[Background] cAMP is produced from ATP by adenylylcyclase. Despite various G-protein coupled receptor (GPCR) signals share cAMP as second messenger, physiologicaleffects of stimulation may not be identical amongreceptors. In addition, the role of cAMP in developing apoptosis is not well understood in neuronal cells, especially during development. [Method] We first examinedthe effects of stimulating two GPCR on neuronal cell apoptosis using neonatal striatal neurons. Then, weinvestigated developmental changes of such effects in striatal neurons obtained from 2 week old mice by a newly developed culture technique in our laboratory. Cultured striatal cells were incubated with isoproterenol or dopamine, then, apoptosis was evaluated. [Result] In neonatal neurons, neither isoproterenol nor dopamine stimulation induced apoptosis. In contrast, in neuronsfrom 2 week old mice, TUNEL staining and DNA fragmentation ELISA revealed that dopaminergic receptor stimulation significantly increased the number of apoptotic cells.Western blot analysis revealed that only isoproterenol stimulation increased phosphorylation of Akt and MAP kinase. In contrast, both isoproterenol and dopamine stimulation increased cAMP. Accordingly, only dopamine stimulation induced cellular apoptosis while isoproterenoldid not, presumably due to cytoprotective effect through Akt and/or MAP kinase activation. [Conclusion] The role of cAMP in developing neuronal apoptosis differs among GPCRas well as in developmental stages. [J Physiol Sci. 2006;56 Suppl:S69]

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Too much increase of the plasma membrane permeability causes necrotic death in the many types of cells. Detail mechanisms of this type of cell death are still unclear. We use amphotericin B (amB) as the membrane pore formation agent and investigate the ionic dependency on the cell death caused by amB. First we apply propidium iodede (PI) to the HeLa cells together with 10 μg/ml amB and observe the staining nucleus by PI using the fluoresent microscope. 2 hours after apllication of amB, we could observe the PI signal in the nucleus, indicating that the large pores were formed after the application of amB. These pores should be larger than the pores formed by amB because PI did not enter into the cells just after amB application. Cl⁻ replacement by gluconate or Cl⁻ channel blocker, DIDS (0.5 mM) inhibited the staining of nulueus after amB application. Next we stained the lysosomes in the HeLa cells by FL-labelled pepstatin A. In the control condition, fluorescent dots were observed around the nucleus and such dots gradually disappered after amB application. Low Cl⁻ condition or DIDS application inhibited the disappearance of the fluorescent dots by amB. These results suggest that the Cl⁻ ions can enter into the cell after amB application and those ions may couse the disruption of lysosome, which enhances the membrane permeability increase by the attacking membrane proteins by the lysosomal enzymes. [J Physiol Sci. 2006;56 Suppl:S69]

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Role of PMCA in smooth muscle contractility was investigated using the bladder isolated from PMCA gene manipulated mice: PMCA4 null mutant (Pmca4^−/−) and PMCA1 and PMCA4 double gene targeted (Pmca1^+/−4^−/−) mice. Western blot shows the loss of PMCA4, a major isoform, but not PMCA1, sarco-endoplasmic reticulum ATPase (SERCA) and Na⁺/Ca²⁺ exchanger (NCX), in the muscle layer preparation of Pmca4^−/− and Pmca1^+/−4^−/−. Half-times of contraction and relaxation upon treatment with 80 mM KCl were determined. Surprisingly, half-times of contraction in Pmca4^−/− and Pmca1^+/−4^−/− muscles tended to be prolonged, when compared with that in WT muscle. Relaxation half-times were also prolonged in the gene manipulated muscles, as expected. On the other hand, inhibition of SERCA or NCX marginally shortened the contraction half-time and prolonged the relaxation half-time in muscles of all tested genotypes. Using relaxation half times, the contribution of PMCA to relaxation was calculated to be 25%, SERCA 20% and NCX 70%. PMCA and SERCA appeared to function additively, but the function of NCX might overlap with those of other components. FuraPE3 signal shows that the basal level of [Ca²⁺]i slightly increased in Pmca1^+/−4^−/− muscle. In summary, the gene manipulation of PMCA indicates that PMCA, in addition to SERCA and NCX plays a role in both excitation-contraction coupling and Ca²⁺-extrusion-relaxation relationship, i.e., Ca²⁺ homeostasis, of the bladder smooth muscle. [J Physiol Sci. 2006;56 Suppl:S69]

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We previously showed that Fyn, a member of Src family tyrosine kinase (Src-TK), was involved in the activation of Rho-kinase (ROK) to develop Ca²⁺ sensitization of vascular smooth muscle contraction induced by sphingosylphosphorylcholine (SPC). Recently we found that SPC can stimulate the formation of stress fibers and filopodia-like protrusions in NIH3T3 fibroblasts through Src-TK/ROK-dependent and independent pathway, respectively. Then, we further investigated the role of Fyn in the SPC-induced formation of stress fibers and filopodia-like protrusions with RNA interference method. Three different Fyn siRNAs were designed and transfected into NIH3T3 cells using Lipofectamine 2000. As control, a non-silencing siRNA and a positive MAPK1 control siRNA were transfected in parallel. The non-silencing AF 488-labeled siRNA was used to monitor transfection efficiency. Our results showed that transfection efficiency was high above 90% and the down-regulation of Fyn expression was confirmed in western blot with concentration-dependency and incubation time-dependency. SPC-induced stress fiber formation was partially inhibited by Fyn siRNAs, but not by other control siRNAs. The formation of filopodia-like protrusions induced by SPC was not affected by Fyn siRNAs. These findings suggest that Fyn plays a role in SPC-induced stress fiber formation, but not in the formation of filopodia-like protrusions in NIH3T3 cells. [J Physiol Sci. 2006;56 Suppl:S70]

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Osmolarity changes created by sucrose during the perfusion of isolated rat submandibular glands (SMG) in vitro alter secretion rates much more than predicted by the osmotic theory of fluid production. However, these are in accord with a theory involving AQP5 feedback control of paracellular fluid transfer (Hill & Shachar-Hill 2002). The changes in transport rate can be predicted with parameters determined earlier for this gland (Murakami et al. 2001) and a model of the SMG system is presented. Experiments were performed with SMG from genetically selected rats that have very low levels of AQP5 as determined by Western blotting (Murdiastuti, K. et al. 2002). The fluid secretion rates after osmolarity changes were those expected for the osmotic theory. We suggest that control of paracellular flow has been lost in these low AQP5 rats which have reverted to osmotic fluid production. Retrograde injection of Hg ions into the duct partially inhibited AQP5, leading to a concentration-dependent reduction in flow rates. However, reduction of fluid secretion after osmolarity changes was still close to that of normal rats. The results suggest the involvement of a feedback loop including AQP5 and paracellular fluid transport. [J Physiol Sci. 2006;56 Suppl:S70]

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We reported that 1 mM of ethanol (relevant to blood level after social drinking) potentiated secretin-stimulated fluid secretion in interlobular ducts isolated from guinea-pig pancreas. In this study, we examined anion composition of the secreted fluid during the hypersecretion. Isolated ducts were cultured overnight, during which time both ends of the ducts sealed. The lumen was punctured and injected with solutions containing BCECF-dextran or ABQ-detxtran (plus Cl-NERF-dextran as ratiometric reference). Luminal pH (pH_L) and luminal Cl⁻ ([Cl⁻]_L) were estimated by microfluorometry. Images of luminal fluorescence were obtained at 1-min intervals and the rate of fluid secretion was calculated from the increment of luminal volume. During stimulation with secretin (10 nM), the rate of fluid secretion was 2.28±0.14 nl min⁻¹ mm⁻² (per unit area of epithelium, n=5, mean±SE), pH_L increased due to HCO₃⁻ secretion, and [Cl⁻]_L steadily decreased. The net Cl⁻ transport calculated from [Cl⁻]_L and luminal volume was nearly zero. Ethanol (1 mM) increased secretin-stimulated fluid secretion to 4.11±0.22. pH_L slightly decreased and [Cl⁻]_L was stable in the presence of ethanol. The net Cl⁻ transport was 0.265±0.042 nEq min⁻¹ mm⁻². The increase of fluid secretion by ethanol was almost equivalent to fluid secretion accompanying Cl⁻ efflux. Ethanol induced Cl⁻ secretion during secretin stimulation without affecting HCO₃⁻ secretion, which resulted in the increase of Cl⁻ of secreted fluid from zero to about 70 mM. [J Physiol Sci. 2006;56 Suppl:S70]

