Barn owls can localize their prey by ear. The physical cues for localization are the interaural time difference (ITD) for the horizontal plane and the interaural level difference (ILD) for the vertical plane. The owl's auditory system processes ITD and ILD in separate parallel pathways. Time is encoded by phase-locked spikes, which are action potentials that occur at particular phase angles of tonal stimuli. To detect ITDs, the owl uses special neural circuits consisting of coincidence detectors and axonal delay lines. Simultaneous arrival of spikes from the two sides causes coincidence detectors to fire. Axonal paths to different coincidence detectors are adjusted such that a coincidence occurs when the sum of acoustic and neural transmission delays is the same for left and right sides. Coincidence detectors respond not only to one particular ITDi and but also to ITDi ± nT, where n is an integer and T is period, because ITDi and ITDi ± nT indicate the same phase difference. This problem, called phase-ambiguity, is resolved in one part of the midbrain where different frequency bands converge on single neurons, endowing them with the ability to discriminate between ITDi and ITDi ± nT. These same neurons also respond selectively to combinations of ITD and ILD, resulting in the creation of spatial receptive fields. Analysis of stimulus induced postsynaptic potentials in these space-specific neurons show that potentials due to input from the ITD pathway and those due to input from the ILD pathway are multiplied. The space-specific neurons are not randomly distributed but organized into a map of auditory space. [Jpn J Physiol 54 Suppl:S2 (2004)]

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Brain activation studies, pioneered by Sokoloff in 1961, are capable of providing "snap shots" of brain activities in terms of specific brain functionality and represent an ultimate tool for the localization based approach to understanding the human mind. Because of its completely non-invasive nature, fMRI, a technique based on T2* detectable MR signal alteration, reflecting a relative reduction of venous blood volume associated with increased regional blood flow in accordance with the Munro-Kellie doctrine, has quickly become the gold standard. A major draw back of fMRI, however, is its significantly high susceptibility to producing "fictitious activation." This is a common pitfall for investigators who lack proper understanding of MR specific signal properties. Use of linear statistical methods such as SPM, especially in highly demanding protocols, i.e. so-called event related fMRI, further accentuates the inherent weakness of fMRI and has resulted in the rather chaotic current situation in the world of brain science. The best remedy for this potential fatal problem is the application of statistical methods capable of automatically separating task-related activations from artificial components. ICS (see http://coe.bri.niigata-u.ac.jp) represents a well-validated, highly reliable technique in this category. The brain is a complex system and its function cannot be described as the liner sum of deterministic functional units. Therefore, absolute localizationism is fated for eventual failure. Nevertheless, because of its capability of performing single session multivariate analysis, ICS fMRI remains to be effective tool for non-linear dynamic analysis in brain science. [Jpn J Physiol 54 Suppl:S2 (2004)]

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To analyze precise localization, number and density of functional membrane molecules at the electron microscopic level, we have established a new approach using SDS freeze-fracture replica labeling (SDS-FRL) method. SDS-FRL enabled us to visualize two-dimensional distribution of various membrane molecules with immunogold labeling. For example, we found different clustering patterns of AMPA, NMDA, kainate, and delta2 receptors in synapses in the rat cerebellum and hippocampus. In the cerebellum, we found AMPA receptors making microclusters in parallel fiber-Purkinje cell synapses with a relatively low density of extrasynaptic receptors. Also, a high variability of AMPA receptor density was observed in these synapses. In climbing fiber-Purkinje cell synapses, AMPA receptors were distributed in a much more homogeneous manner. Combination of electrophysiological measurements of functional AMPA receptors and SDS-FRL revealed that one gold particle for AMPA receptor can roughly represent one functional channel in immature rat climbing fiber-Purkinje cell synapses. In hippocampal CA1 pyramidal cell synapses, AMPA receptors were clustered with a high variability of density in synapses and were distributed in extrasynaptic sites with much higher density than that in the Purkinje cells. NMDA receptors also made clusters in these synapses but with a lower variability of density and a lower relative density in extrasynaptic sites. These results indicate distinct regulation of clustering of ionotropic glutamate receptors between different subunits and cell types. [Jpn J Physiol 54 Suppl:S2 (2004)]

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Since the percentage of the elderly has been increasing at an unprecedented rate, the number of people with physical disorders has likewise increased. The need for assistive technology has greatly increased in order to avoid an increasing burden for the Japanese government. The author has developed one basic research approach for assistive engineering over a period of 34 years, designing several intelligent tools for people with communicative and perceptual disorders. Moreover, he has also found some research problems concerning cognitive physiology, which are hurry up to be solved for designing the tools. The research problems to be solved are as follows, (1) "plasticity" in the human brain, of which findings are useful for designing artificial hearing as well as artificial vision. (2) "association" from somatosensory cortex to auditory/visual cortex for transmitting sounds or images via the somatosensory cortex.(3) "compensation ability" of the visually impaired as well as the hearing impaired for designing sensory substitute devices.(4) "interruption" when two sensory channels are used simultaneously for substituting the lost sensory function.(5) "sensory feedback" to obtain functions of speaking and writing for the sensory impaired using the tools. To obtain the findings for these research problems are not only necessary for designing the assistive tools but also useful for constructing computer pattern recognition, virtual realty environment and sensory system for robot. [Jpn J Physiol 54 Suppl:S2 (2004)]

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With rapid growth in molecular biology, imaging in the molecular function in vivo has become popular. Among these technologies, positron emission tomography (PET) has unique characters for in-vivo imaging of various cellular and molecular functions using variety of suitable radiolabeled pharmaceuticals. In addition, PET permits quantitative analysis of tissue functions, such as regional perfusion metabolic rate of energy substrates, and receptor density.In the brain study, PET enables quantitative assessment of regional cerebral blood flow, cerebral blood volume, and metabolic rate of oxygen. These parameters are quite valuable in the detection of misery perfusion and luxury perfusion in the acute as well as subacute stage of cerebral stroke. PET also permits assessment of glucose utilization and various neuroreceptor functions. These new information has a potential to play key roles for providing new insights into pathophysiology in various neurodegenerative and psychiatric disorders. Furthermore, this may he helpful to find the best strategy for medications.In the cardiac study, PET enables quantitative assessment of regional myocardial blood flow, flow reserve and endothelial function. These parameters may be valuable for risk assessment of coronary artery disease, and also for assessing treatment effects. In addition, metabolic analysis of glucose and oxygen consumption may hold a key role for identifying severity of coronary artery disease.This elegant tool is expected to play an important role for variety of physiological and pathophysiological studies in the near future. [Jpn J Physiol 54 Suppl:S3 (2004)]

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The bioluminescence seen in some biological system are fascinating which occurs due to conversion of chemical energy into light energy. The bioluminescence reaction takes place between luciferase (enzyme) and luciferin (substrate). The bioluminescence also includes fluorescent protein (e.g. GFP) as a part of the system. It widely goes from species like bacteria to fish, and the light emission colors from blue to red in photogenic organism. Though this light emission reaction takes place due to luciferin-luciferase reaction, the chemical structures of enzyme and substrate involving in light emission process are different from one organism to the other. In short, Firefly luciferase cannot catalyze the oxidation of bacterial luciferin. Therefore, the material background and characteristic properties of luciferin and luciferase must be understood in order to sufficiently understand the bioluminescence reaction. The purpose of this seminar is to bring in basic and applied aspects of bioluminescence with the goal of generating new ideas and new approaches for the understanding of basic mechanisms of bioluminescence as well as its practical applications. Especially, I wish to talk about the possibility of bioluminescence systems including fluorescent proteins for analyzing cell dynamics. [Jpn J Physiol 54 Suppl:S3 (2004)]

