Although both the cerebellum and basal ganglia are known to be involved in motor control and learning, the basal ganglia are unique in their dependence on the information of motivation. In order to access the role of midbrain dopamine (DA) system in reward-based decision and learning, we recorded activity of DA neurons in an instrumental conditioning task in which macaque monkeys made a series of behavioral decisions based on the distinct reward expectations. DA neurons responded to the first visual cue appeared in each trial (conditioned stimulus, CS) through which monkeys initiated trial for decision while expecting trial-specific reward probability and volume. The magnitude of CS responses appeared to represent motivational properties rather than the reward expectation, because their magnitude at trials with identical reward expectation had significant negative correlation with reaction times of animal after the CS. DA neurons responded also to reinforcers occurred after behavioral decisions, and the responses precisely encoded positive and negative reward expectation errors (REEs). This supported roles as teaching signals in reinforcement learning theories. The magnitude of CS responses was positively correlated with that to reinforcers. This suggested a modulation of the effectiveness of REEs as a teaching signal by a motivation. For instance, rate of learning could be faster when animals are motivated, while slower when less motivated, even at identical REEs. Therefore, the DA system is critically involved in reward-based decision-making and learning by encoding both motivational signal and reinforcement signal. [Jpn J Physiol 54 Suppl:S51 (2004)]

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'Reinforcement learning' is a computational framework for an autonomous agent to acquire novel behaviors based on 'reward' signal that reports the goodness of its performance. Despite recent progress in reinforcement learning theory, no artificial agent yet implements reinforcement learning as efficiently and robustly as our brain does. In this talk, I present a series of theoretical hypotheses about how reinforcement learning is realized in the brain:1) Within the circuit of cortico-basal ganglia loops, the striatum learns to represent the 'values' of actions, and the its downstream circuit realizes stochastic action selection. 2) The cerebellum, the basal ganglia, and the cerebral cortex are specialized respectively for supervised, reinforcement, and unsupervised learning paradigms, and their combination enables model-based reinforcement learning using contextual information. 3) The ascending neuromodulators signal global signals for learning: dopamine for the error of reward prediction, serotonin for time scale of prediction, noradrenaline for the sharpness of response tuning, and acetylcholine for the speed of memory update. 4) Parallel organizations of the cortico-basal ganglia loops enables flexible selection of different representations, algorithms, and time scales of learning and control. We report our simulation, brain imaging, and neural recording approaches to testing those hypotheses. [Jpn J Physiol 54 Suppl:S51 (2004)]

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Small lipophilic hormones such as steroid and thyroid hormone play a crucial role in normal function and development in the central nervous system. Each hormone binds to specific nuclear receptors (NRs) with similar molecular structure, which binds to specific nucleotide sequences located at promoter region of target gene. Upon binding to DNA, NRs recruit several groups of cofactors (coactivators and corepressors) in a time-dependent manner to regulate transcription. By now, at least three coactivator complexes, which associate with transcriptional activation by NRs, have been characterized. Complexes containing p160 coactivator such as SRC-1 contain histone acetyltransferase activity (HAT), which acetylates histone to unfold chromatin, thereby facilitating the binding the basal transcriptional machinery. Other coactivator complex contains ATP-dependent chromatin remodeling factor, which disrupts histone-DNA association in ATP-dependent manner, thereby induces transcription as for HAT protein complexes. There is another group of coactivator, termed as TRAP/DRIP/ARC complex, which may directly facilitates recruitment of RNA polymerase II-containing complex. Furthermore, there are additional cofactors such as CoAA, which may regulate not only transcription but also other gene regulation events such as DNA repair and splicing. Disruption of such cofactor genes induces various abnormalities in brain. In this symposium, protein trafficking of NRs and cofactors will be shown. Animal models to study such cofactor action will be also introduced. [Jpn J Physiol 54 Suppl:S52 (2004)]

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The steroid receptor coactivator (SRC) family contains three members that can enhance transcriptional activities of nuclear hormone receptors (NRs). To study the role of SRC-1 in brain development and function, we examined the spatial and temporal expression patterns of SRC-1 and characterized the phenotypes of brain development and function in SRC-1 knockout (SRC-1-/-) mice. In the adult, SRC-1 was highly expressed in the various brain areas and preferentially detected in Purkinje cells (PCs) in the cerebellum. SRC-2 was expressed at lower levels in most brain structures where SRC-1 was expressed. However, SRC-3 mRNA was detectable only in the hippocampus. Multiple behavioral tests revealed that SRC-1-/- mice exhibited moderate motor dysfunction. The time-course analysis revealed that the disruption of SRC-1 delayed the PC development at embryonic stages. However, the morphology of SRC-1-/- PCs developed to the same extents of wild type PCs at the neonatal stage when SRC-2 mRNA was significantly elevated in SRC-1-/- PCs, suggesting that SRC-2 may partially compensate the loss of SRC-1 function. In addition, SRC-1 was colocalized with various NRs and other coactivators in the specific brain regions, regulating hormonal action including sexual behavior. These results demonstrate the relative levels of individual SRC expression are dependent on specific brain regions and the degree of overlapping expression may determine their functional redundancy accompanied with hormonal action. [Jpn J Physiol 54 Suppl:S52 (2004)]

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The TRAP/Mediator complex is a metazoan, phylogenetically-conserved, transcriptional regulatory complex. It was originally isolated as a thyroid hormone receptor (TR)-associated protein (TRAP) complex that mediates TR-activated transcription from DNA templates in vitro and probably acts in vivo after the action of other receptor-interacting coactivators involved in chromatin remodeling. The TRAP complex was subsequently re-identified as the metazoan counterpart of the yeast Mediator complex, which is more broadly used for a wide variety of activators. In mammals, solely the TRAP220 subunit mediates ligand-dependent interactions of the complex with nuclear receptors. In contrast to murine Srb7 that is essential for cell viability per se, genetic ablations of murine TRAP220 and TRAP100 subunits have revealed that they are not but are both early embryonic lethal. These studies have also revealed the activator (e.g., nuclear receptor)-specific roles of TRAP220 in various physiological events (such as in adipogenesis and pituitary-thyroid axis) and the roles of TRAP100 in broad transcriptional events. These phenotypic and mechanistic distinctions between TRAP220 and TRAP100 knockouts contrast their many similarities, and indicate the specificity of these subunits (and probably other subunits) in the roles of the complex both for optimal activators functions and for a variety of early developmental and adult homeostasis events in mice. [Jpn J Physiol 54 Suppl:S52 (2004)]

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Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, such as stress response of the target neural cells. These effects are regulated via two receptor systems, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), both of which are ligand-dependent transcription factors. Moreover, various kinds of cofactors interact with these receptors for mediating their transcriptional effects. It has been shown that MR and GR are highly colocalized in the hippocampus that is a target of stress hormone and an especially plastic and vulnerable region of the brain. In this symposium, dynamics of MR and GR in living cultured hippocampal neurons and COS-1 cells will be presented using real time imaging technique with green fluorescent protein (GFP) including fluorescence recovery after photobleaching (FRAP). We will also present the subcelluar distribution of cofactors such as steroid receptor coactivator-1 (SRC-1). Effects of various kinds of substances on the nucleo-cytoplasmic trafficking of these receptors and cofactors, such as ligand, cytoskeletal elements, and carrier proteins of importin a and b, are investigated. Furthermore, we will discuss possible heterodimerization of MR and GR or interaction between corticosteroid receptor and SRC-1 in a single living cell by using fluorescence resonance energy transfer (FRET). Finally, we will show dynamics of GR in hippocampal neurons of GFP-GR knockin mouse. GFP-GR knockin mice may open new windows for analyzing GR functions from celluar to tissue level to yield innovative knowledge. [Jpn J Physiol 54 Suppl:S53 (2004)]

