The perception of ambient temperature is a physiological process critical to the maintenance of body temperature and the avoidance of dangerous thermal extremes. Among six heat-activated channels, TRPV3 and TRPV4 are identified as warm receptors. In addition, P2X₃ receptor, an ATP-gated cation-selective ion channel, was also proposed to have a role in warm sensing. We investigated the effects of warm temperature on intracellular Ca²⁺ by using three lines of cultured cell, fibroblasts (NIH3T3), neuronal cells (NG108-15) and keratinocytes (M308). The warmth stimulus was applied by increasing the temperature of the bath solution from 32 to 38 °C. M308 cells showed rapid increase of cytosolic Ca²⁺ in normal bath solution containing Ca²⁺. Without extracellular Ca²⁺, slow and reduced response was observed, suggesting contribution of Ca²⁺ mobilization from intracellular stores. NIH3T3 cells also responded to the warmth in the normal solution, but not in Ca²⁺-free solution. On the other hand, NG108-15 cells showed both slow Ca²⁺ influx and mobilization. Immunofluorescence microscopy showed expression of TRPV3 and 4 in M308 but not in NIH 3T3 and NG108-15 cells. The expression of four TRPVs was examined by RT-PCR using primers specific for mouse TRPV1-4. These three line of cells may have distinct Ca²⁺ influx pathway in response to warmth stimulus. [J Physiol Sci. 2008;58 Suppl:S78]

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It is well-known that capsaicin activates TRPV1s existing in not only peripheral but also central terminals of primary-afferent fibers; the former activation results in inducing action potentials and the latter one leads to a barrage of the spontaneous release of L-glutamate from nerve terminals to spinal dorsal horn neurons. Although capsaicin is reported to produce a nerve conduction block, this action has not been thoroughly examined yet. We investigated the actions of capsaicin and its related substances on tetrodotoxin-sensitive and fast-conducting compound action potentials (CAPs) recorded from the frog sciatic nerve by use of the air-gap method. Capsaicin reversibly reduced the peak amplitude of CAP in a dose-dependent manner (by 40% at 200 μM). Although a TRPV1 antagonist capsazepine (50 μM) by itself inhibited CAPs, this drug did not affect the capsaicin-mediated inhibition of CAP. A TRPV1 agonist resiniferatoxin (5 μM) had no effect on CAPs. Like capsaicin, dihydrocapsaicin reduced the peak amplitude of CAP in a dose-dependent and reversible manner; this extent was almost comparable to that of capsaicin. Eugenol having the vanillyl group also inhibited CAPs, although this inhibition was less in extent than that of capsaicin. These results indicate that capsaicin inhibits CAPs without an activation of TRPV1s; a double bond existing in the chemical structure of capsaicin but not dihydrocapsaicin does not play a role in the CAP inhibition. [J Physiol Sci. 2008;58 Suppl:S78]

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We previously reported that intracellular Ca²⁺ increased transiently in response to stretch through the activation of mechanosensitive (MS) cation channel in human umbilical vein endothelial cells (HUVEC). However, neither the identity nor the mechanism of activation of the plasma membrane influx pathway that mediates stretch-induced change in [Ca²⁺]_i is known. Since HUVEC expresses several types of mechanosensitive TRP channels including TRPV2, we examined the effects of the targeting suppression of TRPV2 protein expression in HUVEC using TRPV2-specific morphorino-oligo. Western blot analysis and immunocytochemical staining confirmed that TRPV2 was efficiently knocked down by transfection with the morphorino-oligo. In these cells, the stretch induced Ca²⁺ increase measured by fluorescence imaging using fura2 was completely abolished. After the cells were subjected to 20% uni-axial cyclic stretch at 1Hz for 1h, neither a stretch-enhanced stress fiber formation nor a shift in the cell orientation transverse to the strain direction could not be observed. Finally, TRPV2-knocked down HUVEC did not show stretch-induced NO production. These observations strongly suggest that TRPV2 is a responsible ion channel for stretch-induced Ca²⁺ increase, which leads the cytoskeletal reorganization and NO production in HUVEC. Thus, TRPV2 would be a key component of MS channel complex in HUVEC. Furthermore, the result that the production of vasorelaxing factor NO depends on TRPV2 suggests that TRPV2 is involved in hypertrophic heart disease caused by change in blood pressure. [J Physiol Sci. 2008;58 Suppl:S79]

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VSOP is a protein that contains the voltage sensor domain but not pore domain (Sasaki et al, 2006). It exhibits most properties of native voltage-gated proton channels reported in phagocytes and microglia: voltage-dependent gating, proton-selective permeability and pH-regulated gating. Addressing questions how proton permeates and how voltage-dependent gating achieved in VSOP will lead to critical clues to understand general mechanisms of voltage sensor operation and ion permeation. For this purpose, it is important to define whether VSOP operates as monomer or oligomers. In this study, we expressed mouse VSOP in tsA201 cells and performed immunoblots. Antibody was raised against the C-terminal cytoplasmic region of mouse VSOP. Western blot analysis detected a major band, about 30 kDa, corresponding to monomer, whereas weak band corresponding to dimer (about 60 kDa) was also detected. When cysteine residue was introduced into the putative transmembrane region of S4, a key segment for voltage sensing, treatment with H₂O₂ induced upper band shift corresponding to crosslinked dimers. Such crosslinking still occurred when native cysteine was eliminated by point mutation and when the C-terminal cytoplasmic region was removed. Analysis by means of blue native PAGE also detected a band at a region higher than monomer. These suggest that VSOP operates as oligomer rather than monomer. Immunocoprecipitation experiments following cotransfection of myc-tagged VSOP and HA-tagged VSOP also supported this model. [J Physiol Sci. 2008;58 Suppl:S79]

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Voltage-gated proton (H⁺) channels are expressed in many types of cells and considered to play a pivotal role in compensating for pH imbalances across the plasma membrane. Proton channels are characterized by extremely high selectivity for H⁺ and large H⁺ effluxes. The activity of the channels is highly sensitive to temperature, but the temperature-dependence of the open channel conductance has not been determined. We measured the Q₁₀ value of the permeation process using whole-cell recordings of the proton channel in microglia. We developed a rapid temperature-pulse method which could alter ambient temperature within a few ms: the number of open channels should not change during the period. Relationship between the current ratio immediately before and after a temperature-jump and the amount of changes in temperature (dT: -15–+15°C) gave the Q₁₀ value of 1.5 at 25°C. The activation energy (36.9 kJ/mol) for proton permeation was similar to that for other ion channels with water-filled pore. The Q₁₀ value was not affected by potentials and pH gradients across the membrane, the current magnitude or droop (depletion of protonated buffers by H⁺ extrusion). The Q₁₀ value monotonously decreased by increasing temperature from 4 to 49°C. These data suggest that the permeation mechanism of the proton channel has a unique thermodynamic feature. [J Physiol Sci. 2008;58 Suppl:S79]

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An acid-activated ion channel complex PKD1L3/PKD2L1 has been reported as a candidate of sour taste receptor molecules. In mouse tongue, PKD1L3 and PKD2L1 are co-expressed in a subset of taste receptor cells. PKD1L3 and PKD2L1 proteins can interact with each other, and co-expression of the PKD1L3 and PKD2L1 is necessary for their functional cell surface expression. Large currents are evoked by both citric acid and HCl in the PKD1L3/PKD2L1 expressed HEK293 cells. These results suggest that PKD1L3/PKD2L1 channel complex may function as sour taste receptors. Here, we report a unique property of PKD1L3/PKD2L1 channel complex, named as an off-response, which implies that the channel is activated upon the removal of acid stimulus. Acid-induced responses were observed only after the removal of an acidic solution with a pH lower than 3.0. A small increase in pH as low as 1.5 unit appears to be sufficient to activate the PKD1L3/PKD2L1 channels once exposed to the acidic solution with pH 2.5. These results indicate that the PKD1L3/PKD2L1 channel belongs to a novel type of ion channels, named as off-channels, activated by the removal of stimulus. The off-response property of PKD1L3/PKD2L1 channel may explain the physiological phenomena observed in sour taste sensation. [J Physiol Sci. 2008;58 Suppl:S79]