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After complete transection of the optic nerve axotomized retinal ganglion cells (RGCs) survive and regenerate their axons along a piece of grafted peripheral nerve. The regenerated optic axons, when properly guided, make functional reconnections with the target cells in primary visual centers of adult rodents. This procedure of nerve grafting opened up a new field for the functional recovery of damaged CNS tissue of adult mammalian brain. Additional intraocular injections of various neurotrophins further promote the survival and regeneration of RGC axons. As an alternative approach we have recently found that electrical stimulation of the damaged axons of RGCs can significantly promote their survival. We further found that transcorneal electrical stimulation was also effective to promote their survival. From the last October the ophthalmology staffs of our medical school started to evaluate the effect of transcorneal electrical stimulation on various disorders of the RGC and optic nerve as translational research. Now we are getting some promising results to improve visual function after transcorneal electrical stimulation in patients. Electrical stimulation may be generally useful for rescuing damaged CNS neurons and restoring brain functions after injury. [Jpn J Physiol 54 Suppl:S3 (2004)]

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The fact that neuronal electric activity in the brain causes an increase in regional cerebral flood flow has given the basis for the functional imaging of human as well as animal brains. fMRI and PET are well known modalities based on the above. Near-infrared spectroscopy (NIRS), which has been first developed for non-invasive monitoring of brain oxygenation in clinical field. Then, f-NIRS appeared which could detect the changes in blood oxygenation in the brain associated with the neural activity, then, near-infrared optical imaging (NIR-imaging) started to obtain functional optical mapping. However, there seem many difficulties for imaging. In order to overcome the problems associated with NIR-imaging such as the lack of the quantification and poor spatial resolution, we developed a 64-channel time-resolved optical imaging system, by which we could obtain quantitative functional images of human brain activity. Reflectance tomographic images of the changes in oxy-hemoglobin, deoxy-hemoglobin, and total-hemoglobin, associated with neural activation were obtained, and given as absolute concentration changes. Then, the obtained optical functional images were superimposed on 3-D images of the subject's brain reconstructed from MRI. The optical imaging system employed here can be applied to the subjects of all ages and be used at the bedside as well. By simplifying and miniaturizing the imaging system, we could construct a conventional single channel oxygen monitor for clinical use, by which we could quantify the changes of [oxy-, deoxy- and total Hb] during neuronal activation in each subject and, therefore, statistical analysis became possible. [Jpn J Physiol 54 Suppl:S3 (2004)]

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Using single molecule techniques we have been developing, we have recently succeeded in imaging the signal transfer process for the first time in two signalling pathways: (1) the process from Ras to Raf, and (2) various steps from CD59, a GPI-anchored protein, to intracellular calcium responses. The single molecule methods developed here allow for the detection of activation of single Ras molecules in living cells, in addition to the observation of their locations and movement. Such single molecule methods would play key roles in the studies of many other signalling molecules to understand how the cellular signalling network functions as a system. [Jpn J Physiol 54 Suppl:S4 (2004)]

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The current status of the ion channel researches is reviewed, particularly in relation to understanding of pathophysiology of ion channel-related diseases. Whereas ion channels are engaged in various cellular functions, they play the central role in the regulation of electrical activities of excitable cells, which include generation and propagation of action potentials. Further more, ion channels play essential roles in secretion/release of hormones/neurotransmitter and in sensing environmental factors, which include temperature, cell volume, redox status, etc.
Recent advances in molecular genetics have led to discoveries of a number of gene mutations associated with channel-related diseases, but the pathogenetic mechanisms underlying the disorders are not always evident. Recombinant expression of ion channels is now a routine work. Accurate measurements of channel functions are an essential part of the investigation. Use of mutant model animals is useful in understanding effects of the mutations at the tissue/organ levels. But when the system becomes more complicated, it is necessary to put various elements together. Computer simulation will be a powerful tool for such a purpose. One of the most successful applications is the simulation of cardiac cells. Several models have been proposed and they are very helpful to correlate changes in channel property with generation of arrhythmias.
Cooperative studies involving various approaches are necessary to elucidate the pathogenetic mechanisms of ion channel disorders and to develop better methods of their treatments. [Jpn J Physiol 54 Suppl:S4 (2004)]

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Master circadian clock of mammals resides in the hypothalamic suprachiasmatic nucleus (SCN). Recently, brain areas outside the SCN and other peripheral organs are shown to have circadian clock(s). In order to examine the clock organization and its tissue specificity, we monitored clock gene expression rhythms in the SCN and other areas.
Circadian rhythms in single cells are stem from a transcription-translation feedback loop composed of clock genes, Clock, Bmal1, Per and Cry. Dec1 and Dec2 share many properties with Per and Cry. Clock mutation significantly reduced the expression of Dec1 and Dec2 in the SCN and other brain areas, but the suppression patterns were tissue specific. Light effects on Dec and Per expression were also tissue specific.
In the organotypic slice culture of the SCN in Clock mutant mice, single neuronal activities exhibited robust circadian rhythms with lengthened periods. The percentage of rhythmic neurons decreased in the dispersed cell culture, suggesting that relatively small group of clock neurons drive circadian neuronal and molecular rhythms in a large number of non-clock neurons, and increase of the intrinsic period and decrease of cell-to-cell communication may reduce the ability of clock neurons to drive rhythms.
Chronic methamphetamine treatment desynchronizes behavioral rhythm from clock gene rhythms in the SCN. Behavioral rhythms are in phase with clock gene rhythms in some brain areas but not in others, suggesting the hierarchical networks of the master and peripheral clocks can be modified by the treatment. [Jpn J Physiol 54 Suppl:S6 (2004)]

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(1) Entrainment signals to suprachiasmatic (SCN)-independent circadian oscillations. Daily restricted feeding (RF) or methamphetamine (MAP) injection produces anticipatory locomotor activity rhythm and entrains peripheral molecular oscillator. As orexins are neuropeptides that coordinate sleep/wakefulness and motivated behaviors such as food seeking, we tested the involvement of orexin neruons on induction of food anticipatory activity (FAA) rhythm by RF. RF shifted the peak of Fos expression of the orexin neurons from the night to daytime. Genetic ablation of orexin neurons in mice severely reduced acquisition of FAA by RF and MAP-induced oscillation. These results suggest that activity of orexin neuron in the LHA contributes the promotion and maintenance of FAA and MAP-induced oscillation. (2) Out put signals from the SCN to peripheral clock systems. We investigated whether injection of adrenaline/noradrenaline or sympathetic nerve stimulation could induce mPer gene expression in the mouse liver. Acute administration of adrenaline or noradrenaline increased mPer1 expression in the liver. Electrical stimulation of the sympathetic nerves or adrenaline injection caused an elevation of bioluminescence in the liver area of transgenic mice carrying mPer1 promoter-luciferase. Daily injection of adrenaline, administered at a fixed time for 6 days, recovered oscillations of mPer2 and mBmal1 gene expression in the liver of SCN-lesioned mice on day 7. Thus, activation of the sympathetic nerves through noradrenaline and/or adrenaline release was a factor controlling the peripheral clock. [Jpn J Physiol 54 Suppl:S6 (2004)]

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The hypothalamic suprachiasmatic nucleus (SCN) is known to have a pivotal role in the generation of circadian rhythms in mammals. In mice SCN, the clock gene products BMAL1 and CLOCK are basic helix-loop-helix PAS transcription factors that form heterodimers and bind to the E-box enhancers upstream of Per and Cry genes to activate their transcription. The protein products PER and CRY form heterodimers and translocate into the nucleus to inhibit transactivation by CLOCK/BMAL1. One cycle of the molecular loop is assumed to generate a circadian cycle, called a "core loop". The core loop in the SCN may regulate diverse physiological events such as action potential firing rhythms and rhythmic secretion of arginine-vasopression and vasoactive intestinal polypeptide that may ultimately synchronize circadian behaviors. Despite the successful cloning of clock genes, the link between the gene transcription-translation cycles and oscillations in physiological activities of SCN neurons is still unclear. Recent findings that cytosolic Ca²⁺ concentrations oscillate with a circadian profile in SCN neurons (Ikeda et al., Neuron 38:253-63, 2003) provides a novel insight into the cellular consequences underlying physiological activity rhythms in SCN neurons. The circadian Ca²⁺ rhythms are driven by the release of Ca²⁺ from ryanodine-sensitive internal stores and resistant to the TTX-blockade of action potentials. The hypothetical interactions between Ca²⁺ signaling and the core loop are discussed in this presentation with regard to the importance of Ca²⁺ signaling in the organization of circadian rhythms in SCN neurons. [Jpn J Physiol 54 Suppl:S6 (2004)]