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We have studied the relationships between respiration-related behaviors and cortical activation in animal models and human experiment. In animal models, we demonstrated that arousal/yawning response was evoked by microinjections of various agents (L-glutamate, cyanide, Orexin, etc.) into the medial parvocellular subdivision of the paraventricular nucleus (mpPVN) in an anesthetized, spontaneously breathing rat. An arousal shift in ECoG was elicited prior to a yawning response, indicating two distinct neuronal pathways (ascending and descending ones) originated from mpPVN. This notion has been supported by recent morphological experiment. We found those cells double-stained by c-fos and CRF or c-fos and oxytocin in mpPVN, when frequent yawnings were induced, suggesting that CRF cells are responsible for cortical activation, whereas oxcytocinergic neurons cause yawning response. In human experiment, we examined effects of Zen mediation (voluntary abdominal breathing, VAB) on EEG in healthy subjects. We found a unique change in EEG when a subject continued the VAB in the eyes-closed condition. Since the eyes were closed, alpha wave was recorded from the onset of VAB. Spectral analysis of EEG revealed that low-frequency alpha band observed at the beginning of VAB disappeared 6-7 min during VAB, and another peak of the high-frequency alpha band was newly developed 7-8 min after the onset of VAB. Since urinary 5-HT level was elevated after VAB, we hypothesize that such change in alpha wave might be caused by excitation of serotonergic system, activated by rhythmic behavior. [Jpn J Physiol 54 Suppl:S53 (2004)]

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Spontaneous respiratory patters are generated in the brain stem for metabolic, homeostatic purposes. However, the respiratory pattern comes from the complex interaction between metabolic requirements and the nonhomeostatic demands in an awake state. Over the last several years, we have been studying how respiratory patterns can be changed and which neural sources are activated by emotional stages in humans. During anticipatory anxiety produced by administering electrical stimulation to human subjects, positive potential changes were observed in EEGs approximately 350ms after the onset inspiration (RAP: Respiratory Anxiety Potential). The sources generating RAP were estimated in the limbic system by the dipole tracing method (BS-navi). Neural activities in the limbic or paralimbic systems were also examined using an optical recording system in a diencephalon-brainstem-spinal cord preparation from newborn rats. We optically detected spontaneous rhythmic burst activities that initially appeared in the piriform cortex and next migrated to the amygdala. The rhythmic activities tended to synchronize with spinal inspiratory activities. The results suggest that the spontaneous rhythmic activities in the limbic or paralimbic system correlate with respiratory rhythm and may be related to the olfactory sensory system and various emotional changes. [Jpn J Physiol 54 Suppl:S53 (2004)]

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Respiratory motor outputs are involuntary rhythmic activities and also can be voluntarily controlled. In this study, we aimed to analyze the cerebral input organization to respiratory neuron network in cats and rats. For this, we examined the effects of electrical stimulation of cerebrofugal fibers on respiratory neuron activities of ventral respiratory group neurons (VRGs) in the caudal medulla, upper cervical inspiratory neurons (UCINs) of C1-2 spinal segments and phrenic motoneurons (Phr. MNs). Experiments were performed under pentobarbital (cats) or ketamine-xylazine (rats) anesthesia. Animals were immobilized with gallamine triethiodide and artificially ventilated. Bipolar simulation electrodes were localized at the cerebral peduncle (CP) and sensorimotor cortex on the right side. Single or double pulse stimulation of the CP induced short latency facilitation (4-6 ms) and succeeding suppression on respiratory activities of the phrenic nerve and UCINs on the left side, while stimulation of CP predominantly suppressed the right side inspiratory and expiratory VRG activities both in cats and rats. The present results suggest that cerebral excitatory inputs to Phr. MNs and UCINs are not transmitted through VRGs, but rather directly to Phr. MNs and UCINs. [Jpn J Physiol 54 Suppl:S54 (2004)]

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Dramatic changes in a variety of autonomic functions, including blood pressure fluctuation, occur during paradoxical sleep (PS). However, its mechanisms remain to be known. During wakefulness, the lateral hypothalamus and mesopontine tegmental area are considered to have crucial roles in regulation of blood pressure. We have obtained the neurons which increased their activity during PS both from the lateral hypothamlamic area (LHA) and the cholinergic nuclei in the mesopontine tegmentum, the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT). We also found that some of these neurons increased their firing several seconds in advance of the increase in blood pressure during PS, suggesting that both the LHA and LDT are driving the increase in blood pressure during PS. By simultaneous recording of the neurons both from the LHA and LDT, it became clear that the LDT neurons start to fire prior to the LHA neurons in association with blood pressure increase during PS. These results indicate that the cholinergic neurons in the LDT drive the LHA to increase blood pressure during PS. [Jpn J Physiol 54 Suppl:S54 (2004)]

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Skeletal muscle stem cells, also known as muscle satellite cells, are located adjacent to the plasma membrane of myofibers beneath the basement membrane. During muscle regeneration, satellite cells proliferate and then fuse together to form myotubes. Histopathologic analysis has shown that muscle satellite cells differentiate into myotubes and myofibers exclusively , and there has been no evidence that these cells are able to differentiate into nonmuscle cells in vivo. However, both primary cultured mouse myoblasts and the immortalized mouse myoblastic cell line C2C12 differentiate into osteoblasts and adipocytes as well as myotubes under appropriate culture conditions. Although these observations suggest that muscle satellite cells preserve multipotentiality, it has been unclear whether different fates are generated from a single satellite cell. We have now characterized a clone of unmanipulated myogenic cells derived from a single mouse muscle satellite cell and revealed its multipotentiality in vitro. Furthermore, multipotent progenitor cells derived from muscle satellite cells were shown to co-express multiple determination genes under growth conditions. On the basis of these observations, we propose a "stock options" model for the lineage commitment of muscle satellite cells. [Jpn J Physiol 54 Suppl:S54 (2004)]

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Cell body sizes and oxidative enzyme activities of spinal motoneurons innervating the soleus muscle were determined in male rats ranging in postnatal age from 3 to 13 weeks. The mean cell body sizes of motoneurons increased from 3 to 7 weeks of age, while the mean oxidative enzyme activities of motoneurons decreased from 3 to 7 weeks of age. There were no changes in mean cell body size or oxidative enzyme activity of motoneurons from 7 to 13 weeks of age. These results indicate that motoneurons innervating the rat soleus muscle show the adult pattern of cell body size and oxidative enzyme activity at an earlier stage of postnatal growth, 7 weeks of age. Male rats were hindlimb-unloaded from postnatal day 4 to month 3. The rats were sacrificed immediately, 1, 2, and 3 months after hindlimb reloading. Numbers, cell body sizes, and oxidative enzyme activities of the soleus motoneurons were determined. A smaller mean cell body size of alpha motoneurons, but not gamma motoneurons, was observed after hindlimb unloading, but this was recovered 3 months after hindlimb reloading. The mean number or oxidative enzyme activity of gamma or alpha motoneurons did not change by hindlimb unloading. It is suggested that hindlimb unloading inhibits a growth-related increase in the cell body size of soleus alpha motoneurons and that the effects of hindlimb unloading on the cell body size of alpha motoneurons are reversible at an early postnatal stage. [Jpn J Physiol 54 Suppl:S55 (2004)]

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Skeletal muscle hypertrophy consists of both enlargement of muscle fibers and an increase in the number of muscle cells. Recently, it was shown that muscle cell proliferation is induced by some growth factors like FGF and IGF-1. New muscle fibers are formed by proliferation and fusion of myogenic cells. Since the number of satellite cells has been shown to decrease with aging, the existence of myogenic cells other than satellite cells is suggested. The origin of myogenic cells, detected by their positivity for myogenin, which is a transcription factor peculiar to muscular cells, was examined in this study. Histochemical examination revealed, myogenin-positive cells in blood vessels and periphery of muscular tissue and suggested possibility of myogenic cells being carried to muscular tissue through blood. In addition, albumin positive cells, which specifically appeared in an identical distribution in hypertrophied muscle, were also observed. It was suggested from the findings in this study that the hepatic stem cells(HSC) may function as myogenic cells. Furthermore, we examined the transplantation of HSCin an overloaded muscle by tenotomy. After one week, transplanted HSC nuclei were observed in the hypertrophied muscle. These results suggest that the liver may be the source of the myogenic cells in hypertrophied and regenerating muscle following exercise, and that supplementation hepatocyte might have an important role in the adaptation of muscle tissue architecture in response to exercise. [Jpn J Physiol 54 Suppl:S55 (2004)]

