Painless was firstly found in drosophila mutant, and appeared to play a key role in avoidance from noxious heat. Since it belongs to the TRPA1 subfamily, and both mammalian and drosophila TRPA1 are thought to be sensitive to temperature changes, Painless has also been expected to be a heat-activated ion channel. Therefore, we examined the thermal response of Painless expressed in HEK293 cells using Ca²⁺-imaging and patch-clamp methods. Painless-expressing HEK293 cells showed robust intracellular Ca²⁺ increase upon heating. Activation of Painless exhibited transient currents during heat application in a whole-cell patch-clamp mode. I-V relationship displayed dual rectification and extremely high Ca²⁺ permeability. The temperature threshold for activation with or without intracellular Ca²⁺ was around 42.6°C or 44.1°C, respectively. Painless appeared to be activated by heat with physiological intracellular Ca²⁺ concentration. Multiple heat application sensitized Painless by reducing the temperature thresholds in the presence of intra and extracellular Ca²⁺. Interestingly, Painless was insensitive to any known TRPA1 or other typical thermosensitive TRP activators. On the other hand, TRPA1 blockers such as ruthenium red and camphor inhibited the Painless-mediated heat-evoked currents. We conclude that Painless is a heat-sensitive TRP channel regulated by physiological level of intracellular Ca²⁺ ion. [J Physiol Sci. 2008;58 Suppl:S51]

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It is reported that thermosensitive TRP channels (thermo TRPs), TRPV1, TRPV2, TRPA1 and TRPM8 are expressed in adult mouse DRG neurons to exert nociception and/or thermosensation. In general, it is well known that ion channels have distinct roles between embryonic and adult stages, suggesting that the thermo TRPs might also have unexpected specific roles in embryonic neurons compared with those in adult ones. Interestingly, little investigation has been performed to identify when the thermo TRPs are expressed in developing DRG and spinal cord. Identifying expression timing of the channels is necessary to clarify their specific roles in embryo. Therefore, we examined the expression profiles of TRPV1, TRPV2, TRPA1 and TRPM8 in developing mouse DRG and spinal cord by in situ hybridization. Surprisingly, the expression of those thermo TRPs in DRG was already observed in early embryonic stages, and some of them were observed in motor neurons in spinal cord as well, suggesting that those channels might have specific roles in the embryonic stages such as the regulation of cell migration and axon extension. Therefore, we examined whether activation or inhibition of the thermo TRP activities can alter cell migration or axon extension in DRG explant culture from embryonic mice. We found the activation states of thermo TRPs influenced the properties of axon extension. Thus, we for the first time revealed that thermo TRPs have two distinct roles, regulation of neural maturation in embryonic stage and nociception and/or thermosensation in adult stage. [J Physiol Sci. 2008;58 Suppl:S51]

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Background: Plasma-derived lipid mediator sphingosine-1-phosphate (S1P) acts via the G protein-coupled S1P receptor family to regulate a variety of physiological and pathological responses. S1P₁ receptor mediates endothelial cell migration and vascular maturation, promoting vascular integrity. We have previously shown that S1P₂R is distinct from S1P₁R in that it negatively regulates cell migration and endothelial capillary tube formation in vitro. In the present study we investigated the role of S1P₂R in tumor angiogenesis by using S1P₂ knock out (KO) mice. Methods and Results: Lewis lung carcinoma cells were injected into the flank of S1P₂KO and wild type (WT) mice and allowed to grow for 14 days. Tumors in S1P₂KO mice displayed increased numbers of blood vessels, which were associated with much higher numbers of desmin-positive mural cells, and marked upregulation of angiogenic factors including VEGF and the Notch signaling mediator Delta-like ligand 4. Vascular permeability examined by FITC-dextran administration was decreased in S1P₂KO compared with WT mice. Conclusions: S1P₂R negatively regulates tumor angiogenesis in vivo, with inhibition of angiogenic gene expression, mural cell recruitment and vascular integrity. S1P₂R-selective agonist would be a promising candidate for anti-tumor angiogenesis. [J Physiol Sci. 2008;58 Suppl:S52]

