Identification of the functions of the genes at system level is one of the greatest challenges in the post genome-sequence era. To reveal functional significance of the genes expressed in the brain, we have been utilizing a comprehensive behavioral test battery on various kinds of genetically-engineered mice. The behavioral test battery covers a broad range of various behavioral domains, such as learning and memory, sensory-motor functions, emotions and motivation. During the past two years, we have assessed behaviors of more than 30 different strains of mutant mice and, surprisingly, we found at least one significant behavioral abnormality in more than 90% of those strains. This fact not only demonstrates that our strategy is useful in elucidating the functions of the genes expressed in the brain but also implies that a large part of the genes expressed in the brain may have some functions. In this workshop, I'd like to introduce some examples of our work using the test battery on mutant mice and to make a proposal to use it as a "hub" for large-scale neuroscience. Establishing a collaborative network among experts in the field would be necessary for greater success of this approach. The potential issues related to implementing such collaborative network or consortium will be also discussed. [J Physiol Sci. 2006;56 Suppl:S51]

抄録全体を表示

We have been studying neuroendocrinological bases of emotional and socio-sexual behaviors. Our studies using two types of estrogen receptor knockout mice during the last ten years have proven that the knockout mouse model is a powerful tool to delineate the relationship between genes, hormones, and behaviors. Since our main research interest, as behavioral scientists, is to understand brain mechanisms of emotional and socio-sexual behaviors, we have been focusing on developing test paradigms that are able to measure the behaviors of interest most appropriately and reliably. On the other hand, progress in molecular biology has enabled production of enormous numbers of genetically modified animals. As a result, interest and demand for analyses of behaviors as one of the phenotypes of these animals, have rapidly grown for molecular biologists. In this talk, I will first overview our findings in behavioral characteristics of estrogen receptor knockout mice and then discuss the problems in standardization of behavioral testing paradigms. Particularly, I will talk about differences between "how to measure" and "what is measured". [J Physiol Sci. 2006;56 Suppl:S51]

抄録全体を表示

Research Resources Center (RRC) in RIKEN Brain Science Institute (BSI) provides the mouse behavior analysis support service. As a base of experimentation, RRC provides the common laboratories for mouse behavior analysis in the laboratory animal facility in BSI. This laboratory area consists of 12 separated rooms, and one or several kinds of devices for the behavioral experiment are set up in each room. BSI researchers can use these rooms and devices freely following a guidance. The mouse behavior analysis support service section offers user guidance on use, maintains, develops and updates the laboratories and the equipped experimental apparatuses. Another important part of this service is consultation. We help to plan for behavior experiments, paradigms and/or methods, data analysis and so on. Furthermore, a primary behavioral screening of mutant mice based on a "behavioral test battery" is currently provided on a trial basis. In this workshop, the advantage and disadvantage of the mouse behavior analysis support service at BSI will be discussed from the point of view of standardization of the mouse behavior analysis at a large-scale research facility like BSI. [J Physiol Sci. 2006;56 Suppl:S52]

抄録全体を表示

Cerebellum plays an important role in non-declarative memories such as motor memories. Cellular substrate for the cerebellar-dependent memory is long-term depression (LTD). NO-cGMP-PKG (cGMP-dependent protein kinase) pathway is shown to be involved in the induction of cerebellar LTD. NO absorbing reagent prevents cerebellar-dependent learning such as the adaptation of perturbed locomotion and VOR adaptation. Furthermore, each component of NO-soluble guanylate cyclase-cGMP-PKG pathway has been shown to be essential for the induction of LTD. However, the downstream component of PKG was not identified. Recently we molecularly cloned and characterized G-substrate, localized specifically in cerebellar Purkinje cells, as a downstream component of PKG. Further, we have generated a mice lacking G-substrate gene. Homozygous mice are vital as expected from the restricted localization of G-substrate. The behavioral analyses were conducted on the G-substrate gene knockout mice. Significant difference was not observed between the control and G-substrate knockout mice in general behaviors. However, a specific impairment was observed in long-term horizontal optokinetic response (HOKR), a cerebellar-dependent memory, without any impairment in short-term HOKR. In addition, we observed G-substrate, with shuttles between nuclear and cytosol of Purkinje cells. G-substrate may have a role in transcription and translation in the nuclear that is essential for the long-term memory. [J Physiol Sci. 2006;56 Suppl:S52]

抄録全体を表示

Extensive behavioral testing of mice including common laboratory strains and genetically modified lines has revealed that many measures of hippocampus-dependent memory and learning are confounded by reactions of mice towards handling and test conditions. Moreover, very little is known about cognitive activity of mice in their natural environment. Therefore, we have developed transponder-based techniques permitting to assess learning and cognition in social settings, first in outdoor pens and later in large cage units called "Intellicage". Data presented will show how hippocampal malfunction is manifested in the water maze, under outdoor conditions and in Intellicages, and how transponder-based technology can be used for fully automated assessment of memory and learning in large number of mice.The main conclusions for high-throughput phenotyping are:- Mutation, strain and hippocampal lesion effects can easily be discovered within a social group–testing single mice in isolation is methodologically not necessary.- Automated testing is 10 to 40 times more economic than manual testing.- Comparability across labs is improved and standardization is much facilitated.- Transponder-based automated systems can be used for both, screening by laypersons and sophisticated analysis by behavioral experts.Since all testing is done without human interference by computers, different groups can easily share data over the web. This will enable researchers to perform comparative analysis of animals having undergone identical test protocols in different locations. Supported by Swiss National Science Foundation and NCCR "Neural Plasticity and Repair". [J Physiol Sci. 2006;56 Suppl:S52]

抄録全体を表示

The progression mechanism from cardiac dysfunction to advanced heart failure (HF) including dilated cardiomyopathy (DCM) should be clarified to establish a novel treatment. We have identified a gene mutation in δ-sarcoglycan (SG) that causes hereditary DCM in both animals (Sakamoto et al., PNAS, 1997) and humans (Tsubata et al., JCI, 2000). TO-2 hamsters with hereditary DCM show age-dependent cleavage and translocation of myocardial dystrophin (Dys) from sarcolemma (SL) to myoplasm, enhanced SL permeability in situ, and a close relation between Dys loss and hemodynamics. Dys disruption is not an epiphenomenon but directly causes advanced HF, because in vivo transfer of the missing gene to degrading cardiomyocytes ameliorated all of the pathological features and improved the disease prognosis (Kawada et al., PNAS, 2002). Furthermore, acute HF after isoproterenol toxicity and chronic HF after coronary ligation in rats both time-dependently cause Dys disruption in the degrading myocardium (Takahashi et al., CVRes., 2005). Dys cleavage was also detected in human hearts from patients with DCM of unidentified etiology, supporting a scheme of vicious cycle consisting of SL instability, Dys cleavage, and translocation of Dys from the SL to the myoplasm (Toyo-oka et al., PNAS, 2004), irrespective of an acute or chronic process and a hereditary or acquired origin. Activation of endogenous calpain will be discussed for the Dys disruption. [J Physiol Sci. 2006;56 Suppl:S53]

抄録全体を表示

Background and Objectives: Laminin, which is a major component of the extracellular matrix, is known to increase in the myocytes in the diabetic heart and dilated cardiomyopathy. The laminin gamma 1 chain promoter contains a transcriptional element denoted bcn-1 that is inducible and active (Suzuki H, et al. J Biol Chem 271:1996). To elucidate the molecular pathogenesis of the increase of laminin in the cardiac muscle cells of cardiomyopathy, we have carried out the yeast one-hybrid screen using bcn-1 as a bait, and cloned Smarce 1r protein as a molecular partner which interacts with bcn-1 protein. Next, we repeated two-hybrid screen using Smarce 1r as a bait and cloned MLF11P as a partner (Suzuki H, Exp Clin Cardiol 9:2004).Methods: The yeast two-hybrid screen analysis was carried out again using MLF11P protein (amino-acids 1-318) as a bait. A human heart cDNA library was screened by the yeast mating method for overnight culture.Results: We isolated two final positive clones encoded the same protein, which is an alternative-RNA splicing form of the human cardiac troponin I (TnI) protein and we call this as a spliced form of TNI (STNI). The mRNA expression pattern of STNI is heart-specific. The STNI shares several sequence similarities with the human cardiac TNI but lacks troponin T binding protein.Conclusions: We report the heart-specific segment of the human cardiac troponin I isoform which lacks troponin C binding portion. These results suggest that STNI might be involved in the molecular pathogenesis of the increase of laminin in the cardiomyopathy. [J Physiol Sci. 2006;56 Suppl:S53]