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It is well known that mechanical stress induces hypertrophy in skeletal muscles. However, molecular mechanisms responsible for muscular hypertrophy are still unclear. Muscle contraction, as well as mechanical stress on muscular cells, also increases the expression of heat shock protein 72 (HSP72, one of the stress proteins) in muscular cells. Increment in HSP72 expression is also induced by most types of cellular stresses, such as oxygen radicals, the reducing peripheral blood circulation and heat exposure. These observations suggest that some types of cellular stress may induce muscular hypertrophy. Recently, we have discovered heat stress-induced muscular hypertrophy in cultured skeletal muscle cells and in rat skeletal muscles. Evidences suggest that heat stress on muscular cells activates Akt/mTOR/p70S6K signaling pathway and facilitates the proliferation and differentiation of satellite cells. Adult skeletal muscle has a remarkable ability to regenerate following myotrauma. It has been reported that the regeneration of skeletal muscle is largely dependent on satellite cells. Therefore, heat stress-induced muscular hypertrophy may be induced by the activation of the cellular regenerative system in skeletal muscle cells. [Jpn J Physiol 54 Suppl:S55 (2004)]

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Therapeutic hypothermia is well known to even lay citizens as a treatment for brain injury today. The treatment was initially introduced into clinical practice in the 1930s. However, since performing systemic cooling below 30°C caused various severe complications, i.e. infection, cardiac failure and so on. Because of those, hypothermic treatment did not appear to benefit patients with brain and become obsolete by the end of 1980s. In the early 1990s, mild hypothermia (MH) (32~34°C) therapy was demonstrated to have significant neurological protective effects similar to those of the previous deep hypothermic therapy. Since MH does not require rigorous patient management so much as deep hypothermia during hypothermia therapy, it has been widespread dramatically all over the world.
Hypothermic treatment is thought to prevent secondary brain damage induced by ischemic brain insults by inhibiting calcium loading, excessive glutamate release and free-radical derivation. In clinical brain emergence, an early induction of MH could minimize the brain damage and result in better outcome. Results of many laboratory works have supported the beneficial effect of MH. However, in clinical setting, the efficacy of MH remained unproved.
In 2002, two prospective, randomized studies reported improved outcomes when hypothermia was induced in comatose survivors after resuscitation from cardiac arrest. MH was shown to mitigate brain damage significantly when induced before, during, and after cardiac arrest. In today talk, I will review therapeutic hypothermia with an emphasis on mild resuscitative hypothermia. [Jpn J Physiol 54 Suppl:S56 (2004)]

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Kei Nagashima, Dept Physiol, Waseda Univ Sch Human Sci
A reduction in body temperature during fasting in rats
Body temperature (T_b) is determined by the balance between heat production and heat loss. In rats, T_b is linked with heat production during free-feeding. Fasting reduces the production of heat throughout a day; however, T_b is maintained during the dark phase and decreases markedly during the early light phase. The reduction in T_b would protect animals from excessive heat loss, minimizing the temperature difference between the body and environment. Temperature of the tail, reflecting active heat loss, also decreases during the fasting. These results indicate that rats suppress heat loss to maintain T_b. However, the suppression is weaker in the light phase than dark phase, which would be a mechanism involved in the reduction in T_b. Although it still remains unclear if rats control the phase-specific change in thermoregulation, a couple of evidences proving the possibility are presented in this symposium. When the suprachiasmatic nucleus (SCN, the master clock for circadian rhythm) are lesioned, the reduction in T_b does not occur, although metabolic heat production decreases in the same manner as the sham-operated rats. Moreover, when rats are exposed to constant darkness, the reduction is attenuated and the phase of the reduction is delayed. These results suggest that the reduction of T_b occurs in response to the stimulus originating the SCN. Moreover, the light is an important factor for this response. In summary, the reduction of T_b during low energy storage would be a regulated phenomenon linked with biological rhythm. [Jpn J Physiol 54 Suppl:S56 (2004)]

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The thermoregulatory heat production is actively suppressed during the entrance into hibernation, thereafter the body temperature is maintained, for several days or weeks, at a level slightly higher than environment temperature. After this torpid state, onset of arousal is triggered by still-unknown mechanisms, and after several hours of complex, integrated organ specific processes body temperature returns to the cenothermic level. Invivo synthesis of protein and mRNA appears inhibited during hibernation and the early stages of arousal indicating that the genomic requirements for initiation of hibernation and arousal from hibernation all occur in the cenothermic period before the animal enters hibernation. The torpor-arousal cycle repeats for the duration of the hibernation season. Although the hibernation control mechanism is not well understood, hypothermia and the onset of the entrance into hibernation may be triggered by some opioid like substances. The hypothermic action of these substances does not function in active summer animals, suggesting that seasonal and also hibernation specific physiological adaptations are required before hibernation can be initiated and arousal from hibernation can be successfully performed. All of necessaries for arousal from hibernation must have been prepared beforehand, because during deep hibernation protein synthesis seems to be globally suppressed. To confirm this point, we injected hibernating hamsters with radioactively labeled precursors of protein and RNA synthesis, and those substances newly synthesized in the major organs were quantified along the time course of hibernation. [Jpn J Physiol 54 Suppl:S56 (2004)]

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2-deoxy-D-glucose (2DG; for blockade of glycolysis) and alpha-melanocyte stimulating hormone (alpha-MSH) were employed to induce hypothermia in conscious rats. Wistar rats were instrumented chronically with electrodes for measurements of intra-abdominal temperature (Tab), renal sympathetic nerve activity (RSNA), the electrocardiogram and electromyogram, and catheters for systemic arterial and central venous pressures. At least 2 days after surgery, the rats were placed in the temperature controlled metabolic chamber where heat production, Tab, RSNA and hemodynamic functions were monitored continuously. After intravenous (iv) administration of 2DG (750mg/kg), Tab decreased significantly (p<0.05) to a nadir of 2.8 ^oC at 73 min after the administration while heat production remained unchanged throughout the experimental period and heart rate was markedly suppressed by 119 beats/min at 74 min after the 2DG administration. In a separate series of experiment, 8 nmol of alpha-MSH in saline solution was injected into the right lateral ventricle (icv). The Tab decreased immediately to stabilize at 1.6 ^oC at 69 min after the administration; heat production did not change significantly (p<0.05), and heart rate decreased by 124 beats/min (p<0.05) at 33 min. After both 2DG (iv) and alpha-MSH (icv) administration, there was a consistent reduction of Tab without changes in heat production, suggesting that heat loss increased. It is therefore likely that the drug-induced hypothermia was a consequence of acute modification of thermoregulatory system. [Jpn J Physiol 54 Suppl:S57 (2004)]

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We experienced a rare case of a young female patient suffering from hypothermia in a cool environment. There were no overt abnormalities responsible for hypothermia in physical, psychological, neurological and hematological examinations. According to investigations of her thermoregulatory function, we speculated that the threshold core temperatures for thermogenesis of the patient shifted to extremely low levels, which then resulted in hypothermia in a cool environment. In addition, it appeared that there might have been unknown cryogenic substance(s) in the patient blood, since the intraperitoneal injection of the patient's serum into rats produced a pronounced fall in core temperature. In this paper, we discuss the possible existence of cryogenic substance(s) which can induce poikilothermia physiologically or pathophysiologically in humans. [Jpn J Physiol 54 Suppl:S57 (2004)]

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Helicobacter pylori (H. pylori) is a causative agent of gastric diseases ranging from chronic atrophic gastritis to gastric adenocarcinoma. However, little is known about the molecular mechanisms by which H. pylori elicits pathogenic activity. Many strains of H. pylori produce an immunodominant protein called CagA. Recent studies have shown that the cagA-positive H. pylori strains are associated with higher grades of gastric inflammation, and they are thus considered to be more virulent than the cagA-negative strains. Furthermore, epidemiological studies reveal a positive relationship between cagA-positive H. pylori and gastric carcinoma.
CagA is delivered from H. pylori into bacteria-attached gastric epithelial cell via the type IV injection system. The injected CagA localizes the plasma membrane, undergoes tyrosine phosphorylation, and specifically interacts with SHP-2 tyrosine phosphatase in a phosphorylation-dependent manner. Upon complex formation, CagA stimulates SHP-2 phosphatase activity and thereby induces morphological transformation of the cell. Deregulation of SHP-2 by CagA may play an important role in the pathogenesis of cagA-positive H. pylori infection. Tyrosine phosphorylation site of CagA is characterized by the EPIYA motif. There is sequence diversity between Western and East Asian CagA species in their EPIYA-containing regions. Thus, East Asian CagA exhibits stronger SHP-2 binding and greater morphology-transforming activities than Western CagA does. Endemic circulation of H. pylori carrying more virulent CagA in East Asian countries may increase the risk of gastric carcinoma in these geographic areas. [Jpn J Physiol 54 Suppl:S57 (2004)]