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In bovine ciliary muscle, stimulation of M₃-muscarinic receptor opens two types of non-selective cation channel with different unitary conductances of 35 pS and 100 fS which serve as major pathways for Ca²⁺ entry during the sustained contraction. Here we studied the effects of caffeine, an activator of Ca²⁺ release from intracellular stores, on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and NSCC currents. We also examined the existence of STIM1, a putative calcium sensor of the endoplasmic reticulum. Methods Smooth muscle cells enzymatically isolated from the ciliary body of bovine eyes obtained from a local slaughterhouse were used. The [Ca²⁺]_i was recorded using Fluo-4 as the indicator. Results In the normal saline solution containing 2.4 mM Ca²⁺, superfusion of caffeine (10 mM) caused an elevation of [Ca²⁺]_i which showed an initial peak followed by a plateau. In the absence of extracellular Ca²⁺, the initial peak of the caffeine-induced response was only moderately reduced whereas the plateau was abolished.In myocytes under whole-cell voltage clamp at the holding potential of -50 mV, caffeine induced an opening of 100 fS-NSCC whereas it failed to open 35 pS-NSCC. The expression of STIM1 in bovine ciliary muscle was confirmed by RT-PCR and immunofluorescence microscopy. Conclusion Further study is encouraged to examine the possibility that involvement of signals elicited by depletion of intracellular Ca²⁺ store in the activation of NSCCs by muscarinic stimulation. [J Physiol Sci. 2008;58 Suppl:S80]

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In the ciliary muscle, the tonic component of the contraction produced by muscarinic stimulation is highly dependent on Ca²⁺ provided by influx through receptor-operated non-selective cation channels (NSCCs). Here we examined effects of YM-254890, a G_q/11-specific inhibitor, on contraction, NSCC currents and [Ca²⁺]_i elevation induced by carbachol (CCh). Methods Bovine eyes were obtained from a local slaughterhouse. Tension was recorded isometrically from excised ciliary muscle bundles. For the other experiments, myocytes were enzymatically isolated and cultured for 1-5 days. Whole-cell currents were recorded by voltage clamp. The intracellular Ca²⁺ concentration [Ca²⁺]_i was recorded using the Fluo-4 indicator. AlexaFluor-conjugated antibodies were used in the immunostaining. Results In the muscle bundles, superfusion of 2 μM-CCh evoked a sustained contraction. Both phasic and tonic components of this contraction were inhibited by YM-254890 (3-10 μM) in a dose-dependent manner. In the cultured cells, CCh (>0.05 μM) evoked a NSCC current as well as an elevation of [Ca²⁺]_i. Both initial and sustained phases of these CCh-evoked responses were abrogated by YM-254890 (3-10 μM). Immunostaining revealed a dense distribution of G_q/11 in the vicinity of the cytoplasmic side of the plasma membrane of the cultured cells. Conclusion Like its phasic component, the tonic component of the contraction of bovine ciliary muscle is probably under control of signals conveyed from M₃R through a G_q/11-linked mechanism which communicates in some manner with NSCCs. [J Physiol Sci. 2008;58 Suppl:S80]

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α1-adrenergic modulation of volume-regulated chloride current (I_Cl,vol) was examined in mouse ventricular myocytes with the whole-cell patch clamp. Phenylephrine (PE), an α1-adrenergic agonist, inhibited the hypotonicity-induced activation of I_Cl,vol. The inhibition did not occur in the presence of prazosin, an α1-adrenergic antagonist, or in the cells dialyzed with anti-G_q/11 antibody. U-73122, a phospholipase C inhibitor, prevented the PE-induced inhibition of I_Cl,vol, whereas bisindolylmaleimide-I, a protein kinase C inhibitor, was without effect. Interestingly, PE did not affect I_Cl,vol in the cells loaded with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), and the intracellular application of anti-PI(4,5)P₂ antibody reduced the amplitude of I_Cl,vol in control cells. Furthermore, the activation of I_Cl,vol was suppressed by application of wortmannin (WMN), which is an inhibitor of phophatidylinositol kinases, and thus is expected to prevent re-synthesis of membrane PI(4,5)P₂. However, the inhibition of I_Cl,vol by WMN persisted after additional intracellular application of PI(4,5)P₂. Considering that cell-swelling may activate phosphatidylinositol 3-kinase (PI3K) which is sensitive to WMN, we hypothesize that certain phospholipid component(s) converted from membrane PI(4,5)P₂ by the activated PI3K play an essential role in the activation of cardiac I_Cl,vol, and that α1-adrenergically induced depletion of membrane PI(4,5)P₂ level leads to inhibition of I_Cl,vol. [J Physiol Sci. 2008;58 Suppl:S80]

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The activation of a large inward current through the TTX-sensitive Na+ channel (I_Na) accompanied by Ca²⁺ current (I_Ca) activation results in the ventricular action potential. The repolarization of the membrane is mediated by both the inactivation of these two inward current systems and the depolarization-dependent gradual activation of the K⁺ channels (I_Kr and I_Ks). The plateau potential is finally terminated by the repolarization-dependent reopening of the inward rectifier K+ channels (I_K1). Various patterns of spontaneous depolarization occur if the inactivation of I_Na or I_Ca, the activation of I_Kr and I_Ks, or the conductance of I_K1 is incomplete. Additionally, a transient inward current via the Na⁺/Ca²⁺ exchange is induced by the spontaneous Ca²⁺ release from the sarcoplasmic reticulum and potentially triggers the extrasystole under the Ca²⁺ overload condition. These principal mechanisms are modified by the species-dependent expression pattern of ion channels and also by possible intrinsic differences in the channel properties. In the present study, we examine if these mechanisms are applicable to the human ventricular cell models, which is newly developed based on the experimental data obtained from human ventricular myocytes. [J Physiol Sci. 2008;58 Suppl:S80]

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The intensity curve of (1,0) equatorial diffraction examined by x-ray diffraction (BL45XU at SPring-8) was subtracted by the background function and fitted by Gaussian equation. Then (1,0) peak position (d10) and the standard deviation (σ10) were calculated. The lattice spacing disorder (ε10) was evaluated as the ratio of σ10 and d10 (σ10/ d10). Experimentally the ε10 varied with both muscle length and developed force. To examine the length dependence of the ε10, x-ray diffraction image was obtained every 20 msec during isometric contraction in rat papillary muscle. When the isometric force was developed, ε10 increased in proportion to the amount of force development. Taking account of the internal shortening during the isometric contraction, it can contribute to the ε10. Actually at shorter length the ε10 at rest was slightly increased. At this length the resting tension was small, which appeared the small tension on filaments. The smaller tension on filaments can cause the filaments out of order, which may lead the increased ε10. On the contrary the amount of change in ε10 was smaller during isometric contraction at shorter length. The developed tension became also smaller, which may affect the smaller change of ε10. Therefore it is suggested that the ε10 can be affected by both the longitudinal tension on filaments and the developed tension. [J Physiol Sci. 2008;58 Suppl:S81]

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Arginine-vasopressin (AVP) neurons are osmosensory neurons in the brain. So far, it is known that AVP neurons detect an increase in plasma osmolarity by an intake of salt and water and then release vasopressin to regulate plasma osmolarity. Although AVP neurons can regulate plasma osmolarity, it is not known whether they can respond to a hypotonic challenge with activation of Cl ⁻ channels and thereby regulate their cell volume. In this study, therefore, we examined functional expression of volume-sensitive Cl⁻ channels and its role in AVP neurons isolated from AVP-enhanced GFP transgenic rats. Swelling-induced currents exhibited phenotypical characteristics of volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel, such as outward rectification, inactivation kinetics at large positive potentials, low-field anion selectivity sequence and inhibition by Cl⁻ channel blockers relatively specific to VSOR. Cell cross-sectional area measurements showed that the cells exhibit a regulatory volume decrease (RVD) and the RVD response was inhibited by VSOR Cl⁻ channel blockers. On the other hand, the amount of AVP secreted was found to be increased in response to osmotic swelling and the swelling-induced AVP secretion was enhanced by the presence of VSOR Cl⁻ channel blockers. These results show that VSOR Cl⁻ channel is expressed in rat AVP neurons and is involved in the RVD response and AVP secretion in hypotonic conditions. [J Physiol Sci. 2008;58 Suppl:S81]