抄録全体を表示

Dilated cardiomyopathy (DCM) is characterized by cardiac dilation and systolic dysfunction, which often leads to severe heart failure and sudden death. However, little is known about the pathogenic mechanism for DCM, and no therapeutical method is established at present except for cardiac transplantation. We created a knock-in mouse model of DCM caused by a deletion mutation δK210 in cardiac troponin T and explored its molecular pathogenic process and potential pharmacotherapy. Mutant mice developed enlarged hearts with bi-ventricular dilation and systolic dysfunction and suffered sudden death frequently, recapitulating human DCM with this mutation. Skinned cardiac muscle fibers from mutant mice showed a decreased Ca²⁺ sensitivity of force generation, confirming our previous hypothesis that decreased contractility of cardiac muscle is a primary pathogenic mechanism of this mutation. Surprisingly, however, intact cardiac muscle fibers from mutant mice showed no significant reduction in isometric force per cross-sectional area. Analyses of Fura-2 loaded cardiomyocytes revealed that this was due to an increase in the amplitude of intracellular Ca²⁺ transient. Biochemical analyses, including DNA microarray, strongly suggested that Ca²⁺ transient was increased through down-regulation of a specific isoform of phosphodiesterase (PDE4B) and associated increase in cAMP in cardiomyocytes of mutant mice, which could compensate for the decreased myofilament Ca²⁺ sensitivity but at the same time would increase the risk for arrhythmia leading to sudden death due to a Ca²⁺ overload. [J Physiol Sci. 2006;56 Suppl:S53]

抄録全体を表示

OLETF rat is a model of spontaneous non-insulin-dependent diabetes mellitus (DM), accompanying diastolic dysfunction associated with abnormal Ca²⁺ handling and decrease in sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) expression. The aim of this study was to examine whether SERCA2a gene transfer can restore left ventricular (LV) function in this DM in terms of LV mechanical work and energetics. DM rats were randomized to receive adenovirus carrying SERCA 2a gene (DM+Serca) or β-galactosidase (DM+Gal), or saline (DM+Sa) by catheter-based (cross-clamping) technique. LV mechanoenergetics was assessed in cross-circulated whole heart preparations 3 days after the infection. In DM, end-systolic pressure (ESP_0.1) was low and end-diastolic pressure (EDP_0.1) was high at 0.1 ml intraballoon water. In DM+Serca, however, ESP_0.1 increased and EDP_0.1 decreased. LV relaxation rate in DM+Serca was faster than that of DM+Gal/Sa groups. Oxygen cost of LV contractility in DM was higher than that of normal, indicating energy wasting in Ca²⁺ handling during E-C coupling. The O₂ cost of LV contractility decreased to non-DM rat level in DM+Serca, although it remained high in DM+Gal and DM+Sa. These results indicate that SERCA2a overexpression by SERCA2a gene transfer improves not only LV mechanics but also energetics in DM rat hearts. [J Physiol Sci. 2006;56 Suppl:S54]

抄録全体を表示

Is respiratory rhythm generated by pace-maker neurons or by network mechanisms? Although this longtime and important question has not been solved yet, recent studies provide evidence supporting pace-maker theory at least under some experimental conditions and clarified characteristics of respiratory pace-maker neurons. In respiratory rhythm generation, a term of pace-maker neurons indicates neurons that possess cellular property intrinsically producing rhythmic burst (but not tonic) activity. In pace-maker theory, it is hypothesized that intrinsic burst generating neurons are embedded in a network composed of neuronal population with same active phase and contribute to produce basic synchronized rhythm, whereas inhibitory synaptic connections are not necessary for the rhythm generation. At least two respiration-related rhythm generators have been suggested to exist in the medulla and to produce intrinsic periodic burst activity. One is an inspiratory (Insp) neuronal network (i.e., Insp rhythm generator), which is localized predominantly in the pre-Boetzinger complex of the ventrolateral medulla. Another is a pre-inspiratory (Pre-I) neuronal network (i.e., Pre-I rhythm generator), which generates activity prior to Insp bursts and is located in the more rostral ventrolateral medulla including the para-facial respiratory group (pFRG). We have shown that pFRG-Pre-I neurons serve as a primary rhythm generator, triggering inspiratory burst periodically in the brainstem-spinal cord preparation from newborn rat. In this presentation, we focus on mainly burst generating properties of Pre-I neurons and the ionic and synaptic mechanisms. [J Physiol Sci. 2006;56 Suppl:S54]

抄録全体を表示

Many smooth muscles exhibit spontaneous electrical and mechanical activity. This has been considered to "myogenic" activity for many years, however, it is in fact generated by specialized cells, namely interstitial cells of Cajal (ICC). In the gastrointestinal tract, a network of myenteric ICC (ICC-MY) generates pacemaker potentials to initiate spontaneous electrical activity. Intramuscular ICC (ICC-IM) augment the depolarizations by generating ongoing unitary potentials (UPs). However, corporal ICC-IM may be dominant pacemaker as they create a greatest frequency in spontaneous activity. ICC-like cells have been found in other smooth muscle organs and may play a similar role. In the urethra, ICC-like cells generate spontaneous transient depolarizations (STDs), which sum to activate L-type Ca channels. UPs and STDs result from the opening of Ca-activated Cl⁻ channels. UPs solely depend on inositol 1, 4, 5-trisphosphate (InsP₃)-dependent Ca release, whilst the generation of STDs requires both InsP₃- and ryanodine-receptors. In corpus cavernosum (CC), where smooth muscle cells are capable of generating spontaneous depolarizations, ICC-like cells express cyclooxygenase 2 and spontaneously produce prostaglandins to reinforce spontaneous activity. Therefore, although ICC or ICC-like cells in different regions or tissues share many similarities, they also have a significant diversity. Interaction between ICC and smooth muscle cells result in a further heterogeneity of spontaneous activity, and thus develops characteristics of individual smooth muscle organs. [J Physiol Sci. 2006;56 Suppl:S54]

抄録全体を表示

Computers have become the necessities of life science with improvements of information technologies. Thus, Systems Biology, whose aim is to describe the whole biological interactions as a mathematical model and understand the whole mechanisms by simulation, has been emerged. We have started developing a comprehensive cardiac model (Kyoto model), and a cardiac sinoatrial node pacemaker model is developed with the same set of equations as the ventricular cell model. The model successfully reconstructs the experimental action potentials at various concentrations of external Ca²⁺ and K⁺, and reaches steady-state and the sensitivity analyses can be applied to a variety set of parameters. Increasing the amplitude of L-type Ca²⁺ current (I_CaL) prolongs the duration of the action potential and thereby slightly decreases the spontaneous rate. On the other hand, a negative voltage shift of I_CaL gating by a few mV markedly increases the spontaneous rate. When the amplitude of sustained inward current is increased, the spontaneous rate is increased irrespective of the I_CaL amplitude. When the spontaneous activity is stopped by decreasing I_CaL amplitude, the resting potential is around -35 mV over 1-15 mM [K⁺]_o because of the presence of the background non-selective cation current. The unique role of individual voltage- and time-dependent ion channels is clearly demonstrated and distinguished from that of the background current by calculating an instantaneous equilibrium potential during the course of the spontaneous activity. [J Physiol Sci. 2006;56 Suppl:S55]