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The gastric proton pump, H⁺,K⁺-ATPase, is responsible for gastric acid secretion and is almost exclusively found in the parietal cells of stomach. Under physiological conditions, this pump generates and maintains a proton gradient in excess of a million-fold across the luminal membrane. This pump consists of the catalytic α- (114 kDa) and non-catalytic glycoprotein β-subunits (33 kDa as a protein core). Correct assembly between them is essential for the functional expression of the pump at the cell surface. To study the trafficking and cellular regulation of membrane proteins, good culture systems are required. However, at present, there are no cell lines retaining the function of parietal cells. Recently, we constructed stable cell lines expressing the α- and/or β-subunits from human renal HEK-293 cells. Here, we studied the quality control mechanism of the proton pump on the ER, and the fate of the α- and β-subunits before and after the cell surface expression by immunofluorescence, pulse chase labeling, and cell surface labeling with Sulfo-NHS-SS-biotin. The α-subunit was expressed at the cell surface only in the presence of the β-subunit, whereas the β-subunit can travel to the cell surface even in the absence of the α-subunit. The α/β complex expressed at the cell surface was active for ion transport. The unassembled α-subunit was retained in the ER, polyubiquitinated and degraded by the proteasomes. These findings suggest that the ubiquitin/proteasome system is important in controlling the cell surface expression of functional α/β holoenzymes. [Jpn J Physiol 54 Suppl:S58 (2004)]

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The epithelial Na⁺ channel (ENaC), composed of three homologous subunits (α, β, and γ), is shown to be molecularly expressed in the apical membrane of surface cells of the distal colon. We have recently demonstrated that electrophysiological properties of an amiloride-sensitive macroscopic Na⁺ current in surface cells, which are freshly isolated from rectal colon of rats fed a normal Na⁺ diet resemble those reported for heterologously expressed αβγ-ENaC and that the currents are most likely responsible for electrogenic Na⁺ transport in rat rectum (Inagaki et al., Am.J.Physiol. Cell Physiol. 286 (2): C380-C390, 2004). Using the standard whole-cell patch-clamp technique, we have found that the surface cells also exhibit an inwardly rectifying anion current, which is activated by membrane hyperpolarization, has an anion selectivity sequence of Cl⁻ > I⁻, and is blocked by external Zn²⁺. RT-PCR analysis showed the presence of transcripts of rClC-2 in rat rectal mucosa. Electrophysiological properties of the native currents were similar to those of whole-cell currents recorded from HEK-293 cells heterologously expressing rClC-2 cloned from rat rectal mucosa. We also demonstrated that both ENaC and ClC-2-like currents were recorded in the same surface cells. Collectively, these results provide the electrophysiological evidence for functional coexpression of ENaC and ClC-2-like channel in rat rectal surface cells. Possible role of ClC-2-like channel in electrogenic Na⁺ transport in the distal colon is also discussed. [Jpn J Physiol 54 Suppl:S58 (2004)]

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Proteases are involved in many biological and physiological processes in the human body. Previously we demonstrated that a serine protease prostasin increased the activity of epithelial sodium channels (ENaC) when the two are co-expressed in Xenopus oocytes. In a mouse cortical collecting duct cell line (M-1 cells), we found that aldosterone increased the secretion of prostasin into culture media, and that prostasin then stimulated 22Na uptake. TGF-β1 has been shown to antagonize the natriferic action of aldosterone in rat collecting ducts. However, the precise mechanisms by which TGF-β1 inhibits the aldosterone-induced sodium entry at the apical membrane are not fully understood. Recently we isolated 2.8kb rat prostasin promoter and studied the transcriptional activity of prostasin promoter in M-1 cells. We found that TGF-β1 substantially decreased the transcriptional activity of prostasin promoter in a dose-dependent manner in M-1 cells. In addition, we found that TGF-β1 inhibited the aldosterone-induced increase in prostasin protein expression in M-1 cells. These results indicate that the possibility that prostasin is an important target molecule for the natriferic and natriuretic hormones to modulate the sodium reabsorption through ENaC in the kidney. [Jpn J Physiol 54 Suppl:S58 (2004)]

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Na+ reabsorption in the renal distal tubule is finely regulated by various factors such as aldosterone, vasopressin and extracellular osmolality to maintain body Na+ content and blood pressure. The amiloride-sensitive epithelial Na+ channel (ENaC) in the apical membrane contributes to transepithelial Na+ rebasorption as the rate limiting step in the distal nephron. Although we indicated that extracellular hypotonicity stimulates Na+ rebasorption through induction of ENaC gene expression, the mechanism behind this is still under investigation. We studied a physiological role of regulatory volume decrease (RVD) for sensing the extracellular hypotonicity in renal epithelial A6 cells. Extracellular hypotonicity induced the RVD-dependent tyrosine dephosphorylation of focal adhesion kinase (FAK) and paxillin by SRC homology 2 domain (SH2)-containing tyrosine phosphatase (SHP-2). This phenomenon was reproduced by decreasing cytosolic Cl- concentration ([Cl-]c) under an isotonic condition. These observations suggest that extracellular hypotonicity might be converted to the decrease in [Cl-]c through RVD which regulates the SHP-2-dependent FAK dephosphorylation, and indicate the importance of cytosolic Cl- in signal transduction for osmotic shock. Furthermore, hypotonicity caused the SHP-2-dependent stimulation of Na+ reabsorption through gene expression for adaptation to a new environment. Supported by JSPS 15659052 (YM), 15590189 (NN). [Jpn J Physiol 54 Suppl:S58 (2004)]

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Epidemiological data shows that cholesterol cholelithiasis is a disease that demonstrates one of the highest increases in incidence among digestive diseases during the past 50 years. Impairment of gallbladder contractions causes bile stasis, and stasis of supersaturated bile in the gallbladder favors cholesterol crystallization and gallstone formation. However, it remains unclear if the etiology of gallstone formation is multifactorial or genetic. We recently determined the transcriptional start site of the human CCK-A receptor (R) gene and found two polymorphisms (-81A/G, -128G/T). We also cloned the genomic structures of CCK-AR in rats and mice, and generated CCK-AR gene knockout (-/-) mice. Sludge and gallstone formation were significantly higher in CCK-AR(-/-) mice than in CCK-AR(+/+) mice at 12 and 24 months of age. The plasma cholesterol levels, daily food intake, and body weight were not significantly different between CCK-AR(+/+) and (-/-) mice. Sludge and gallstone formation were not observed at 6 months of age. The homozygote (GG/TT) polymorphism of the human CCK-AR gene was associated with a significantly higher percentage of body fat. As the incidence of cholesterol gallstones has been correlated with obesity, polymorphism of CCK-AR enhanced gallstone formation is likewise a cause, indirectly, via obesity. In conclusion, a decrease in the strength of gallbladder contractions, possibly induced by alterations in the CCK-AR gene, as well as by CCK-AR gene polymorphism, was shown to promote gallstone formation. [Jpn J Physiol 54 Suppl:S59 (2004)]

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel present in epithelial membranes. In pancreatic ducts the CFTR plays the pivotal role in the apical bicarbonate transport. Loss of its function due to CFTR mutations causes cystic fibrosis (CF). Mild mutations may result in dysfunction of the CFTR that causes a variety of atypical diseases, such as chronic pancreatitis (CP). The sweat chloride concentration was elevated in about 50% of CP patients due to the failure of chloride reabsorption in sweat ducts. Therefore, we studied DNA of 65 CP patients and 162 healthy controls (C) for 29 CFTR mutations and 3 polymorphisms (poly T, TG repeats, and M470V). Compared with Caucasians, Japanese had a higher frequency of the (TG)12 with the (TG)11:(TG)12 of roughly 1:1. Genotype analysis revealed two major haplotypes, the (TG)12-M470 (31%) and (TG)11-V470 (51%); the former was known to express a smaller amount of intact CFTR proteins and the latter to produce proteins with lower intrinsic activity. These genetic backgrounds, together with a high association of Q1352H (12.3% in CP vs. 3.7% in C) or R1453W (6.2% vs. 3.1%), may explain the association of CFTR dysfunction and CP in Japan where CF is very rare. It remains to be studied how CFTR dysfunction leads to CP. As the CFTR regulates other ion transporters, such as the chloride-bicarbonate exchange and sodium-bicarbonate cotransport, the investigation on their molecular interaction with CFTR may provide a physiological background for understanding pathogenesis of CF and CP. [Jpn J Physiol 54 Suppl:S59 (2004)]