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S1P plays a critical role in vascular maturation in vivo during the mammalian development. Recently, we demonstrated for the first time that S1P stimulated neo-angiogenesis in ischemic hindlimbs of mice. In the previous study, we daily injected S1P solution locally into the hindlimb muscle. In the present study, we successfully generated a slow release type of S1P by taking advantage of three different poly(lactic-co-glycolic-acid)(PLGAs), and tested its efficacy in the mouse ischemic hindlimb where femoral artery was ligated and removed unilaterally. The amounts of S1P released from S1P-containing PLGA microparticles in a physiological medium were quite constant over 15 days in PLGA 5005. In contrast, S1P release from PLGA7505 and PLGA7510 with longer half lives than PLGA5005 sharply declined after the initial burst. Local injections of PLGA5005-S1P microparticles every 3 days, but not PLGA7505-S1P or PLGA7510-S1P, into ischemic muscle stimulated the blood flow recovery over 28 days after femoral arteriectomy, as determined with a laser Doppler imager. PLGA5005-S1P was effective in the wide range of doses. In contrast, PLGA7505-S1P and PLGA7510-S1P were less effective or non-effective in stimulating the blood flow recovery. Injections of PLGA5005-S1P stimulated angiogenesis, as determined with anti-CD31 immunohistochemistry. These results suggested that PLGA5005-S1P could be potentially useful as a therapeutic agent for stimulating post-ischemic angiogenesis. [J Physiol Sci. 2008;58 Suppl:S52]

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Rho-kinase (ROK)-mediated Ca²⁺-sensitization of vascular smooth muscle (VSM) contraction plays a critical role in abnormal VSM contraction. We previously found that sphingosylphosphorylcholine (SPC) induces the ROK-mediated Ca²⁺-sensitization, dependently on cholesterol in serum and VSM tissue and through the activation of Src family tyrosine kinases (Src-TKs). In this study, we examined the involvement of membrane rafts, a cholesterol-enriched membrane microdomain, in the SPC/ROK-mediated Ca²⁺-sensitization. Immunocytochemistry and membrane fractionation revealed that SPC induced the translocations of Fyn, a member of Src-TKs, and ROK from cytosol to membrane rafts, which were labeled with caveolin-1 and cholera toxin subunit B. In addition, β-cyclodextrin, which selectively deprived VSM membrane of cholesterol and thereby removed caveolin-1 from membrane rafts, inhibited the SPC-induced translocation of Fyn and ROK and VSM contraction, without affecting Ca²⁺-induced contraction. In contrast, eicosapentaenoic acid (EPA), an inhibitor of Src-TKs translocation, inhibited the SPC-induced translocation of Fyn and ROK without affecting the localization of caveolin-1. Moreover, EPA selectively abolished the SPC-induced Ca²⁺-sensitization, without affecting the Ca²⁺-induced contraction. These findings suggest that membrane rafts play a pivotal role in abnormal VSM contraction. [J Physiol Sci. 2008;58 Suppl:S52]

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Actin stress fibers play important roles in various cellular functions, including cell motility, morphogenesis and tumorigenicity. It is known that Rho and its effector Rho-kinase (ROCK) play a critical role in stress fiber formation and are implicated to be involved in the stress fiber formation induced by sphingosylphosphorylcholine (SPC) and lysophosphatidic acid (LPA). However, except for Rho, the upstream mediator(s) for the ROCK-mediated stress fiber formation is still unknown. In this study, we provide the first direct evidence that Fyn, a member of Src family tyrosine kinase, acts as a novel signaling molecule in the actin stress fiber formation in NIH3T3 fibroblasts. Either LPA or SPC activated Fyn and induced stress fiber formation, which was blocked by pharmacological inhibition and gene silencing of Fyn, or dominant negative Fyn. Overexpressed constitutively active Fyn colocalized with focal adhesion kinase at the both ends of F-actin bundles and triggered stress fiber formation, only the latter of which was abolished by ROCK inhibition. SPC, but not LPA, also induced another form of actin cytoskeleton reorganization, filopodia-like protrusion formation, which was not mediated by Fyn and ROCK. Thus, Fyn appears to act downstream of LPA and SPC to specifically stimulate stress fiber formation mediated by ROCK in fibroblasts. [J Physiol Sci. 2008;58 Suppl:S52]