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Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel that plays an important role in bicarbonate transport in the exocrine pancreas. Accumulating evidence suggests that CFTR mutations are associated with a subset of patients with chronic pancreatitis. We have identified a new point mutation (L1156F) in exon 18 in patients with chronic pancreatitis. In order to elucidate a disease associated with this mutation, we examined the function of L1156F-CFTR. The mutation was introduced in pCMV-wild type (WT) CFTR plasmid by site directed mutagenesis. Chloride channel activity was measured in HEK293 cells expressing either WT or L1156F-CFTR by whole cell current recording. The amount of CFTR proteins expressed was analyzed by immuno-blotting using an anti-CFTR antibody. When stimulated with 10μM forskolin, WT and L1156F generated a chloride current of 1534±72 and 476±45 pA (n=5), respectively. The introduction of the L1156F mutation did not affect the expression of CFTR protein compared with the WT. In conclusion the L1156F mutation reduces the CFTR chloride current by 69%. The lack of lung and intestinal symptoms and the chronic pancreatitis in these patients further highlight that low activity of CFTR is sufficient for normal lung and intestinal function and the particular susceptibility of the pancreas to mutations in CFTR. [J Physiol Sci. 2006;56 Suppl:S71]

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For gastric acid (HCl) secretion, protons are actively secreted by H⁺,K⁺-ATPase in the apical membrane of gastric parietal cells, but it has not been established what molecule contributes to Cl⁻ secretion. K⁺-Cl⁻ cotransporter (KCC) plays a significant role in epithelial transport and cell volume regulation. Four KCC isoforms have been cloned. KCC1, 3, 4 are widely expressed and KCC2 is neuron specific. However, the expression of KCC isoforms in stomach has not been reported. Here we examined whether the KCC isoforms are expressed in gastric parietal cells. Western blot analysis showed that KCC3 and KCC4 were expressed in isolated gastric mucosa of rats and mice. Immunohistochemistry in the isolated gastric mucosa showed that KCC3 was expressed in the basolateral membrane and KCC4 was expressed in the apical membrane of the gastric parietal cells. Interestingly, KCC3 and KCC4 were abundantly expressed in the parietal cells located at luminal segment of the gland. Because luminal segment parietal cells are much more active in HCl secretion than those of the basal segment, KCC3 and KCC4 may be involved in the mechanism of HCl secretion. We constructed the T-REx system for KCC3 in the LLC-PK1 cells. Tetracycline-induced expression of KCC3 protein significantly increased ouabain-sensitive Na⁺,K⁺-ATPase activity. [J Physiol Sci. 2006;56 Suppl:S71]

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Rat non-gastric H⁺,K⁺-ATPase is highly expressed in the distal colon and may be associated with K⁺ conservation in the colon. But physiological function of human non-gastric H⁺,K⁺-ATPase (ATP1AL1) has not been clarified yet. Here, we have cloned a normal type (NT) and a novel splicing valiant deleting exon4 (Δexon4) of ATP1AL1. Then, the stable cell lines expressing gastric H⁺,K⁺-ATPase β-subunit (gHKβ) were transfected with the pcDNA4/His-ATP1AL1 cDNA (NT or Δexon4) construct. The activities of the K⁺-dependent ATPase and the ⁸⁶Rb⁺ uptake of ATP1AL1 were estimated by subtracting 1 mM ouabain-sensitive activity from 5 μM ouabain-sensitive activity. These activities of ATP1AL1 were also measured by using 100 μM SCH 28080, an inhibitor of gastric H⁺,K⁺-ATPase. We found that Δexon4 is expressed in the plasma membrane of the cells. Similar to the case for NT, the gHKβ was required for expression of Δexon4. We found that Δexon4 has no activities of the K⁺-dependent ATPase and the ⁸⁶Rb⁺ uptake. When NT and Δexon4 were co-transfected into the stable cell lines expressing gHKβ, the ⁸⁶Rb⁺ uptake activity was significantly lower than that in the cells transfected with NT alone. Apparently, Δexon4 had no effect on the level of expression of NT in the cells. These results suggest that Δexon4 exerts a dominant negative effect on NT. [J Physiol Sci. 2006;56 Suppl:S71]

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I_K1 is known to be responsible for the late rapid repolarization phase of action potential in cardiac ventricular myocytes, yet its role in the slow repolarization phase remains unclear. The amplitude of I_K1 is determined by voltage-dependent block of the channel by internal spermine (SPM) and Mg²⁺. During repolarization, the release from the Mg²⁺ block in the presence of SPM induces a significant transient component of I_K1 in voltage-clamp experiments. We developed a new model of the I_K1 channel, which includes the high- and low-affinity modes of blocks by SPM and by Mg²⁺ (Yan & Ishihara, J. Physiol. 563, 2005), and examined the participation of this dynamic gating of I_K1 channel to action potential repolarization by incorporating it into the Kyoto cardiac ventricular cell model. The model shows that the Mg²⁺-induced transient component of I_K1 appears during the slow phase of repolarization. Thus, changes in the I_K1 density significantly alter the action potential duration (APD), as has been demonstrated in experiments. A decrease in the Mg²⁺ concentration or an increase in the SPM concentration prolongs APD by reducing the transient component of I_K1. Under this pathological condition, if the rapid component of delayed rectifier K⁺ current is blocked, APD is markedly prolonged. This model study predicts that the internal spermine and Mg²⁺ are important factors affecting the occurrence of early afterdepolarization and arrhythmia. [J Physiol Sci. 2006;56 Suppl:S72]

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The slow response of myocardial contractility to stretch has been linked to a nitric oxide mediated stretch-induced increase in ryanodine receptor Ca²⁺ release, revealed as an elevated diastolic Ca²⁺ spark rate in rat ventricular myocytes exposed to 10% stretch for 10 min (Vila Petroff et al. 2001). Here, we investigate whether stretch-induced changes in Ca²⁺ spark rate may occur more rapidly. Isolated rat ventricular myocytes were exposed to 10% axial stretch using computer-controlled piezo-manipulated carbon fibres, attached to the centre and one end of a cell. Controlled and reversible stretch was applied selectively to a half-cell only, allowing the non-stretched part to serve as control. Diastolic spark rate was studied using a Zeiss 510 system and software detection of signal deviation from background by > 2 S.D. Within 10 s, axial stretch transiently increased Ca²⁺ spark rate by 31 ± 6.5% (n = 8, p < 0.05), followed by return to background levels within 1 min. The response was not blunted by 1 mM L-NAME (nitric oxide synthase inhibitor; n = 7). We conclude that: i) axial stretch acutely raises diastolic Ca²⁺ spark rate in rat ventricular myocytes; ii) underlying mechanisms differ from those involved in the slow response to stretch. This study is supported by the British Heart Foundation and Eisai Co., Ltd. [J Physiol Sci. 2006;56 Suppl:S72]

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The effect of β-adrenergic stimulation on cardiac Na/Ca exchange current (I_NCX) has been controversial. Recently, 25–400% increase by isoproterenol of I_NCX was reported. To reexamine this effect, we measured I_NCX in voltage-clamped guinea-pig, mouse and rat ventricular cells. When I_NCX was defined as a 5 mM Ni²⁺-sensitive current in guinea-pig ventricular myocytes, 1 μM isoproterenol apparently augmented I_NCX by about 32%. However, this increase was probably due to contamination of the cAMP-dependent Cl⁻ current (I_CFTR), because Ni²⁺ inhibited the activation of I_CFTR by 1 μM isoproterenol, with a half-maximum concentration of 0.5 mM under the conditions where I_NCX was suppressed. 5 or 10 mM Ni²⁺ did not inhibit I_CFTR activated by 10 μM forskolin, an activator of adenylate cyclase, suggesting that Ni²⁺ acted upstream of adenylate cyclase in the β-adrenergic signaling pathway. Furthermore, in a low Cl⁻ bath solution, 1 μM isoproterenol did not significantly alter the amplitude of Ni²⁺-sensitive I_NCX at +50 mV, which was close to the reversal potential of I_CFTR. No change in I_NCX amplitude was induced by 10 μM forskolin. When I_NCX was activated by external Ca²⁺, it was not significantly affected by 1 μM isoproterenol in guinea-pig, mouse or rat ventricular cells. We concluded that β-adrenergic stimulation does not have significant effects on I_NCX in guinea-pig, mouse or rat ventricular myocytes. [J Physiol Sci. 2006;56 Suppl:S72]