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We investigated the spontaneous electrical and mechanical activities in the ileum of W/W^v mutant mice, in which ICC at myenteric region (ICC-MY) is deficient but ICC at deep muscular region (ICC-DMP) is present. In the isolated 1-cm long segment, electrical slow waves (SW) and action potentials (AP), the force generated in the muscle in the longitudinal direction and the intraluminal pressure was recorded simultaneously. In +/+ mice, rhythmic circular and longitudinal muscle activities were synchronously recorded. SW and AP were also recorded associated with mechanical activities. In W/W^v mice, SW could not be recorded, but irregular mechanical activities associated with AP could be recorded. Tetrodotoxin (TTX) or L-NAME significantly decreased the interval of the longitudinal muscle contractions and increased their regularity. These results suggested that the strong suppression mediated by ICC-DMP and enteric motor nerves on the autonomic rhythmicity was working in the mouse ileum in the absence of ICC-MY. To reveal the contribution of ICC-DMP and enteric motor nerves to changes in gut spontaneous motility in experimental colitis, electrical and mechanical activities were recorded simultaneously in the isolated ileum. Colitis was induced by the provision of dextran sodium sulfate (DSS: m.w. 40,000) in the drinking water at a concentration of 3% for 7 days in +/+ and W/W^v mutant mice. The changes in frequency, regularity and patterns of spontaneous motilities were compared between +/+ and W/W^vmutant mice. The effects of L-NAME, hexamethonium and TTX were also compared. [Jpn J Physiol 54 Suppl:S59 (2004)]

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Thromboxane synthase (TXS), a downstream enzyme of cyclooxygenase-2, catalyses the conversion of prostaglandin H₂ to thromboxane A₂ (TXA₂), which has been shown to induce platelet aggregation and vasoconstriction. We found that irinotecan, an antitumor drug, indirectly stimulates Cl⁻ secretion in isolated rat colonic mucosa via release of TXA₂ from the subepithelial layer. We also found recently that platelet activating factor causes the TXA₂-mediated Cl⁻ secretion in the rat colon. Herein we investigated whether TXS and TXA₂ are related to human colorectal cancers. The specimens were obtained from surgical resection of patients in accordance with the recommendations of the Declaration of Helsinki. Informed consents were obtained from all patients at Toyama Medical and Pharmaceutical University Hospital. We found that both mRNA and protein of TXS were highly expressed in human colorectal carcinoma, and its expression was associated with that of transcription factor NF-E2. TXS was also highly expressed in human colonic cancer cell lines such as HT-29, KM12-L4, T-84 and WiDr. In KM12-L4 cells, the cell proliferation was significantly inhibited by the disruption of TXS protein by a specific antisense oligonucleotide. Direct addition of STA₂, a stable analogue of TXA₂, accelerated the proliferation the cells. After the disruption of TXS protein in the KM12-L4 cells, STA₂ still induced the cell proliferation. These results suggest that TXA₂ is involved in the formation of human colorectal cancer. [Jpn J Physiol 54 Suppl:S60 (2004)]

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To elucidate the mechanism responsible for the ischemia-induced norepinephrine (NE) efflux from cardiac sympathetic nerve endings, we applied dialysis technique to the heart of anesthetized cats and monitored myocardial interstitial NE levels during 120 min-coronary occlusion and after reperfusion. We locally administered neuronal blockades to the ischemic region through a dialysis probe and compared the ischemia-induced increase of interstitial NE in the presence and absence of these blockades. 1) Coronary occlusion markedly increased interstitial NE levels in the ischemic region, but not in the non-ischemic region. After reperfusion, NE levels decreased in the ischemic region, and there was no significant difference in tyramine-induced NE efflux between the ischemic and non-ischemic regions. 2) ω-Conotoxin GVIA (N-type Ca²⁺ channel antagonist) suppressed the increase in interstitial NE levels within 20 min of occlusion, but did not suppress NE increase after 20 min of occlusion. 3) Desipramine (inhibitor of uptake₁ carrier) suppressed the increase in interstitial NE levels at 20-60 min of occlusion, but NE levels gradually increased and reached the same level as those in the vehicle group at 120 min of occlusion. 4) Addition of TMB-8 (intracellular Ca²⁺ release inhibitor) to desipramine further suppressed the increase in interstitial NE levels during the 60-120 min of occlusion. Our results suggest three different mechanisms of ischemia-induced NE efflux to operate subsequently during the course of myocardial ischemia. [Jpn J Physiol 54 Suppl:S60 (2004)]

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We had previously reported that activation of histamine H₃-receptors (H ₃R) on cardiac adrenergic nerve terminals decreases norepinephrine (NE) overfow from ischemic hearts and alleviates reperfusion arrhythmias. Thus, we used transgenic mice lacking H ₃R (H₃R^−/-) to investigate whether ischemic arrhythmias might be more severe in H3R^−/- hearts than in hearts with intact H ₃R (H₃R^+/+). We report a greater incidence and longer duration of ventricular fibrillation (VF) in H₃R^−/- hearts subjected to ischemia.VF duration was linearly correlated with NE overfow, suggesting a possible cause-effect relationship between magnitude of NE release and severity of reperfusion arrhythmias. Thus, our findings strengthen a protective antiarrhythmic role of H₃R in myocardialischemia. Since malignant tachyarrhythmias cause sudden death in ischemic heart disease, attenuation of NE release by selectiveH₃R agonists may represent a new approach in the prevention and treatment of ischemic arrhythmias. [Jpn J Physiol 54 Suppl:S60 (2004)]

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Purpose: Despite the importance of vagal efferent activity during acute myocardial ischemia (AMI) such as an antifibrillatory effect, changes in the endogenous acetylcholine (ACh) release in the ischemic myocardium remain unknown. We examined whether AMI disrupted vagal nerve terminal function. Methods: In anesthetized cats, a dialysis probe was implanted into the left ventricular free wall. The ACh content in the dialysate was measured as an index of myocardial interstitial ACh level. We examined the effects of AMI induced by a left anterior descending coronary artery occlusion on myocardial ACh release in cats with and without vagotomy. In different cats with vagotomy, we examined the effects of Na⁺ channel blocker (tetrodotoxin), intracellular Ca²⁺ antagonist (3,4,5-trimethoxybenzoic acid 8-(dietyl amino)-octyl ester), N-type Ca²⁺ channel blocker (ω-conotoxin GVIA), L-type Ca²⁺ channel blocker (verapamil), P/Q-type Ca²⁺ channel blocker (ω-conotoxin GVIIC), and Na⁺/Ca²⁺ exchange inhibitor (KB7943). Results: AMI increased myocardial ACh level from 0.68±0.12 to 12.3±3.3 nM (mean±SE, P<0.01). Vagotomy did not inhibit the ACh release. Inhibition of intracellular Ca²⁺ mobilization suppressed the ACh release (4.4±0.9 nM). None of Na⁺ channel blockade, N-, L-, P/Q-type Ca²⁺ channel blockade, and Na⁺/Ca²⁺ exchange inhibition suppressed the ACh release. Conclusion: AMI increased myocardial ACh release in the ischemic myocardium via the intracellular Ca²⁺ mobilization. [Jpn J Physiol 54 Suppl:S61 (2004)]

