Hiromichi Takano

Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor

1 Membrane potential recordings, made from the circular smooth muscle layer of the gastric antrum taken from mutant mice which lacked the inositol trisphosphate (InsP3) type 1 receptor, were compared with those obtained from the stomach of control (wild‐type) mice. 2 Immunostaining of gastric muscles indicated that the distribution and form of c‐kit positive cells were similar in wild‐type and mutant mice. 3 Smooth muscles from wild‐type mice generated slow waves that in turn initiated spike potentials, while those from mutant mice were either quiescent or generated irregular bursts of spike potentials. In the presence of nifedipine, slow waves with reduced amplitude were generated in wild‐type mice, while all electrical activity was abolished in mutant mice. 4 Acetylcholine depolarized and sodium nitroprusside hyperpolarized the membrane in muscles from both types of mice, being more effective in wild‐type mice. Noradrenaline produced similar hyperpolarizations in both types of mice. 5 Transmural nerve stimulation evoked inhibitory junction potentials (IJPs) in both wild‐type and mutant mice. In wild‐type mice, the IJPs were reduced in amplitude by nitroarginine and converted to a cholinergic excitatory junction potential (EJP) by apamin. In mutant mice, the IJPs were unaffected by nitroarginine or atropine but were abolished by apamin. 6 It is concluded that in antral smooth muscle, the expression of InsP3 type 1 receptors may be causally related to the generation of slow waves but not to the generation of action potentials. A lack of InsP3 receptors attenuates cholinergic excitatory and nitrergic inhibitory responses but does not alter the response to noradrenaline.

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.

Properties of spontaneously active cells distributed in the submucosal layer of mouse proximal colon

Intracellular electrical activity was recorded from smooth muscle tissues of the mouse proximal colon, and the impaled cells were visualized by injection of neurobiotin. Slow potentials with initial fast and subsequent plateau components (plateau potentials), generated at a frequency of 14.8 min−1, were recorded from oval‐shaped cells with bipolar processes. Periodic bursts of spike potentials (4.6 min−1) and bursts of oscillatory potentials (4.3 min−1) were recorded in circular and longitudinal smooth muscle cells, respectively. Nifedipine (0.1 μm) abolished the bursts of spike and oscillatory potentials and reduced the duration of plateau potentials. The plateau potentials were abolished by 1 μm nifedipine. The plateau potentials were also abolished by cyclopiazonic acid (an inhibitor of Ca2+ uptake into internal stores) or 2‐aminoethoxydiphenyl borate (an inhibitor of inositol 1,4,5‐trisphosphate receptor‐mediated Ca2+ release), and were inhibited by bis‐(aminophenoxy) ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (a chelator of intracellular Ca2+). Carbonyl cyanide m‐chlorophenylhydrazone (a mitochondrial protonophore) abolished plateau potentials, and its action was not mimicked by oligomycin (an inhibitor of mitochondrial ATPase). It is concluded that in mouse proximal colon, submucosal c‐kit‐positive bipolar cells spontaneously generate plateau potentials with rhythms different from those generated by smooth muscle cells. The plateau potentials are generated through activation of voltage‐gated Ca2+ channels, which are coupled to the release of Ca2+ from the internal stores and the handling of Ca2+ in mitochondria.

Alteration of the properties of gastric smooth muscle in the genetically hyperglycemic OLETF rat

Membrane responses were recorded from isolated gastric smooth muscle of Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats, using microelectrode techniques. At the age of 68-76 weeks, the blood sugar level was 181 mg/dl in LETO rats and 350 mg/dL in OLETF rats. In both rats, the membrane potential was stable in fundus muscle and spontaneously active with generation of slow waves in antrum muscle. The resting membrane potential was about - 46 mV in fundus and - 55 mV in antrum muscles of LETO rats, and the values were 3-7 mV lower in OLETF rats. The slow waves were generated regularly in LETO rats, while they were irregular and of small amplitude in OLETF rats. Transmural nerve stimulation evoked a cholinergic excitatory junction potential and following inhibitory junction potential in LETO rats, and only an inhibitory junction potential of smaller size was generated in most of OLETF rats. The acetylcholine-induced depolarization was greater in OLETF than in LETO rats. The level of hyperpolarization produced by noradrenaline was similar between OLETF and LETO rats. Thus, the reduction of the resting membrane potential, weakening of spontaneous activity, impairment of cholinergic transmission and cholinergic supersensitivity were associated with hyperglycemia. These alterations were considered due to the development of diabetes mellitus.