抄録全体を表示

Many physiological functions show circadian rhythms controlled by suprachiasmatic nucleus (SCN) in mammals. From the SCN, ca. 24 hr rhythmicity accompanying the time of day information (phase) is conveyed to the other area of the brain and peripheral organs. Arg-vasopressin (AVP) containing neuron is one of the output neuron from the SCN. VIP and glutamate increased AVP release from the SCN, meanwhile suppressed by GABA (via GABAa receptor) and melatonin (MT2 receptor). The rPer2 mRNA products (CCG protein) promote the rBmal1 mRNA increase, which concerns the AVP mRNA transcription. In the SCN, before and after the 8-hr advance of the LD cycle, AVP mRNA and AVP peptide in the SCN remain coupled with time. After the light pulse (800 lux, 15 min at ZT 22), AVP mRNA increased in the SCN, but not in the paraventricular nucleus (PVN). Following the melatonin injection (1 mg/kg, i.p.), AVP content in the SCN decreased at both light and dark period, while no changes were observed in the PVN. In the SCN, rPer2 mRNA increased at both light and dark period. In the PVN, rPer2 decreased and increased at light and dark period, respectively. While in the pineal gland, rPer2 mRNA increased at 180 min after the melatonin injection. The melatonin-AVP reciprocal system indicates that PVN is temporally coupled with the SCN clock activity. The AVP and melatonin on the locomotor activity and body temperature rhythms are interesting theme. Body temperature decrease caused by the melatonin application (i.p.) during the night is considered to be a decrease of AVP in the SCN. Functional significance of AVP in the SCN would be discussed. [J Physiol Sci. 2006;56 Suppl:S55]

抄録全体を表示

Recently, we observed that olfactory stimulation with scent of grapefruit essential oil caused excitations of sympathetic nerves innervating the epididymal (white) adipose tissure, interscapular (brown) adipose tissue and adrenal gland and inhibition of a parasympathetic nerve innervating the stomach. In contrast, olfactory stimulation with scent of lavender essential oil inhibited sympathetic nerves innervating the epididymal adipose tissue, interscapular adipose tissue and adrenal gland and excitated a parasympathetic nerve innervating the stomach. These findings suggest that the scent stimulation with grapefruit oil elevates lipolysis, thermogenesis, adrenaline secretion, thus, the blood glucose and the blood pressure, and suppression of food intake, and that the scent stimulation with lavender oil induced opposite responses. In the experiments examined this, we found evidences suggesting that these are the cases. Moreover, we observed that bilateral electrolytic lesions of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian clock, and histaminergic blockers eliminated these responses. That is, a histamine H3-antagonist, thioperamide, abolished the effects of lavender oil and a histamine H1-receptor-antagonist, diphenhydramine, suppressed the effects of grapefruit oil. Anosmic treatment with zinc sulfate eliminated all of the changes due to olfactory stimulations. These findings suggest that olfactory stimulations with scents of grapefruit and lavender oils affect appetite and metabolism via the functions of the SCN and histaminergic nerve. [J Physiol Sci. 2006;56 Suppl:S55]

抄録全体を表示

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a ubiquitous neuropeptide in the central and peripheral nervous systems. Previously we reported that PACAP potentiates both insulin release from pancreatic β-cells and insulin action in adipocytes, contributing to reduction of blood glucose and energy storage. PACAP38 is localized in pancreatic islets and serves as an endogenous amplifier of glucose-induced insulin secretion via VPAC2 and PAC1 subtypes of PACAP receptors. In an adipocyte cell-line, 3T3-L1 cells, PACAP enhances insulin-stimulated glucose uptake via PAC1 receptors and promotes adipocyte differentiation. In contrast, PACAP stimulates secretion of glucagon and catecholamine and glucose output from the liver, causing elevation of blood glucose. Thus, PACAP regulates the glucose and energy metabolism at multiple processes in several tissues. In this symposium, we present three novel effects of PACAP in the metabolism. (1) The action of PACAP to protect islet cells against lipotoxicity and glucotoxicity. (2) PACAP knock out mice exhibits decreased fat mass and increased insulin sensitivity, suggesting a role of PACAP to facilitate adiposity and decrease insulin sensitivity. (3) PACAP promotes feeding behavior by activating neuropeptide Y neurons in the hypothalamic arcuate nucleus, a feeding center.Based on these well known and newly identified metabolic effects of PACAP, we discuss a possible therapeutic use of PACAP receptor subtype-specific agonists and/or antagonists in the treatment of metabolic syndrome. [J Physiol Sci. 2006;56 Suppl:S56]

抄録全体を表示

Many molecules compose the brain-adipose axis that controls appetite. Using a subtraction cloning assay, we searched genes which were activated by a PPAR-gamma activator and identified nesfatin, a secreted protein of unknown function, which was expressed in the appetite-control hypothalamic nuclei and adipose in rats. Intracerebroventricular (icv) injection of nesfatin caused a dose-dependent decrease in food intake, and icv injection of its antibody stimulated feeding. The structure of nesfatin possesses several cleavage sites that may undergo processing by prohormone convertase (PC), and nesfatin was co-localized with PC-2 and PC-3. Western blot analysis demonstrated the presence of nesfatin-1 in the hypothalamic extract. Icv injection of nesfatin-1, but not nesfatin-2 or -3, produced satiety, and injection of an antibody neutralizing nesfatin-1 stimulated feeding. Chronic icv injection of nesfatin-1 reduced body weight, and rats gained body weight after chronic icv administration of an antisense morpholino-oligonucleotide against the nesfatin gene. The present data provide evidence that nesfatin is a novel, secreted anorexigenic molecule in the hypothalamus. [J Physiol Sci. 2006;56 Suppl:S56]

抄録全体を表示

Weight gain is associated with infiltration of fat by macrophages, suggesting they are an important source of inflammation in obese adipose tissue. We have recently developed an in vitro co-culture system composed of adipocytes and macrophages and examined the molecular mechanism whereby these cells communicate. Co-culture of differentiated 3T3-L1 adipocytes and macrophage cell line RAW264 results in marked up-regulation of pro-inflammatory cytokines such as MCP-1 and TNF-&alpha and down-regulation of anti-inflammatory cytokine adiponectin. Such inflammatory changes are induced by the co-culture without direct contact, suggesting the role of soluble factors. A neutralizing antibody to TNF-&alpha, which occurs mostly in macrophages, inhibits the inflammatory changes in 3T3-L1, suggesting that TNF-&alpha is a major macrophage-derived mediator of inflammation in adipocytes. Conversely, FFAs may be an important adipocyte-derived mediator of inflammation in macrophages because the production of TNF-&alpha in RAW264 is markedly increased by palmitate, a major FFA released from 3T3-L1. The inflammatory changes in the co-culture are augmented by use of either hypertrophied 3T3-L1 or adipose stromal vascular fraction obtained from obese ob/ob mice. We postulate that a paracrine loop involving FFAs and TNF-&alpha between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes in the adipose tissue. This study suggests the pathophysiologic implication of the intimate crosstalk between adipocytes and macrophages in the development of inflammatory changes in obese adipose tissue and thus the metabolic syndrome. [J Physiol Sci. 2006;56 Suppl:S56]