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Articular chondrocytes play a crucial role in the production and maintenance of extracellular matrix, which is influenced by mechanical and osmotic environment. The present study investigated the mechanisms underlying activation of I_Cl,vol in rabbit chondrocytes using whole-cell patch-clamp method. Exposure of chondrocytes to the hyposmotic solution resulted in a cell swelling, which was accompanied by the activation of I_Cl,vol. Bath application of tyrosine kinase inhibitor genistein partially and reversibly blocked I_Cl,vol but its inactive analogue daidzein had little effect. On the other hand, intracellular application of tyrosine phosphatase inhibitor orthovanadate via a recording pipette gradually activated an outwardly rectifying current with a reversal potential of -20.3 mV close to the predicted Cl⁻ equilibrium potential of -18.3 mV even under isosmotic condition. This orthovanadate-evoked current was almost completely abolished by the Cl⁻ channel blocker DIDS and was also largely reduced by cell shrinkage caused by exposure to hyperosmotic solution. Pretreatment of chondrocytes with genistein significantly prevented the activation of Cl⁻ current by orthovanadate, suggesting that the basal activity of tyrosine kinase is required for the orthovanadate-evoked activation of Cl⁻ current. Our results strongly suggest that tyrosine phosphorylation is involved in the activation process of I_Cl,vol in rabbit articular chondrocytes. [J Physiol Sci. 2008;58 Suppl:S81]

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Using RT-PCR analysis, the expression of mRNAs for candidates of ATP-releasing pathways was examined. In both control and osmotically swollen astrocytes, amplification of cDNA fragments of expected size was observed for connexins (Cx32, Cx37, Cx43), pannexin 1 (Px1), the P2X7 receptor, MRP1 and MDR1, but not for CFTR. Though the molecular identification of volume-sensitive outwardly rectifying (VSOR) chloride channels and maxi-anion channels are remained to be done, electrophysiological studies proved that both of them could be activated in astrocytes under the stress of hypotonicity. Blockers of exocytotic vesicular release, gap junction hemi-channels, CFTR, MRP1, MDR1, the P2X7 receptor, and VSOR chloride channels failed to affect the massive ATP release from astrocytes. In contrast, the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium (50 μM), a blocker of the maxi-anion channel which has recently been shown to constitute a pathway for ATP release from several other cell types. Thus, we propose that the maxi-anion channel serves as a major pathway for swelling-induced ATP release from cultured mouse astrocytes. [J Physiol Sci. 2008;58 Suppl:S81]

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A pineal body is neurosecretory organ secreting melatonin in the night, but the characteristic and the details of the secretion control remain unclear due to small size of the principal cell (pinealocyte). In this study, whole-cell patch-electrodes were applied to pinealocytes of the rodents (ddy mouse, Wistar rat), and the characteristics of the receptors mediating autonomic neural control were analyzed. Block preparations of about 200-micron thickness were made from the pineal gland isolated from the animal, and various drugs were applied rapidly to the pinealocyte using the Y-tube method. The results show; 1. The nicotinic ACh receptors are expressed in the pinealocyte, causing membrane depolarization in response to application of nicotine. 2. The magnitude of nACh-mediated responses increased significantly during the development of the animal. The nicotinic response of the pineal gland showed obvious inward rectification, while no inter-cellular couplings were detected causing undesired space clamp condition. 3. During application of muscarine to the pinealocyte, inward currents with slow rise time and oscillatory changes in the amplitude were evoked in the cell, which recovered in several minutes after the wash out of muscarine. It is concluded that both nicotinic and muscarinic receptors are expressed in the pinealocyte, which may influence cellular metabolism of the pinealocytes leading changes in melatonin secretion. Supported by grant from Smoking Research Foundation (Japan) [J Physiol Sci. 2008;58 Suppl:S82]

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Midbrain substantia nigra pars reticulata (SNr) consists mostly of GABAergic neurons, of which spontaneous firing rate is among highest in the brain, suggesting that demand for energy source such as oxygen and glucose is very high. Indeed, severe hypoxia suppresses the firing rate of SNr GABAergic neurons both in slice and acutely dissociated conditions. We have shown that ATP-sensitive potassium (K_ATP) channels exert a critical role in the hypoxic response of these neurons. On the other hand, when the extracellular glucose concentration was moderately lowered, the firing rate of most SNr neurons was increased in a K_ATP channel-independent manner, and some exhibit periodical, abrupt increases in the firing rate (up to 120Hz) in slice (multiminutes oscillation). Although molecular mechanisms underlying the glucose-sensitive responses remain unknown, no such oscillation was detected when neurons were dissociated, suggesting that interaction between multiple cellular components is important. Actually, the oscillation was blocked by low extracellular Ca²⁺ in slice, although it was not affected by blockade of EAA- and GABA-mediated transmissions. In the present study, we provide electrophysiological and anatomical data suggesting that glycine participates in the glucose sensitivity of the SNr GABAergic neurons both in juvenile and adult stages. [J Physiol Sci. 2008;58 Suppl:S82]

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In mammalian kidneys, NHE3 (luminal type of Na⁺/H⁺ exchangers) mediates both net bicarbonate reabsorption at the proximal tubule and H⁺ excretion for urine acidification at the thick ascending limb. We investigated localization of NHE3 along the nephron and the level of expression in response to acidification. Methods: Expression of NHE1-4 mRNAs was assessed by in situ hybridization through the nephron, such as proximal convoluted tubule (PCT), proximal straight tubule (PST), discending thin limb (DTL), cortical and medullary thick ascending limbs (cTAL and mTAL), distal convoluted tubule (DCT), and collecting ducts (CD). Abundance of NHE3 mRNA was assessed by the same technique under the condition of chronic metabolic acidosis. Results: Blood pH and [HCO₃⁻] were significantly decreased from 7.37 (control, n=5) to 7.17 (acidosis, n=5) and from 23.4 to 12.3 (mmol/L), respectively, on the second day of the experiment, but they returned to a normal level on the 6th day. In control, NHE1 mRNA was ubiquitously present along the nephron. NHE2 mRNA strongly expressed at the TAL and DCT. NHE3 mRNA was present at the PCT, DTL and TAL. NHE4 mRNA was present through the distal part of the nephron. Under chronic (6 d) metabolic acidosis, expression of NHE1, 2, 4 mRNAs were unchanged, but NHE3 mRNA increased dramatically only at mTAL. Conclusion: NHE3 at the mTAL, NOT at the PCT, may play an important role for body pH regulation at least during acidosis. [J Physiol Sci. 2008;58 Suppl:S82]

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To elucidate functional properties of human astrocytoma cell lines, U-251MG, U-87MG, and KINGS-1, we examined the expression of neurotransmitter receptors with Ca²⁺ imaging. Those cells were loaded with Ca²⁺ sensitive dye (fura-2 AM), and subjected to measurement of the Ca²⁺-dependent fluorescence changes using a specific imaging device. The cells which showed the increase in [Ca²⁺]_i during the administration of a neurotransmitter were classfied as positive. We tested the effects of amino acid (glutamate), catecholamine (noradrenaline), autacoid (histamine), and others (ATP, acetylcholine) on each cell line. The astrocytoma cells showed positive responses during the administration of histamine, acetylcholine, noradrenaline, and ATP. The amplitude of Ca²⁺ response was dose-dependent (10 nM–1 mM), and significantly reduced by each specific antagonist. Surprisingly, Ca²⁺ measurement indicated that those cells showed a negative sensitivity to glutamate (100 μM), which had been reported to be an important mediator between glial cells and neuronal cells. Those results suggest that the loss of glutamate receptor is involved in an initial step of the functional disorder of communication between astrocytes and neurons in the human brain. [J Physiol Sci. 2008;58 Suppl:S82]