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It is well accepted that gap junctions (GJs) play a pivotal role in the intercellular spread of electrical flows between cardiac myocytes. Dysfunction of GJs can thus be one of major arrhythmogenic factors. In this study, we have obtained the evidence that in both type I and II diabetic hearts, myocardial intercellular communication through GJs is impaired via PKC-mediated hyper-phosphorylation of the GJ protein Cx43. Western blot and immunohistochemical analyses of the PKC-activated diabetic hearts indicated that the expression level of Cx43 is significantly reduced compared with control hearts, with altered distribution of sparse or sporadic pattern at the intercalated disk. These alterations were ameliorated by treatment with lysosomal inhibitors as well as PKC inhibitors, but could not be prevented by the proteosmal inhibitor ALLN. These results strongly suggest that facilitated lysosomal degradation of Cx43 via PKC-mediated hyper-phosphorylation may underlie the down-regulation of Cx43 protein in rat diabetic hearts. This mechanism may in part account for the reported vulnerability of rat diabetic hearts to ventricular fibrillation. [J Physiol Sci. 2006;56 Suppl:S73]

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Present study was designed to demonstrate intracellular radial gradients of myoglobin oxygen saturation and ATP in single cardiomyocytes with elevated oxygen demand. Intracellular ATP concentration was indirectly assessed from intracellular free Mg²⁺ concentration using a Mg²⁺ sensitive ratiometric fluorescent dye mag-fura-2 (MF2). A bright field fluorescence microscopy was newly manufactured for simultaneous measurements of myoglobin light absorption (spectrophotometry) and MF2 fluorescence (fluorometry). Uncoupler of oxidative phosphorylation (1 μM CCCP) was used to increase oxygen flux. While significant intracellular gradients of myoglobin oxygen saturation was demonstrated in CCCP treated cells at physiological extracellular Po₂, no significant heterogeneity was found in MF2 fluorescence. In contrast, in cardiomyocytes treated with 0.5 mM iodoacetamide (a blocker of creatine kinase) in addition to CCCP, gradients of MF2 fluorescence from the sarcolemma to the cell core (indicating radial gradients of ATP) were demonstrated. Such MF2 fluorescence gradients were not demonstrated when extracellular oxygen concentration was elevated to 20%. From these results, it is concluded that significant gradients of ATP may be produced in the isolated single cardiomyocyte when mitochondrial oxygen consumption is moderately elevated at physiological extracellular Po₂. However, such potential ATP heterogeneities are effectively buffered in the normal cardiomyocyte by ATP supplementations from creatine phosphate. Thus, present results propose a new function of creatine kinase in the myocardium. This study was supported by JSPS KAKEN #15390061. [J Physiol Sci. 2006;56 Suppl:S73]

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We previously demonstrated that excitatory agonists such as noradrenaline (NA) and membrane depolarization induce Ca²⁺-dependent activation of Rho GTPase in vascular smooth muscle (VSM) cells, resulting in inhibition of myosin phosphatase (MP) through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit MYPT1/MBS. We found that phosphoinositide 3-kinase class IIα (PI3K-C2α) plays an essential role in NA-induced Rho activation and contraction in differentiated VSM primary cultured-cells. In the present study we show that ionomycin, a Ca²⁺ ionophore, induced contraction with stimulated phosphorylation of MYPT1. Ionomycin-induced MYPT1 phosphorylation and contraction was inhibited by the Rho kinase inhibitor Y-27632. Silencing PI3K-C2α, but not PI3K p110α, expression by small interfering RNA (siRNA) in differentiated VSM cells inhibited ionomycin-induced phosphorylation of MYPT1, consequent reinforcement of 20-kDa myosin light chain (MLC) phosphorylation and contraction. Consistent with this, the PI3K inhibitors Wortmannin and LY294002 inhibited both MLC phosphorylation and contraction. These findings indicate an essential role of PI3K-C2α in Ca²⁺-dependent, Rho/Rho kinase-mediated negative control of MP and VSM contraction. [J Physiol Sci. 2006;56 Suppl:S73]

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Background: Identification of dynamic and static characteristics of a given biological system promotes the understanding of the system behavior under a variety of circumstances. Purpose: To estimate the dynamic and static characteristics of a baroreflex neural arc from pressure input to efferent sympathetic nerve activity, we developed a system identification method using a neural cascade. Method: A "neuron" used in a neural network can represent the dynamic linear element followed by a nonlinear transfer function. By connecting two neurons in series, we can represent a system comprised of dynamic-linear (L1), static-nonlinear (NL), and dynamic-linear (L2) subsystems. Because the contamination of noise to the observed output resulted in biased estimates of the system characteristics, we added an iterative noise cancellation procedure where the noise was estimated by an autoregressive model. Results: In a simulation study, the neural cascade effectively identified the dynamic and static characteristics of an L1-NL-L2 system. The baroreflex neural arc is known to have derivative characteristics followed by a sigmoidal nonlinearity. When applied to the actual input-output data of the baroreflex neural arc obtained from rabbits, the neural cascade could identify the derivative characteristics followed by the sigmoidal nonlinearity. Conclusion: The neural cascade proposed in the present study may provide a useful method to simultaneously identify the dynamic and static characteristics of a biological system. [J Physiol Sci. 2006;56 Suppl:S73]

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Estrogen deficiency in the menopause is associated with an increased cardiovascular risk. Endogenous estrogen has been suggested to exert vasoprotective effects through decreasing vascular oxidative stress. To investigate the mechanism of the decreasing in oxidative stress by estrogen, we examined superoxide production and antioxidant enzyme expression in aorta in ovariectomized Dahl salt-sensitive (DS) rats. Female DS rats (8 weeks old) were ovariectomized (OVX group) or sham-operated (sham group). Estrogen pellets were subcutaneously implanted in ovariectomized rats for estrogen treatment (OVX + E group). After 4 weeks of salt-loading (8% NaCl diet), blood pressure was increased in OVX group compared with sham and OVX + E groups. Superoxide production in aortic ring was higher in OVX group than in sham and OVX + E groups. Increase in superoxide production was abolished by pretreatment with diphenyleneiodonium, a NADPH oxidase inhibitor. Expression of mRNA of p22phox, a NADPH oxidase subunit, increased in aorta from OVX group compared with sham group. In contrast to p22phox, mRNA expressions of antioxidant enzymes extracellular superoxide dismutase (ecSOD) and glutathione peroxidase (GPX) were decreased in OVX rats. Expression levels of p22phox, ecSOD and GPX in OVX + E rats were not different from that in sham group. These data suggest that estrogen deficiency in ovariectomized DS rats enhances oxidative stress through increased NADPH oxidase expression and decreased antioxidant enzymes, and promotes vascular injury by salt-loading. [J Physiol Sci. 2006;56 Suppl:S74]

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Acute hypoxia elicits a biphasic ventilatory response, initial augmentation and subsequent depression. Although oxygen-sensitive channels of type I cells in the carotid body are considered to be involved in the initial augmentation, the underlying cellular mechanism for the subsequent depression, hypoxic ventilatory decline (HVD), has not been fully elucidated. The purpose of the present study is to examine the role of an ATP-sensitive potassium channel, Kir6.2, in the hypoxic ventilatory response including HVD in the mouse. We serially measured minute ventilation volume (Ve) of the Kir6.2-knockout mouse (Kir6.2-/-: n = 5) exposed to hypoxia (12% O₂ in N₂: 10min) in an unanesthetized unrestrained state by whole body plethysmography in the 2nd and 4th postnatal weeks. Percent changes from the baseline Ve in the room air were calculated and compared with that in the C57BL/6 mouse (n = 10). In the 2nd postnatal week, there was no difference in the hypoxic ventilatory response between the C57BL/6 and Kir6.2-/- mice. Meanwhile, in the 4th week, the initial augmentation lasted longer, and HVD was much weaker in the Kir6.2-/- than in the C57BL/6. It is concluded that Kir6.2 is involved in the hypoxic ventilatory response including HVD in an age-dependent manner in the mouse. [J Physiol Sci. 2006;56 Suppl:S74]

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Our previous studies showed that frequent yawns can be evoked by microinjection of L-glutamate into the paraventricular nucleus (PVN) in an anesthetized, spontaneously breathing rat. The yawning response was characterized by an arousal shift in the ECoG to lower voltage and faster rhythms. We focused the present study upon frequent yawns in human. Yawns occurred once a few minutes for more than 5 minutes in this study. We monitored electroencephalogram (EEG) and cerebral blood flow (CBF) using near-infrared spectroscopy (NIRS). We found a shift in the EEG to lower voltage and faster rhythms and an increase of CBF in ventromedial prefrontal cortex (PFC) during and immediately after yawning responses. These results suggest that the activation of ventromedial PFC may be associated with an arousal/yawning response in human. [J Physiol Sci. 2006;56 Suppl:S74]