抄録全体を表示

In order to investigate how β-stimulation affects the contractility of cardiac muscle, x-ray diffraction from cardiac muscle in the left ventricular free wall of a mouse heart was recorded in vivo. This is the first x-ray diffraction study on a heart in a living body. After the R-wave in electrocardiograms, the ratio of the intensities of the equatorial (1,0) and (1,1) reflections decreased for about 50 msec from a diastolic value of 2.1 to a minimum of 0.8, and then recovered. The spacing of the (1,0) lattice planes increased for about 90 msec from a diastolic value of 37.2 nm to a maximum of 39.1 nm, and then returned to the diastolic level, corresponding to about 10% stretch of sarcomere. Stimulation of β-adrenergic receptor by dobutamine (20 μg/kg/min) accelerated both the decrease in the intensity ratio, which reached a smaller systolic value, and the increase in the lattice spacing. However, the intensity ratio and spacing at the end-diastole were unchanged. The recovery of the lattice spacing during relaxation was also accelerated. The mass transfer to the thin filaments at systole in a β-stimulated heart was close to the peak value in twitch of frog skeletal muscle at 4 °C, showing that the majority of cross-bridges have been recruited with few in reserve. [J Physiol Sci. 2006;56 Suppl:S57]

抄録全体を表示

Clinical trials have demonstrated the adverse long-term effect of inotropic drugs for the treatment of heart failure. Because most of these drugs exert their effects via the increase of intracellular calcium concentration, people have sought a new class of agents working independently of the calcium handling mechanism. We have studied the function of cardiac myosins with various molecular structures to elucidate their role in diseased conditions, but, at the same time, such studies suggested the possible new mechanisms for modulating cardiac contractility at the crossbridge level. Studies on mutant myosins implicated in familial hypertrophic cardiomyopathy, replacement of a single amino-acid located in the C-terminus thus being far from the functional domains responsible for ATP hydrolysis or actin-binding introduced severe functional defect. On the other hand, myosin light chains seem to modulate crossbridge kinetics without changing ATPase activity of myosin. These calcium-independent (downstream) mechanisms for the modulation of cardiac contractility will be discussed with additional observations. [J Physiol Sci. 2006;56 Suppl:S57]

抄録全体を表示

To analyze the fundamental mechanism of the Frank-Starling's law, we studied the left ventricular (LV) transmural difference in the time course of crossbridge dynamics (CBD) and the end-diastolic myosin filament lattice spacing (EML) in the isolated crystalloid-perfused isovolumically contracting rat hearts (n=11) paced at 2 Hz using X-ray diffraction at SPring-8. We recorded x-ray diffraction patterns of the epicardial (EPI) and deeper (DEEP) myocardium region of the LV free wall along with LV pressure (LVP) at the end-diastolic pressure of either 0 or 20 mmHg by adjusting LV volume. We analyzed transmural CBD from the X-ray diffraction patterns according to the transmural variations of regional myofilament orientation. The developments of CBD of both EPI and DEEP and normalized LVP were synchronous during contraction, but not during relaxation. The CBD decay was significantly faster in DEEP than EPI. The LVV increase significantly potentiated LVP development and more prolonged the CBD decay in EPI than DEEP associated with more reduction of EML in EPI than DEEP. From studies on CBD and EML, we were able to propose a possible underlying mechanism for the Frank-Starling's law in the whole heart. [J Physiol Sci. 2006;56 Suppl:S57]

抄録全体を表示

We demonstrate using synchrotron x-ray radiation that length-dependent mechanisms influence cross-bridge cycling in in situ rat hearts, and then describe the effects of ischaemia-reperfusion on contractility. All experiments were performed in real time using synchrotron radiation at Spring-8 and x-ray diffraction techniques. Mass transfer of myosin to actin during contraction was inferred from the change in diffraction intensity ratio (intensity of 1,0 reflection / 1,1 reflection) derived from the myosin-actin filaments in fibres. Sustained volume loading by infusion of sodium lactate (40-60 ml/h for <5 min) evoked a rightward shift in left ventricular volume and significant decreases in epicardial myosin spacing, consistent with sarcomere stretching. Increases in stroke volume were correlated with increases in mass transfer and myosin spacing change. Local comparisons of intensity ratio and myosin spacing cycles during ischaemia-reperfusion indicate that 1) mass transfer in the damaged area was reduced by moderate ischaemia, and sometimes unsynchronised, 2) compensatory increases in contractility were detected in non-ischaemic areas (and during reperfusion), and 3) myosin spacing increases were reduced and delayed or reversed under severe ischaemia. These findings suggest that non-functional fibres are stretched under high cardiac output during acute ischaemia. [J Physiol Sci. 2006;56 Suppl:S58]

抄録全体を表示

The growth cone is a motile structure located at the distal tip of the nerve fiber. The 2.5S nerve growth factor (NGF) promotes the axonal growth and the survival of sensory neurons by reacting with their growth cones. We observed the behavior of single molecules of NGF on the growth cone of sensory neurons by using a fluorescent NGF, Cy3-NGF. Upon the application of 0.4 nM of Cy3-NGF, the growth cones responded within one minute of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied less than 5% of the estimated total binding sites on a single growth cone, were required to initiate the motile responses. After binding to the receptor, Cy3-NGF displayed lateral diffusion on the membrane of the growth cones. The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin and the movements could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were internalized in the vicinity of the central region of the growth cone during this rearward trafficking, as Cy3-NGF remained in the growth cone after the removal of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF-receptor complex is an essential step to the accumulation and endocytosis of NGF at the growth cone. [J Physiol Sci. 2006;56 Suppl:S58]

抄録全体を表示

Local translation in neuronal dendrites plays a key role in activity-dependent synaptic modifications, and is needed for long-term synaptic plasticity. RNA granules, which consist of clusters of ribosomes and RNAs, are responsible for transport of mRNAs to the dendrites and local translational control. We identified RNG105 (RNA granule protein 105) as a novel component of the RNA granules in dendrites of hippocampal and neocortex neurons. The RNG105-localizing RNA granules contain mRNAs, the translational products of which play key roles in synaptic plasticity. RNG105 is an RNA-binding protein and has ability to repress translation both in vitro and in vivo. Time-laps fluorescence imaging revealed that dissociation of RNG105 from the RNA granules is induced by BDNF (brain-derived neurotrophic factor) stimulation. In contrast, even after the BDNF stimulation, ribosomes remain in/near the RNA granules. The RNG105 dissociation is concomitant with the induction of local translation of the mRNAs located in the RNA granules. These findings suggest that RNG105 is a translational repressor in the RNA granules and becomes dissociated from the granules by synaptic stimulation, which cancels the translational repression of the mRNAs in the RNA granules. We also want to show our recent progress in the study of RNG105 knockout mice and identification of RNG105-associated components of the RNA granules. [J Physiol Sci. 2006;56 Suppl:S58]

抄録全体を表示

Single-molecule imaging is a useful technique for analyzing functions and interactions of protein molecules. I will show some instances how this technique is applied to the biological studies. The first example is the study of chaperonin assisted protein folding. GroEL mediates the folding of nascent or denatured proteins in the E.coli collaborating with co-chaperonin GroES. We visualized GroEL-GroES interaction at the single molecule level [1]. Release of GroES from GroEL occurred after a rag period (–3s), that was not recognized in previous bulk-phase studies. Furthermore, we succeeded in observing the refolding of denatured GFP in the GroEL-GroES complex, and found that GFP could not start to refold for 3s after GroES binding. This observation suggests the presence of a new kinetic intermediate "cis ATP*-complex" in the GroEL-GroES reaction pathway. It is important for the efficient encapsulation of nonnative protein into the GroEL cavity. The second example is the transport of mRNA within a living cell. Fluorescently labeled mRNA was injected into the nuclei of living cells and was visualized by fluorescence microscopy. The injected mRNA molecule were in equilibrium of two states, Brownian motion with diffusion coefficients of 0.2 μm²/s and corralled in a restricted area for 20 s. These results suggested that mRNA travels from the site of synthesis to nuclear pore by diffusion process. REFERENCES [1] Taguchi, H., T. Ueno, H. Tadakuma, M. Yoshida, and T. Funatsu (2001) Nature Biotechnol. 19: 861-865. [2] Ueno T., H. Taguchi, H. Tadakuma, M. Yoshida and T. Funatsu (2004) Molecular Cell, 14: 423-434. [J Physiol Sci. 2006;56 Suppl:S59]