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Various homeostatic systems are under the control of the circadian clock. Recently we found that fasted rats kept under a light-dark cycle showed a decrease in body temperature (Tcore) only during the light phase. In the fasting condition, in the dark phase Tcore was maintained normal by a suppression of heat loss mechanism despite the reduction of metabolic heat production. In contrast, the response was weakened in the light phase, decreasing Tcore greatly, but in this period cold escape behavior is facilitated, probably for the compensation of Tcore decrease. These modulations of thermoregulatory effectors, depending on the time of the day, are considered as adaptive responses to the lack of food. Interestingly, in the rats with the lesion of the hypothalamic suprachiasmatic nucleus in which daily rhythms of Tcore and activity were abolished, body temperature was unchanged by food deprivation. Very similar loss of Tcore modulation was observed in those mice lacking the criptochrome genes (Cry1^−/-/Cry2^−/-) during food restriction. These results suggest that the circadian clock is a key structure for coordinating the controls of body temperature, energy metabolism, and behavior. [Jpn J Physiol 54 Suppl:S7 (2004)]

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Behavior of human circadian rhythms could be interpreted according to the two oscillator regime: one for circadian pacemaker driving temperature/plasma melatonin rhythm, and the other for rest-activity rhythm, which are tentatively called Osc I and Osc II, respectively. Osc I can be regarded as collective pacemakers in the suprachiasmatic nucleus. On the other hand, Osc II might be synergetically organized by peripheral oscillators. Recently, we have developed a phase oscillator model consisting of mutually coupled Osc I and II, and an extra-oscillator representing an overt rest-activity rhythm. Feedbacks are introduced to the model, which are devised to modulate the coupling strength dependent on a coherence between the oscillators and the overt rest-activity rhythm. Considering these macroscopic oscillators consisting of cellular or molecular oscillators, these feedbacks deliver the macroscopic coherence to a molecular level. At a molecular level, transcription/translation feedback loops involving the clock genes are apparently believed to generate the rhythms. However, accumulated evidence suggests that such transcription/translation loops are open to the intracellular signaling pathways and inter-cellular environments. Based on this knowledge, an oscillator model has been developed for rodent's molecular clock. This model includes the CREB-dependent transcription mechanism, where the phospholyration level of CREB is also under circadian regulation. Our models could provide a novel framework toward integrative modeling of the hierarchical circadian system because of their structure open to the other layers of the hierarchy. [Jpn J Physiol 54 Suppl:S7 (2004)]

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White adipose tissue plays an important metabolic role by storing triacylglycerol in periods of energy excess and releasing free fatty acids and glycerol during energy deprivation. Furthermore, adipocytes secrete a number of hormones that regulate metabolic homeostasis. The process of adipocyte differentiation has been characterized in cultures of cell lines of preadipocytes. In vitro adipogenesis requires a sequence of events, including growth arrest of proliferating preadipocytes, coordinated reentry into the cell cycle with limited clonal expansion, and growth arrest associated with terminal differentiation. Insulin and insulin-like growth factoir-1 promote differentiation of preadipocyes and it has been demonstrated that PI 3-kinase activity plays an important role in adipogenesis. However, the mechanism by which the PI 3-kinase /Akt signal is relayed to the nucleus to activate adipocyte differentiation is unknown. Akt phosphorylates the forkhead transcription factor Foxo1 and inhibits its transcriptional activity. Foxo1 is induced in the early stages of adipocyte differentiation but that its activation is delayed until the end of the clonal expansion stage. Constitutively active Foxo1 prevents the differentiation of preadipocyte due to early induction of cyclin-dependent kinase inhibitor, p21/Cip. In contrast, dominant-negative Foxo1 restores adipocyte differentiation of embryonic fibroblasts from insulin receptor-deficient mice. These data suggest that Foxo1 plays an important role in the integration of hormone-activated signaling pathway with the complex transcriptional cascade that promotes adipocyte differentiation. [Jpn J Physiol 54 Suppl:S7 (2004)]

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Role of Adipocytokine in Metabolic SyndromeIichiro Shimomura, M.D., Ph.D.Department of Medicine and Pathophysiology, Graduate School of Frontier Bioscience, Graduate School of Medicine, Osaka University. The adipose tissue produces and secretes many bioactive substances, which we conceptualized as Adipocytokine. Adiponectin is an adipocytokine identified by screening the adipose-specific genes in human fat. Adiponectin mRNA is expressed exclusively in adipose tissues. The adiponectin mRNAs and its plasma levels are reduced in obesity, type 2 diabetes and atherosclerosis. Adiponectin exhibited the anti-diabetic effects targeting to skeletal muscle and anti-atherogenic moieties functioning on various vascular cells. Therefore, hypoadiponectinemia stands at upstream of metabolic syndrome, hence, become a direct target of the drug intervention to tackle the life style-related disease. Visfatin is a new adipocytokine identified as gene expressed more abundantly in the human visceral fat than subcutaneous fat. In KKAy mice, the mRNA was increased markedly in visceral fat, with the increased concentration of the protein in plasma. In human, its plasma levels showed strong correlation with the accumulation of visceral fat. As the function, the recombinant protein accumulated triglyceride in adipocytes, at the similar strength to the effect of insulin. We conclude that Vistafatin is another adipocytokine, which production is augmented in the accumulated visceral fat, and which aggravates the triglyceride accumulation in the adipose tissue of whole body. [Jpn J Physiol 54 Suppl:S8 (2004)]

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Obesity-linked diseases such as diabetes and cardiovascular disease are sharply increasing in developed and developing countries. Heterozygous PPARgamma or CBP knockout mice were protected from high-fat diet induced obesity and insulin resistance (Mol. Cell 4:597, 1999; Nature Genetics 30:221, 2002). We then carried out systematic gene profiling analysis of these mice and found that adiponectin/Acrp30 was overexpressed. Functional analyses including generation of adiponectin transgenic or knockout mice have revealed that adiponectin serves as an insulin sensitizing adipokine (Nature Medicine 7:941, 2001). In fact, obesity-linked down regulation of adiponectin was a mechanism whereby obesity can cause insulin resistance and diabetes. We further studied the mechanism of adiponectin action and found that adiponectin can activate AMP kinase pathway and PPARalpha pathway, leading to fat combustion and amelioration of insulin resistance (Nature Medicine 8:1288, 2002). Recently, we have cloned adiponectin receptors in the skeletal muscle (AdipoR1) and liver (AdipoR2), which appear to comprise a novel cell-surface receptor family (Nature 423:762, 2003). The expression of AdipoR1/R2 appears to be regulated by several physiological and pathophysiological states such as cell differentiation,fasting/refeeding and hyperinsulinemia, and correlated with adiponectin sensitivity. Adiponectin receptor agonists and adiponectin sensitizers should serve as versatile treatment strategies for obesity-linked diseases such as diabetes. [Jpn J Physiol 54 Suppl:S8 (2004)]