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We examined the effect of local shoulder skin warming on blood flow of trapezius muscle and shoulder skin in eight elderly subjects (73±2 yr), using a steaming pad (12cmx20cm, Kao Corp.). Muscle regional oxygenation that reflects muscle blood flow was measured as tissue oxygenated Hb concentration (dHbO₂μmol/L) by non-invasive near-infrared spectroscopy (NIRO-120, Hamamatsu Photonics). Skin temperature (9 points) and skin blood flow of shoulder were also measured with thermocouples and laser Doppler flowmeter(Advance), respectively. Subjects sit on the chair for 10 min (control period) followed by shoulder warming with the steaming pad (30min) and removal of the pad (20min) in the thermoneutral environment (25°C, 45%RH). By warming, shoulder skin temperature increased from 33.1 to 40.0°C. The regional oxygenation of trapezius muscle increased by 3.8 μmol/L, and the sholder skin blood flow increased from 2.9 to 8.5 ml/min/100g. Local skin temperatures increased at toe (+0.9°C), hand (+0.5°C), chest (+0.7°C) and finger (+0.3°C), whereas, leg temperature fell by 0.7 °C. Tympanic temperature was unchanged. Systolic blood pressure decreased significantly from 137 to 123 mmHg, whereas diastolic pressure and heart rate did not change. There was a negative correlation between initial mean blood pressure and the increment of trapezius HbO₂. These results suggest that the thermal (steaming) stimulus on shoulder skin can elicit increases in the regional circulation of the muscle and the skin and in skin temperature of the periphery of extremities. [J Physiol Sci. 2008;58 Suppl:S53]

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The acupuncture effect of manual stimulation at the acupoints Neiguan (PL-6) and Zu-sang-li (ST-36) on mean arterial blood pressure (MAP) was investigated in rats anesthetized with i.p. pentobarbital injection followed by continuous infusion of 1% pentobarbital-PBS-solution (0.5-0.8ml/h) intravenously. Ventilation of the animal was artificially applied and body temperature was kept at normal level.Manual acupuncture (MA) was applied for 30 s with a frequency of 2 HZ by inserting a needle into the left side of PL-6 and ST-36 in the fore limb and hind limb. Three different types of MA were applied: 1) twisting needle from left to right; 2) lifting and thrusting needle; and 3) combination of 1) and 2). MAP was used as an indicator to judge the effect of MA. The results showed that the MAP was decreased but fluctuated in intact rats, and intensively decreased in all cases after vagotomy during MA at PL-6, ST-36, respectively. The reduction of MAP was slightly apparent during MA with type 1, and reinforced significantly during MA with type 2 and 3 at PL-6 or ST-36. The MAP response was significant higher during MA at PL-6 rather then St-36. Meanwhile, the reductions of MAP were completely abolished during MA at either PL-6 or ST-36 after spinalization. The above results suggest that MA at either PL-6 or ST-36 can reduce the blood pressure in anesthetized rats, and modulatory site for such an effect by MA in its central pathways is superspinal. (Financial support: FSMCST: 06DZ19732; 973 Program: 2005CB523306) [J Physiol Sci. 2008;58 Suppl:S53]

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The effects of acupuncture-like stimulation of a hindlimb on heart rate were examined in anesthetized rats. An acupuncture needle, having a diameter of 0.34 mm, was inserted into the skin and underlying muscles at a depth of about 5 mm and twisted right and left twice every second for 1 min. Acupuncture-like stimulation produced a decrease in heart rate. These bradycardiac responses were completely abolished after severance of the femoral and sciatic nerves ipsilateral to the stimulation. In addition, acupuncture-like stimulation to muscles alone decreased heart rate significantly, but stimulation to skin alone did not produce significant changes in heart rate. To clarify the efferent pathways of the acupuncture-like stimulation-induced bradycardia, the autonomic nerves to the heart were severed. The response was not influenced by bilateral severance of the vagal nerves at the cervical level, but was abolished by bilateral stellectomy. Furthermore, acupuncture-like stimulation induced a decrease in the activity of the cardiac sympathetic efferent nerve as well as decrease in heart rate. From these results, it appears that the decrease in heart rate induced by acupuncture-like stimulation of a hindlimb is a reflex response. The afferent pathway is composed of hindlimb muscle afferents while the efferent pathway is composed of cardiac sympathetic nerves. [J Physiol Sci. 2008;58 Suppl:S53]