抄録全体を表示

Microtubule, a polarized hollow tube having plus and minus ends, is highly dynamic structure repeating growth and shortening, especially at its plus end. In cells, the parameters of microtubule dynamics are spatio-temporally regulated by a number of microtubule-binding proteins that stabilize or destabilize microtubules, and thereby asymmetrical microtubule networks are generated. The generation of a polarized microtubule organization is critically important for proper cellular functions, such as cell division and migration. To explain how microtubules set up and make contacts with cellular structures, a "search-and-capture" mechanism has been proposed, in which the microtubule plus ends dynamically search for and capture specific sites, such as mitotic kinetochores and cell cortex. To date, several classes of proteins called "microtubule plus-end-tracking proteins" or "+TIPs" have been shown to be associated with "growing" microtubule plus ends in a wide range of organisms from fungi to humans to play critical roles in the "search-and-capture" mechanism. Some of +TIPs highlight every growing microtubule plus ends, while some of them accumulate at the microtubule-capturing structure created at the specialized sites, and the complex formation of these molecules serves a link between the microtubule plus end and variety of cellular structures. In this talk, I will introduce the tools to visualize microtubule dynamics in living cells and overview our current understanding of the +TIPs. [J Physiol Sci. 2006;56 Suppl:S59]

抄録全体を表示

Synapses undergo activity-dependent changes not only during development but also in adulthood. Mechanisms underlying these dynamic changes have been studied intensively. However, it is largely unknown how the strength of once matured synapse is maintained stably in the brain. We show that significant weakening in the strength of excitatory climbing fiber (CF) to Purkinje cell (PC) synapse followed chronic inhibition of neuronal activity by tetrodotoxin or persistent blockade of postsynaptic AMPA receptors by their selective antagonist, NBQX. These treatments reduced glutamate concentration transients at synaptic clefts and decreased the frequency of quantal excitatory postsynaptic current (EPSC). In contrast, neither the amplitude of quantal EPSC nor the release probability was changed. Our morphological examination demonstrates selective reduction of CF innervation at PC shaft dendrites after NBQX-treatment. We thus conclude that in the mature cerebellum, AMPA receptor-mediated neuronal activity in PCs maintains CF's functional release sites and its innervation of PC shaft dendrites. [J Physiol Sci. 2006;56 Suppl:S59]

抄録全体を表示

Parallel fibers (PFs), axons of cerebellar granule cells, transmit multimodal sensory information via mossy fiber inputs originating from the pontine nuclei. Each PF forms synapses with multiple Purkinje cells (PCs) whose plasticity is believed to play cardinal roles in motor learning. In addition, PF-PC synapses were reported to be dynamically reorganized in an activity-dependent manner even in adulthood. Several lines of evidence have suggested that the δ2 glutamate receptor (GluRδ2) is one of the key molecules that regulate functions of PF-PC synapses; GluRδ2-null mice show ataxia and loss of long-term depression (LTD), a putative cellular model of cerebellar information storage. In addition, GluRδ2-null mice display half the number of PF-PC synapses. Despite their importance, the mechanisms by which GluRδ2 participates in PF-PC synaptic functions have been elusive, mainly because it is not activated by glutamate analogs. To gain insight into GluRδ2's mechanisms, we developed a Sindbis virus vector that express a wild-type GluRδ2. By introducing this virus vector into GluRδ2-null cerebellum in vivo, we could rescue several abnormal phenotypes, such as impaired LTD and enhanced paired-pulse facilitation of excitatory postsynaptic currents at PF-PCs synapses. This virus-based method has several advantages over non-viral conventional gene expression methods and transgenic techniques. Based on studies expressing a mutant GluRδ2, in which several functional domains were mutated, GluRδ2's mechanisms in controlling PF-PC synapses will be discussed. [J Physiol Sci. 2006;56 Suppl:S60]

抄録全体を表示

Loss of responsiveness to an eye deprived of vision is mediated by rapid functional disconnection followed by slow axonal rearrangement within kitten and rodent visual cortex (Antonini and Stryker, 1993; Antonini et al., 1999). We hypothesized that extracellular serine proteases (tissue-type plasminogen activator (tPA)-plasmin system) act to degrade cell-adhesion molecules or extracellular matrix proteins to permit earlier synaptic remodeling dependent on visual experience. Indeed, functional ocular dominance (OD) plasticity was reversibly impaired in tPA knockout mice (KO) (Mataga et al, PNAS, 2002). Moreover, we identified a robust anatomical change in layer 2/3 of mouse visual cortex by brief monocular deprivation (MD) (Mataga et. al., Neuron, 2004). Protrusions on the apical dendrite of pyramidal cells increased steadily in number with postnatal age, but were rapidly and transiently lost after MD only during the physiological critical period (CP). Targeted disruption of tPA or its upstream regulation by glutamic acid decarboxylase (GAD65) reversibly prevented MD-induced spine pruning in vivo. The tPA-plasmin system may, thus, rapidly couple physiological perturbation of sensory input to early structural rearrangement of synaptic input. [J Physiol Sci. 2006;56 Suppl:S60]

抄録全体を表示

To examine the plastic changes in orientation selectivity of developing visual cortex, we manipulated visual experience of kittens for 1-7 weeks under a freely moving condition, mounting cylindrical-lens-fitted goggles to present uni-axially elongated images of their environments, or spherical-lens-fitted goggles to present a stationary black-and-white oriented grating. We performed intrinsic signal optical imaging from those animals to reconstruct cortical representation of orientation preferences. For exposure to either a dynamic or stationary single orientation through the goggles, the extreme over-representation of exposed orientation was found in the visual cortex immediately after 1-2 weeks of continuous goggle rearing. For kittens exposed to a dynamic single orientation for a longer time, reorganized orientation maps were preserved although the degree of over-representation was somehow reduced. However, for kittens persistently exposed to a stationary oriented grating for long time, the over-representation effect almost disappeared or in some cases the orientation maps paradoxically exhibited the over-representation of the orientation orthogonal to the exposed orientation. It is suggested that the consolidation of reorganized orientation maps requires the experience of moving visual stimuli with behavioral relevance. [J Physiol Sci. 2006;56 Suppl:S60]

抄録全体を表示

We are surrounded by rich environment which include varieties of social properties. Our brains are handling such information for choosing and performing behaviors. Social meaning of each behavior is always changing time to time so that the brain has to take much effort to extract social parameters mined in the environment. Although we are highly social animal, we know very little about how the brain is manipulating such social parameters. There is almost no neurophysiological study focused on social brain functions. Therefore we tried to see how social parameters are affecting on brain function at single neuronal level by using Japanese monkeys known for their complex social behaviors. Two Japanese Macaques were used. We implanted multiple electrodes chronically (24 and 30 for each monkey) in prefrontal and parietal cortices. During the experiment, we altered monkeys' relative spatial positions so as to manipulate their social spaces. In some cases, their peri-personal spaces were made either overlapped or separated. Their behaviors were monitored by using motion capture technique and video. These techniques allowed monkeys relatively free movement during recording. We found that monkeys altered their behaviors dramatically simply by changing social structure between them. While monkeys were showing social interaction, prefrontal and parietal neurons showed varieties of response patterns depending on social context. Prefrontal neurons tended to respond earlier and parietal responses followed, suggesting two areas are managing different aspects of social functions in the brain. [J Physiol Sci. 2006;56 Suppl:S61]