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Peroxisome proliferators-activated receptor (PPAR)γ was reported to express in vascular endothelial cells (VECs) and was involved in the regulation of coagulation and fibrinolytic system. To examine the effects of PPARγ agonist, troglitazone (TRO) and telmisartan (TMS), on the expression of fibrinolytic and anticoagulant factors, the mRNA expressions of PAI-1, t-PA, u-PA and thrombomodulin (TM) in cultured VECs were measured using the comparative RT-PCR method. Cultured VECs were established from LETO rat's thoracic aorta by the explant method. To determine the concentrations of TRO and TMS used for activation of PPARγ, concentration-dependent activation of PPARγ by TRO and TMS was determined by ELISA measuring binding activity of PPARγ to coactivator (CBP). TRO and TMS concentration-dependently activated PPARγ with the EC₅₀ values of 3.5 μM and 52 μM, respectively. RT-PCR revealed that PPARγ mRNA was constitutively expressed in cultured VECs. The mRNA expression of PAI-1 was significantly decreased by treatment with TMS (50 μM), but not with TRO (10 μM). These result suggest that TMS possibly promotes fibrinolytic activity by downregulation of PAI-1 through PPARγ activation in VECs. [J Physiol Sci. 2008;58 Suppl:S83]

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Signaling through receptor tyrosine kinases (RTKs) is implicated in the control of various cellular functions. Immediately after activation, these RTKs are rapidly translocated from cell surface into the endosomal compartment. Then, these are sorted into lysosomes for degradation. Cbl-interacting protein of 85 kDa (CIN85) is a multiadaptor protein containing three Src homology 3 (SH3) domains, a proline-rich region and a coiled-coil domain. We have previously shown that CIN85 is involved in regulation of ligand-induced endocytosis of epidermal growth factor (EGF) receptors. To analyze the biological function of CIN85 in vivo, we generated the CIN85 knockout (KO) mouse. No significant effects of the mutation was detected on energy metabolism parameters (body weight, food consumption, feces production). CIN85 KO mice do not show any morphological phenotypes in the spleen, thymus or kidney. However, the corpus callosum (commissure of the cerebral hemispheres) was absent. Now we are analyzing the effect of this mutation [J Physiol Sci. 2008;58 Suppl:S83]

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Muscimol conjugated with fluorescent or MRI-contrast agents may be useful for visualizing inactivated brain areas if they act as agonists of GABA-A receptor. Recently, some N-acyl muscimol conjugated with a marker molecule have been reported to bind to GABA receptors. However, it remains unclear whether they can activate the GABA-A receptor. In this study, we synthesized two N-acyl muscimol derivatives and purified them thoroughly. Their pharmacological effects on neurons were examined by whole-cell patch clamp in vitro and extracellular recording in vivo. Muscimol (m.w. 114.1) and the smallest derivative N-acetylmuscimol (m.w. 156.1) evoked inward currents with the threshold concentrations of 0.05 and 200 µM, respectively. Evoked currents were potentiated by diazepam and inhibited by SR-95531, indicating that the GABA-A receptor was activated. On the other hand, a more bulky derivative N-biotin-6-aminohexanoylmuscimol (m.w. 453.6) did not evoke any currents even at concentrations up to 1 mM. Although a previous study (Biomaterials 26, 1895-1903, 2005) showed that N-biotin-6-aminohexanoylmuscimol activates GABA-A receptor, we failed to confirm this observation. This discrepancy might arise from the difference in the grade of purification to remove residual unreacted muscimol. Our experiments suggest that N-acylation impaired the agonistic property of muscimol on the GABA-A receptor. Supported by MEXT18500311 (HI), MEXT19390055 (JN), and MEXT19002010 (YM) [J Physiol Sci. 2008;58 Suppl:S83]

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It is known that the role of GABA_A-R mediated actions is important for early CNS development. The GABA_A-R mediate actions can regulate radial migration of cortical plate (CP) cells. GABA content is decreased in the brain of GAD67-GFP knock-in mouse. Therefore, these mice are useful to examine the effects of ambient GABA on CP cells. Since taurine, a partial agonist of GABA_A-R, is the most abundant free amino acid in developing brain, the roles of ambient taurine and GABA in activation of GABA_A -Rs on CP cells were investigated using GAD67-GFP knock-in mice. The CP cells were labeled by electroporation of mRFP gene. There was no significant difference in the distribution of labeled cells among genotypes of GAD67-GFP knock-in mice. GABA_A-R mediated currents were then recorded. Dose-response relationships of evoked GABA current had no differences among genotypes. Ambient taurine was decreased by the i.p. injections of taurine metabolism blocker, D-cysteine sulfinate (D-CSA). The radial migration of CP cells was facilitated in D-CSA injected homozygous GAD67-GFP knock-in mice, in which ambient taurine could give rise to GABA_A-R-mediated tonic currents. Chronic application of GABA_A-R blocker to homozygous fetal ventricle also facilitated the radial migration of CP cells. These result indicated that taurine may be endogeneous co-agonist for GABA_A-R that could play critical role in neocortical development. [J Physiol Sci. 2008;58 Suppl:S83]

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P2X receptor channels are extracellular ATP gated cation channels. We previously observed in Xenopus oocytes expression system that the P2X₂ channel current at the steady state after ATP application shows gradual increase upon hyperpolarization, showing presence of voltage-dependent gating in spite of the lack of a transmembrane voltage sensor domain. The analyses of tail current amplitude and voltage-dependent activation time constant revealed that the G-V relationship was shifted towards depolarized potentials in accordance with the increase in [ATP] and the activation kinetics was accelerated; implying that the gating mechanism at the steady state is dependent on both voltage and [ATP]. We hypothesized that the binding itself of negatively charged ATP to the binding pocket of P2X₂ could be a source of this mechanism and analyzed K71A/R, K69A/R, R290A/K, K308A/R mutants of the ATP binding region. Having lowered sensitivity to ATP (10 to 400 fold), none showed any [ATP] dependent acceleration of kinetics but all were differed in activation time constant corresponding to WT responding to various [ATP]. Other than K69R no remarkable [ATP] dependent shift of G-V relationship was observed. These findings suggest that residues in the ATP binding site are involved in voltage and [ATP] dependent gating mechanism and the ATP binding is a critical step for voltage dependency. [J Physiol Sci. 2008;58 Suppl:S84]

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Purinergic P2X4 receptor is a non-selective ATP-gated channel, and is distributed in various tissues including vascular endothelial cells (ECs). Our previous studies demonstrated that P2X4 in ECs mediates flow-induced Ca²⁺ influx and subsequent NO production, and regulates vascular remodeling. Our fluorescence microscopic observation showed that P2X4 channels are predominantly localized on the intracellular vesicles, and that a very small fraction is present at the cell surface where P2X4s should sense extracellular ATP. The mechanisms involved in the up-regulation of cell surface P2X4 remains unclear. To address this issue, we investigated the intracellular dynamics of P2X4 during sensing stimulus. We found that ligand treatment or flow shear stress increased the amount of channels biotinylated by membrane-impermeable reagents, namely P2X4 at the cell surface, by a factor of 1.5–2. Consistently, we observed massive Ca²⁺-dependent exocytosis of GFP-tagged P2X4 on the plasma membrane in response to ligand treatment or shear stress using total internal reflection fluorescence microscopy. Furthermore P2X4 currents evoked by ATP were increased after a second repetitive ligand treatment. These results suggest that the number of cell surface P2X4 of ECs is up-regulated by shear stress, leading to an apparent increase in the sensitivity to external ATP. [J Physiol Sci. 2008;58 Suppl:S84]