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We investigated metabolic changes in prefrontal and parietal cortices and EEG changes during a long-term breath holding (BH) in professional divers. He/she performed BH for 2 to 7 minutes following a preparatory period of approximately 5 minutes. Such BH procedures were repeated 3 times in this study. We measured metabolic changes in cerebral cortex using near-infrared spectroscopy (NIRS) and made spectral analysis of EEG before, during and after BH. Concentration of deoxygenated hemoglobin (deoxyHb) showed a gradual increase during BH in both prefrontal and parietal cortices. There is a linear relationship between the maximal level of deoxyHb and the duration of BH. In contrast, concentration of oxygenated hemoglobin (oxyHb) of BH decreased along with prolonged duration of BH. Voluntary abdominal breathing (VAB) was performed during the preparatory period before BH. Spectral analysis of EEG was focused on the high-frequency alpha (HF-alpha) band (10-13Hz) in this study. The higher power of HF-alpha band was observed before and immediately after the onset of BH, and thereafter the HF-alpha power exhibited a gradual decrease until the end of BH. Since previous study showed that VAB produced increase in HF-alpha power and urinary 5-HT level (fumoto et al, 2004), we suggest that the higher HF-alpha power evoked before BH may contribute to the tolerance to cerebral hypoxia during BH. [J Physiol Sci. 2006;56 Suppl:S75]

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Ventilatory long-term facilitation (vLTF) following acute intermittent hypoxia (AIH) has been reported to variable extent in unanesthetized rats. However, none of these studies reported sleep-state, a critical variable in many physiological functions. We hypothesized that vLTF would be preferentially expressed in sleeping vs. awake Lewis rats following AIH. The sleep-wake state of unrestrained rats was determined from implanted EEG and nuchal EMG electrodes. Tidal volume (VT), frequency (f), minute ventilation (VE) and CO2 production (VCO2) were determined in unanaesthetized male Lewis rats via plethysmography before, during and after AIH (five, 5-min exposures, 10.5% O2; 5-min normoxic intervals) or acute sustained hypoxia (25-min exposures, 10.5% O2, ASH). VE, VT and f in quiet wakefulness (QW) or NREM sleep were normalized to its own baseline value during the corresponding state during baseline, pre-hypoxia conditions. LTF was observed in VE after AIH, but not ASH. Following AIH in NREM, VE gradually increased and reached maximum level at 20 min post-hypoxia, remaining at that level for at least 60 min (26.6±5.2% baseline). The main contributor to vLTF was VT (13.5±2.3%), with a lesser increase in f (7.4±1.7%). The corresponding increase in VE/VCO2 was 35.5±2.4% baseline. In QW, significant vLTF was not observed. The duration, magnitude, and pattern in vLTF in NREM were similar to phrenic LTF in anesthetized rats. In conclusion, vLTF is highly state and pattern sensitive in unanesthetized rats. (Supported by NIH HL65383, HL07654, HL68255). [J Physiol Sci. 2006;56 Suppl:S75]

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Chemical compounds in breath originate mainly from volatile metabolic products in health and diseases. Among them, hydrogen and methane are believed to represent the conditions of gastro-intestinal systems in the presence of bacterial flora. On the other hands, carbon monoxide and nitric oxide are well-known bioactive chemicals to play important roles in physiological and pathophysiological conditions. Because matrix analysis based on simultaneous measurements of exhaled chemicals with such low mass numbers may give us important information of physical conditions, we conducted the following experiment.Nine hundreds and forty two subjects aged from 20 to 88 years old volunteered for the present study. After tooth brushing and gargling, fasted subjects breathed purified artificial air via a mouthpiece in a sitting position for 12 min, during which exhaled air was collected via one-way valve into the Douglas bag. Minute ventilation volume and concentrations of carbon dioxide and oxygen in exhaled air were monitored by respiratory metabolic analyzer with the chemical sensors. Concentrations of hydrogen, methane, carbon monoxide and nitric oxide in the bag were analyzed by gas chromatography and chemiluninescence methods. We will present the summarized results of the relationships between minute expired volumes of these compounds and such physical conditions as age, gender, laboratory data, scores of health-related questionnaires, diseases and disorders by using the multivariate analysis. [J Physiol Sci. 2006;56 Suppl:S75]

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Thixotropy is a passive property of the skeletal muscle that depends on the muscle's immediate history of contraction and length change. We showed that inspiratory muscle thixotropy affects the end-expiratory position of the rib cage. The present study aimed to test whether changes in end-expiratory chest wall volume (Vcw) occurs after thixotropic inspiratory muscle conditioning in normal subjects (n = 32). We first examined effects of the conditioning on end-expiratory Vcw of succeeding five breath cycles with respiratory induction plethysmography. Subjects participated in the conditioning at three different Vcw (60% inspiratory capacity [IC] + end-expiratory Vcw of baseline breathing [EEB], EEB, and residual volume [RV]) giving one of two levels of inspiratory effort (no effort or maximal inspiratory effort) with airway closure in the sitting position. End-expiratory Vcw increased after conditioning at 60% IC + EEB and decreased after conditioning at RV. We then measured the time course of changes in spirometrically determined IC, which confirmed thixotropic changes in end-expiratory Vcw. A decrease in the IC was found at 60 s after conditioning performed at the higher volume. However, the decrease disappeared until 180 s. Conditioning at the lower volume was followed by an increase in the IC, which was maintained even after 180 s. Furthermore, the thixotropic inflation/deflation of the chest wall was proved by the helium-dilution FRC and esophageal pressure measurements. In conclusion, thixotropy conditioning of inspiratory muscles changes end-expiratory Vcw. [J Physiol Sci. 2006;56 Suppl:S75]

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We examined whether the chemoreceptor reflex in prepro-orexin knockout mice was blunted or not, and if so, whether supplementation of exogenous orexin restore the abnormality. In addition, we studied whether pharmacological blockade of orexin in the wild-type mice resulted in a similar abnormality. A cannula for intracerebroventricular injection to the lateral ventricle was implanted to the isoflurane-anesthetized mice together with electrodes for recording electroencephalogram and electromyogram. Ventilation was recorded by whole body plethysmography after recovery period of at least 7 days. After recording of baseline breathing for 1 hr, orexin-A, -B, an orexin receptor antagonist, or vehicle was intracerebroventriculary injected and hypercapnic or hypoxic gas mixture was introduced into the recording chamber for 10 min. Data were examined for only awake period because sleeping distorts chemoreflex sensitivity. Hypercapnic ventilatory responses but not hypoxic responses were attenuated in orexin knockout mice as compared to those in the wild-type littermates. Similar abnormality was reproduced in wild-type mice treated with orexin antagonist. Intracerebroventricular injection of orexin partially restored the hypercapnic chemoreflex in the mutant mice. Our findings suggest that orexin plays a crucial role for CO₂-sensitivity at least during awake periods. [J Physiol Sci. 2006;56 Suppl:S76]

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Background: Vascular endothelial cells (VECs) contribute to keep the patency of vasculature through the regulated expression and secretion of various molecules having either anti-coagulatory or high fibrinolytic activity. tPA, the primary PA in the vasculature, is secreted from VECs as an active form and express fibrinolytic activity in blood. In blood there also exist its specific inhibitor of PAI-1. Though the impaired tPA secretion as well as the elevated plasma concentration of PAI-1 are considered to be risk factors for thrombosis, precise mechanism underlying in tPA secretion is not clarified. Here, we analyzed the dynamics of tPA secretion from its containing granules and its modulation by PAI-1 using total internal reflection fluorescence microscopy(TIRFM). Method: An established cell-line of VECs was cultured and transfected with tPA-GFP. The dynamics of tPA-GFP secretory granules near the plasma membrane was analyzed by TIRFM. Results: 1) The dynamics of tPA-GFP granules including its opening and tPA-GFP secretion were successfully monitored by TIRFM. 2) Once tPA-GFP granules open, they kept open and tPA-GFP was released slowly. The secreted tPA-GFP was detected as tPA-GFP-PAI-1 complex in cultured medium. 3) The velocity of tPA release was facilitated by supplementary added PAI-1, which resulted in the increase in tPA-PAI-1 complex in supernatant. Conclusion: tPA-GFP is beneficial tool to investigate its exocytotic dynamics. PAI-1 seems to facilitate tPA release as an inactive complex-form, which suppresses fibrinolytic activity on VECs. [J Physiol Sci. 2006;56 Suppl:S76]