抄録全体を表示

Recently, it is claimed that automatic simulation of inner state in other's brain by observation of action is very important neural mechanism for social interaction. The idea is on the lines of simulation theory. Mirror neurons, which were found in the ventral premotor cortex and inferior parietal cortex of the monkey, are considered to be neuronal correlates of this simulating mechanism. On the other hand, self-other representation in the brain should be necessary in social interaction. Recent imaging experiments suggest that motor control system may be involved in recognition of agency of action or ownership of one's own body parts. We speculate the mirror neurons in the parietal cortex of the monkey would be also involved in monitoring own body action. Actually, we found that some neurons related to the hand manipulation task in the parietal cortex responded to the movie of own hand action. These neurons were also active during observation of other's hand action. Further, we also found that this visual response was less active in the delayed feedback than in the real time. These results suggest that matching between efference copy and sensory feedback (visual and somatosensory) may occur in the parietal cortex. This may be neural bases of self-other distinction. Further, I will also present visual-somatosensory bimodal neurons have visual receptive field on the corresponding other's body parts. I would like to discuss functional property of inferior parietal cortex for the self-other distinction and matching. [J Physiol Sci. 2006;56 Suppl:S61]

抄録全体を表示

Social stimuli during the neonatal and juvenile periods are known to affect various aspects of physiological and behavioral development in rodents. For example, maternal behavior and partner preference in the adulthood are determined by the social environments in which the animals are reared. These phenomena are so called "non-genomic transmissions". In this study, we investigated the long-lasting influences of earlier weaning on adulthood behavioral traits in rats and mice. Subjects were weaned from their mothers one week earlier than the normal weaning period. To assess anxiety levels at ages 8 weeks and 22 weeks, both early weaned and control mice pups were subjected to several behavioral tests, and it was found that the early-weaned animals had a sustained increase in anxiety levels compared to the control groups. Social behavioral tests performed in other sets of mice revealed that early weaned pups engaged in more fights under several conditions, including co-housing them with other mice, although neither isolation-induced aggression nor territorial aggression differed from normally weaned mice. In addition, early weaned females repressed maternal behavior. Similar results were obtained in rats as well. Concurrently, neurochemicals that are responsible for the behavioral and endocrine responses to stress were affected by the early weaning manipulations. These results suggest that the absence of mother-pup interactions during the last several days of pre-weaning period may lead to a persistent increase in anxiety and aggression during adulthood in rodents. [J Physiol Sci. 2006;56 Suppl:S61]

抄録全体を表示

Interpersonal activity is essential in making humans the uniquely social beings that we are. The underlying neural grounds of the social interaction are the fusiform region, amygdala, superior temporal sulcus, orbitofrontal and dorsolateral prefrontal cortex, which are of great interest to neuroscientists. To anticipate possible futures and coordinate thought and action for achieving desired outcomes, the prefrontal lobes play a pivotal role. The dorsolateral prefrontal cortex is essential to guide the behavior by thought and language, while the orbital and ventromedial regions have been considered to be the neural correlates for affective evaluation of the consequences of our action. The brain region that is implicated in gaze processing, STS, has repeatedly been activated when viewing gaze in the normal brain. We have presented a case, MJ, in a recent report, with a circumscribed lesion in the right superior temporal gyrus, due to a cerebrovascular accident, who manifested a puzzling difficulty in obtaining eye-contact. As the STG comprises a part of the STS, we investigated her ability in processing gaze. Indeed, MJ demonstrated a unique impairment in discriminating gaze direction, which is the first neuropsychological evidence that establishes STS as a gaze processor, so often implicated in animals and human neuroimaging studies. [J Physiol Sci. 2006;56 Suppl:S62]

抄録全体を表示

Ca²⁺/calmodulin-dependent protein kinase 4(CaMK4)is a protein kinase that activates the transcription factor, cAMP response- element binding protein (CREB). CaMK4 has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. However, functions of CaMK4 in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. To gain more insight into behavioral significance of CaMK4, we subjected CaMK4 -/- mice to a battery of behavioral tests, including neurological screen, light/dark transition, open field, elevated plus maze, social interaction, rotarod, hot plate, prepulse inhibition, Porsolt forced swim, 8-armed radial maze, Barnes maze, fear conditioning, latent inhibition, and passive avoidance tests. CaMK4 -/- mice exhibited increased social interaction in home cage. They did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. These results indicated selective and specific involvement of CaMK4 in regulating emotional behaviors. [J Physiol Sci. 2006;56 Suppl:S62]

抄録全体を表示

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is expressed abundantly in the nuclei of neurons and thought to regulate gene expressions mediated by the transcriptional factors such as CREB. We have been characterized CaMKIV comparing with other subtypes CaMKI and CaMKII in the hippocampal neurons. The basal CaMKIV activity is kept low by constant inactivation by associated protein phosphatase 2A, and the activation is transient because calcineurin inactivate CaMKIV after the neuronal stimuli, while the activities of CaMKI and CaMKII are sustained. In other words, CaMKIV is hard to be switched on and is easy to be switched off. Based on this fact, we thought the functions of CaMKIV are reflected in the animals after the chronic stimulation rather than the acute one. Recently, we found that chronic treatments of the rats with antidepressants increased CaMKIV activity and CREB phosphorylation in the prefrontal cortex and the hippocampus, suggesting the importance of CaMKIV in the effects of antidepressants. These results led us to perform the behavioral assessments of anxiety, depression and the sensitivity to antidepressants in CaMKIV-knockout mice by some experimental paradigms including the forced swim test, the tail suspension test and the novelty-suppressed feeding test. From the results of these experiments, it was suggested that CaMKIV is involved in some of the depression-related behaviors and the sensitivities to antidepressants. [J Physiol Sci. 2006;56 Suppl:S62]

抄録全体を表示

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine/threonine protein kinase that is abundant in the brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. Several behavioral abnormalities of CaMKIIα mutant mice were reported, but systematic assessments of CaMKIIα mutant mice have not been well conducted. To analyze the behavioral effects of CaMKIIα deficiency, we subjected CaMKIIα heterozygous knockout mice to a comprehensive behavioral test battery. The mutant mice showed increased locomotor activity, markedly decreased anxiety, decreased depression-related behavior, increased offensiveness, selective and severe spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. To identify the mechanism underlying the behavioral abnormalities of CaMKIIα mutant mice, gene expression analysis and biochemical analysis of the brain of the mutant mice were conducted. The potential involvement of CaMKIIα in pathogenesis/pathophysiology of psychiatric disorders, such as schizophrenia, bipolar disorder, and personality disorders, will be discussed. [J Physiol Sci. 2006;56 Suppl:S63]

抄録全体を表示

Previous studies have shown that αCalcium/Calmodulin dependent protein kinase II(αCaMKII) plays important roles in not only learning and memory but also aggressive and fear response in mice. To further understand roles of αCaMKII in brain function, we have generated transgenic mice overexpressing αCaMKII in forebrain. Interestingly, these mutant mice showed increase in anxiety in open field and elevated zero maze tests and increase in offensive aggression in resident-intruder test. Increase in anxiety observed in these transgenic mice suggests that expression level of CaMKII positively correlates with expression of anxiety related behavior. We next examined effects of administration of selective serotonin reuptake inhibitor (SSRI) on anxiety related behavior observed in these mutant mice. Treatment of these transgenic mice with SSRI suppressed anxiety-related behavior in both tests. These results raise the possibility that these mutant mice is a mouse model of anxiety disorder, that allows to develop therapeutic drugs . In addition, we have examined the expression profile of these mutant mice with or without the treatment of SSRI and tried to find out the anxiety-related genes. [J Physiol Sci. 2006;56 Suppl:S63]

