Shinsuke Nakayama

Requirement of ryanodine receptors for pacemaker Ca2+ activity in ICC and HEK293 cells.

Intracellular Ca2+ ([Ca2+]i) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca2+ release from intracellular Ca2+ stores produces [Ca2+]i oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca2+]i oscillations in ICCs are preserved in the presence of dihydropyridine Ca2+ antagonists, which suppress Ca2+ activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca2+]i oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca2+]i oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP3Rs. The reconstituted [Ca2+]i oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.

Measurement of Spontaneous Oscillatory Magnetic Field of Guinea-Pig Smooth Muscle Preparation Using Pico-Tesla Resolution Amorphous Wire Magneto-Impedance Sensor

New measured results of spontaneous oscillatory bio-magnetic field generated from preparations of guinea-pig smooth muscle tissue are presented in this paper using the pico-Tesla MI sensor. It is confirmed that the changes in the bio-magnetic field activity precisely synchronized with extracellular electric activity for stomach musculature of the guinea-pig. It is, however, noteworthy that these two activities provide different biological information. A phase lag of around 2 s of the magnetic signal pulse against the extracellular electric signal pulse is newly found. It is also shown that Ca2+ channel activity of the smooth muscle tissue (taenia caeci) can be evaluated using the MI sensor. Both electric and magnetic activities depend on the functionality of the living tissues. Therefore, the pico-Tesla MI sensor has the potential for non-invasive detection use in wide range of biology and medicine, including non-invasive evaluation use of the stem cells development.

In Vitro Formation of Enteric Neural Network Structure in a Gut‐Like Organ Differentiated from Mouse Embryonic Stem Cells

Using an embryoid body (EB) culture system, we developed a functional organ‐like cluster—a “gut”—from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited spontaneous contractions but did not exhibit distinct peristalsis‐like movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (c‐kit [a transmembrane receptor that has tyrosine kinase activity]‐positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified; however, enteric neural ganglia were absent in the spontaneously differentiated ES gut. By adding brain‐derived neurotrophic factor (BDNF) only during EB formation, we for the first time succeeded in in vitro formation of enteric neural ganglia with connecting nerve fiber tracts (enteric nervous system [ENS]) in the ES gut. The ES gut with ENS exhibited strong peristalsis‐like movements. During EB culture in BDNF+ medium, we detected each immunoreactivity associated with the trk proto‐oncogenes (trkB; BDNF receptors) and neural crest marker, proto‐oncogene tyrosine‐protein kinase receptor ret precursor (c‐ret), p75, or sox9. These results indicated that the present ENS is differentiated from enteric neural crest‐derived cells. Moreover, focal stimulation of ES guts with ENS elicited propagated increases in intracellular Ca2+ concentration ([Ca2+]i) at single or multiple sites that were attenuated by atropine or abolished by tetrodotoxin. These results suggest in vitro formation of physiologically functioning enteric cholinergic excitatory neurons. We for the first time succeeded in the differentiation of functional neurons in ENS by exogenously adding BDNF in the ES gut, resulting in generation of distinct peristalsis‐like movements.

Ca2+ channel properties in smooth muscle cells of the urinary bladder from pig and human

Ca(2+) channel properties of pig and human bladder smooth muscle were investigated utilizing standard whole-cell patch clamp techniques. Both the amplitude obtained and the current density of Ca(2+) channel current evoked by step depolarization were larger in human than in pig myocytes. The inward currents were sensitive to an L-type Ca(2+) channel antagonist, nifedipine, the effects of which were not significantly different between species. In both species, prior application of ATP (0.1 mM) had no effect on activation of this voltage-sensitive channel current, while a muscarinic receptor agonist, carbachol (0.1 mM), significantly attenuated the amplitude of this current. Furthermore, inclusion of GDP-beta-S or Heparin in the pipette abolished or had no effect on the suppression of Ca(2+) current by carbachol, respectively. These results forward the pig as a good model for the human in detrusor Ca(2+) channel properties, especially with regard to neural modulation, although voltage-sensitive Ca(2+) channels seem to make greater contribution in human bladder physiology.