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.

Voltage-operated Ca2+ currents and Ca2+-activated Cl– currents in single interstitial cells of the guinea-pig prostate

To investigate the expression of ‘T‐type’ and ‘L‐type’ voltage‐operated Ca2+ channels in single interstitial cells of the guinea‐pig prostate.

Properties of submucosal venules in the rat distal colon

Venules within the gut wall may have intrinsic mechanisms for maintaining the circulation even upon the intestinal wall distension. We aimed to explore spontaneous and nerve‐mediated contractile activity of colonic venules.

Mechanical responses evoked by nerve stimulation in gastric muscles of mouse lacking inositol trisphosphate receptor.

Alteration of mechanical responses elicited by transmural nerve stimulation (TNS) was investigated in pylorus muscle of stomach isolated from mutant mice lacking expression of IP, type-1 receptor. In wild and mutant mice. TNS inhibited spontaneous contractions and generated an off-response at the cessation. The effects of inhibitors of neurotransmission revealed that in wild mice, acetylcholine and nitric oxide were involved as excitatory and inhibitory mediators, respectively. In mutant mice, a lack of nitroxidergic component with associated attenuation of cholinergic transmission was found. The off-response was inhibited by apamin in both mice. In mutant mice, spantide-sensitive excitatory response appeared in the presence of apamin. Acetylcholine and substance P enhanced while noradrenaline and sodium nitroprusside inhibited spontaneous contractions, in both wild and mutant mice; the actions were weaker in mutant mice than in wild mice for any agonists. The results indicate that pylorus smooth muscles receive cholinergic excitatory and nitroxidergic and non-adrenergic non-cholinergic inhibitory projections, and a lack of IP, type-1 receptor results in an impairment of cholinergic and nitroxidergic components, with no alteration of non-adrenergic non-cholinergic inhibitory projections. In addition, the mutation induces a substance P projection which is not detected in wild mice.

Pranidipine enhances relaxation produced by endothelium-derived relaxing factor in carotid artery.

The effects of pranidipine, a novel dihydropyridine-type Ca(2+)-channel antagonist, on acetylcholine-induced endothelium-dependent relaxation were investigated in isolated carotid artery of the guinea-pig. In arteries contracted with high-K(+) solution ([K(+)](0)=28.8 mM) containing noradrenaline, the relaxation was inhibited by N(omega)-nitro-L-arginine, indicating an involvement of endothelium-derived relaxing factor. Pranidipine (10(-9)-10(-7) M) augmented the relaxation in a concentration-dependent manner. Sodium nitroprusside produced a relaxation in arteries contracted with high-K(+) solution containing noradrenaline, in an endothelium-independent manner, and the relaxation was enhanced by pranidipine. 1H-[1,2,4] oxadiazolo [4, 3-a] quinoxalin-l-one (ODQ), an inhibitor of nitric oxide-sensitive guanylate cyclase, attenuated the relaxation produced by acetylcholine or sodium nitroprusside. In the presence of ODQ, pranidipine did not enhance the acetylcholine-induced relaxation. The relaxation produced by endothelium-derived hyperpolarizing factor was inhibited by pranidipine, with no alteration of the hyperpolarization. Thus, pranidipine augments the nitric oxide-induced relaxation, possibly by enhancing the mechanisms related to cyclic GMP.

Junctional transmissions in smooth muscle of the guinea pig duodenum and their modulation by the prokinetic agent DQ-2511.

Junctional transmissions and their modulation by DQ-2511, a novel prokinetic agent, were investigated in smooth muscle of the guinea pig duodenum. Transmural nerve stimulation evoked a cholinergic excitatory junction potential, a nonadrenergic, noncholinergic inhibitory junction potential, and a substance P mediated slow depolarization. DQ-2511 showed dual effects on the slow depolarization: a low concentration (10–9 mol/l) enhanced and high concentrations (>10–6 mol/l) tended to inhibit. The depolarization produced by exogenously applied substance P was enhanced by low concentrations of DQ-2511. The results suggest that the prokinetic actions of DQ-2511 involve an enhancement of transmission from substance P neurons due to an increase in sensitivity of postjunctional receptors to substance P.