抄録全体を表示

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant protein kinase in the central nervous system and a key mediator of intracellular Ca²⁺ in response to various stimuli. CaMKII is involved in many neuronal functions including the regulation of neuronal activity. Previous studies reported episodes of epileptic seizure in CaMKIIα knock-out mice and manifestation of epileptic discharges in cultured neurons treated with anti-sense oligonucleotide to CaMKIIα. However, which protein function of CaMKII, i.e., protein kinase activity, calmodulin-binding capacity or multimeric structure interacting with other proteins, is responsible for stabilizing neuronal activity remains to be elucidated yet. To clarify specifically the role of protein kinase activity of CaMKII, we engineered knock-in mice with the inactivated α subunit of CaMKII by replacing Lys-42 with Arg-42. CaMKIIα protein level was unchanged, but CaMKII activity was specifically decreased in these mutant mice. Spontaneous death rate was higher, and pentylenetetrazole injection resulted in higher seizure-induced mortality in homozygous mutants. Spontaneous seizure was sporadically observed in homozygous and heterozygous mutants, but rarely in wild type controls. Cytochrome oxidase staining revealed decreased neuronal activity in nucleus accumbens in homozygous mutants. These results indicate that protein kinase activity of CaMKII, i.e., protein phosphorylation by CaMKII, is important for maintaining basic and normal neuronal activity in vivo. [J Physiol Sci. 2006;56 Suppl:S63]

抄録全体を表示

The attachment of a ubiquitin (Ub) to a substrate serves as an important regulatory modification implicated in receptor endocytosis, virus budding, gene transcription, DNA repair and replication, etc. The discovery of Ub-binding domains (UBDs) has indicated how Ub can regulate such distinct cellular functions. We have recently cloned two novel Ub-binding domains named UBM (Ub binding motif) and UBZ (Ub binding Zn finger), which are evolutionarily conserved in Y-family translesion polymerases (pols). These domains are required for binding of pols to Ub, their accumulation in replication factories and interaction with monoubiquitinated PCNA. In addition, Ub-binding domains of Y-family polymerases play essential roles for in vivo translesion synthesis, which is the major pathway by which mammalian cells replicate across DNA lesions. Interestingly, novel Ub-binding domains are found in several other proteins implicated in regulation of DNA repair and replication.In addition to binding Ub, several UBDs promote monoubiquitination of host proteins. Biochemical, biophysical and mathematical evidence support the concept whereby monoubiquitination of the Ub binding proteins facilitates intramolecular interactions with the UBDs, thus preventing them from binding in trans to ubiquitinated targets. Monoubiquitination of Ub-binding proteins thus represents a regulatory mechanism that inhibits their capacity to bind to and control functions of ubiquitinated targets in vivo. [J Physiol Sci. 2006;56 Suppl:S66]

抄録全体を表示

In the stomach, protons are actively secreted by the gastric H,K-ATPase, but it is unclear what molecule contributes to the secretion of Cl⁻. We have previously shown that CLC-2 may not be responsible for the Cl⁻ transport in the gastric acid secretion. However, we found that CLC-5 was expressed in the gastric parietal cells. Here, we examined the interaction between CLC-5 and the gastric H,K-ATPase. We constructed a tetracycline-regulated expression system of CLC-5 in the HEK293 cells stably expressing the gastric H,K-ATPase. The H,K-ATPase activity and the ⁸⁶Rb⁺ transporting activity were examined by using SCH 28080, a K⁺-competitive inhibitor of the H,K-ATPase. Expression of CLC-5 in the HEK293 cells significantly increased the H,K-ATPase activity by 24.1 ± 8.9% (n = 6) and ⁸⁶Rb⁺ transporting activity by 28.2 ± 5.9% (n = 5). The expression level of H,K-ATPase in the plasma membrane was not affected by CLC-5 in the HEK293 cells. Furthermore, we found that expression of CLC-5 significantly up-regulated the phosphorylation level of H,K-ATPase by 48 ± 18% (n = 4). These results suggest that CLC-5 may be a modulatory subunit of the gastric H,K-ATPase. [J Physiol Sci. 2006;56 Suppl:S66]

抄録全体を表示

Our recent study indicates that hypotonicity-induced decreases in intracellular Cl⁻ concentration ([Cl⁻]_i) could act as a signal to regulate Na⁺ reabsorption through changes in αENaC mRNA expression in renal epithelial A6 cells. This result suggests that the change of [Cl⁻]_i is one of the important signals to cell function. However, currently reported techniques for the measurement of [Cl⁻]_i by using halide-specific fluorescent dyes lack sufficient sensitivity and accuracy. One reason for problems in the use of these dyes for measurement of [Cl⁻]_i during hypotonicity-induced regulatory volume decrease (RVD) is a change of intracellular dye concentration during RVD, since a fluorescent intensity of these indicators depends not only on [Cl⁻]_i but also on intracellular concentration of dyes. In this study, we have developed a new method for measuring [Cl⁻]_i by using a cell analyzer Quanta. This flow cytometer can simultaneously measure the exact cell volume by Coulter principle and the fluorescent intensity. The concentration of Cl⁻ in A6 cells diminished during RVD by 72% (from 47.3 mM to 13.3 mM). This reduction of [Cl⁻]_i was blocked by inhibition of RVD with quinine (K⁺ channel blocker) or NPPB (Cl⁻ channel blocker). These results suggest that a change in external osmolality is converted into the change in [Cl⁻]_i, and that the change of [Cl⁻]_i is the primary hypotonic signal in A6 cells. This work was supported by Grants-in-Aid from JSPS (17390057, 17590191 and 17790154). [J Physiol Sci. 2006;56 Suppl:S66]

抄録全体を表示

The effect of extracellular ATP on adipogenesis was investigated using 3T3-L1 cell line. Incubation of cells with ATP (1-100 μM) for 5 min induced membrane ruffling and migration (chemokinesis). In this cell line, growth arrest is required before initiation of differentiation, and growth-arrested post-confluent cells can be converted to adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or post-confluent cells. In contrast, proliferating cells (density<50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triglyceride accumulation, compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP prior to the addition of the adipogenic hormones, gene expression of adipose protein 2 was markedly increased in 6 days, while the expression level stayed very low without ATP pretreatment. These results suggest that extracellular ATP renders preadipocytes responsive to the adipogenic hormones during the growing phase. [J Physiol Sci. 2006;56 Suppl:S67]

抄録全体を表示

We previously identified sphingosylphosphorylcholine (SPC) and Fyn as upstream signal molecules of Rho-kinase-mediated Ca²⁺-independent abnormal contraction of vascular smooth muscle (VSM) and found that eicosapentaenoic acid (EPA) can selectively inhibit such abnormal events without affecting Ca²⁺-dependent normal VSM contraction by blocking the translocation of Fyn to plasma membrane. Moreover, we reported that EPA was clinically and highly effective in preventing vasospasm after subarachnoid hemorrhage. However, EPA is limited to oral administration and thus unsuitable for clinically serious patients unable to ingest orally. We therefore screened novel compounds which could inhibit Ca²⁺-independent abnormal VSM contraction and could substitute for EPA. Tension study of VSM showed that several compounds inhibited SPC-induced abnormal VSM contraction, which was comparable to the effects of EPA. These results suggest that these newly found compounds would be the candidates for novel therapeutic drugs for vasospasm which could substitute for EPA. [J Physiol Sci. 2006;56 Suppl:S67]

抄録全体を表示

Rho-kinase (ROK) is being regarded as the critical signaling molecule of the Ca²⁺-independent contraction of vascular smooth muscle (VSM). We recently identified that the sphingosylphosphorylcholine (SPC) induced Ca²⁺-independent contraction of VSM by activating ROK and that SPC activated ROK via activating Src family tyrosine kinase (Src-TK). Since VSM expresses Fyn and c-Src among Src-TK, we analysed which Src-TK is involved in this SPC/ROK-mediated Ca²⁺ sensitization. The inhibitors of Src-TK (PP1 and PP2) abolished all the reactions of Ca²⁺-independent contraction, activation of Src-TK and ROK, and tyrosine phosphorylation of p60 protein induced by SPC. SPC induced the translocation of Fyn from cytosol to the plasma membrane of VSM cells, but not that of c-Src. In order to examine directly the ability of Fyn to induce Ca²⁺-independent contraction, we made recombinant Fyn proteins using a baculovirus system. In beta-escin permeabilized VSM, constitutively active Fyn induced Ca²⁺-independent contraction which was inhibited by Y27632, while dominant negative Fyn inhibited the contraction induced by U46619+GTP. These findings suggest that Fyn tyrosine kinase plays a pivotal role in the SPC-induced and ROK-mediated Ca²⁺-independent contraction. [J Physiol Sci. 2006;56 Suppl:S67]