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The recovery process of sympathetic neuronal function (SN) and β-adrenoceptors (AR) after reperfusion following transient ischemia is not fully understood. We investigated (1) serial changes in SN and AR after transient ischemia and (2) effect of brief ischemia before sustained ischemia on SN in rat hearts. Methods: (1) Left coronary artery of Wistar rats was ligated for 15-min followed by reperfusion. SN and AR were assessed by dual-tracer method of ¹³¹I-metaiodobenzylguanidine (MIBG) and ¹²⁵I-cyanopindolol (ICYP). (2) Five-min coronary occlusion before the 30-min occlusion was performed in Wistar rats and cardiac MIBG uptake was determined 3 days after ischemia. Cardiac interstitial norepinephrine (iNE) during 30-min coronary occlusion was measured using microdialysis method. Results: (1) Decrease of MIBG uptake in ischemic region was modest at day 1 after reperfusion and was most severe at days 7 and 14. It was partially restored at day 28. In contrast, the ICYP uptake was severely decreased until day 3 and recovered thereafter. (2) MIBG uptake in ischemic region was greater in rats with 5-min ischemia before 30-min ischemia than in rats without it, associated with lower levels of iNE in rats with brief ischemia. Conclusion: Impairment of cardiac SN after transient ischemia is sustained for a long period and its recovery course is quite different from that of AR. A brief episode of ischemia before prolonged ischemia inhibits deterioration of SN after ischemia, phenomenon of neural preconditioning. [Jpn J Physiol 54 Suppl:S61 (2004)]

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Several epidemiological studies show that estrogen replacement therapy (ERT) protects against the development of Alzheimer's disease (AD) in postmenopausal women. ERT may slow AD progression by reducing the release of Aβ, the primary constituent of amyloid plaques and the major determinant of AD pathogenesis, into brain parenchyma. This Aβ burden is reversible with post-surgical estrogen replacement. In addition, a recent study demonstrates an inverse relationship between the levels of 17β-E2 and Aβ42, the more amyloidogenic Aβ variant, in the cerebrospinal fluid of female AD patients. Our recent study demonstrated that, like estrogen, testosterone increases the secretion of the nonamyloidogenic APP fragment and decreases the secretion of Aβ. This result raises the possibility that testosterone supplementation in elderly men may be protective in the treatment of AD.
Production of Aβ peptides from β-amyloid precursor protein (APP) requires sequential proteolytic cleavages by the β- and γ-secretases, mostly occurring in the trans-Golgi Network (TGN) where APP molecules predominantly reside. We recently demonstrated that estrogen may exert by regulating APP trafficking from the TGN and hence reduce the availability of APP for the secretase cleavages. These data suggest a novel mechanism through which estrogen exerts its anti-Aβ effects in estrogen-responsive tissues and illustrate how altering the kinetics of a protein's transport can influence its metabolic fate. It is our goal to identify regulatory factors that reduce Aβ generation and to elucidate potential therapeutic targets in AD. [Jpn J Physiol 54 Suppl:S61 (2004)]

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The discovery of stem cells in the adult brain has generated tremendous interest in identifying factors that could prompt them to promote neurogenesis and repair in vivo. During pregnancy, the functions of several physiological systems in the mother are altered in order to promote fetal growth and rearing of the newborn. We show here that the production of new neuronal progenitors in the forebrain subventicular zone is stimulated during pregnancy in female mice, and that the effect is mediated by the hormone prolactin. The neuronal progenitors then migrate to produce new olfactory interneurons, an effect likely important for maternal behaviour as olfactory discrimination is critical for recognition and rearing of offspring. Remarkably, neurogenesis occurs even in females that mate with sterile males, implying that forebrain neurogenesis is a maternal adaptation to pregnancy that anticipates a need during the postpartum period. [Jpn J Physiol 54 Suppl:S62 (2004)]

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Oxytocin plays critical roles in mammalian labor and lactation. The hormone is essential for the induction of normal labor through uterine contraction and for normal development of offspring through lactation in mammals. We recently discover a new function of oxytocin in causing plastic changes in hippocampal synapses during motherhood. Mother is "smarter". Oxytocin receptors were strongly expressed in the hippocampus of female mice. In oxytocin-perfused hippocampal slices, one-train tetanus stimulation induced long-lasting long-term potentiation (L-LTP) and phosphorylation of cyclic AMP-responsive element binding protein (CREB), and MAP kinase inhibitors blocked these inductions. An increase in CREB phosphorylation and one-train tetanus-induced L-LTP was observed in the multiparous mouse hippocampus without oxytocin application. Furthermore, intracerebroventricular injection of oxytocin in virgin mice improved long-term spatial learning in vivo, whereas an injection of oxytocin antagonist in multiparous mice significantly inhibited the improved spatial memory, L-LTP and CREB phosphorylation. In this symposium, I will review these findings and discuss the role of oxytocin in improvement of hippocampus-dependent learning and memory during motherhood. [Jpn J Physiol 54 Suppl:S62 (2004)]

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Oxytocin (OT) within the olfactory bulb (OB) has been known to play pivotal roles in the induction of maternal behaviour, olfactory recognition of offspring and the preservation of social recognition. However, the cellular mechanisms of OT' actions in the OB remain to be elucidated. Microcircuits in the OB are relatively simple and include the prominent reciprocal dendrodendritic synapse between mitral and granule cells. Glutamate released from mitral cell dendrites activates the dendrites of granule cells, which in turn mediate GABAergic dendrodendritic inhibition back onto mitral cell dendrites. This feedback inhibition at the reciprocal synapses regulates mitral cell activity. In virgin rats, pup odours are aversive and maternal behaviour is not elicited until this aversion is overcome. We have indicated that OT released at parturition modulates olfactory inputs at the level of the OB and thereby facilitates maternal behaviour. Whether OT acts directly on mitral cells, on granule cells or on both has yet to be determined. By using primary OB cultures and whole-cell recording techniques, we have demonstrated that OT facilitates spontaneous EPSCs in granule cells by both pre- and postsynaptic mechanisms and depresses spontaneous IPSCs in mitral cells by a presynaptic mechanism. Taken together with in vivo electrophysiology and in situ hybridization histochemistry, the results suggest that OT predominantly facilitates dendrodendritic interactions between mitral and granule cells by acting on both cells, thereby depressing mitral cell firing. [Jpn J Physiol 54 Suppl:S62 (2004)]

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Degeneration of neurofilaments is considered as the marker of Wallerian degeneration of peripheral nerves. It is also true that neurofilaments are the first to regenerate when the regeneration process begins. Presently, the neurofilaments can only be detected by destructive methods such as electron microscopy and biochemical methods. In this report, we propose H-2 double-quantum filter (DQF) NMR as a potential technique to measure the neurofilaments non-destructively. The signals of water in the epineurium, endoneurium, and axon of sciatic nerve are resolved in the H-2 DQF NMR spectrum, due to their different quadrupolar splittings (V), and the dependence of the DQF signal intensity of each of the signals on the creation time (τ) of their DQ coherences. We have observed the Wallerian degeneration of rat sciatic nerve for 4 days, and measured signal intensity of the DQF spectrum of the intraaxonal water compartment. The intensity of the intraaxonal signal (V= 9 Hz at τ= 30 ms) started to decrease 24 hrs after the incision, and almost disappeared at day 4. The remaining signal observed is mostly that of the water in the epineurium, which has its maximal value at τ of 3 ms. The time-course of the degeneration as detected by H-2 DQF NMR agrees well with the TEM images obtained by Jeol JEM-1200EX also confirmed the degeneration process of the neurofilaments. From these results, we can conclude that H-2 DQF NMR can be used to quantify the degeneration process of the neurofilaments in the axon. [Jpn J Physiol 54 Suppl:S64 (2004)]

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Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the oxidation of acyl-CoA thioesters to the corresponding trans-2-enoyl-CoA. The C(1)=O of the acyl-CoA is hydrogen-bonded to the ribityl-2'-OH of FAD and the main chain amide N-H of Glu376 in MCAD, and the hydrogen bonds are important in the catalysis. The details of the hydrogen bonds were investigated through the use of artificial FADs by Raman and FT-IR spectroscopies. 8-NH₂-FAD-MCAD binds a substrate octanoyl-CoA, but cannot oxidize it, thus the complex is a valuable sample for exploration of the details of enzyme-substrate complex. FT-IR difference spectra between non-labeled and [1-¹³C]octanoyl-CoA were measured free in solution and bound to 8-NH₂-FAD-MCAD. The 1668-cm⁻¹ band of C(1)=O stretch of octanoyl-CoA free in solution shifted to 1626 cm⁻¹ in the bound form. This 42-cm⁻¹ downward shift reflects an appreciable contribution of a polarized form of the C(1)=O moiety in the enzyme-bound acyl-CoA, and can be explained by the hydrogen bonds of C(1)=O in the active site. The similar downward shifts have been observed by resonance Raman method in the C(1)=O stretch of octenoyl-CoA in MCAD purple complex (reduced MCAD-product complex). We estimated the hydrogen bond enthalpy change (ca. 15 kcal/mol in both cases) for the transfer of the substrate and product from aqueous solution to the active site in oxidized and reduced MCAD, respectively, based on the shift in frequency. [Jpn J Physiol 54 Suppl:S64 (2004)]