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CFTR channel is a good model for studying mechanism of NBD gating engine which is common in ABC trasnporter superfamily because we can observe motion of the NBD engine as channel gating in a real time base using patch-clamp technique. There is convincing evidence that opening of the CFTR channel is associated with dimerization of CFTR's two NBDs (NBD1 and NBD2). Contrary to wild-type (WT) CFTR, which usually opens for hundreds of milliseconds, hydrolysis-deficient mutants can open for minutes. Interestingly, we observed that WT-CFTR occasionally shows long-lasting openings similar to that of hydrolysis-deficient mutants. We investigated the mechanism of the occasional long-opening events shown in WT-CFTR. WT-CFTR showed a fast single exponential relaxation in the macroscopic current relaxation after a rapid removal of ATP in excised inside-out patches. However, mutations in Y1219 which were supposed to interact with the adenine ring of ATP at NBD2, induced an additional significant slow phase in the current relaxation. The increases of fraction and time constant of the Y1219 mutation-induced slow phase were correlated with ATP dose-response curve shift shown in each Y1219 mutant, which suggested that less ATP binding affinity in NBD2 caused more long-opening of CFTR channel. We concluded that Y1219 residue mediated the stable binding of ATP molecule to NBD2 and the instability of ATP binding to NBD2 caused long-opening of CFTR channel presumably via a failure of ATP hydrolysis. [J Physiol Sci. 2008;58 Suppl:S84]

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Modulation of the spontaneous myogenic contraction of the lateral oviduct by biogenic monoamines octopamine and serotonin was studied in the cricket Gryllus bimaculatus. Octopamine and serotonin caused an increase and a decrease in the frequency of myogenic contraction, respectively. Application of the octopamine receptor antagonist phentolamine and the serotonin receptor antagonist ketanserin attenuated the effects of octopamine and serotonin, respectively. The excitatory action of octopamine could be mimicked by application of 8-bromo-cAMP, a membrane-permeable cAMP analog. GsMTx-4, a peptide toxin that possesses specific blocking activity for the stretch-activated (SA) channel decreased the frequency of myogenic contraction. In order to determine whether SA channel is involved in the modulation of myogenic contraction, single SA channel currents were recorded from the cell-attached membrane patches in myocytes isolated from the cricket lateral oviduct. Application of octopamine and serotonin on myocytes increased and decreased the open probability (Po) of the SA channels, respectively. Phentolamine and ketanserin reduced the octopamine- and serotonin-induced effect, respectively. An increase in Po was also observed by bath application of 8-bromo-cAMP. These results indicate that SA channel is involved in the modulation of the myogenic contraction by octopamine and serotonin and that cAMP signaling pathway is involved in the excitatory action of octopamine. [J Physiol Sci. 2008;58 Suppl:S84]

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The bacterial MscL (mechanosensitive channel of large conductance) is one of the best studied Mechanosensitive (MS) channels owing to its resolved 3D structure by X-ray crystallography. In addition MscL is known to be activated solely by tension in the membrane and a tension sensitive region of MscL has been determined. However, how tension leads to structural changes towards channel opening remains to be solved, mainly because we do not know the precise profile of tension generation during membrane stretch. To address this issue, we investigated the effects of the membrane perturbing amphipath chlorpromazine (CPZ) on the MscL activation using the patch-clamp technique. CPZ is known to be an MS channel activator that is supposed to preferentially insert in the inner leaflet of the lipid bilayer. Application of micromolar concentrations of CPZ decreased the threshold tension to activate MscL in a dose-dependent manner resulting in a leftward shift of the open probability vs. tension curve. It is suggested that CPZ systematically modifies tension profile in the bilayer, thus could be a useful tool to explore the biophysical mechanism of MscL activation by membrane tension. [J Physiol Sci. 2008;58 Suppl:S85]

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Signaling through receptor tyrosine kinases (RTKs) is implicated in the control of various cellular functions. Immediately after activation, these RTKs are rapidly translocated from cell surface into the endosomal compartment. Then, these are sorted into lysosomes for degradation. We have previously shown that degradation of EGF receptor (EGFR) is controlled by ubiquitination of activated EGFR and CIN85 by Cbl, a RING-type E3 ubiquitin ligase. On the other hand, TrkA, a member of RTK family, is also internalized after activation by NGF and targeted for degradation. Here we report that the TrkA are ubiquitinated in response to NGF. The ubiquitination of TrkA is induced by Nedd4-2, HECT-type E3 ubiquitin ligases. We also found that Cbl promotes degradation of activated TrkA through ubiquitination. Our data provide that Nedd4-2 and Cbl promote the downregulation of TrkA through ubiquitination. [J Physiol Sci. 2008;58 Suppl:S85]

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Short-chain fatty acids (SCFAs) are the major anions in the lumen of the large intestine. SCFAs are produced by bacterial fermentation of undigested carbohydrates and proteins. Luminal SCFAs are not only absorbed as nutrients, but also influence various physiological and pathophysiological functions of the gastrointestinal tract. Such effects of SCFAs in the intestinal lumen are considered to be induced via the activation of specific receptors and/or via absorption in epithelial cells. However, the sensing mechanism of SCFAs in the intestinal lumen is currently unclear. Nowadays, SCFA receptors were identified from orphan G-protein coupled receptors, specifically GPR41 and GPR43. We have reported the expression of GPR43 in the human colon. In this study, we investigated the expression of GPR41 in the human colon used by RT-PCR analysis, Western blot analysis and immunohistochemistry. As a result, we have discovered GPR41 mRNA and protein were expressed in mucosa of human colon, and the co-localization study demonstrated that GPR41-immunoreactive enteroendocrine cells contained peptide YY, but not serotonin. These results are similar to another SCFA receptor, GPR43. Therefore, it is considered that SCFAs are recognized by GPR41- and/or GPR43-immunoreactive enteroendocrine cells, which secrete peptide YY. Peptide YY is also known to be an important appetite control hormone, inhibiting food intake as a satiety signal. There is a possibility that luminal SCFAs may influence appetite control. [J Physiol Sci. 2008;58 Suppl:S85]

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Channerhodopsin-1 (ChR1) and -2 (ChR2) are archaeal type rhodopsins with seven transmembrane domains (ChR1, ABCDEFG and ChR2, abcdefg), and are involved in the phototaxic and photophobic behavior of one of green algae, Chlamydomonas reinhardtii. The ChR1 generates H⁺-carried photoreceptor current with a maximum absorbance at 510 nm whereas the ChR2 is a light-switched non-selective cation channel with 460 nm maximal action spectrum. To identify the critical domains involved in wavelength sensitivity, homologous recombinations were made between ChR1 and 2 apoproteins at regions connecting the putative transmembrane domains using overlap extension PCR method. The photocurrents of these recombinants were evoked at 477 nm and 532 nm under whole cell patch clamp of transfected HEK293 cells. The photocurrent showed a preference to 477 nm for the recombinants including the fifth transmembrane domain of ChR2. On the other hand the preference was changed to 532 nm when this domain was derived from ChR1. It is suggested that the fifth transmembrane domain of channelrhodopsins may play a critical role in the wavelength preferences. [J Physiol Sci. 2008;58 Suppl:S85]

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The kidney macula densa (MD) controls the tubuloglomerular feedback system and stimulates renin release from the granular cells through sensing the change of luminal NaCl concentration ([NaCl]) in proportion to changes in glomerular filtration rate. Although moderate/low water permeability of luminal membrane of MD cells has been reported, its molecular identity is still unknown. To identify molecular machineries of the transmembrane water permeability, we examined a functionally intact MD cell line (NE-MD: Yasuoka Y et al, Jpn J Physiol 2005) with RT-PCR, western blotting, Immunohistochemistry (IHC). Antibodies against neuronal nitric oxide synthase (nNOS) and a renal outer medullary potassium channel (ROMK) confirmed the authenticity of NE-MD cells. RT-PCR revealed AQP1 mRNA expression in NE-MD cells, but not AQP2, AQP3, and AQP4 mRNAs. Immunoblots with anti AQP1 antibody revealed a positive, but weak 28-kDa band corresponding to very much stronger bands of both red blood cells and AQP1-transfected HeLa cells. IHC of the mouse kidney cortex showed the strong staining in the proximal tubule and the much weaker staining in the macula densa. These data suggest that although the expression level of AQP1 protein is low, AQP1 may facilitate water transport of MD cells, if any, to promote the cell volume change upon changes in luminal [NaCl]. [J Physiol Sci. 2008;58 Suppl:S86]

