Inhibition of the internal Ca2+ store pump with cyclopiazonic acid depolarized the membrane and inhibited the plateau component of pacemaker potentials. 2-Aminoethoxydiphenyl borate (an inhibitor of IP3-induced Ca2+ release) and carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial protonophore) depolarized the membrane and abolished pacemaker potentials. Low [Ca2+]o solution reduced the frequency and rate of rise of pacemaker potentials, and the effects were mimicked by BAPTA-AM (an intracellular Ca2+ chelator). 4,4′-Diisothiocyanatostilbene-2,2′-disulphonic acid and low [Cl–]o solution inhibited the plateau component of pacemaker potentials. Depolarization of the membrane with high [K+]o solutions increased the frequency and reduced the dV/dtmax of pacemaker potentials. During high-[K+]o-induced depolarization, cyclopiazonic acid abolished pacemaker potentials. Caffeine, forskolin, papaverine, 8-bromo-cGMP and (±)S-nitroso-N-acetylpenicillamine (SNAP) inhibited the plateau component, with no alteration of the primary component. It is concluded that the primary and plateau components of pacemaker potentials are related to voltage-gated Ca2+ influx and Ca2+-activated Cl– channels, respectively, and cyclic nucleotides inhibit mainly the latter. Pacemaker potentials may be generated by the release of Ca2+ from internal stores through excitation of inositol 1,4,5-trisphosphate receptors, coupled with Ca2+ uptake into mitochondria.

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.

Potassium channels activated in the endothelium-dependent hyperpolarization in guinea-pig coronary artery

1 Properties of the endothelium‐dependent hyperpolarization evoked by acetylcholine (ACh) in smooth muscle of the guinea‐pig coronary artery were investigated using conventional microelectrode techniques. 2 ACh hyperpolarized the membrane in an endothelium‐dependent manner. The hyperpolarization comprised two components: an initial and a slow hyperpolarization. The former appeared during application of ACh, while the latter occurred after withdrawal of ACh. 3 Indomethacin and diclofenac, inhibitors of the enzyme cyclo‐oxygenase, blocked only the slow hyperpolarization, indicating that this potential was produced by endothelial prostanoids. 4 Clotrimazole and SKF 525a, known inhibitors of the enzyme cytochrome P450, inhibited both the initial and the slow hyperpolarizations, suggesting that these chemicals acted as non‐selective inhibitors of arachidonic acid metabolism. Inhibition of the lipoxygenase pathway of arachidonic acid metabolism by nordihydroguaiaretic acid had no effect on either component of the hyperpolarization. 5 The slow hyperpolarization was inhibited by 4‐aminopyridine (4‐AP; 10−4‐10−3 M) and glibenclamide (10−6 M). The initial hyperpolarization was greatly inhibited by charybdotoxin (CTX; 5 × 10−8 M) and partially inhibited by apamin (10−7 M), but was not inhibited by glibenclamide (10−5 M). Ba2+ (10−4 M) depolarized the membrane and increased the amplitude of both components of the ACh‐induced hyperpolarization. 6 Hyperpolarizations produced by Y‐26763, a K+ channel opener, were inhibited by glibenclamide, but not by 4‐AP. 7 The results indicate that the slow hyperpolarization is produced by endothelial prostanoids through activation of 4‐AP‐sensitive K+ channels (possibly delayed rectifier type). The initial hyperpolarization is produced mainly through activation of CTX‐sensitive K+ channels (possibly Ca2+‐sensitive type).

MECHANISMS OF EXCITATORY TRANSMISSION IN CIRCULAR SMOOTH MUSCLES OF THE GUINEA PIG SEMINAL VESICLE

PURPOSE Cellular mechanisms of excitatory neuromuscular transmission in circular smooth muscles of the seminal vesicle were investigated. MATERIALS AND METHODS Circular smooth muscles of the seminal vesicle of the guinea pig were isolated. Changes in membrane potential produced by transmural nerve stimulation were recorded using intracellular microelectrode techniques. Changes in the intracellular Ca ion concentration induced by transmural nerve stimulation were measured in preparations loaded with Ca indicator fura-PE3. Responses produced by bath applied norepinephrine and alpha,beta-methylene adenosine triphosphate (ATP) were also examined. RESULTS Transmural nerve stimulation evoked excitatory junction potentials that triggered action potentials and also caused transient increases in [Ca2+] (Ca transients). Nifedipine abolished action potentials, leaving underlying excitatory junction potentials unchanged, and reduced the amplitude of Ca transients. Excitatory junction potentials were blocked by alpha,beta-methylene ATP or guanethidine but not by phentolamine. A train of transmural nerve stimulation evoked oscillatory changes in membrane potential and [Ca2+], which were abolished by phentolamine or inhibited by nifedipine. Nifedipine insensitive components were abolished by cyclopiazonic acid. Norepinephrine depolarized the membrane and elicited oscillatory potentials with an associated elevation in [Ca2+]. These responses were inhibited by nifedipine and abolished by additional application of cyclopiazonic acid. Transient depolarization with an associated increase in [Ca2+] was elicited by alpha,beta-methylene ATP and [Ca2+] responses but no potential changes were inhibited by nifedipine. CONCLUSIONS Circular smooth muscles of the guinea pig seminal vesicle receive a projection of sympathetic nerves that release norepinephrine to initiate slow depolarization through the activation of alpha-adrenoceptors. These nerves also release ATP to elicit excitatory junction potentials. Neurally released norepinephrine and ATP are increased [Ca2+] by the influx of Ca2+ through L-type Ca2+ channels and also by the release of Ca2+ from internal stores.