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Although there are many clinical case reports, electroacupuncture for cardiovascular diseases is not a sure cure. To increase the efficacy of electroacupuncture, in prior study we demonstrated that electroacupuncture at Zusanli reset the arterial baroreflex neural arc to lower sympathetic nerve activity, which moved the operating point of baroreflex toward lower arterial pressure and sympathetic nerve activity (Am J Physiol Heart Circ Physiol. 2006, 291(1), H318-26). However, the strength of electroacupuncture we used was relatively weak. Therefore, in this study, we have systematically investigated the effects of electroacupuncture on sympathetic nerve activity and arterial pressure from low (<1 Hz) to high (100 Hz) stimulation frequency in anesthetized rabbits (n=8). The sympathetic nerve activity and arterial pressure decreased in response to weak (<5Hz) electroacupuncture. However, during moderate stimulation (5-10 Hz), they first decreased and turned to increased their baseline levels within 1 min. During high stimulation (10-100 Hz), they increased without the initial drops. Computer simulation study based on these results suggests that effects of electroacupuncture on the sympathetic nervous and arterial pressure responses are characterized by low-pass filter with low intensity threshold (gain<0), high-pass filter with moderate intensity threshold (gain>0) and low-pass filter with high intensity threshold (gain>0). These findings may have a merit to develop closed-loop electroacupuncture system. [J Physiol Sci. 2008;58 Suppl:S53]

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Pancreatic duct cells produce HCO₃⁻-rich isotonic fluid secretion. HCO₃⁻ transport across the apical membrane are thought to be mediated by CFTR and SLC26 anion transporters. We previously examined changes in pH_i upon removal and restoration of luminal Cl⁻ in the presence of HCO₃⁻ in interlobular pancreatic ducts isolated from slc26a6 null mice. In slc26a6 null ducts, HCO₃⁻-efflux mode of apical [Cl⁻]_o-[HCO₃⁻]_i exchange was decreased while HCO₃⁻-influx mode of apical [Cl⁻]_i-[HCO₃⁻]_o exchange was increased, suggesting the uni-directionality of slc26a6-mediated HCO₃⁻ transport. To investigate the stoichiometry of slc26a6 Cl⁻-HCO₃⁻ exchange, we examined net transport of HCO₃⁻ and Cl⁻ by measuring luminal pH (pH_L) and fluid secretory rate in sealed ducts of which the lumen was injected with BCECF-dextran. Duct lumen was filled with HCO₃⁻-free, Cl⁻-rich (150 mM) solution and the superfusate was switched from the HCO₃⁻-free, Cl⁻-rich solution to the standard HCO₃⁻-buffered solution. This protocol would instantaneously generate a favorable gradient for apical Cl⁻-HCO₃⁻ exchange. Switching the superfusate caused transient decrease of pH_L (CO₂ diffusion) followed by continuous increase due to HCO₃⁻ secretion. The changes in pH_L were accompanied with transient increase of fluid secretory rate in wild-type ducts, while transient fluid absorption was observed in most of slc26a6 null ducts. Calculation of net HCO₃⁻ and Cl⁻ fluxes assuming ([HCO₃⁻] + [Cl⁻] = 150 mM) suggests that slc26a6 mediates 1Cl⁻-2HCO₃⁻ exchange and that a 2Cl⁻-1HCO₃⁻ exchanger is up-regulated in slc26a6 null ducts. [J Physiol Sci. 2008;58 Suppl:S54]