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Obesity is closely associated with the metabolic syndrome including diabetes, dyslipidemia and hypertension. The best predictor of these morbidities is not total body fat mass but quantity of visceral fat. Glucocorticoids play a pivotal role in regulating fat metabolism, function and distribution, and its action on target tissue depends not only on circulating levels but also on intracellular concentrations. Locally-enhanced action of glucocorticoid in adipose tissue has been implicated in the molecular basis of the metabolic syndrome. Evidence has accumulated that enzyme activity of 11β-hydroxysteoid dehydrogenase type 1 (11β-HSD1), which regenerates active glucocorticoids from inactive forms within cells, and plays a central role in regulating intracellular glucocorticoid concentrations, is commonly elevated in fat depots from obese individuals. This suggests a role for adipose glucocorticoid (adiposteroid) in obesity and the metabolic syndrome. We have demonstrated that 11β-HSD1 knockout mice resist visceral fat accumulation and insulin resistance even on a high-fat diet. We also have revealed that adipose-specific 11β-HSD1 transgenic mice, which show increased enzyme activity to a similar extent seen in obese humans, develop distinguished visceral obesity with insulin resistance, dyslipidemia and hypertension. Recent reports have shown that selective inhibitors of the enzyme can ameliorate diabetes in genetically-diabetic obese mice. These data highlight the importance of adiposteriod activation in the pathophysiology of the metabolic syndrome. [Jpn J Physiol 54 Suppl:S8 (2004)]

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Ligand-gated ion channels, including the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), are key components in many important signal transduction and amplification pathways. The IP₃R forms homotetrameric Ca²⁺ channels in the endoplasmic reticulum (ER) with an overall mass of 1,252 kDa. Binding of the two co-agonists IP₃ and Ca²⁺ induces the IP₃R channel to open, which results in Ca²⁺ release from the ER into the cytoplasm. This process is crucial for neuronal transmission via Ca²⁺ signaling and for many other functions that relate to morphological and physiological processes, such as memory and development. We analysed the three-dimensional structure of the ligand-free form of the receptor based on single particle technique¹ using an automatic particle picking system, which is capable of identifying particles even in very noisy images of vitrified specimens². We propose a model which explains the complex mechanism for the regulation of Ca²⁺ release by co-agonists, Ca²⁺, inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of inositol 1,4,5-trisphosphate binding domain. This research is the collaboration with Dr. Fujiyoshi's group and Dr. Mikoshiba's group.
1. C.Sato et al. J. Mol. Biol. 336, 155-164, 2004.
2. T.Ogura and C.Sato. J. Struct. Biol. 145, 63-75, 2004. [Jpn J Physiol 54 Suppl:S9 (2004)]

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A biological cell, such as a nerve cell regulates a cell signalling mainly by ion channels. Water movement through membrane should therefore be strictly separated from the movement of ions. This means water channels must be highly specific for water to prevent any ions. The studies on function of a water channel, aquaporin-1 for the last decade put us some puzzling questions. For answering the puzzling questions, structure of aquaporin-1 was analysed at a resolution of 3.8 Å by one of the third generation cryo-EM and electron crystallography ¹.
The muscle-derived electric organ of the Torpedo electric ray is highly enriched in acetylcholine (ACh) receptor-containing membranes which have been a major source of material for structural study of the receptor. The membranes are readily converted into tubular crystals, having helical symmetry. By imaging of the tubular crystals in thin films of amorphous ice structure of ACh receptor was analysed at a resolution of 4Å ². The ACh receptor has a cation-selective pore, delineated by a ring of five subunits. In each subunit, four membrane-spanning segments, M1-M4 were predicted and the second membrane-spanning segment, M2, shapes the lumen of the pore and forms the gate of the channel. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When ACh enters the ligand-binding domain, it triggers rotations of the inner parts of the domains of alpha-subunits. These rotations are communicated through the inner (M2) helices and open the pore by breaking the girdle apart. [Jpn J Physiol 54 Suppl:S9 (2004)]

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Ciguatera is a human intoxication caused by the ingestion of a variety ofreef fish. More than 20,000 victims suffer annually from ciguatera, makingit one of the largest-scale food poisoning of nonbacterial origin.Ciguatoxins are characterized as principal causative toxins of thepoisoning. Upon ingestion, these toxins cause various disorders involvingthe neurological, gastrointestinal and cardiovascular systems through thepotent activation of voltage-sensitive sodium channels. Ciguatoxins arelipophilic, cigar-shaped molecules, and consist of 13 fused ether rings,which spans over 3 nm.
In 2001, we achieved the first total synthesis of CTX3C, one of the mostimportant congeners of ciguatoxins, starting from D-glucose. To investigatethe detailed structure-activity relationship, various CTX3C analogs wereprepared from the synthetic intermediates. Evaluation of the activities ofthese analogs revealed that structural modulation of the central region ofthe molecule significantly changed the biological functions. In thispresentation, we will report chemistry and biology of ciguatoxins and theiranalogs. [Jpn J Physiol 54 Suppl:S9 (2004)]

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Crystal structure of channel proteins opened a new era that channel function can be related to structure in atomic resolution. To build up a dynamic (functioning) picture of ion channels from atomic (static) coordinates, several methods have been applied: Conformational changes upon gating were detected by spectroscopic methods; ion distributions in the selectivity filter were resolved by crystallography. Among them availability of atomic coordinates for the pore gave a chance to simulate permeation process using molecular dynamics method. These calculations reached to a time range of nanosecond, which is far from the average time of ion permeation through the pore (microsecond). To fill the gap, several methods have been proposed. We reviewed these methods and presented our approaches on gating and permeation processes. For ion permeation, coupling between permeation and gating was made used to isolate elementary steps of ion permeation. In HERG potassium channel the fast inactivation was sensitive to permeating ion-species and concentrations. Thus, occupancy probability of ions in the selectivity filter was estimated from the inactivation kinetics. Similar method was applied to KcsA potassium channel, in which fast gating is also sensitive to concentration of ion species. Based on these observations, a permeation model was constructed. [Jpn J Physiol 54 Suppl:S10 (2004)]

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The importance of being connected at heart with others.Yasutoshi YasudaProfessional Go playerA junior high school student took his own life 10 years ago due to school bulling. His life could have been saved if he had talked about his problems to someone. The news of this suicide set me off on my Go program to help others. Since then, I have interacted with more than 200,000 people including many foreigners, through Go. I would like you to think about how important it is to be connected at heart with others through my experiences. [Jpn J Physiol 54 Suppl:S10 (2004)]

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Acquiring knowledge and skills is a form of behavioral modification.It could be understood by the framework of operant conditioning.Motivation, incentive, and other everyday concepts related toeducation can be uniquely explained in terms of schedules ofreinforcement, three-term contingency, and other behavioristic ideas.I would like to introduce these ideas and to discuss their usefulnessin improving teaching skills. [Jpn J Physiol 54 Suppl:S10 (2004)]

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The Physiology Education Sharing Group, a special interest group of the Physiological Society of Japan, has launched the "Physiology: the first step" project (http://physiology1.org).