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FMRFamide-gated Na⁺ channel (FaNaC) is the peptide-gated sodium channel belonging to the epithelial Na⁺ channel/degenerin family. In the Aplysia kurodai FaNaC (AkFaNaC), external Ca²⁺ and Mg²⁺ affect the channel differently (Furukawa et al, 2006). Ca²⁺ but not Mg²⁺ blockes the channel, high Mg²⁺ increases the currents, and Mg²⁺ but not Ca²⁺ enhances the desensitization. In the present study, Mg²⁺ was found to inhibit Ca²⁺ block of AkFaNaC. The concentration-response relationship by FMRFamide was shifted to the left by Mg²⁺, and shifted to the opposite direction by Ca²⁺. The maximum acitivatable current was significantly enhanced by Mg²⁺, but noticeably reduced by Ca²⁺. Because two aspartate residues (D552 and D556) are conserved in the presumed pore region of FaNaCs, we examined the mutant channels in these sites. Most of the actions of Mg²⁺ and Ca²⁺ on AkFaNaC were either abolished or depressed in a mutant D552N. In D552N, Mg²⁺ also blocked the channel. At D556, we made three mutants (D556N, D556C, D556E) but none of them were functional. We propose that D552 is involved in a binding site of divalent cations, and that the binding of divalent cations to the site affects the affinity and/or efficacy of FaNaC as well as the channel block. [J Physiol Sci. 2008;58 Suppl:S86]

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Straight-chain n-alcohols are known to modulate some neuronal activities through direct binding to the ligand-gated ion channel. It has not been established, however, whether effects of alcohol on the receptor-induced current responses are due to a direct interaction with the agonist binding or are secondary to a perturbation of the membrane lipids. Octanol is one of the long chain alcohols and known to block the gap junction composed of two hemichannels. To compare the effects of octanol on receptor-induced responses of neurons, we applied 30-100 μM 1-octanol to Aplysia ganglion cells and tested with various receptors of ionotropic and metabotropic types using conventional voltage-clamp method. Application of octanol reduced significantly and reversibly nicotinic type of Na⁺-current response, another nicotinic type of Cl⁻-current response, GABA_A type of Cl⁻-current response, and dopamine-induced fast Na⁺-current response. In contrast, the same concentration of octanol did not reduce M₂ type of K⁺-current response, D₂ type of K⁺-current response, H₂ type of K⁺-current response, FMRFamide-induced K⁺-current response, D₁ type of Na⁺-currrent response, nor serotonin-induced Na⁺-currrent response. The former 4 types of receptors are known to be ionotropic and the latter 6 types to be metabotropic. Therefore, all types of ionotropic receptor responses examined so far were selectively inhibited by octanol. Keinetic analysis suggested that the inhibition of octanol of these responses is not caused by competition at their receptors. [J Physiol Sci. 2008;58 Suppl:S86]

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To explain to entrain seasonal day length change, there is a model of two separate circadian oscillators in SCN of nocturnal rodents: an E oscillator, which entrain to the sunset, and M oscillator, which entrain to dawn. It is thought that two oscillators regulate onset and offset of activity rhythm respectively. It turns out that these oscillators separate under specific LD conditions (DLDL5:7:5:7, LD18:6 etc), and mPer1 expression shows bimodal rhythm then in a whole SCN. At this time, the role to regulate the peripheral tissue of E/M oscillator is examined by observing the rhythm of clock gene expression of the mouse liver. We investigated the rhythm of liver through Lumicycle using Bmal1::luciferase and Per1::luciferase transgenic mice which were housed individually in cage equipped with running wheel under the specific LD conditions. Even when the bimodal rhythm was formed in SCN, unimodal rhythm was always formed in liver. Moreover, the phase of this rhythm was preferred to be entrained mainly by E oscillator. Since the feeding rhythm is thought to be used as the major factor to form circadian rhythm in the liver, we considered that feeding start by switch-on of E oscillator deliver some information to reset the liver clock. [J Physiol Sci. 2008;58 Suppl:S86]

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Endogenous machinery oscillating about 24hrs called circadian clock ticks internal body time and controls many physiological phenomena. The goal of circadian-modulated drug administration or "chronotherapy" is to find times for drug delivery that result in increasing main and reducing side effect. We still have difficulties to make transfer chronotherapeutic idea from laboratory to clinical situation, as we do not have highly sensitive and easily applicable method of each individual's body time detection yet. We've already invented "molecular time-table method" which utilize expression amount of cycling genes in target organ like liver to detect body time (Ueda et al., PNAS, 2004). To make this method more applicable, we decided to use blood because blood is frequently collected in hospitals. We collected mice blood every 4hr for two days under both light (12hrs)-dark and constant dark conditions. We detected more than 1,000 peaks of substances using capillary electrophoresis-time of flight mass spectrometry (CE-TOFMS), and we searched for corresponding peaks from samples to samples by informatics approach. We then selected cycling substances with statistical filters. As a next step, we tested whether we can detect the body time from simply one time-point sample by measuring the amount of cycling substances. We also asked whether the method works for detection of rhythm disorder. [J Physiol Sci. 2008;58 Suppl:S87]

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Infants spend most of their time with his/her mother. On the basis of this, it has long been speculated that the circadian rhythms of the infant and his/her mother should synchronize with or entrain each other. However, the empirical evidences supporting this hypothesis are scarce except for the studies by sleep diaries, which have recently been criticized for their unreliability. In the present study, we investigated whether there is any relation between the circadian rhythms of the infants and their mothers with more reliable and objective measure. Specifically, we measured the frequency of body movements of infants during 3-18months after birth and their mothers continuously for 72 hrs with actigraph, the reliability of which as a measurement instrument of activity has been well documented. Then, the strength of circadian rhythm was quantified for the infant and his/her mother separately, on the basis of the Periodogram calculated from the actigraphic data. The developmental courses of the strength of circadian rhythms of the infant and his/her mother, and the relation between them were analyzed.The results revealed a significant positive correlation between the strength of the circadian rhythms of the infant and his/her mother; the infants of mother with weak circadian rhythm also had a weak circadian rhythm. This indicates that there does exist some relation between the circadian rhythms of infants and their mothers. [J Physiol Sci. 2008;58 Suppl:S87]

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We have shown that circadian body temperature (T_b) rhythm is finely regulated, but feeding condition affects largely the T_b rhythm. During fasting, T_b gradually decreases in the light (inactive) phase, whereas T_b in the dark (active) phase is maintained at the free-feeding level. The purpose of this study was to elucidate the mechanism involved in the altered thermoregulation between dark and light phase during fasting. Male ICR mice (2-3 mo old) were housed at an ambient temperature (T_a) of 27°C in a 12:12-h light-dark cycle. After 48-h fasting, cold exposure (T_a=20°C) was carried out at light phase (ZT1-4) or dark phase (ZT13-16). T_b (biotelemetry) and oxygen consumption (VO₂, indirect calorimetry) were measured. c-Fos immunostaining was also performed after the cold exposure. During the cold exposure with fasting, T_b decreased by 5.5 ± 2.0°C at light phase, and VO₂ remained unchanged. In contrast, at dark phase, T_b decreased by only 2.0 ± 0.5°C with an increase in VO₂ (p<0.05), which was greater than that in the light phase (p<0.05). The number of c-Fos-immunoreactive cells after the cold exposure with fasting were greater at the dark phase than the light phase in the medial preoptic area, dorsomedial hypothalamus, paraventricular hypothalamic nucleus, and arcuate nucleus. These results indicate that fasting attenuates thermoregulatory response to the cold, and the response differs among time of the day. The difference between the dark and light phase would be partially due to the neuronal activity in the hypothalamus. [J Physiol Sci. 2008;58 Suppl:S87]