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The digital light processing (DLP) technique is used to form images through the projector. A key device for the DLP is an array of micromirrors (digital micromirror device: DMD). Firstly, we illuminated microscopic specimens using a DMD. To this end, a light source and a concave mirror of a DLP projector were replaced with a diode laser and a dichroic mirror for effective excitation and detection of the fluorescence. With the modified DLP projector mounted in the place of the light source for a microscope with a x60 objective lens and an image-intensified camera, an epi-fluorescence image of the HeLa cells stained with tetramethylrhodamine was clearly observed. Under this condition, the illumination area could be modified freely by a personal computer. Individual micromirrors of the DMD changed the angle of reflection digitally and rapidly, thus formed pixels with a gray scale on the screen. We, next, attempted to employ a DMD as a novel scanner for the confocal microscope, since a single mirror in the DMD shared a function similar to the confocal pin hole. When micromirrors were controlled to form a light spot containing 5x5 pixels for spatial scanning, optical slicing was achieved in a 4 μm thickness. The thickness of optical slice was variable by changing the number of pixels in the scanning-spot. The plasma membrane and other cellular organelles may be distinguishable clearly by using this novel microscope system. [Jpn J Physiol 54 Suppl:S64 (2004)]

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Effects of a time-varying magnetic field on signal transduction of cultured bovine adrenal chromaffin cells. Ikehara, T.¹, Teramoto, T.², Houchi, H.², Yamaguchi, H.⁴, Hosokawa, K.¹, Kinouchi, Y.³, Yoshizaki, K.¹ and Miyamoto, H.¹¹Dept. Physiol., ²Dept. Pharmacy, Sch. of Med., ³Dept. Electric. & Electronic Engr., Fac. Engr., Univ. Tokushima, Tokushima 770-8503, ⁴Dept of Environ. Physiol., Fac. Human Life Sci., Tokushima Bunri Univ., Tokushima 770-8513, Japan. Effects of a time-varying magnetic field on chromaffin cells were examined. The magnetic flux density was varied intermittently from 0.07 -1.5 T at an interval of 3 sec. An increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) was stimulated by addition of bradykinin or acetylcholine (ACh) to a medium. The stimulator-induced increase in [Ca²⁺]i in Ca²⁺-free medium was inhibited by 2 hr exposure to the magnetic field. In normal medium, the exposure did not affect the ACh-induced increase, but slowed the decay phase of [Ca²⁺]i after the peak. Also, the exposure affected the cellular ATP content and oxygen uptake. These results suggest that exposure to the magnetic field affect not only ER but also other organells, and the influences by the exposure on these organells might suppress the increase in [Ca²⁺]i. [Jpn J Physiol 54 Suppl:S65 (2004)]

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We have developed the system to analyze temporal changes of multiple fluorescent components (either intrinsic or extrinsic) in single cells, based on the recording of two-dimensional (excitation x emission wavelengths) spectra. Excitation wavelength was switched using band-pass filters, and emitted fluorescence in a wide range of wavelength was recorded simultaneously with a multi-channel spectrometer. Fluorescence components were extracted from a set of spectral data by the blind spectral decomposition algorithm. In the present study, this system was applied to analyze the fluorescent signals from single mammalian oocytes stained with voltage-sensitive dyes. Several components were separated from time series of 2D spectra recorded from voltage-clamped oocytes whose plasma membrane were stained with a styryl dye, di-8-ANEPPS. Two of them showed a large and opposite voltage dependence, and their ratio could be a good index of membrane potential. The system was further applied to the oocytes in which the endoplasmic reticulum (ER) was stained with another styryl dye, di-18:2-ANEPPS, to investigate the changes in the ER membrane potential during Ca²⁺ release. The oocytes were loaded with Ca²⁺-sensitive fluorescent dye, to monitor cytoplasmic Ca²⁺ simultaneously. The spectral decomposition separated fluorescence components of these dyes successfully, and the result suggested a lumen-negative change in the membrane potential upon IP₃-induced Ca²⁺ release from the ER. [Jpn J Physiol 54 Suppl:S65 (2004)]

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Human serum albumin (HSA) is a mixture of human mercaptalbumin (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form), i.e., a protein redox couple. Therefore, commercial albumin preparations may also have a different degree on protein redox state. We examined the redox state of commercial albumin preparations from various sources by our HPLC method.
Eight kinds of commercial HSA preparations were obtained from Sigma Co. and from Calbiochem. Co. Those samples were analyzed by an HPLC system equipped with a Shodex-Asahipak ES-502N column. To determine the value for each fraction of albumin, the obtained HPLC profiles were subjected to numerical curve fitting.
An HPLC profile of HSA showed three peaks corresponding to HMA (reduced form), HNA-1 (reversible oxidized form) and HNA-2 (irreversible oxidized form), respectively. Product No. A1653 from Sigma was the product corresponding to Cohn Fraction five, which is a starting material from pooled sera. Values (%) for HMA, HNA-1 and HNA-2 were 37.6, 44.3 and 13.1%, respectively. In contrast, those values for the final product (A3782), which was prepared through lyophilized and defatted processes, were 12.7, 30.2 and 38.6%, respectively, and it was contained dimer remarkably (17.0%). These results suggested that the heterogeneity of redox state of commercial HSA preparations appears to occur during manufacturing process of HSA from large-scale pooled blood. [Jpn J Physiol 54 Suppl:S65 (2004)]

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Electron-transferring flavoprotein purified from the anaerobic bacterium Megasphaera elsdenii contains one molecule of coenzyme FAD and can bind additional one molecule of FAD to become the holoprotein. The additionally bound FAD exhibits an anomalous absorption spectrum which shows a main absorption band at 400 nm with a shoulder at 450 nm. The flavin released from holoprotein by potassium bromide or guanidine hydrochloride was identified to be normal FAD judging from its absorption spectrum and the retention time on reverse phase chromatography. This result indicates that the unusual spectrum is not due to a chemical modification of the flavin ring but the flavin environment in the protein. The apoprotein was prepared from the holoprotein by cycles of ultrafiltration and dilution with guanidine hydrochloride solution. Dilution of the guanidine-denatured apoprotein with a buffer solution containing FAD resulted in the holoprotein which showed the unusual absorption spectrum like the original holoprotein. This result excludes the possibility that unknown small molecules contained in the holoprotein affect the flavin absorption spectrum. Mammalian electron-transferring flavoprotein contains one FAD and one AMP, unlike M. elsdenii protein. AMP-free mammalian protein, prepared by denaturation and renaturation, showed under acidic conditions an absorption spectrum similar to the unusual spectrum of M. elsdenii protein. This observation suggests a common flavin environment between mammalian and M. elsdenii proteins. [Jpn J Physiol 54 Suppl:S65 (2004)]

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Caveolin, a major structural component of caveolae, is well known to inhibit the function of molecules involved growth factor signal. However, caveolin may exceptionally stimulate insulin signal as implicated in an in vitro experiment using purified recombinant insulin receptor kinase and short caveolin-1 peptides. We have examined the effect of overexpressing caveolin-3, the muscle specific subtype that is not endogenously expressed in the liver, by the use of HepG2 cells, human hepatoma cells, because the liver plays a major role in insulin signal. We used adenovirus harboring caveolin-3 (Ad.cav3) or green fluorescent protein (control, GFP). Caveolin-3 overexpression did not alter the expression of endogenous insulin receptor in HepG2 cells, but significantly increased the phosphorylation of insulin receptor induced by insulin (Ad.cav3 132+18%, control, 100+16%, p<0.05, n=4). Serine phosphorylation of Akt1, a downstream molecule of insulin signal, was also enhanced upon insulin stimulation (Ad.cav3 167+42%, control, 100+10%, p<0.05, n=4). When adenovirus was injected intravenously in mice, GFP was accumulated abundantly in the liver. Putting together, our data suggest that adenovirus-mediated caveolin-3 overexpression augments insulin signal in hepatocytes; this strategy may be utilized in future gene therapy for improving hepatic glucose metabolism in diabetes. [Jpn J Physiol 54 Suppl:S66 (2004)]