In this project, the proposal of presenting information "discretely, non-ambiguously and accurately " was applied to physiology education. For example, in "Cardiac cycle: the first step", only the basic essential information, namely the contraction/relaxation of the ventricle wall, pressure of the atrium, ventricle, and artery, the opening/closing of the atrioventricular and arterial valves, and blood flow in the left heart system are illustrated and presented in a step-by-step manner. After self-studying, twenty-six first-year blinded medical students, randomly assigned to either the new material or a conventional material with a typical figure and text, were given the same quiz (30 two-item choice questions asking basic essential information). The number of correct answers, scored by a blinded rater, was 25.7 ± 3.7 (mean ± SD) in the conventional group and 29.4 ± 1.1 in the first-step group (p < 0.01). Other organs, such as the nervous system, kidney, hormones, immune system and respiration, are also presented in a similar manner. The project also provides numerous achievement quizzes to give students an opportunity to actively use their newly gained knowledge. The website is being accessed about 40,000 times a year, and has been ranked highly by various search engines both internationally and domestically. [Jpn J Physiol 54 Suppl:S11 (2004)]

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When electrically stimulated, lingual nerve (LN) elicits a variety of responses mediated via parasympathetic reflex mechanismin the orofacial area. These include vasodilatation, salivation, lacrimation and mydriasis. We examined whether the trigeminal spinal nucleus (Vsp) forms part of the central mechanism by which electrical stimulation of the central cut end of the LN evokes parasympathetic reflex vasodilatation in the lower lip and mydriasis in artificially ventilated, cervically vago-sympathectomized cats deeply anesthetised with α-chloralose and urethane. Local anesthesia of Vsp by microinjection of lidocaine (reversible local anesthesia; 2%; 1 μl/site) reversibly and significantly reduced the ipsilateral-LN-evoked parasympathetic reflex vasodilatation and mydriasis. Unilateral microinjection of kainic acid (irreversible neurotoxic damage; 10 mM; 1 μ l/site) into Vsp ipsilateral to the stimulated LN led to an irreversible reduction in the reflex vasodilatation and mydriasis but had no effects on the vasodilatation and mydriasis elicited by stimulation of the contralateral LN. Electrical stimulation of Vsp elicited vasodilatation and mydriasis in an intensity- and frequency-dependent manner, regardless of whether systemic arterial blood pressure rose or fell. These results strongly suggest that Vsp is an important bulbar relay for LN-evoked parasympathetic reflex vasodilatation and mydriasis in the cat. [Jpn J Physiol 54 Suppl:S11 (2004)]

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The vanilloid receptor 1-like receptor (VRL-1), a capsaicin receptor homologue, is activated by high temperatures with a threshold >52°C. By VRL-1 immunhisotchemistry, the distribution of high-threshold heat nociceptors was examined in the trigeminal ganglion (TG). In the rat TG, VRL-1-immunoreactive (ir) neurons were abundant (14%) and had medium-sized to large cell bodies. The development of VRL-1-ir neurons was also examined in knockout mice for neurotrophin receptors and Brn-3a, a member of the POU family of transcription factors. In the knockout mouse for trkA (nerve growth factor high-affinity receptor), VRL-1-ir neurons almost disappeared. In addition, the loss of trkC (neurotrophin-3 high-affinity receptor) decreased the number of VRL-1-ir neurons. However, such neurons were abundant in the knockout mouse for trkB (brain-derived neurotrophic factor and neurotrophin-4 high-affinity receptor). On the other hand, VRL-1-ir neurons disappeared in the Brn-3a knockout mouse.
These findings suggest that high-threshold heat nociceptors have medium-sized to large cell bodies in the TG. Their development probably depends on nerve growth factor, neurotrophin-3 and Brn-3a. [Jpn J Physiol 54 Suppl:S11 (2004)]

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Presence of many autonomic small ganglia in oro-facial regions has been reported in mammals. However, localizations of the ganglia and their morphological properties have not been known in detail. In the present study we investigate whole localizations of cranial autonomic ganglia, pathways of their postganglionic fibers, their characteristic histological properties and their postulated functions. A microdissection using a whole-mount acetylcholinesterase histochemistry revealed whole localizations of ganglia clearly, and a histological analysis indicated that the ganglia were parasympathetic in nature. Two kinds of neurons, a large and a small one, were readily discernible in silver-gold staining preparations. Many ganglia contained both types of neurons, while, considerable number of microganglia consisted of only the small neurons. In injections of tracers into glandular regions, both small and large neurons were retrogradely labeled. While, injections into gland-free regions resulted in labeling in the small neurons exclusively, and the labeled neurons were found in two or more microganglia. In nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase stained preparations, almost all small neurons were intensely labeled. These findings suggest that blood vessels in the cranio-oro-facial regions were innervated by the small nitrergic neurons and the neurons may regulate local circulation grossly. [Jpn J Physiol 54 Suppl:S12 (2004)]

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Salivary secretion is reflexly evoked by taste and mechanical stimulation of the oral region. Flow rate of saliva is mainly regulated by the parasympathetic nerve innervating the salivary glands. In the present study we undertook in vivo and in vitro experiments for analyzing activity of the parasympathetic neurons innervating the submandibular gland of rats. The following results were obtained: 1) vigorous salivary secretion was produced by electrical stimulation of the parasympathetic nerve at a frequency of 10-20 Hz, which corresponds to salivation when rats chewed foodstuffs, groomed, and were exposed to heat; 2) the preganglionic parasympathetic nerve showed reflex responses to taste and mechanical stimulation applied to the oral region, tonic firing (5-29 Hz), and phasic firing (up to 50-80 Hz); 3) we also studied activity of the superior salivatory neurons (soma of the parasympathetic nerve) in brainstem slice preparations of neonatal rats using the whole-cell patch-clamp technique, and found the tonic firing pattern and phasic firing pattern; 4) the latter firing pattern was invariably found in the neurons innervating the anterior part of the tongue, which are responsible for vasodilatation; 5) the superior salivatory neurons received glutamatergic excitatory synaptic inputs, and the NMDA component was larger than the non-NMDA component in the neurons innervating the salivary glands. These characteristics may be involved in determining the tonic and low firing properties of the parasympathetic nerve innervating the salivary glands. [Jpn J Physiol 54 Suppl:S12 (2004)]

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The electrical excitability of nerve and muscle is due to the voltage-sensitive ion channels in the plasma membrane. Natural toxins that selectively block the voltage-sensitive channels have provided valuable information about the functional diversity of the voltage sensitive channel superfamily. Also, natural toxins have been extensively used to elucidate drug receptor functions. Reversible protein phosphorylation plays an essential role in controlling wide range of cellular events such as smooth muscle contraction, cell division, cell signalling, differentiation and metabolism. Selective inhibitors of protein kinase and phosphatase are widely used as first-choice reagents for elucidating physiological role of protein phosphorylation and dephosphorylation. The key points of the intracellular signalling cascades, including NF-κB and DNA polymerase can be found by applying a selective modulator to intact-cell preparations, where it affects a particular cellular response. From this viewpoint, a variety of physiologically active substances have been extensively studied by pharmacologists, physiologists and biochemists, because they interact with specific sites. This symposium focuses on research that has given insight into the cellular and molecular mechanisms of action of natural products isolated from marine organisms, terrestrial plants and microorganisms, and has revealed the molecular regulatory mechanisms of signal transduction through the use of these compounds. [Jpn J Physiol 54 Suppl:S12 (2004)]

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Dysiherbaine (DH) is a novel marine sponge-derived amino acid and is highly epileptogenic in mice. Administration of DH induced long lasting convulsive behaviors with ED₅₀ values of 13 pmol/mouse, i.c.v., and 0.97 mg/kg, i.p. which is 5-7 time more potent than that of domoic acid. In rat brain synaptic membranes DH displaced binding of [³H]kainic acid (KA) and [³H]AMPA with Ki values of 26 and 153 nM, respectively. In contrast, DH did not displace the NMDA receptor ligand [³H] CGS-19755. DH also displaced [³H]KA from the recombinant GluR5 and GluR6 at Ki value of each 0.74 and 1.2 nM, respectively. In whole-cell voltage clamp recordings from cultured rat hippocampal neurons DH evoked inward currents from both AMPA and KA receptors with EC₅₀ values of 9.7 μM and 210 nM, respectively. Additionally DH activated mGlu5 but not mGluR1. In the heteromerically expressed KA receptors, GluR5-KA2, in HEK 293 cells DH evoked desensitizing inward current. However, the inward current arose again after removal of DH. Application of glutamate to this "activated" receptor can further elicit the desensitizing inward current. Surprisingly, non-desensitizing inward current was gated by CNQX, a classically defined antagonist. These results demonstrated that DH can activate only high affinity GluR5 site in the heteromerically assembled GluR5/KA2, and that for the first time the low affinity subunit, KA2, can gate channel currents individually upon application of the agonists in quite unexpected ways. [Jpn J Physiol 54 Suppl:S13 (2004)]