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Students of today in Japan suffering from psychosomatic complaints have problems in their basic lifestyle habits. The aim of our study was to clarify the physiological sources underlying their psychosomatic complaints. We examined 15 healthy students (7 men, 8 women) of 21-22 years old. To evaluate the level of psychosomatic symptoms, we developed a self-assessment Psychosomatic Complaints Questionnaire for this study consisting of two categories: 5 items on physical symptoms and 5 items on mental symptoms. Subjects estimated their psychosomatic conditions twice a day (08.00 h & 20.00 h). Cortisol, melatonin and Growth Hormone (GH) were measured by FEIA (Fluorescence Enzyme Immunoassay). Salivary samples were taken from each subjects 5 times a day in `Salivette` tubes at home. The samples were centrifuged and frozen at -20 degrees until analysed. Physiological variables were assessed by means of a standardized score.Our results showed that the self-assessment scores were related to the level of cortisol, melatonin and GH profiles, though in the limited number subjects. Subjects of high self-assessment score in the morning tended to display marked peak, especially in GH profiles. The psychosomatic symptoms might be associated with the physiological hormone rhythm related to their basic lifestyle habits. These results suggest that GH is effective as an index of a biological rhythm, as GH cannot be easily influenced by stress or light stimulus unlike cortisol or melatonin. [J Physiol Sci. 2008;58 Suppl:S87]

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In the present study, we examined the effects of acute light-phase advance on the expression of the arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene, Per 1 gene and Per 2 gene in the suprachiasmatic nucleus (SCN) as well as the galanin gene in the preoptic area (POA), using AVP-eGFP transgenic rats. The expression of the galanin gene in the POA was measured as a homeostatic sleep-related parameter. The acute phase advance was achieved by shortening the dark period from 12 hours to 4 hours, and animals were sacrificed 12 hours after the beginning of the dark period. mRNA levels of AVP-eGFP, Per1 and Per 2 in the SCN were significantly decreased by the acute light-phase. In contrast, the expression of the galanin gene in the POA was significantly increased by the acute light-phase advance. These results suggest the possibility that expressions of circadian and homeostatic sleep-related genes in the hypothalamus can be monitored simultaneously in relation to various circadian and homeostatic manipulations, and the evaluation may be useful for studying conditions of a desynchrony of circadian and sleep patterns, such as jet-lag. [J Physiol Sci. 2008;58 Suppl:S88]

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Cells also display 24-hour rhythms throughout the body .These are entrained by signals from a central clock located in the suprachiasmatic nucleus(SCN) of the hypothalamus, which is reset daily by light. Topoisomerase gene1 (Top1) is an abundant nuclear enzyme expressed throughout the cell cycle. It has circadian expression in cells. We study analysised the function and transcriptional regulation of the mouse Topoisomerase gene1 (mTop1). First we cloned mTop1 promoter region about 1100bp of the DNA upstream of the exon1. Real-time monitoring of the reporter activities of the mTop1 promoter showed the activities oscillated over an opproximately 24-hour cycle. This result suggested that there are responsive sequences for the rhythmic expression of mTop1 in the 1100bp region that we cloned. Using deletion analysis of the mTop1 promoter in NIH3T3 cells we try to find the response elements for the circadian oscillation of the mTop1 gene. In addition, the region between nucleotides -127 and -82 perhaps contains a strongly negative regulatory element in NIH3T3 cells. [J Physiol Sci. 2008;58 Suppl:S88]

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The body temperature shows the circadian rhythmicity with high temperature in the active stage and low in the sleep stage. However, in human, the temperature rhythm disorder appears with a menstrual cycle. Most of middle-aged climacteric women accuse various sicknesses to be called menopausal disorders. The cause of such a temperature disorder is considered to be the secretion fall of the female hormone, but the detailed mechanism is not clear. In this study, we focus on such temperature disorder seen in menopausal disorders and investigated the relationship between menopausal disorders and the biological clock using model mice with menopausal disorders.We used the mice which removed the ovary (OVX mice) for model of menopausal disorder. We evaluated validity as the menopausal disorders model of the OVX mice by measuring ultradian rhythm of the temperature. As a result, the rise of the basal body temperature and an appearance of the ultradian rhythm were observed in the OVX mouse, and these temperature disorders tended to be improved by chronic administration of the estrogen. Next we examined the relationship between the temperature disorder in the menopausal disorders model mice and the biological clock. SCN lesioned mice did not show ultradian rhythm either the ovary was removed or not. Therefore, SCN affected an appearance of the ultradian rhythm in menopausal disorders, namely the control of a sudden temperature change such as a hotflash and a chill was suggested. [J Physiol Sci. 2008;58 Suppl:S88]

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Mammalian clock proteins, such as CLOCK:BMAL1 heterodimer that drives circadian gene expression, likely control circadian biological system via transcriptional-translational-posttranslational feedback loops. We previously reported on a circadian periodically fluctuating CLOCK kinase (p45PFK) in the master clock, rat suprachiasmatic nuclei (SCN) . p45PFK phosphorylated CLOCK:BMAL1 in vitro. In the present study, we found that p45PFK in the rat SCN showed robust CLOCK:BMAL1 rhythmic kinase activities with differential circadian patterns exhibiting peaks correlated with circadian nucleocytoplasmic localization/phosphorylation patterns of CLOCK:BMAL1. Gene silencing of p45PFK in NIH-3T3 fibroblast, as a clock model, damped circadian Per2 expression via suppression of nuclear BMAL1 accumulation. Moreover, a p45PFK major phosphorylation site-deficient BMAL1 mutant did not accumulate in the nucleus and could not rescue circadian Per2 expression in Bmal1-/- cells. These findings reveal that p45PFK is an essential clock-regulating rhythmic CLOCK:BMAL1 kinase via controlling BMAL1:CLOCK nuclear entry. [J Physiol Sci. 2008;58 Suppl:S88]

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Mammalian circadian pacemaker is located in suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is entrained by light-dark cycle; the photic information is transmitted via the retino-hypothalamic tract (RHT). The main neurotransmitter of the tract is glutamate. Glutamate or its agonist N-methyl-D-aspartate (NMDA) induce light-pulse type phase shift in vivo and in vitro. Measuring AVP releasing rhythm in the SCN slice culture, we examined the effect of EGTA on NMDA-induced phase shift. The pulse application of NMDA induced phase at subjective night. This phase shift was attenuated in the presence of EGTA. These results suggest that the calcium influx would be required for the effect of NMDA. Furthermore, a calcium ionophore ionomycin also induced phase shift at subjective night. On the other hand, the application of EGTA alone induced phase shift during subjective day, but not during subjective night. Changes in extra cellular calcium concentration would affect circadian oscillation in the SCN. [J Physiol Sci. 2008;58 Suppl:S89]

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The members of the p160 family of coactivators, which include SRC-1, GRIP1, and pCIP, are involved in the transactivation of nuclear hormone receptors by binding to them directly and recruiting CBP/p300 cofactors. Transcription of the Bmal1 gene is activated by an orphan nuclear receptor, ROR alpha, and repressed by Rev-Erb alpha via ROR-responsive elements (ROR-RE) located in the promoter/enhancer region of the Bmal1 gene. In a previous study, we demonstrated that the basal and ROR-alpha-mediated transcription of Bmal1 was enhanced by SRC-1, GRIP1, and pCIP via ROR-RE in the Bmal1 gene. To search for factors that enhance the amplitude of the clock gene oscillation, we used a real-time oscillation monitoring system and demonstrated that the amplitude of the Bmal1 oscillation was enhanced by the p160 family of coactivators without changing the phase and period. To better understand the functional involvement of the p160 coactivators in enhancing the amplitude of clock gene oscillation, we constructed a dominant negative pCIP expression construct (pCIP-DN). Co-transfection with pCIP-DN reduced the amplitude of the Bmal1 and Cry1 oscillation in NIH3T3 cells. These results showed that p160 coactivators play important roles in the formation of circadian oscillation. [J Physiol Sci. 2008;58 Suppl:S89]