Cellular mechanisms of nitric oxide‐induced relaxation of corporeal smooth muscle in the guinea‐pig

The cellular mechanism of nitric oxide (NO)‐induced relaxation in corporeal smooth muscle (CSM) of the guinea‐pig was investigated. Changes in the intracellular concentration of calcium ions ([Ca2+]i), membrane potential and isometric tension were measured. CSM cells exhibited spontaneous depolarizations and transient increases in [Ca2+]i (Ca2+ transients) which were accompanied by contractions. This spontaneous activity was abolished by nifedipine (10 μm). NO released by 3‐morpholino‐sydnonimine (SIN‐1, 10 μm) hyperpolarized the membrane and prevented the generation of spontaneous depolarizations. SIN‐1 also abolished Ca2+ transients and associated contractions. These effects of SIN‐1 were blocked by 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μm), an inhibitor of guanylate cyclase. Noradrenaline (NA, 1 μm) increased [Ca2+]i to levels similar to those produced by high potassium‐containing solution (high K+ solution, [K+]o= 40 mm), however, NA‐induced contractions were three times greater in amplitude than those induced by high K+ solution. In NA precontracted preparations, SIN‐1 inhibited 80 % of the contraction and decreased [Ca2+]i by 20 %. In contrast, nifedipine reduced [Ca2+]i by 80 %, while the level of contraction was decreased by only 20 %. SIN‐1‐induced reduction in [Ca2+]i but not the tension effect, was abolished by pretreatment with cyclopiazonic acid (CPA, 10 μm). In high K+ precontracted preparations, SIN‐1 inhibited 80 % of the contraction and reduced [Ca2+]i by 20 %. Nifedipine, however, largely abolished increases in both [Ca2+]i and tension under these circumstances. These results suggest that decreasing the sensitivity of contractile proteins to Ca2+ is probably the key mechanism of NO‐induced relaxation in CSM of the guinea‐pig.

Hyperpolarization‐induced dilatation of submucosal arterioles in the guinea‐pig ileum

The effects of inhibition of acetylcholine (ACh)‐induced hyperpolarization on dilatation of submucosal arterioles were investigated in the guinea‐pig ileum. In smooth muscles of the arterioles depolarized by Ba2+ (0.5 mM) to about −40 mV, ACh (3 μM) repolarized the membrane to about −65 mV (hyperpolarization), irrespective of the absence or presence of L‐Nω‐nitroarginine (L‐NOARG, 0.1 mM) and diclofenac (1 μM), and increased the diameter (dilatation). Combined application of charybdotoxin (CTX, 50 nM) and apamin (0.1 μM), inhibitors of some types of K+‐channels, abolished the ACh‐induced hyperpolarization and dilatation. 18β‐Glycerrhetinic acid (18β‐GA, 30 μM), a known inhibitor of gap junctions, depolarized the membrane to about −36 mV, either in the absence or in the presence of Ba2+, with no associated contraction of the arterioles. In the presence of 18β‐GA, ACh‐induced hyperpolarization was abolished, however the dilatation was inhibited only partially, with associated inhibition of constriction produced by Ba2+ and NA. 18β‐GA inhibited the dilatation produced by sodium nitroprusside, an NO donor. The ACh‐induced hyperpolarization and dilatation were abolished in the presence of 2‐aminoethoxydiphenyl borate (30 μM), an inhibitory modulator of inositol trisphosphate receptor‐mediated Ca2+ release from intracellular stores. It is concluded that in submucosal arterioles, hyperpolarizations produced by ACh have causal relationship to the arteriolar dilatation. 18β‐GA did not induce parallel relationship between hyperpolarization and dilatation produced by ACh. 18β‐GA may have unidentified inhibitory effects on agonist‐mediated actions, in addition to the inhibition of gap junctions.

Modulators of internal Ca2+ stores and the spontaneous electrical and contractile activity of the guinea‐pig renal pelvis

The role of internal Ca2+ stores in the generation of the rhythmic electrical and contractile activity in the guinea‐pig proximal renal pelvis was examined using intracellular microelectrode and muscle tension recording techniques. Ryanodine (30 μM) transiently increased contraction amplitude, while caffeine (0.5 – 3 mM) reduced contraction amplitude and frequency. Contractility was also reduced by 2‐aminoethoxy‐diphenylborate (2‐APB 60 μM), xestospongin C (1 μM), U73122 (5 μM) and neomycin (4 mM), blockers of IP3‐dependent release from Ca2+ stores. 60 mM K+ saline‐evoked contractions were reduced by caffeine (1 mM), U73122 (5 μM) and neomycin (4 mM), but little affected by ryanodine or 2‐APB (60 μM). Spontaneous action potentials consisting of an initial spike followed by a long plateau were recorded (frequency 8.6±1.0 min−1) in small urothelium‐denuded strips of proximal renal pelvis. Action potential discharge was blocked in 75 and 35% of cells by 2‐APB (60 μM) and caffeine (1 mM), respectively. In the remaining cells, only a truncation of the plateau phase was observed. Cyclopiazonic acid (CPA 10 μM for 10 – 180 min), blocker of CaATPase, transiently increased contraction frequency and amplitude. Action potential durations were increased 3.6 fold. Contraction amplitude and frequency slowly declined during a prolonged (>60 min) CPA exposure. We conclude that the action potential in caffeine‐sensitive cells and the shoulder component of caffeine‐insensitive action potential arise from the entry of Ca2+ through Ca2+ channels. The inhibitory actions of modulators of internal Ca2+ release were partially explained by a blockade of Ca2+ entry.