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Puffer fish accumulates tetrodotoxin (TTX), a potent Na⁺ channel blocker, in its tissues. In this talk, two possible mechanism for resistibility of puffer fish against TTX is proposed:(1) TTX resistant Na⁺ channel, (2) detoxification by a soluble TTX and saxitoxin (STX) binding protein. First, K_d for ³H-STX to brain membranes and to skeletal muscle membranes of puffer fish Fugu pardalis have been estimated to be 190-fold and 460-fold respectively larger than those to corresponding membranes of rat, by a rapid filtration assay, while these values for ³H-PbTx-3 have been estimated to be one-third and half of those to rat, respectively. A cDNA, encoding an entire voltage-gated Na⁺ channel α-subunit (fMNa1, 1880aa) from skeletal muscle of F. pardalis was obtained. In fMNa1 protein, the residues for ion selective filter and voltage-sensor, and the charged residues in SS2 regions of domain I-IV were conserved, but the aromatic amino acid (Phe/Tyr), commonly locating in SS2 region of domain I of TTX-sensitive Na⁺ channels, was replaced by Asn. With this particular criterion, fMNa1 protein is proposed to be a TTX-resistant Na⁺ channel. Second, a novel soluble glycoprotein (PSTBP, 200 kDa) that binds to TTX and STX was obtained from plasma of F. pardalis. PSTBP possessed a B_maxof 10.6 nmol/mg protein and a K_d of 14.6 nM for ³H-STX in equilibrium binding assays. ³H-STX (10 nM) binding to PSTBP was half-inhibited by the presence of TTX and STX at 12 μM and 8.5 nM, respectively. Further, extremely potent novel STX analog, zetekitoxin, from Panamanian frog Atelopus zeteki will be also presented. Ref: BBRC,267,2000,403. EJB,268,2001,5937. [Jpn J Physiol 54 Suppl:S13 (2004)]

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X-ray crystallography is the potent tool for understanding the interactions between molecule and molecule. Here, I report the crystal structure of the complex between the protein phosphatase 1(PP1) catalytic subunit and its inhibitor, calyculin A, which shows direct evidence for understanding the general mechanism of the PP1 inhibition.
PP1 is one of the four major enzymes that dephosphorylate serine and threonine residues of proteins in the cytosol of eukaryotic cells. The catalytic subunit of PP1 (PP1c) is subjected to inhibition by various toxins such as okadaic acid, microcystin-LR (MCLR), calyculin A, and so on. X-ray crystallographic works of the PP1c complexed with MCLR or okadaic acid provided detailed views of the interaction between the enzyme and the toxins. Several binding models for calyculin A have been proposed on the basis of these crystal structures. However, calyculin A has very flexible backbone structure, and these models were purely speculative.
Calyculin A is located in two of the three grooves on the molecular surface in an extended form ^[1]. This is the first observation to note that the inhibitor adopts not a pseudocyclic conformation but an extended conformation in order to form a complex with the protein. The amino acid terminus of calyculin A contributes in a limited manner, to the binding to PP1c, which is consistent with findings from the studies of dose-inhibition analysis.

^[1] Kita, A., Matsunaga, S., Takai, A., Kataiwa, H., Wakimoto, T., Fusetani, N., Isobe, M., Miki, K. (2002). Structure, 10, 715-724. [Jpn J Physiol 54 Suppl:S13 (2004)]

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Bioactive natural products acting specifically on its molecular targets are expected as promising drug leads in clinical use and/or useful biological probes for basic studies in life sciences.
Two phenalenone compounds, sculezonones A and B, isolated from a marine-derived fungus inhibit DNA polymerases alpha and beta or gamma (1), while an oxylipin compound, plakevulin A, obtained from a marine sponge is an inhibitor of DNA polymerases alpha and gamma (2). An oxyindole alkaloid, speradine A, separated from a marine-derived fungus inhibits Ca-ATPase (3). Two taxoids with different functionalities from taxol, taxuspines D and L, isolated from a Japanese yew inhibit microtubule depolymerization (4), whereas a bicyclic peptide, moroidin, and its related peptides, celogentins, obtained from seeds of a higher plant is an inhibitor of tubulin polymerization (5). Some macrolides otained from marine sponges are F-actin destabilizers, while a macrolide, amphidinolide B, separated from a marine dinoflagellate activates actomyosin ATPase (6). A tetracyclic alkaloid, sieboldine A, isolated from a club moss is an inhibitor of acetylcholinesterase(7).
In this symposium, the structures and activities of these natural products will be described.
References
(1) Biochemistry 2002, 41, 7610-7616. (2) Tetrahedron 2003, 59, 1137-1141. (3) Tetrahedron 2003, 59, 3227-3230. (4) Medical Research Reviews 2002, 22, 305-328. (5) J. Org. Chem. 2001, 66, 6626-6633. (6) J. Org. Chem. 2002, 67, 6585-6592. (7) Org. Lett. 2003, 5, 3991-3993. [Jpn J Physiol 54 Suppl:S13 (2004)]

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Microbial and plant secondary metabolites are the treasury of useful chemotherapeutic compounds, enzyme inhibitors, and other bioactive compounds. We initiated the screening of signal transduction inhibitors of small molecular weight in 1990s. NF-κB is the transcription factor that promotes transcription of inflammatory cytokines, cell adhesion molecules, and inhibitor of apoptosis proteins. Therefore, inhibitors of NF-κB function should be useful as anti-inflammatory and anticancer agents. They should be also useful to study the role of NF-κB in various biological phenomena. We have designed a new NF-κB inhibitor, DHMEQ, based on the structure of epoxyquinomicin isolated from Amicolatopsis. DHMEQ was shown to inhibit NF-κB activity at the level of nuclear translocation. Cell adhesion to the endothelial cells would be involved in the etiology of atherosclerosis and blood-borne metastasis. DHMEQ inhibited the expression of adhesion molecules in endothelial cells. It is likely that macrophages play important roles in induction of inflammation and progression of tumors. DHMEQ inhibited the production of nitrogen oxide, the secretion of inflammatory cytokines such as IL-1β, IL-6, IL-12, and TNF-α, and the phagocytotic activity in macrophages. It suppressed rheumatoid anthritis and prostate carcinoma in mice. Thus, NF-κB was shown to play important roles in cell adhesion to endothelial cells, activation of macrophages, and progression of tumors. Since DHMEQ shows very little toxicity in mice, it is being developed as an anticancer agent. [Jpn J Physiol 54 Suppl:S14 (2004)]

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Higher intelligent behaviors of humans consist of various neuropsychological elements. Among them, amodal imagery without overt sensory input and/or motor output is important for simulating the real world in mind. A series of our human noninvasive studies with combined techniques of functional magnetic resonance imaging and repetitive transcranial magnetic stimulation show that the rostral part of the premotor cortex (Brodmann area 6), which is situated between the prefrontal cortex and primary motor area and often regarded as the bridge between cognition and motor control, plays a key role in mental-operation without overt motor control. In addition, the medial and lateral part of the rostral premotor cortex is functionally specialized for different cognitive functions likewise for motor functions. The medial premotor cortex is relevant for internally generated sequential operation of abstract representation, whereas the lateral premotor cortex is closely related to spatial manipulation of concrete representation. As a common computational process shared by the motor and cognitive behaviors in these regions, we envisage functions to simulate the outcome of rule-based operations. If an arbitrary stimulus-to-response linkage function of the premotor cortex could cover a response output to the memory system instead of overt motor execution, identical control principles could apply more widely to nonmotor executive behaviors. [Jpn J Physiol 54 Suppl:S14 (2004)]