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In mammals, an auto-regulatory transcription and translation feedback loop consisting of several clock genes and their protein products is assumed to be the molecular mechanism of circadian oscillation in the suprachiasmatic nucleus (SCN) of the hypothalamus. In the present study, we tried to stop the circadian oscillation with a protein synthesis inhibitor, Cycloheximide (CHX), treated in the cultured mouse SCN and analyzed the process of reappearance of the oscillation. Per1-Luc transgenic mice carrying a luciferase reporter of the clock gene, Per1 , expression were used. Coronal brain sections were obtained from mice under a light-dark cycle, and the bilateral SCNs were cultured on a membrane in a Petri dish. After monitoring the bioluminescence rhythms for 3 to 4 cycle, CHX was applied to the culture medium for 6 h up to 48 h and washed out. Circadian bioluminescent rhythms from the entire SCN culture and single SCN cells were measured before and after CHX application. The circadian oscillation in the SCN was terminated by CHX applied for more than 18 hours. On the other hand, the circadian bioluminescent rhythm showed a bimodal pattern for the first several cycles after 6 h CHX treatment and withdrawal. By measuring the circadian rhythms in individual SCN cells, it turned out that the bimodality reflected two groups of circadian rhythms, which were 180 degree out of phase. Differential responses of the circadian rhythms in individual SCN cells to CHX suggest the different mechanisms underlying the circadian oscillation. [J Physiol Sci. 2008;58 Suppl:S89]

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A transcriptional-translational feedback loop has been considered as a model for the intracellular rhythm generation in the mammalian circadian clock, in which the protein products of clock genes, PERs and CRYs repress their own transcription. In order to clarify the dynamics of molecular loop, we investigated effects of inhibitors to the several steps of feedback processes. The Bmal1-luc transgenic mice were used, in which the firefly luciferase was expressed under the control of clock gene Bmal1- promoter. The suprachiasmatic nucleus of the hypothalamus (SCN) slices was cultured in the serum-free medium containing Luciferin, and the bioluminescence was monitored continuously. After 5 days in culture, inhibitors of transcription, translation, protein's nuclear export or protein phosphorylation were added into the medium, and analyzed the circadian Bmal1-luc rhythms during and after the treatment of an inhibitor. A protein synthesis inhibitor applied for more than 12 hours stopped the circadian oscillation in the SCN reversibly. Treatment with inhibitors of transcription, protein's nuclear export or protein phosphorylation induced phase shifts in the Bmal1- expression rhythms. From these results, the roles of each step of the transcriptional-translational feedback loop are discussed on the circadian rhythm generation. [J Physiol Sci. 2008;58 Suppl:S89]

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The effects of 4 sessions of scheduled exposure to novel environment with a running-wheel were examined in mice on re-entrainment of Per1 gene expression rhythms in the SCN, liver, lung and skeletal muscle to 8 h phase-shifted light-dark (LD) cycles. Per1 expression was monitored by a bioluminescence reporter introduced into mice (Per1-luc). The mice were subjected to 8 h phase advanced or delayed LD with exposures (Exposed) or without (Control) to the novel environment. Three cycles later, they were released into constant darkness (DD). The SCN and peripheral tissues were sampled between 1200 and 1600 h in the baseline LD cycles (Baseline) and from 16 to 21 h after the onset of DD. In the phase-advance shift, the Per1-luc rhythm of the cultured SCN in both the exposed and control groups phase-advanced significantly and almost re-entrained to the new LD cycles. The lung and skeletal muscle in the control group phase-delayed significantly but did not fully re-entrained. In the exposed group, the lung and skeletal muscle fully re-entrained. The liver in both groups did not phase-shift significantly. In the phase-delay shift, the SCN and all peripheral tissues examined phase-delayed significantly and almost re-entrained to the new LD cycles. These results suggest that scheduled novel environment with a running-wheel accelerates re-entrainment of the peripheral clocks in the lung and skeletal muscle but not in the liver in the phase-advanced LD cycles. [J Physiol Sci. 2008;58 Suppl:S90]

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Circadian timing system and the cell division cycle are frequently deregulated in cancer. We examined the differences of carcinogenicity and circadian activity rhythmicity by the time of day after the injection of carcinogen. Diethylnitrosamine (DEN), which is widely used to promotes hepatic carcinoma, was injected (i.p., 200 mg/kg) on rats kept under the light-dark cycle at zeitgeber time (ZT) 3:00, 9:00, 15:00 and 21:00. Before and after the injection of DEN, locomotor activity rhythm was recorded until 10th week. After the activity recording, livers of same specimens were histochemically analyzed using H-E and GST-P immunostaining, early detection marker of carcinogenicity, positive cells. After the DEN injection, decrease of activity mass on the next day was common in any of the injection time. The GST-P positive cells were abundant when the DEN was injected at 9:00 and 21:00 compared that at 15:00 and 3:00. The ratio of activity during the dark and light was greatest when the DEN was injected at 3:00. The effect of DEN on carcinogenicity and locomotor activity rhythm are not uniform by the administration time in a day. The activity levels during the dark period might relate with the hepatic carcinogenicity. [J Physiol Sci. 2008;58 Suppl:S90]

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Glycine is now used as a chemical for promotion of sleep in humans. It has been demonstrated that glycine administration up to 2g/kg causes the increase of NREM sleep in sleep disturbed rat and hypothermia (0.6 C decrease) in rat. The administration of benzodiazepine not only caused the sleep but affected the circadian rhythm; it blocked the light-induced phase advance of activity rhythm and elevation of Per1 gene expression in the suprachiasmatic nucleus (SCN) in hamsters. In the present experiment, we examined the possibility whether glycine affect light-induced phase shift and c-fos expression in the SCN and retina of mice. Glycine dose-dependently decreased the body temperature of mice (1C decrease with 2 g/kg). After extended light exposure for 2 hrs in the night under LD cycle mouse was kept under constant dark condition. Glycine (0.5-2 g/kg) pretreatment before light exposure dose-dependently attenuated light-induced phase delay. Glycine (2g/kg) pretreatment slightly attenuated light-induced increase of Fos immunoreactivity in the mouse SCN. Further experiment should elucidate whether glycine administration itself causes the phase shift or not. The present results suggest that high dose of glycine may attenuate the light-induced phase shift of circadian clock. [J Physiol Sci. 2008;58 Suppl:S90]

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Oexin and non-orexin waking-active neurons in the posterior hypothalamus were identified in non-anesthetized, head-restrained mice, by using extracellular single unit recordings alone or in combination with neurobiotin juxtacellular labeling and orexin (hypocretin) immunohistochemistry. Orexin neurons were characterized by biphasic broad spikes, whereas non-orexin neurons were characterized by either biphasic narrow or triphasic broad spikes. All orexin and non-orexin waking-active neurons exhibited slow (< 10 Hz) tonic discharges during wakefulness and ceased firing shortly after the onset of EEG synchronization, the EEG sign of sleep (drowsy state). They remained silent during slow-wave sleep, but displayed transient discharges during paradoxical (or rapid eye movement) sleep. During the transition from sleep to wakefulness, both orexin and triphasic non-orexin neurons fired in clusters prior to the onset of EEG activation, the EEG sign of wakefulness, and responded with a short latency to an arousing sound stimulus given during sleep. The activity of all waking-active neurons preceded the return of muscle tonus at the transition from paradoxical sleep to wakefulness. These results suggest that the activity of orexin and non-orexin waking-active neurons in the posterior hypothalamus plays an important wake-promoting role and that their activity antagonizes cortical deactivation and loss of muscle tone. [J Physiol Sci. 2008;58 Suppl:S90]

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