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Neuronal nitric-oxide synthase (nNOS) is regulated by Ca²⁺-dependent binding of calmodulin (CaM) and kinase-dependent phosphorylation. We demonstrate here that nNOS is phosphorylated and inhibited by a constitutively active form of Ca²⁺/CaM-dependent protein kinase I (CaM-K I_1-293) in vitro. Substitution of Ser⁷⁴¹ to Ala blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser⁷⁴¹ by Ser to Asp mutation resulted in decreased binding of and activation by CaM since the mutation was within the CaM-binding domain. Phosphorylation of wild-type but not Ser⁷⁴¹Ala mutant nNOS by CaM-K I_1-293 decreased CaM binding to the enzyme. CaM-K I_1-293 also gave phosphorylation of nNOS at Ser⁷⁴¹ in transfected cells, which was determined using phosphorylation site-specific antibody against phospho-Ser⁷⁴¹ nNOS, resulting in 60-70% inhibition of nNOS activity. Furthermore, we obtained evidence that wild-type CaM-K I but neither CaM-K II nor CaM-K IV phosphorylated nNOS at Ser⁷⁴¹ in transfected cells. We have previously demonstrated that CaM-K II directly phosphorylates nNOS at Ser⁸⁴⁷ and can attenuate the catalytic activity of the enzyme in neuronal cells. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser⁷⁴¹ on nNOS in neuronal cells. [Jpn J Physiol 54 Suppl:S66 (2004)]

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'Regulators of G-protein-signalling (RGS)' are a family of proteins, which accelerate intrinsic GTP-hydrolysis on α subunits of trimeric G-proteins and play crucial roles in the physiological regulation of G-protein mediated cell signaling. If RGS proteins were active unrestrictedly, it would completely suppress various G protein-mediated signallings as has been seen in the over-expression experiments of RGS proteins. Therefore, it is quite important to understand how the actions of RGS proteins are regulated in the physiological conditions. We recently revealed in cardiac myocytes a physiological control of RGS proteins. The voltage-dependent formation of Ca²⁺/calmodulin (CaM) facilitated the GTPase-activity of RGS protein via removing intrinsic inhibition mediated by a kind of phospholipid, phosphatidylinositol-3,4,5,-trisphosphate (PI(3,4,5)P₃). This modulation of RGS-action underlies a temporal behavior of G-protein-gated K⁺ (K_G) channels, called 'relaxation',. Further examination using protein-lipid co-sedimentation assay, we detected the specific interaction between RGS4 and PI(3,4,5)P₃ (but not other PIPs), which was decreased by Ca²⁺/CaM. The allosteric modulation is exclusively performed within RGS domain. We identified the clusters of positively charged residues in RGS domain as a candidate of the molecular switch of PI(3,4,5)P₃/CaM-modulation. Because the residues are conserved in almost all RGS protein subtypes, the allosteric modulation of RGS proteins should be important in the physiological control of G-protein signaling by various RGS proteins. [Jpn J Physiol 54 Suppl:S66 (2004)]

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Mutations in the gene encoding for the renal tight junctional protein paracellin-1 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, an autosomal recessive disorder of renal calcium and magnesium handling. To elucidate the magnesium and calcium permeability by paracellin-1, FLAG-tagged paracellin-1 was stably expressed in Madin-Darby canine kidney (MDCK) cells. Expression and localization of paracellin-1 were determined by immunoblotting and immunofluorescence microscopy. The protein co-localized with tight junctional proteins, occludin and ZO-1. Paracellin-1 expression affected neither the levels of occludin nor ZO-1. In vitro binding assay showed that the COOH-terminal domain of paracellin-1 was associated with ZO-1. In similar fashion, immunoprecipitation by anti-FLAG antibody showed that paracellin-1 was associated with ZO-1 in FLAG-tagged paracellin-1-expressing MDCK cells. Next, we examined the effect of paracellin-1 expression on transepithelial electrical resistance (TER) and calcium permeability. Paracellin-1 expression increased TER twice compared with non-expression cells. In contrast to TER, paracellin-1 expression increased calcium transport in the apical-to-basolateral direction. Increase of magnesium concentration diminished the calcium transport. These results suggest that paracellin-1 creates calcium- and magnesium-selective channels and has no relation to determination of TER. [Jpn J Physiol 54 Suppl:S66 (2004)]

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Proximal tuble cells which were isolated from the rat kidney and maintained in culture medium grew to conflence at day 4. Gap junctional intracellular communication (GJIC) also developed, which was indicated by the dye-coupling and electrical coupling between adjacent cells. Calmodulin-Ca²⁺ complex (CaM-Ca) blocked the GJIC. NOC 7(10μM), one of NO donors, also blocked GJIC. These results suggerst that intracellular Ca²⁺ and NO production regulates gap junction channels. It was known that cadmium (Cd) accumulates in the kidney and causes renal injury. Dye coupling between cells was significantly reduced 30 minutes after exposure to 100 μM Cd and disappeared completely after 60 minutes. Howecer, the Cd exposure did not bigin to affect the cell morphology until 60 minutes after exposure. The inhibiting effect of Cd on GJIC is avoidance from nephrotoxicity by Cd. [Jpn J Physiol 54 Suppl:S67 (2004)]

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We have previously reported that the 40 pS K channel is the most frequently observed K channel in cultured human renal proximal tubule cells under the control condition, and that the activity of this channel is maintained at least in part by the PKG-mediated phosphorylation. In this study, we examined whether a well-known activator of the cGMP/PKG pathway, nitric oxide (NO), would be involved in the regulation of this K channel, using the patch-clamp technique. In cell-attached patches, a non-selective inhibitor of NO synthase (NOS), L-NAME (100 μM), reduced channel activity, which was mimicked by an nNOS- and iNOS-specific inhibitor, TRIM (100 μM). These results were supported by the data that mRNAs of nNOS and iNOS, but not eNOS were detected by RT-PCR. Furthermore, an inhibitor of soluble guanylate cyclase, ODQ (10 μM), also reduced channel activity. SNP, an NO donor, of 10 μM enhanced channel activity, which was abolished by a PKG inhibitor, KT5823 (1 μM). In contrast, SNP of a higher dose (1 mM) suppressed channel activity, which was not restored by 8Br-cGMP (100 μM). Membrane potential recorded with the slow whole-cell configuration showed that L-NAME depolarized the cells by 10 mV whereas a low dose of SNP hyperpolarized by 7 mV. In conclusion, the endogenous NO produced by nNOS and/or iNOS in these cells would contribute to the maintenance of basal activity of this K channel and hence the potential formation via a cGMP/PKG-dependent mechanism, while a high dose of NO impaired channel activity independent of cGMP/PKG. [Jpn J Physiol 54 Suppl:S67 (2004)]

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Temperature sensing ability is underlain by TRPV, TRPM and TRPA cation channels.Here we show heat-evoked activation of the TRPM2 channel, which has been reported to be activated by β-NAD⁺, ADP-ribose, and redox status change. TRPM2 activation had threshold of approximately 35°C and was maximally induced at about 36°C. When heat stimulus was given in the presence of intracellularly applied β-NAD⁺ or ADP-ribose, TRPM2 activity was dramatically potentiated, compared to the activity induced by heat, β-NAD⁺ or ADP-ribose alone. Furthermore, cyclic ADP-ribose elicited similar enhancement of heat-evoked TRPM2 activity. The heat-evoked TRPM2 responses were also observed in inside-out configuration, indicating direct gating of TRPM2 by heat. Similar native heat-evoked channel responses were observed in rat insulinoma RIN-5F cells. TRPM2 was expressed in RIN-5F cells, and co-expressed with insulin in mouse pancreatic β-cells. These results indicate that body temperature regulates Ca²⁺ entry into cells that express TRPM2 and that TRPM2 functions as the first molecular target of cyclic ADP-ribose on plasma membrane. [Jpn J Physiol 54 Suppl:S67 (2004)]

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