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A novel cell separation method which can continuously separate the cells according to their densities has been developed for application to the transfusion medicine. In the past the performance of this method was examined on separation of human buffy coat, peripheral blood and co-cultured cell suspensions. Flow cytometry analysis on separation of human buffy coat revealed that CD34-positive cells, which were assumed to be hematopoietic progenitor cells, were distributed around density = 1.065. Recently, the colony-forming cell assay was performed on human hematopoietic progenitor cells separated from peripheral blood by the present method. Five polymer media with densities of 1.060, 1.065, 1.070, 1.075 and 1.080, prepared with sterile isotonic Percoll media and PBS, were used for the separation, and the fractionated cells were cultured in a methylcellulose-based medium containing hSCF, hGM-CSF, hIL-3 and hEPO. Colony-forming unit-erythroid (CFU-E) were appeared on day 7. Burst-forming unit-erythroid (BFU-E), CFU-GM (granulocyte, macrophage) and CFU-GEMM (granulocyte, erythroid, macrophage, megakaryocyte) were observed on day 14. BFU-E, CFU-GM and CFU-GEMM were colonies that were found in the fourth fraction (density = 1.075), among those no CD-34 positive cells being detected by flow cytometry analysis. These results suggest that the method might enable to harvest many types of human hematopoietic progenitor cells according to minute differences in their densities. [J Physiol Sci. 2006;56 Suppl:S76]

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Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin. In this report, the interaction of nonadecapeptide derived from staphylokinase with plasminogen, was investigated. The effects of synthetic peptides on plasminogen activation were estimated by using a chromogenic substrate assay and ¹²⁵I-labeled plasma clot lysis assay. The binding of peptides to Glu-plasminogen was estimated by using IAsys resonant mirror biosensor. The synthetic nonadecapeptide (SAK22-40) corresponding to Glu22-Leu40 of SAK amino acid sequence did not show any PA activity in the presence of plasmin. However, SAK22-40 enhanced Glu-plasminogen activation by t-PA. SAK22-40 bound to plasminogen in a concentration-dependent manner. Although this binding ability was not inhibited in the presence of anti-K1-K3 (plasminogen fragment containing kringle 1 to 3 domains) IgG or anti-K4 (plasminogen fragment containing kringle 4 domain) IgG, it was partially inhibited by anti- mini plasminogen IgG. The substitution of Lys35 to Ala in SAK22-40 did not show the enhancement of PA activity by t-PA. The t-PA activity was enhanced in the presence of cultured endothelial cells, and it was further enhanced by SAK22-40. These findings indicate that the synthesized nonadecapeptide, SAK22-40, binds to B-chain of Glu-plasminogen and enhances PA activity by t-PA. The mechanism by which SAK22-40 enhances t-PA activity seems to be different from that by which COOH-terminal Lys of fibrin does. [J Physiol Sci. 2006;56 Suppl:S77]

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In mammals including humans, a brain-gut hormone, cholecystokinin (CCK) mediates the satiety effect via CCK-A receptor (R). However, we generated CCK-AR gene deficient (-/-) mice and found that the daily food intake, energy expenditure, and gastric emptying did not change compared with those of wild-type mice. We also generated CCK-BR (-/-), CCK-AR (-/-) BR (-/-) mice. Daily food intake and ghrelin contents in the blood and the stomach were investigated. Male mice at 6-8 months of age were used. Mice, deprived of food for 18 hr with free access to water, were injected i.p. (0.1ml/mouse) with either vehicle or CCK-8 (0.3, 1.0, and 3.0 n mol/mouse). Thereafter, daily food intake was measured. Additional animals were sacrificed by guillotine, the blood was obtained, and the stomach was removed to measure ghrelin contents. Administration of CCK-8S significantly decreased food intake in wild-type and in CCK-BR (-/-) mice. However, no significant inhibition was observed in CCK-AR (-/-) and in CCK-AR (-/-) BR (-/-) mice. When mice injected with vehicle were compared, food intake in CCK-AR (-/-) BR (-/-) mice was significantly lower than that in wild-type and CCK-BR (-/-) mice. Moreover, the ghrelin contents in the blood as well as in the stomach were significantly lower in CCK-AR (-/-) BR (-/-) mice than wild-type mice. CCK-Rs may be involved in the regulation of ghrelin biosynthesis and secretion. [J Physiol Sci. 2006;56 Suppl:S77]

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Despite the importance of short chain fatty acids (SCFA) in maintaining the ruminant physiology, the mechanism of SCFA absorption is still not fully studied. Therefore, this study was carried out to elucidate the mechanism of SCFA transport in the caprine rumen epithelia by investigating the possible involvement of monocarboxylate transporter 1 (MCT1) as well as to delineate the precise localization and the level of MCT1 protein along the caprine gastrointestinal tract. To achieve these objectives, molecular studies including: RT-PCR, Western blotting, and immunohistochemistry as well as functional studies (in vivo and in vitro) were used. RT-PCR studies revealed the presence of mRNA encoding for MCT1 in all regions of the caprine gastrointestinal tract. By immunoblotting analysis on membrane protein extract, a 45-kDa-band corresponding to MCT1 was detected in all parts of the forestomach and abomasum as well as along the entire length of the intestine. The MCT1 protein level was found most abundantly in forestomach, at intermediate levels in large intestine and abomasum, and lower levels in small intestine. MCT1 is immunolocalized mostly to the stratum basale and stratum spinosum of the caprine rumen epithelia. The MCT1 inhibitor, pCMB exerted a significant influence on acetate flux in vitro as well as short-chain fatty acids absorption in vivo. The results obtained provide evidence, for the first time, for transepithelial transport of SCFA via MCT1 across the caprine ruminal epithelia. [J Physiol Sci. 2006;56 Suppl:S77]

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Disordered eating has been investigated extensively. The large bowel has been studied less. The objective is to find its influence on appetite. Particularly, the relationship between large bowel fermentation and appetite is not known. Hundred patients with no or some colons and the age sex BMI matched controls were given a breakfast followed by lactulose. The hydrogen breath and appetite levels were determined. Energy intake of all patient groups was less than their matched controls. At 180 min, in proctocolectomised group less hunger and higher satiety levels than that of controls group were observed. The satiety levels of subtotal/hemi-colectomised significantly less at 240 and 330 min. The breath hydrogen level at -30 min and 60 min is positively correlated with the satiety levels at 120 min proctocolectomy group. The breath hydrogen level -30 min and 60 min is negatively correlated with the satiety levels at 240 min subtotal removal of the rectum group. The breath hydrogen level -30 min is positively correlated with the satiety levels at 180 min, 300 min and 330 min in total rectal removal group. The subjects who have had large bowel surgery had lower hunger and higher satiety levels than controls implicated that large bowel plays a role in appetite regulation. The breath hydrogen influences the satiety in proctocolectomy and total rectal removal positively and negatively in sub/hemi colectomy, and the hunger only in sub/hemi colectomy in one time point. [J Physiol Sci. 2006;56 Suppl:S78]

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Alcoholic beverages contain several congeners. N-methyltyramine (NMT) was isolated from beer as a factor to stimulate gastric acid secretion. We examined whether NMT stimulated pancreatic secretion in conscious rats. Methods: Male Wistar rats (330g) were prepared with cannulae for draining bile and pancreatic juice separately, with two duodenal cannulae, with a gastric cannula, and with an external jugular vein cannula. The rats were placed in modified Bollman-type restraint cages. After the 4-day recovery period, the experiments were conducted in unanesthetized rats. Different concentrations of NMT solutions (5, 25, and 50 micro-g/kg/3ml/3min) were infused into the stomach. NMT content in beer was 2mg/L, so that if 50kg body weight man consumes a bottle of beer, 25 micro-g/kg NMT will be ingested. To examine the mechanism, the effects of proton pump inhibitor, CCK-BR antagonist, CCK-AR antagonist and atropine were administered prior to the NMT infusion. The effect of intravenous infusion of NMT (2.5 micro-g/rat) was also determined. Results: Intragastric administration of NMT significantly increased pancreatic exocrine secretion in a dose dependent manner. Atropine abolished the stimulatory effect of NMT, but others did not. Intravenous infusion of NMT did not affect pancreatic secretion. Conclusions: NMT stimulates pancreatic secretion via cholinergic gastro-pancreatic reflex. Therefore, the stimulatory effect of beer on pancreatic secretion was produced by not only ethanol but also a congener, NMT. [J Physiol Sci. 2006;56 Suppl:S78]