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When we try to solve a task, brain cortical areas that have different but necessary functions to the task are selectively activated depending on the task. Their activation automatically produces a system function that is tuned to the task realization. The selection of those areas is dependent on the task and changes when the task changes. We should think that the brain searches for a new combination of necessary areas every time it encounters a new task. So, in our model, we assume a set of functional parts that correspond to the cortical areas with different functions, and a control system that searches for a combination of those areas. The function of control system is alike to that of attention working on the internal behavior. We call the idea of system function formation as the task dependent functional parts combination model (TDFPC). The specific feature of TDFPC model is an incremental nature of resolvable task types by an acquisition of new functional circuit for the available functional parts set. The possible range of realizable task function increases depending on the type and the number of available functional parts. In this study, we present a computer simulation result of TDFPC application on a set of navigation tasks, and evaluate a possibility of the model as the basic principle of brain intelligence. [Jpn J Physiol 54 Suppl:S14 (2004)]

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It is generally believed that the prefrontal cortex has a critical role for intelligence, which could be closely related to the ability of response selection. However, prefrontal patients and monkeys do not show severe deficit in a simple task situation. To uncover the prefrontal dysfunction, more demanding task is required. We trained Japanese monkeys to perform a go/no-go task where they had to use selective attention to one aspect of a compound visual stimulus (a moving random pattern of colored dots) to make the correct response. In this task, the monkeys should make a go or no-go response depending on either the color or the motion direction of the compound stimulus. In previous studies we have showed that the neuronal network system in the prefrontal cortex is hierarchically organized to generate appropriate behavioral codes and top-down attention can modulate the activity level of these long-term stimulus-response codes depending on task requirements with suppressing irrelevant codes. Based on the results, here we propose a model that can explain how irrelevant stimulus-response codes compete with relevant stimulus-response codes in prefrontal cortex. To test the plausibility of the model, reversible inactivation studies were done in various sub-regions in prefrontal cortex of the monkeys. Inactivation was not effective in less competitive situations, but severely effective in a competitive situation, which had been expected by our model. [Jpn J Physiol 54 Suppl:S15 (2004)]

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From the system theoretic point of view, methods for exploring neural mechanism could be categorized into those that use (1) the transient response of the neural system to an input, (2) the response of the neural system to additional perturbation, and (3) the steady state properties of neural system when there is a steady input. By observing these responses and properties, we can explore the neural mechanisms of brain functions. In this talk, I present examples for approaches (1) and (2). (1) We analyzed population behaviors of face-responsive neurons in the IT cortex. To clarify their transient responses, we applied Principal Component and Mixture of Gaussian analyses to single neuron responses recorded by Sugase et al. (1999). We found that the population vectors of neural responses split into three clusters corresponding to human faces, monkey faces and other simple shapes in the early phase of the response (90-140msec) and that these three clusters split into sub-clusters corresponding to finer categorizations in the later phase (140-190msec). (2) We investigated the neural mechanism for transcranial magnetic stimulation (TMS). We found that the temporal properties of visual percept suppression induced by TMS can be well explained by either an isolated single neuron model (Kamitani et al, 2001) or a simple excitatory-inhibitory balanced network. Secondly, we show that sustained inhibitory effect of subthreshold TMS is reproduced by the network model, but not by the single neuron model. Then, we conclude that the TMS can be used as a powerful tool to perturb neural computation in population level rather than single neuron level. [Jpn J Physiol 54 Suppl:S15 (2004)]

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Recent progress in the imaging techniques has made it available to real-timely monitor the dynamics of signaling transduction and cellular function. We have studied the spatial and temporal imaging and control of cellular functions by combining real-time imaging with protein transduction and gene expression. We have developed a new method called Protein Therapy to deliver proteins, peptides and biologically active compounds into eukaryotic cells by poly-arginines protein transduction domain (PTD). Functional peptides fused with 11-arginine (11R) could efficiently penetrate across the plasma membrane and target to specific subcellualr compartments. By real-time imaging we could detect the spatiotemporal transduction of 11R-FITC in live neurons and Hela cells. With such spatial and temporal control of the delivery of functional peptides we could specifically regulate the intracellular signaling and control the physiological and pathological functions. Introduction of calcineurin autoinhibitory peptide with 11R-PTD potently inhibited CaN phosphotase activites and efficiently prevented glutamate-induced neuronal death. Similarly transduction of calpastatin peptide fused with 11R inhibited the activity of calpain. Further we monitored the dynamic movement and morphological changes of mitochondria by expressing mitochondria-targeted fluorescent protein in cultured hippocampal neurons. We found that distinctive mitochondrial changes were regulated by channel specific Ca2+ signaling and these changes control neuronal survival and death. [Jpn J Physiol 54 Suppl:S15 (2004)]

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Local contact with astrocytes via integrin receptors elicited protein kinase C (PKC) activation in individual dissociated neurons. This activation, initially focal, soon spread throughout the entire neuron. We demonstrated pharmacologically that the arachidonic acid cascade, triggered by the integrin reception, is responsible for the global activation of PKC. Local astrocytic contact also facilitated excitatory synaptogenesis throughout the neuron, a process which could be blocked by inhibitors of both integrins and PKC. Thus, propagation of PKC signaling represents an underlying mechanism for global neuronal maturation following local astrocyte adhesion. [Jpn J Physiol 54 Suppl:S16 (2004)]

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Robust entry of calcium ion is initiated locally at synapses upon receipt of packets of glutamate released from presynaptic terminals. This event is thought to trigger stimulation of a variety of neuronal signaling pathways that are critical in associating synaptic events with higher cognitive functions. Activation of synaptic CaMKII has been shown to play a significant role in increasing AMPA current density at postsynaptic sides. Here we show that other Ca²⁺-dependent signals are induced in parallel to facilitate signaling from synapse-to-nucleus and from synapse-to-actin cytoskeleton. Together, an ensemble of Ca²⁺-mediated biochemical switches may confer bimodal stability to synaptic signaling events, thereby enabling efficient storage of information both at the synaptic as well as at cellular levels. [Jpn J Physiol 54 Suppl:S16 (2004)]

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The resolution and the width of view in the light microscope are quite limited in all three spatial dimensions. However, by trading one dimension off, a sectional view can be obtained with the highest possible detectability. We evaluated the sectioning property of the slit scanning method and applied it to functional studies of the cellular activity. Individual molecules of tetramethyl rhodamine were easily visualized under the slit light illumination. Narrowing the slit width from 30 to 1 μm greatly enhanced the contrast of the single molecule image. The Q-dot nanocrystals (<50 nm) were also easily visible at a single particle level. Fluorescent beads of 1 μm in diameter appeared as 0.2 μm spots when placed at the window for the slit light emergence. They appeared as rings of variable sizes when placed a few micron distant from the emergence window, indicating that the thickness of the slit light was smaller than 1 μm. Scanning the cell with the thin sheet of light, we recorded fluorescent images of the cell at the shallow and deep parts. MIN6 cells with their secretory vesicles stained with the green fluorescent protein tagged to insulin showed clear shape of the vesicles. Hippocampal neurons dissociated and cultured on a coverslip in the medium containing FM1-43 showed rapid uptake of the dye into fine vesicles in the neurites upon stimulation. These fluorescent vesicles increased in number with the number of stimulation. Later, they showed trafficking activity along the neurite shaft. [Jpn J Physiol 54 Suppl:S16 (2004)]