Voltage sensitivity of slow wave frequency in isolated circular muscle strips from guinea pig gastric antrum

In circular muscle preparations isolated from the guinea pig gastric antrum, regular spontaneous electrical activity (slow waves) was recorded. Under normal conditions (6 mM K+), the frequency and shape of the slow waves were similar to those observed in ordinary stomach smooth muscle preparations. When the resting membrane potential was hyperpolarized and depolarized by changing the extracellular K+ concentration (2-18 mM), the frequency of slow waves decreased and increased, respectively. Application of cromakalim hyperpolarized the cell membrane and reduced the frequency of slow waves in a dose-dependent manner. Cromakalim (3 microM) hyperpolarized the membrane, and slow waves ceased in most preparations. In the presence of cromakalim, subsequent increases in the extracellular K+ concentration restored the frequency of slow waves accompanied by depolarization. Also, glibenclamide completely antagonized this effect of cromakalim. In smooth muscle strips containing both circular and longitudinal muscle layers, such changes in the slow wave frequency were not observed. It was concluded that the maneuver of isolating circular smooth muscle altered the voltage dependence of the slow wave frequency.In circular muscle preparations isolated from the guinea pig gastric antrum, regular spontaneous electrical activity (slow waves) was recorded. Under normal conditions (6 mM K+), the frequency and shape of the slow waves were similar to those observed in ordinary stomach smooth muscle preparations. When the resting membrane potential was hyperpolarized and depolarized by changing the extracellular K+ concentration (2-18 mM), the frequency of slow waves decreased and increased, respectively. Application of cromakalim hyperpolarized the cell membrane and reduced the frequency of slow waves in a dose-dependent manner. Cromakalim (3 μM) hyperpolarized the membrane, and slow waves ceased in most preparations. In the presence of cromakalim, subsequent increases in the extracellular K+ concentration restored the frequency of slow waves accompanied by depolarization. Also, glibenclamide completely antagonized this effect of cromakalim. In smooth muscle strips containing both circular and longitudinal muscle layers, such changes in the slow wave frequency were not observed. It was concluded that the maneuver of isolating circular smooth muscle altered the voltage dependence of the slow wave frequency.

Serotonin Augments Gut Pacemaker Activity via 5-HT3 Receptors

Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HTs actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca2+ and electric activities in ICC were performed by employing fluorescent Ca2+ imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca2+ antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca2+ oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT3 receptor antagonist suppressed spontaneous Ca2+ activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT3 receptor agonist, restored it. GR113808, a selective antagonist for 5-HT4, and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT3 receptors, had little effect on ICC Ca2+ activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT3 receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT3 receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome.

Pacemaker phase shift in the absence of neural activity in guinea‐pig stomach: a microelectrode array study

Gastrointestinal (GI) motility is well organized. GI muscles act as a functional syncytium to achieve physiological functions under the control of neurones and pacemaker cells, which generate basal spontaneous pacemaker electrical activity. To date, it is unclear how spontaneous electrical activities are coupled, especially within a micrometre range. Here, using a microelectrode array, we show a spatio‐temporal analysis of GI spontaneous electrical activity. The muscle preparations were isolated from guinea‐pig stomach, and fixed in a chamber with an array of 8 × 8 planar multielectrodes (with 300 μm in interpolar distance). The electrical activities (field potentials) were simultaneously recorded through a multichannel amplifier system after high‐pass filtering at 0.1 Hz. Dihydropyridine Ca2+ channel antagonists are known to differentiate the electrical pacemaker activity of interstitial cells of Cajal (ICCs) by suppressing smooth muscle activity. In the presence of nifedipine, we observed spontaneous electrical activities that were well synchronized over the array area, but had a clear phase shift depending on the distance. The additional application of tetrodotoxin (TTX) had little effect on the properties of the electrical activity. Furthermore, by constructing field potential images, we visualized the synchronization of pacemaker electrical activities resolving phase shifts that were measurable over several hundred micrometres. The results imply a phase modulation mechanism other than neural activity, and we postulate that this mechanism enables smooth GI motility. In addition, some preparations clearly showed plasticity of the pacemaker phase shift.

Co-contribution of IP₃R and Ca²⁺ Influx Pathways to Pacemaker Ca²⁺ Activity in Stomach ICC:

Intracellular Ca2+ oscillations in interstitial cells of Cajal (ICCs) are thought to be the primary pacemaker activity in the gut. In the present study, the authors prepared small tissues of 100-to 300-µm diameter (cell cluster preparation) from the stomach smooth muscle (including the myenteric plexus) of mice by enzymatic and mechanical treatments. After 2 to 4 days of culture, the intracellular Ca2+ concentration ([Ca2+]i) was measured. In the presence of nifedipine, a dihydropyridine Ca2+ channel antagonist, spontaneous [Ca2+]i oscillations were observed within limited regions showing positive c-Kitimmunoreactivity, a maker for ICCs. In the majority of cell cluster preparations with multiple regions of [Ca2+]i oscillations, [Ca2+]i oscillated synchronously in the same phase. Asmall number of cell clusters (8 of 53) showed multiple regions of [Ca2+]i oscillations synchronized but with a considerable phase shift. Neither tetrodotoxin (250 nM) nor atropine (10µM) significantly affected [Ca2+]i oscillations in the presence of nifedipine. Low concentrations (40µM) of Ni2+ had little effect on the spontaneous [Ca2+]i oscillation, but SK&F96365 (40µM) and Cd2+ (120µM) terminated it. Applications of either 2-aminoethoxydiphenyl borate (10µM) or xestosponginC(10µM) completely and rather rapidly (~2 min) abolished the spontaneous [Ca2+]i oscillations. The results suggest that pacemaker [Ca2+]i oscillations in ICCs are produced by close interaction of intracellular Ca2+ release channels, especially inositol 1,4,5-trisphosphate receptor (InsP3R) and Ca2+ influx pathways, presumably corresponding to store-operated type channels. Reverse transcription polymerase chain reaction examinations revealed expression of TRPC2, 4, and 6, as well as InsP3R1 and 2 in ICCs.

Consequences of metabolic inhibition in smooth muscle isolated from guinea-pig stomach

1 In smooth muscle isolated from the guinea‐pig stomach, cyanide (CN) and iodoacetic acid (IAA) were applied to block oxidative phosphorylation and glycolysis, respectively. Effects of IAA on generation of spontaneous mechanical and electrical activities were systematically investigated by comparing those of CN. Spontaneous activity ceased in 10–20 min during applications of 1 mm IAA. On the other hand, application of 1 mm CN also reduced the spontaneous activity, but never terminated it. In the presence of CN the negativity of the resting membrane potential was slightly reduced. 2 When spontaneous activity ceased with IAA, the resting membrane potential was not significantly affected. Also, before ceasing, the amplitude and duration of the spontaneous electrical activity were significantly reduced. The amplitude of the electrotonic potential was, however, not changed by IAA. Further, glibenclamide did not prevent the effects of IAA. These results suggest that, unlike cardiac muscle, activation of metabolism‐dependent K+ channels in stomach smooth muscle does not seem to play a major role in reducing and terminating spontaneous activity during metabolic inhibition. 3 Carbachol‐induced contraction transiently increased, and subsequently decreased gradually during application of IAA. 4 After 50 min application of IAA, when there was no spontaneous activity, the concentrations of phosphocreatine (PCr) and ATP measured with 31P nuclear magnetic resonance decreased to 60 and 80% of the control, respectively, while inorganic phosphate (Pi) concentration paradoxically fell to below detectable levels. During subsequent prolonged application of IAA, high‐energy phosphates steadily decreased. On the other hand, after 50 min CN application, [PCr] and [ATP] decreased to approximately 30 and 80% of the control, respectively, while [Pi] increased by 2.6‐fold. 5 In the presence of either CN or IAA, spontaneous mechanical and electrical activities were reduced or eliminated, although amounts of high‐energy phosphates sufficient to contract smooth muscle remained. It can be postulated that some mechanism(s) related to energy metabolism, but not including ATP‐sensitive K+ channels, plays an important role in generating spontaneous activity in guinea‐pig stomach smooth muscle. During metabolic inhibition the energy metabolism‐dependent mechanism(s) would preserve high‐energy phosphates, and consequently cell viability, by stopping spontaneous activity.

Recent advances in studies of spontaneous activity in smooth muscle: Ubiquitous pacemaker cells

The general and specific properties of pacemaker cells, including Kit-negative cells, that are distributed in gastrointestinal, urethral and uterine smooth muscle tissues, are discussed herein. In intestinal tissues, interstitial cells of Cajal (ICC) are heterogeneous in both their forms and roles. ICC distributed in the myenteric layer (ICC-MY) act as primary pacemaker cells for intestinal mechanical and electrical activity. ICC distributed in muscle bundles play a role as mediators of signals from autonomic nerves to smooth muscle cells. A group of ICC also appears to act as a stretch sensor. Intracellular Ca2+ dynamics play a crucial role in ICC-MY pacemaking; intracellular Ca2+ ([Ca2+](i)) oscillations periodically activate plasmalemmal Ca2+-activated ion channels, such as Ca2+-activated Cl(-) channels and/or non-selective cation channels, although the relative contributions of these channels are not defined. With respect to gut motility, both the ICC network and enteric nervous system, including excitatory and inhibitory enteric neurons, play an essential role in producing highly coordinated peristalsis.