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We found that organic solutes potently compress the myofilament lattice of skinned skeletal muscle. This compressing effect cannot be ascribed to the ordinary osmotic compression due to the filtration of the solutes by the lattice, because some solutes smaller than ATP in molecular weight could effectively compress the muscle. Another candidate for the compressing force comes from the entropic aggregating force of the macromolecules constituting the myofilament lattice. As Asakura-Oosawa theory and its derivatives describe, the aggregation of macromolecules in the presence of smaller particles depends on the exclusion of the particles from the very vicinity of the macromolecule surface. To estimate the effective exclusion volume for the organic solutes in the myofilament lattice, we examined the compressing effects of a series of organic molecules from mono- and poly-hydric alcohols. The results clearly indicated that the compressing efficiency of the alcohols depends primarily on the number of CH₂ group that is not directly attached by the hydroxyl group. That is, the unitary component for the compressing effect is a single CH₂ group. Since the molecular size of the CH₂ group is very close to that of the water molecule (OH₂; 0.31 nm³), any macromolecular aggregation force due to the exclusion of solutes would not work, unless each CH₂ group forms a larger complex with surrounding water molecules or water molecules form a stable cluster larger than the bare CH₂ group. The possibility of the CH₂-OH₂ complex formation and the stable water cluster formation will be discussed. [J Physiol Sci. 2006;56 Suppl:S78]

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In the present studies, the diameter of single skeletal myofibrils was measured under various physiological conditions by AFM. Our AFM instrument is not suitable for precise diameter measurements of specimen having the diameter range of micrometer like single myofibrils. Therefore, we modified our AFM system by incorporating precise piezo systems so as to be useful for the present purpose. Single myofibrils were prepared by homogenizing glycerinated muscle fibers of the rabbit psoas muscle. Myofibrils were fixed on the surface of cover slip coated with aminosilanes. A small glass sphere was adhered to the tip of AFM cantilever to smoothly touch to the surface of myofibrils. The piezo stage could be moved in the horizontal and the vertical directions in the range of 10 μm with the accuracy of ± 2 nm. The diameter of myofibrils was determined by comparing the distance moved for the glass sphere fixed to AFM cantilever to touch the top surface of myofibrils and that to touch the surface of the cover slip. The error of the diameter measurements was < ± 5 nm. The diameter of single myofibrils thus obtained was 1.119 ± 0.042 μm (n=5) in the relaxed state and 1.026 ± 0.039 μm (n=5) in the rigor state. Thus the diameter of myofibrils in the rigor state was smaller by 8% than that in the relaxed state. We also further examined the diameter of single myofibrils under other physiological states. The results thus obtained will be compared with the data for the lattice spacing of actomyosin filaments of muscle fibers reported by X-ray diffraction studies. [J Physiol Sci. 2006;56 Suppl:S79]

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It is generally accepted that the contraction of muscle fiber is reversibly inhibited by 2,3-butanedione monoxime (BDM). Since the muscle contraction takes place by the interaction between thin actin filaments and myosin heads of thick filaments, it was suggested that BDM directly inhibits the actin-myosin interaction. But detail mechanisms of the suppression of contraction by BDM are still unknown. In this study, we examined how BDM affects the transverse stiffness of myofibrils and muscle fibers by AFM. Myofibrils and muscle fibers prepared from psoas muscle of rabbit were used. The transverse stiffness was measured as in our previous studies. Modified AFM cantilevers having a glass rod attached to the tip of AFM cantilever were prepared and used for measurements to minimally alter the actin-myosin lattice structure. The tip of AFM cantilever was approached to the surface of preparations attached to coverslip, and force-curves were measured to obtain the transverse stiffness. The transverse stiffness of myofibrils decreased in the order of rigor state > contracting state > contracting state (+BDM) > relaxed state. Remarkably the transverse stiffness of myofibrils was much greater in contracting state (+BDM) than in relaxed state. For muscle fibers, the transverse stiffness decreased in the same order as above for myofibrils. These results indicate that cross-bridges are formed in contracting state (+BDM), and suggest that the suppression of muscle contraction by BDM is not simply due to the inhibition of cross-bridge formation. [J Physiol Sci. 2006;56 Suppl:S79]

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Interstitial cells of Cajal distributed in myenteric region (ICC-MY) generate spontaneous electrical activity called pacemaker potential in gastrointestinal tract. Pacemaker potentials recorded from mouse small intestine have two components: rapidly rising primary component and following plateau component. In the present study the effects of temperature on the generation of pacemaker potentials were studied with conventional microelectrode techniques. Elevation of temperature increased the frequency and maximum rate of rise (dV/dt_max) of pacemaker potentials and decreased their duration, without affecting the resting membrane potential and amplitude. CPA (3μM), an inhibitor of internal Ca pump, abolished pacemaker potentials at around 28°C, while CPA only shortened the duration of pacemaker potentials with the depolarization of the membrane at around 40°C. KCN (30μM), an inhibitor of metabolic process in mitochondria, decreased the frequency of pacemaker potentials without changing the amplitude and dV/dt_max. These results suggest that the sensitivity to temperature is different between two components (primary and plateau) of pacemaker potentials. The primary component may be generated by the activation of temperature sensitive Ca permeable channels. On the other hand, the plateau component seems to be generated by temperature insensitive mechanisms. The results also suggest that mitochondrial function seems to be uninvolved in the formation of two components of pacemaker potentials. [J Physiol Sci. 2006;56 Suppl:S79]

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In contrast to cardiac myocytes, sympathetic stimulation does not largely enhance L-type Ca²⁺ channel current in smooth muscle cells. In the present study, we assessed possible mechanisms underlying this discrepancy, using a whole-cell clamp technique. In guinea-pig detrusor cells, only L-type Ca²⁺ channels occur. During depolarizations of large positivity, the conformation of the majority of Ca²⁺ channels is converted from the normal (O₁) to a second open state (O₂), in which Ca²⁺ channels do not, or only slowly inactivate during depolarization. This feature of the O₂ state produces U-shaped inactivation. In order to estimate the population of Ca²⁺ channels that can be converted to the O₂ state, we applied a paired pulse protocol: Test steps with and without preconditioning step (+80 mV, 4s) were alternately applied. Extracellular application of genistein decreased the amplitudes of both conditioned and unconditioned test inward currents (A_cond and A_uncond), accompanied by significant reduction of A_cond/A_uncond, while genistin, an inactive analogue, did not. Intracellular application of genistein caused similar or more pronounced effects, when ATP was removed from the patch pipette. This result is consistent with the fact that ATP antagonizes the inhibitory effect of genistein on tyrosine kinase activity. It is concluded that even under normal conditions smooth muscle L-type Ca²⁺ channels are already in a "stimulated mode° due to a tyrosine-kinase-related mechanism(s). [J Physiol Sci. 2006;56 Suppl:S79]

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Ca_V1.2 (α1C) and Ca_V1.3 (α1D) are pore-forming subunits of cardiac L-type Ca²⁺ channels. We have previously shown that Ca_V1.3 activates and inactivates at more negative voltages than those of Ca_V1.2, thus contributing to the threshold and the duration of the pacemaker action potential in SA nodal cells. To elucidate molecular regions underlying the unique voltage-dependence of Ca_V1.3 in comparison with Ca_V1.2, we examined chimeras of Ca_V1.2 and Ca_V1.3, and found that repeat II of Ca_V1.3 contains a critical domain for the negative shift of its voltage-dependence. In the present study, we further localized the critical regions of the unique voltage-dependence in repeat II of Ca_V1.3. We introduced a series of point mutations in Ca_V1.2 and Ca_V1.2/Ca_V1.3 chimera. The mutant Ca²⁺ channels were transiently expressed in BHK6 cells and analyzed in patch-clamp experiments. As a result, F618L at the outside of three Rs in IIS4 reversed the negative shift of the steady-state inactivation of Ca_V1.2/Ca_V1.3 chimera channel, indicating that F618 is one of regions underlying the voltage-dependence of Ca_V1.3. These results suggest that the difference in a single amino acid in IIS4 between Ca_V1.2 and Ca_V1.3 confers the unique voltage-dependence in cardiac L-type Ca²⁺ channels. [J Physiol Sci. 2006;56 Suppl:S80]