抄録全体を表示

Introduction : GLP-1, a cAMP mobilizing agonist, is an insulinotropic peptide released from the intestinal L cell in response to a meal. However, the underlying mechanisms of the stimulatory effect of GLP-1 on insulin secretion remain fully elucidated. Aim: The present study was conducted to examine whether GLP-1 can activate PKCalpah and PKCepsilon in INS-1 cells at a substimulatory concentration of glucose. Methods: We employed either GFP or DsRed-tagged proteins related to PKC signaling pathway using epifluorescence microscopy and total internal reflection fluorescence microscopy. Results: We first showed that GLP-1 translocated endogenous PKCalpah and PKCepsilon from the cytosol to the plasma membrane. Then we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), as a maker of PKC activation. GLP-1 translocated GFP-tagged MARCKS from the plasma membrane to the cytosol and the GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors. The above observations were verified in three different ways using live cell imaging technique.In addition to these results, PKC inhibitors reduced forskolin-induced insulin secretion in INS-1 cells and rat islet beta cells. Conclusion: GLP-1 can activate PKCalpah and PKCepsilon, and the GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose. [J Physiol Sci. 2006;56 Suppl:S67]

抄録全体を表示

Ghrelin, isolated from the human and rat stomach, is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). We have reported that GHS-R was expressed in rat pancreatic islets and that ghrelin suppressed glucose-induced insulin release via activation of voltage-dependent delayed rectifier K⁺ channels and attenuation of glucose-induced action potentials, leading to suppression of glucose-induced Ca²⁺ signaling in β-cells. In this study, we aimed to determine the involvement of cyclic AMP productions, another major signalling pathway for insulin release, in the ghrelin-induced suppression of insulin release. Both GHS-R blockade and anti-ghrelin antiserum markedly enhanced 8.3 mM glucose-induced insulin release in rat perfused pancreas and isolated islets. GHS-R blockade and anti-ghrelin antiserum also enhanced 8.3 mM glucose-induced cyclic AMP productions in rat islets. Conversely, exogenous ghrelin (10 nM) suppressed insulin release and cyclic AMP productions. In the presence of either dibtyryl cyclic AMP or adenylate cyclase inhibitor MDL12330A, ghrelin failed to attenuate glucose-induced insulin release. This study suggests that ghrelin suppresses glucose-induced cyclic AMP production as well as cytosolic Ca²⁺ response. These abilities of ghrelin to impede cyclic AMP and Ca²⁺ signaling routes at least partly account for the inhibition of glucose-induced insulin release. [J Physiol Sci. 2006;56 Suppl:S68]

抄録全体を表示

Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction. The well-established mode for its regulation is to phosphorylate the 20kDa myosin light chain (MLC20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC20. In order to study this non-kinase activity of MLCK and its active site. We engineered two MLCK fragments, one contained the myosin-binding domain but devoid of a catalytic domain and another further deleted a calmodulin (CaM) domain. The former fragment stimulated myosin ATPase activity by Vmax= 5.53 ± 0.63- fold with Km = 4.22 ± 0.586μM (n = 4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. We failed to observe the stimulation with the latter fragment. Similar stimulating effect were obtained by measuring the ATPase activity of phosphorylated myosin, HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. We conclude that the non-kinase stimulation of MLCK are involved in the mode for activation of myosin. The CaM domain is one of the active site for non-kinase activity of MLCK fragment. [J Physiol Sci. 2006;56 Suppl:S68]

抄録全体を表示

Sphingosine-1-phosphate (S1P) is a plasma lysophospholipid with diverse activities, and is released in a large amount from activated platelets. Our laboratory as well as others have identified the existence of the G protein-coupled S1P receptor family, which include ubiquitously expressed S1P1, AGR16/Edg5/S1P2 and S1P3. In an attempt to get more insight into roles of S1P in vivo, we have generated transgenic (TG) mice that overexpress a major S1P synthetic enzyme sphingosine kinase 1 (SPHK1) in diverse tissues, with up to several ten fold increases in the SPHK1 activity. Although previous reports suggested the involvement of SPHK1 in cell proliferation and transformation, the TG mice show normal growth and no obvious increase in spontaneous malignancy. Importantly, TG mice with a high but not a low level of SPHK1 expression in the heart show age-dependent, progressive cardiac fibrosis with development of dilated cardiomyopathy in a limited population. Transgenic heart tissues show elevated activities in both Rac1 and RhoA small molecular weight G proteins and enhanced superoxide generation in responses to phorbol ester. Treatment of TG mice with an HMG-CoA reductase inhibitor or an antioxidant N-2-mercaptopropyonylglycine, but not an angiotensin II type 1 receptor blocker, resulted in alleviation of cardiac fibrosis. These results provide evidence for a pathophysiological role of SPHK1 and probably S1P. [J Physiol Sci. 2006;56 Suppl:S68]

抄録全体を表示

[BACKGROUND] Experimental autoimmune uveoretinitis (EAU) is an organ-specific autoimmune disease that is induced in animals sensitive to retinal antigens. The recruitment of leukocytes is crucial for ocular inflammation in EAU, whereas target cells and effector cells have not yet been clearly characterized.[PURPOSE] Isolation and characterization of target cells in EAU[METHODS] EAU was induced in B10 mice by immunization with 50 μg human interphotoreceptor retinoid binding protein peptide 161-180 in emulsion with CFA supplemented with 3.5 mg/ml M. tuberculosis(1:1,vol/vol). Disease severity was assessed clinically by funduscopic examination. Retinal cells and ocular infiltrating cells were obtained by enzyme digestion from the eyecups of normal and EAU mice,respectively, and were separated into different types of cells by Percoll density gradient centrifugation. To assess which types of cells were target and effector cells in EAU, we determined cytotoxic activity of infiltrating cells against retinal cells by ⁵¹Cr release assay.[RESULTS] Two kinds of retinal cells were isolated as ⁵¹Cr incorporationg cells. They appeared to be retinal pigment epithelial (RPE) cells and monocytic cells judging from their morphological features under an electron microscope. The identification of effector cells responsible for EAU is under investigation in our laboratory.[CONCLUSIONS] We isolated RPE cells and monocytic cells as monodispersed growing cells or a candidate for target cells in EAU. [J Physiol Sci. 2006;56 Suppl:S68]

抄録全体を表示

Short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, are the products of bacterial fermentation from indigestible dietary fibers in the large intestine. SCFAs have been known to play a variety of physiological and pathophysiological roles for intestine. We have recently reported the mechanism of SCFA-induced responses to the motility of the rat distal colon (Ono et al. Jpn J Physiol 65: 69-76, 2004; Mitsui et al. Neurogastroenterol Motil 17: 585-594, 2005). These results suggest that SCFAs are sensed at mucosa and modulate the colonic motility through the enteric nervous system as a neural reflex. However, the mechanism of the sense of SCFA is currently unknown. In 2003, two orphan G protein-coupled receptors, GPR41 and GPR43, have been identified as the SCFA receptors (Brown et al. J Biol Chem 278: 11312-11319, 2003; Le Poul et al. J Biol Chem 278: 25481-25489, 2003). The present study shows the localization of GPR43 in the rat intestine by RT-PCR, Western blotting and immunohistochemistry. The results of the present study indicate that GPR43 is expressed by enteroendocrine cells and mucosal mast cells in the rat intestine. [J Physiol Sci. 2006;56 Suppl:S69]

抄録全体を表示