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Cyclic ADP-ribose (cADPR), a mediator involved in Ca²⁺ signaling, has the characteristic 18-membered ring consisting of an adenine, two riboses and a pyrophosphate, in which the two primary hydroxyl groups of the riboses are linked by a pyrophosphate unit. The analogues of cADPR can be used quite effectively in proving the mechanism of cADPR-mediated Ca²⁺ signaling pathways. These analogues are also expected to be lead structures for the development of drugs. Therefore, A variety of cADPR analogues modified in the base, sugars, and pyrophosphate moieties have been designed and synthesized. We designed cyclic ADP-carbocyclic-ribose (cADPcR), in which the ring oxygen in the "northern" ribose is replaced by a methylene unit. Since they lack the unstable N1-ribosyl linkage of cADPR, this carbocyclic analogue should be resistant to both enzymatic and chemical hydrolysis, while still preserving most of the functional groups of cADPR. We first developed an efficient chemical total synthetic method for cADPR analogues to accomplish the synthesis of cADPcR. Biological evaluations of cADPcR showed that cADPcR actually functions as a stable mimic of cADPR.The structure-activity relationship of cADPR analogues including cADPcR will be discussed. [Jpn J Physiol 54 Suppl:S17 (2004)]

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In 1993, we found that an NAD⁺ metabolite, cyclic ADP-ribose (cADPR), is a second messenger for Ca²⁺ mobilization for insulin secretion in pancreatic beta-cells and proposed the CD38-cADPR signal system for insulin secretion by glucose. We demonstrated that CD38, originally identified as a cell surface marker of lymphocyte differentiation, has both ADP-ribosyl cyclase and cADPR hydrolase activities and that ATP, produced by glucose metabolism, competes with cADPR for the binding site, Lys-129, of CD38, resulting in the inhibition of cADPR hydrolysis and thereby causing cADPR accumulation in beta-cells. cADPR then binds to the FK506-binding protein 12.6 (FKBP12.6 or calstatin 2) in the ryanodine receptor (RyR), dissociating the binding protein from RyR to release Ca²⁺ from the endoplasmic reticulum. Thus, cADPR acts as a second messenger for Ca²⁺ mobilization to secrete insulin in response to glucose stimulation. To confirm the hypothesis, we produced CD38 knockout mice by homologous recombination and demonstrated that CD38 disruption impairs the glucose-induced increases in cADPR, intracellular Ca²⁺ concentration, and insulin secretion. We also produced FKBP12.6 knockout mice and the mice showed impaired insulin secretion by glucose stimulation in vitro and in vivo. Recently, various physiological phenomena from animal to plant cells, in addition to those of pancreatic beta-cells, become understandable in terms of the CD38-cADPR signal system for Ca²⁺ mobilization. [Jpn J Physiol 54 Suppl:S17 (2004)]

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Human serotonin 5A (5-HT_5A) receptors couple to multiple signaling cascades including the inhibition of cADPR. When the 5-HT_5A receptors were expressed in undifferentiated C6 glioma cells, activation of 5-HT_5A receptors inhibited ADP-ribosyl cyclase and cyclic ADP ribose formation via Gi/Go. Cyclic ADPR is produced from NAD+ by ADP-ribosyl cyclase and targets type-II ryanodine receptors to release Ca²⁺. Therefore, cADPR is suggested as an intracellular Ca²⁺ modulating second messenger. The formation of cADPR is regulated by the phosphorylation of ADP-ribosyl cyclase, dependent on either cAMP- or cGMP-dependent protein kinase. Since inhibition of adenylyl cyclase activity and cAMP accumulation by 5-HT_5A receptors was observed, cAMP-dependent protein kinase was predicted to be also inhibited which in turn might attenuate ADP-ribosyl cyclase activity. Therefore, the dual inhibitions of ADP-ribosyl cyclase due to both direct and indirect effect of 5-HT_5A receptors may affect cellular functions related to cADPR. On the contrary, activation of 5-HT_5A receptors transiently opened the K⁺ channels, presumably due to the increase in intracellular Ca²⁺ after formation of inositol (1,4,5) trisphosphate. Taking these competing actions on Ca²⁺ signaling, 5-HT_5A receptors are likely to tune the intracellular Ca²⁺ level. In fact, 5-HT-induced outward K⁺ currents were significantly augmented by cyclosporine, an inhibitor of ryanodine receptor-induced Ca²⁺ mobilization. Impairment of these multiple signaling might therefore cause complex disabilities, leading to the various psychological disorders. [Jpn J Physiol 54 Suppl:S17 (2004)]

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In olfactory receptor cells, it is well established that cAMP and IP₃ act as second messengers during odor responses. We, however, have shown that cAMP-increasing odorants induce odor responses even after complete desensitization of the cAMP-mediated pathway suggesting that at least one cAMP-independent pathways contribute to the generation of odor responses. In attempt to identify a novel second messenger, we investigated the possible role of cyclic ADP-ribose (cADPR) in olfactory transduction. The turtle olfactory cells responded to dialysis with cADPR with an inward current and an increase of the intracellular Ca²⁺ concentration, [Ca²⁺]i. Flooding of cells with 100 μM cADPR from the pipette also induced an inward current without changes in [Ca²⁺]i in Na+-containing and Ca²⁺-free Ringer solution. In a Na+-free and Ca²⁺-containing Ringer solution, cADPR induced only a small inward current with a concomitant increases in [Ca²⁺]i. Inward currents and increase in [Ca²⁺]i induced by cADPR were completely inhibited by removal of both Na⁺ and Ca²⁺ from the outer solution. The experiments suggest that cADPR activates a cation channel at the plasma membrane, allowing inflow of Na⁺ and Ca²⁺ ions. The magnitudes of the inward current responses to cAMP-increasing odorants were greatly reduced by prior dialyses of a high concentration of cADPR or 8-bromo-cyclic ADP-ribose (8-Br-cADPR), an antagonist. It is possible that the cADPR-dependent pathway contributes to the generation of olfactory responses. [Jpn J Physiol 54 Suppl:S18 (2004)]

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Transient increases or oscillations of intracellular Ca²⁺concentration occur during fertilization of animal eggs from invertebrates to mammals, which is the necessary event in the egg activation. The Ca²⁺ is thought to be derived from intracellular store via inositol 1,4,5-triphosphate receptor/channel (IP3R), or via both of IP3R and ryanodine receptor/channel (RyR). The IP3R is thought to be activated by IP3 in many cell types, and to be functional in fertilization of egg cells. On the activation of RyR, the group of H.C.Lee showed that the derivative of NAD caused a Ca²⁺-transient in the egg homogenate of sea uechins in 1987, and identified cADPR as the derivative in 1989. Now it is showed that cADPR activates RyR and causes the Ca²⁺ release in many cell types. The RyR system exists in mammalian, ascidian and sea urchin eggs, but its behavior in fertilization is still unclear. The RyR is thought to be modulate by cADPR and secondarily, by a cGMP, which activates ADP-ribosyl cyclase through the action of a cGMP-dependent protein kinase to produce cADPR. We measured the cGMP, cADPR and IP3 contents of sea urchin eggs during the early stage of fertilization and compared with the Ca²⁺ rise in the presence or absence of inhibitors against guanylate cyclase. These results suggest that the RyR pathway including cGMP and cADPR is not solely responsible for the initiating the Ca²⁺-transient, but contributes to the IP3 production. [Jpn J Physiol 54 Suppl:S18 (2004)]

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Cyclic ADP-ribose (cADPR) is an endogenous modulator of ryanodine receptor Ca2+ releasing channels in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient Cyclic ADP-ribose (cADPR) is an endogenous modulator of ryanodine receptor Ca2+ releasing channels in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular calcium concentration, and/or secondarily potentiates cytosolic calcium increases that are the result of depolarization-induced calcium influx through voltage-activated calcium channels. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies potassium currents, including KCNQ/M channels, rather by a calcium-independent fashion. cADPR is synthesized by both membrane-bound and cytosolic forms of ADP-ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Stimulation of muscarinic and beta-adrenergic receptors activates membrane-bound ADP-ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes, as in cardiac myocytes by beta-adrenergic receptors. These results suggest that cADPR might be involved in M1-muscarinic inhibition of both KCNQ2/3 and merg1 channel, and that cADPR is a second messenger in calcium and non-calcium signalings in the nervous system. [Jpn J Physiol 54 Suppl:S18 (2004)]

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