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The synthetic ciguatoxin, CTX3C has four distinct effects on Na channels:1) to speed up time-to-peak Na current (I_Na), 2) to suppress peak I_Na amplitude, 3) to shift the activation curve in the hyperpolarizing direction, and 4) to delay recovery from "slow inactivation." In this study, we explored possible sites modulating CTX effects. Mutant channels N434A, L437A or A438K lacked effect 3 of CTX3C. These sites in D1S6 selectively modulate activation mechanisms via CTX binding. In separate experiments, we systematically replaced positively charged amino acids (Arg or Lys) with a neutral residue (Gln) in each S4 segment of the four domains. In all domains, neutralization of positive charges did not suppress the hyperpolarizing shift of the activation curve induced by the toxin (effect 3). However, CTX3C(3μM) did not suppress I_Na in the four mutants with neutralized charges in D2 (lack of effect 2). Interestingly, some of the D2 mutants even exhibited an increase in I_Na in the presence of CTX3C: I_Na grew progressively larger as the location of the mutations approached the extracellular face of the membrane. CTX3C is a membrane-spanning molecule having a length of 30 Å and possessing a relatively constrained structure. Thus, CTX3C may affect the voltage sensor by accessing the sodium channel from its exterior surface through the membrane lipid phase. [J Physiol Sci. 2006;56 Suppl:S80]

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Heterotetramers of two kinds of KCNQ subunits form voltage-dependent K channels called M channels in the brain neurons. The outward current from M channels repolarizes the action potential. It is reported that point mutations in KCNQ3 cause a neuronal channelopathy. In this study, we primarily examined the functional consequences by mutations. Whole-cell currents were measured from the mutant KCNQ3 channels expressed in HEK293 cells. 1-1) Homomeric channels composed of mutant KCNQ3 lacked currents. 1-2) Heteromeric channels composed of wild-type KCNQ2 and mutant KCNQ3, which were mimicked to the native M channels, were lower in conductance than those composed of wild-type KCNQ2 and wild-type KCNQ3. 1-3) The activation curve of heteromeric channels composed of wild-type KCNQ2 and mutant KCNQ3 was shifted to the right than that composed of wild-type KCNQ2 and wild-type KCNQ3. We also reconstituted the three-dimensional protein structure of the mutant K channels. 2-1) The selectivity filter of wild-type KCNQ3 channel was structurally supported by chemical bonds. 2-2) The mutation, however, lead to a loss of chemical bonds. Such a structural alteration may result in the lower conductance in mutant KCNQ3 channels. [J Physiol Sci. 2006;56 Suppl:S80]

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An inwardly rectifying K channel with inward conductance of 40 pS is the most frequently observed K channel in cultured human proximal tubule cells. We have previously reported that the activity of this K channel was modulated, at least in part, by nitric oxide (NO) which was mainly derived from inducible NO synthase (iNOS) in these cells. Since cytokines are known to enhance the expression of iNOS mRNA, we explored the effects of interleukin-1β (IL), tumor necrosis factor-α (TNF) and interferon-γ (IFN) on the K channel activity, using the patch-clamp technique and RT-PCR. After 24-h incubation of cells with each cytokine, iNOS mRNA was significantly increased by IFN (100 U/ml) but not by IL (10 U/ml) and TNF (200 U/ml). In cell-attached patches using the IFN-treated cells, a NOS substrate, L-arginine (500 μM) suppressed channel activity, whereas a NOS inhibitor, L-NAME (100 μM) stimulated it. These observations were in sharp contrast to the case with control cells where L-arginine was stimulatory and L-NAME was suppressive. Acute effects of cytokines on channel activity were tested in cell-attached patches using control cells. Addition of IL to the bath suppressed channel activity, which was not restored by the subsequent addition of L-arginine. On the other hand, addition of IFN stimulated channel activity and this stimulatory effect was not abolished by L-NAME. TNF had little effect on channel activity. These results suggested that IFN affected K channel activity through the NO-dependent and -independent pathways whereas the effect of IL was NO-dependent. [J Physiol Sci. 2006;56 Suppl:S81]

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Cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial fluid and electrolyte transport as a Cl⁻ channel and also as a regulator of other transporters. CFTR channel function is widely deviated among several different CFTR epithelia whereas any CFTR subtype has never been identified. Recent evidence suggests that ion channels can target to membrane lipid raft and associate with other regulatory proteins. In this study, we have investigated the role of cholesterol, an important component of lipid raft, in the regulation of CFTR channel function using patch-clamp technique. In cell-attached patches, cholesterol-depletion by cyclodextrin treatment inhibited CFTR channel activity Po from 0.35±0.03 to 0.15±0.02 with decreasing burst duration (T_b) from 2.3±0.3 to 0.31±0.03 msec and interburst duration (T_ib) from 3.4±0.6 to 1.8±0.03 msec (n = 5). On the contrary, cholesterol-enrichment by treatment with cyclodextrin-solubilized cholesterol slowed the CFTR channel gating with prolonging T_b to 5.6±1.1 msec and T_ib to 10.8±3.5 msec (n = 5). These results indicate that cholesterol plays a critical role in regulation of CFTR channel kinetics. We also found that CFTR channels expressed in Hi-5 insect cell line which is known not to synthesize cholesterol exhibited a similar gating kinetics to those in cholesterol-depleted NIH3T3 cells. We speculate that CFTR channels localize to lipid raft and are tissue-specifically modulated. [J Physiol Sci. 2006;56 Suppl:S81]

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A molecular entity for Ca²⁺-dependent Cl⁻-transport has not been well characterized in rat salivary glands. We previously identified a new member of the CLCA family, rCLCA, which we isolated from rat ileum by a PCR-based strategy. Here, we report that rCLCA isoforms are expressed in the submandibular gland (SMG). The full length of rCLCA mRNA is 3.3 kb, and the predicted ORF encodes a 903-a.a. protein. The amino acid sequence of rCLCA protein has 83% homology to murine CLCA1 and 2. Transient transfection of HEK293 cells with rCLCA cDNA resulted in a marked Ca²⁺-dependent Cl⁻-conductance with an outward rectification in the I-V relationship. Intense immunostaining was detected in the striated ducts of SMG, but not in the acinar cells. Immunoblot of the membrane fraction of the SMG yielded N-glycosylated 137- and 90-kDa bands. Interestingly, we also found the expression of a truncated isoform (60 kDa, presumably 514 a.a.) including the N-terminal 455 a.a. of the full-length form. Comparison of this mRNA with that of the full-length form revealed that exon 10 is truncated in this isoform. The full-length of rCLCA is likely to be responsible for modulation of Ca²⁺-dependent Cl⁻ transport in the ductal cells, although the function of the trancated isoform was not defined. [J Physiol Sci. 2006;56 Suppl:S81]

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Objectives: Aquaporins (AQPs) are 6 trans membrane proteins facilitating water transport via a plasma membrane and are involved in water, urea, glycerol or lipid transport. It has currently demonstrated that AQPs are located in intracellular organelle and their localization is changed by cell stimulation. In this study, we investigated AQP6 in the rat parotid gland.Methods: Plasma membrane and secretory granule fractions were isolated from the rat parotid glands using parcoll gradient. mRNA expression was determined by RT-PCR. Protein expression was determined by western blotting analysis. Morphological localization was observed by confocal microscopy and electron microscopy with immunolabelled ultrathin-cryosection. Results and Discussion: In RT-PCR, 262 bp band of AQP6 was detected in parotid acinar cells. In western blotting analysis using anti-AQP6 antibody, only 29 kDa protein was detected in plasma membrane and secretory granule membrane fractions of the parotid gland. In confocal microscopy, immunofluorescence with anti-AQP6 antibody was positive at the apical sites, especially cell-cell junctional region, in parotid acinar cells. When the glands were stimulated by β-agonist, AQP6 was observed to accumulate at the apical sites. These results suggest that AQP6 expresses in the apical sites and secretory granule membrane in the rat parotid gland as a monomeric form. The change of localization of AQP6 appears to be involved in exocytosis. [J Physiol Sci. 2006;56 Suppl:S81]

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Previously, we have shown that Ca oscillations modulate the activities of ion channels and the fluctuation of membrane potentials in human mesenchymal stem cells (hMSCs). We have also found an ATP autocrine/paracrine signaling pathway as the mechanism for Ca oscillations. In this study we further investigated whether Ca dependent transcription factors are regulated by Ca oscillations by examining the localization of NFAT and NF-κB through immunocytochemical experiments. NF-κB was found to be more abundant in the cytosol than in the nucleus in most undifferentiated hMSCs (140/151 cells). On the other hand, nuclear staining of NFAT was detected in more than 80% of hMSCs (40/48 cells). The nuclear translocation of NFAT was also confirmed by Western blotting of the cytoplasmic and nuclear fractions. These findings suggest that NFAT is constitutively activated in hMSCs. When an ATP autocrine and Ca oscillations signaling pathway was blocked, the NFAT signals in the nucleus were reduced, indicating the involvement of Ca oscillations in the regulation of NFAT activation. Using adipogenic techniques we differentiated hMSCs into adipocytes. In these adipocytes, no spontaneous Ca oscillations were observed (32/32 cells). In the immunocytochemical experiments, the nuclear staining of NFAT was not prominent. Thus, we concluded that Ca oscillations might play an important role in NFAT activation in undifferentiated hMSCs but not in derived adipocytes. [J Physiol Sci. 2006;56 Suppl:S82]

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Recent data have shown that inward rectifier K⁺ channel Kir7.1, localized in the basolateral membrane of rat distal nephron (JASN 2000; 11; 1987-1994), may be involved in the development of renal K⁺ excretion (Kidney Int 2003; 63; 969-975). To further assess the role of Kir7.1 in kidney K⁺ excretion, we have generated transgenic (Tg) mice expressing dominant-negative mutant of Kir7.1 (dnKir7.1 Tg/+). Wild-type (WT) and dn Kir7.1 Tg mice were placed in metabolic cages and their water balance and urine osmolality and concentrations of urine electrolytes (Na⁺, K⁺, Cl⁻) were examined. Both WT and Tg mice fed a K⁺-free diet developed hypokalemia (2.4 and 1.7 mEq/l, respectively) at 3 days and after. Water intake was increased in WT, but not in Tg mice. On the other hand, Tg mice fed a normal diet demonstrated a significant polydipsia and polyuria, with a relatively lower urine osmolality as compared to WT mice. Further, urinary K⁺ excretion of WT and Tg mice increased in proportion to that their water increase. There was no significant difference in renal K⁺ excretion between WT and Tg mice. In conclusion, Tg mice being insufficient with the basolateral Kir7.1 of the kidney distal nephron probably drink more water to compensate for the ability to excrete a K⁺ load. [J Physiol Sci. 2006;56 Suppl:S82]

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Neurological research has traditionally emphasized neuronal components of the nervous system. Recent findings suggest, however, that astrocytes harbor greater (patho-)physiological significance than previously thought. Known to provide mechanical support and chemical control of the neuronal environment (neurotransmitter clearance, extracellular K+ siphoning, etc.), newer data now additionally tie astrocytes to pharmaceutical pathways (Nature 433:73) and neurological disorders such as epilepsy (Nat. Med. 11:973), calling for a clearer molecular description of astrocyte function. The inwardly-rectifying K+ channel Kir4.1 (KCNJ10) is exclusively expressed in astrocytes in the brain. Due to its central role in astroglial K+ buffering, Kir4.1 is likely a critical element of astrocyte performance. We examined the effects of various neuroactive and neurotoxic compounds on human Kir4.1 using heterologous expression and the whole-cell configuration of the patch-clamp technique and found that the antiarrhythmic/antimalarial agent quinidine reversibly blocked Kir4.1 current in a time-dependent manner at concentrations slightly above the therapeutic range. Voltages positive to the Nernstian potential strongly enhanced Kir4.1 block, but frequency and number of the supplied depolarizing pulses seemed to be of little importance. It is conceivable that electrochemical alterations caused by astroglial ion channel block are in fact responsible for numerous so-far unexplained neurological effects of commonly administered therapeutic agents. [J Physiol Sci. 2006;56 Suppl:S82]

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Mechano-sensitive channels (MSCs) have been implicated to mediate the mechano-sensation of various types of cells. It has been well established that, in prokaryotes, MSCs can be activated directly by tension in the membrane. In contrast, the mechanism for eukaryotic MSCs activation has remained unclear. Since isolated eukaryotic MSCs in liposome could not be activated, it is proposed that eukaryotic MSCs may form molecular complex with other accessory proteins such as cytoskeletons for their function. To address this issue, we have investigated lateral mobility of hSAKcaCs (human stretch activated and Ca²⁺-activated big K channels), which we have recently identified, in the plasma membrane of living cells. We have expressed GFP-tagged hSAKcaCs in HeLa cells and observed individual channels by single fluorophore video imaging using total internal reflection fluorescence microscopy. Most of hSAKcaCs exhibited simple diffusion (diffusion coefficient, 0.4 μm²/s on average), but approximately 25% of the channels was almost stationary. The observation of single channels of hSAKcaC simultaneously with RFP-tagged paxillin showed that these immobilized channels were specifically localized on/near focal contacts. These results suggest that hSAKcaCs may associate with the force-transmitting modules including adhesion molecules and cytoskeletons to form a mechano-sensing device. [J Physiol Sci. 2006;56 Suppl:S82]

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The striatum plays an important role in linking cortical activity to basal ganglia outputs. We conducted the Ca²⁺ imaging study to investigate the spontaneous activities of the striatum. Corticostriatal slices of 10-25 days old Sprague Dawley rats were stained with Fura-PE3-AM to measure intracellular Ca²⁺ concentration ([Ca²⁺]_i) using a cooled-CCD imaging system. Long lasting spontaneous [Ca²⁺]_i transients, which lasted up to about 250 s, were observed. Most cells exhibited irregular frequencies, but some exhibited oscillatory feature. A pairwise correlation analysis revealed that some cells appear to belong to a correlated network. Administration of TTX or of CNQX + AP5 did not block the [Ca²⁺]_i transients. Therefore, the action potentials and the excitatory synaptic inputs in the striatal network were not involved in induction of the [Ca²⁺]_i transients. In contrast, the number of active cells, which exhibited the [Ca²⁺]_i transients, was greatly reduced by the intracellular Ca²⁺ store depletor, thapsigargin, and was reduced little by the administration of the Ca²⁺-free saline. Therefore, the intracellular Ca²⁺ store is likely to contribute to the [Ca²⁺]_i transients. In the mouse, which expressed green fluorescent protein (GFP) in astrocytes (GFAP-GFP mouse), both the GFP positive cells and the GFP negative cells exhibited the [Ca²⁺]_i transients. These results suggested that both astrocytes and neurons may exhibit the long lasting spontaneous [Ca²⁺]_i transients in the striatum. [J Physiol Sci. 2006;56 Suppl:S83]

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Dendritic spines are the major sites of excitatory synaptic transmission, and their morphogenesis plays a pivotal role in neuronal development and plasticity. Recent studies suggest the correlation between spine morphology and spine functions. Diversity in synapse functions is related to the diversity in spine morphology. As one of the candidate which control spine morphology, we focused on a major actin-binding protein in the brain, drebrin A (DA). We hypothesized that spine morphology depends on DA, and we analyzed spine ultrastructure in terms of DA localization using immunoelectron microscopy. We have recently reported that only 75% of spines are DA immuno-positive in adult rat cortex. We first analyzed if there was any morphological differences between DA immuno-positive spine (DPS) and DA immuno-negative spines (DNS). We found that DPS were larger in the spine head area and in the post synaptic density (PSD) length. Next, we analyzed if overexpression of DA changed spine morphology, using DA transgenic mice (Tg) that overexpressed DA in the forebrain. Similar to wild type mice (WT), DPS were larger than DNS in Tg; however, spine head area of DPS was significantly smaller than that of WT. The amount of DA within each spine did not change between two genotypes. These data suggest that overexpression of DA leads to smaller DPS with higher concentration of DA. There were no differences between two genotypes in the ratio of DPS to DNS, the spine density, and the DA localization within spines. These results indicate that DA is involved in spine morphology in vivo although its molecular mechanism is not yet clarified. [J Physiol Sci. 2006;56 Suppl:S83]

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TRPV4 is one of the member of themo-TRP channels. We screened brain-TRPV4 mRNA expressions by in situ hybridization, and found hippocampus strongly expressed TRPV4 in addition to choloid plexus, where TRPV4 expression has been reported. We speculated that TRPV4 might be an important ion channel to regulate hippocampal functions. Therefore, we established a new dissociated neural culture system from neonatal mice hippocampi. TRPV4 protein was localized in soma and dendrites in the culture. We also examined whether functional-TRPV4 was expressed in hippocampal neurons by a Ca²⁺ imaging method with Fura2. The hippocampal neurons responded to the all reported stimulus, such as heat (>32 °C), hypotonic stimulus and 4αPDD, but no response was observed in the TRPV4-mutant neurons. We considered that body temperature activates brain-TRPV4, and the activation might contribute to slight depolarization of the resting membrane potential (RMP). Next, we compared the RMP between wild type and TRPV4-mutant neurons at 37 °C, and found the wild type RMP was approximately 5 mV higher than the TRPV4-mutant RMP. We also performed current-injection experiments in both neurons, and found that TRPV4-mutant neurons required much bigger currents to get their firing. We conclude that TRPV4 is activated by body temperature in hippocampus, and produces proper environments for their firing. [J Physiol Sci. 2006;56 Suppl:S83]

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