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In the resting state of gastric parietal cells, most of H⁺,K⁺-ATPase is located in intracellular tubulovesicles. Upon stimulation, the tubulovesicles fuse with the apical surface membrane. Here, two types of gastric vesicles (intracellular tubulovesicles (TV) and stimulation-associated vesicles (SA) derived from apical surface membrane) were prepared from hog gastric mucosa. In the TV sample, 60% of H⁺,K⁺-ATPase were found in the detergent (1% CHAPS)-resistant membrane microdomains (DRM) and 40% of H⁺,K⁺-ATPase were in non-DRM. In the SA sample, 30% of H⁺,K⁺-ATPase were in the DRM and 70% of H⁺,K⁺-ATPase were in the non-DRM. Phospholipids were abundant in the DRM of the TV, while those were present both in the DRM and non-DRM of SA. Cholesterol was abundantly present in the DRM of TV and SA. Expression level of caveolin-1, a caveolae marker, in TV was much higher than that in SA. In contrast, expression level of flotillin-2, a raft marker, in SA was higher than that in TV. The H⁺,K⁺-ATPase activity in TV was significantly reduced by treatment of the vesicles with methyl-β-cyclodextrin (MβCD), and the MβCD-decreased activity was significantly restored by exogenous addition of cholesterol. These results suggest that H⁺,K⁺-ATPase is present in intracellular caveosome (TV) and in the raft- and non-raft-domains in the apical surface membrane (SA) of parietal cells, and that cholesterol is necessary to maintain the pump activity. [J Physiol Sci. 2008;58 Suppl:S54]

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Human non-gastric H⁺,K⁺-ATPase, ATP1AL1, mediates Na⁺ or H⁺ efflux and K⁺ influx. It is reported that four amino acid residues of M3/M4 loop in Na⁺,K⁺-ATPase are essential for ouabain binding. To examine functions of the extracellular M3/M4 loop of ATP1AL1, the amino acid residues of ATP1AL1 were replaced with corresponding amino acid residues of Na⁺,K⁺-ATPase (Q334T, D337E, S338A, I339V). A tetra-mutant (TEAV) and four triple-mutants (EAV, TEA, TEV, TAV) were constructed, and they were transfected with HEK293 cells. Expression level of all ATP1AL1 mutants was not significantly different from that of wild-type, and the endogenous Na⁺,K⁺-ATPase level was unchanged by these transfections. In all of the mutant-expressing cells, 10 μM ouabain- sensitive K⁺-ATPase activity was not significantly different from that of wild-type. On the other hand, 3 mM ouabain-sensitive K⁺-ATPase activity of TEV, TEA and TEAV mutants was significantly decreased compared with that of wild-type. In the molecular dynamics simulation, we found that M3/M4 loop is indirectly involved in the closed ion gate formed from M1/M2 loop of ATP1AL1, and that the gate of TEV, TEA and TEAV mutants was unstable or disrupted. These results suggest that M3/M4 loop in ATP1AL1 is not essential for ouabain sensitivity, but that it may be important for stabilization of closed ion gate. [J Physiol Sci. 2008;58 Suppl:S54]

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We found that K⁺-Cl⁻ cotransporter 4 (KCC4) was highly expressed in the luminal gastric parietal cells, in which gastric acid is actively secreted. To clarify distribution and function of KCC4 in gastric parietal cells, two types of gastric vesicles, stimulation-associated vesicles (SA) derived from surface apical membrane and intracellular tubulovesicles (TV), were prepared from hog gastric mucosa. KCC4 was predominantly expressed in SA but not in TV. Gastric proton pump (H⁺,K⁺-ATPase) was expressed both in SA and TV. KCC4 was co-immunoprecipitated with H⁺,K⁺-ATPase in the lysate of SA. Both KCC inhibitor (DIOA, 10 μM) and H⁺,K⁺-ATPase inhibitor (SCH 28080, 10 μM) significantly reduced the ATP-dependent Cl⁻ uptake in SA by 78.0 ± 9.8% and 71.3 ± 11.8% respectively. The inhibition of DIOA plus SCH 28080 on the Cl⁻ uptake was not additive, indicating that the Cl⁻ transporting activity of KCC4 is positively regulated by H⁺,K⁺-ATPase. On the other hand, H⁺ uptake and ATP-hydrolyzing activity of H⁺,K⁺-ATPase in SA were significantly inhibited by DIOA (10 μM), suggesting that H⁺,K⁺-ATPase activity is positively regulated by KCC4. Thus, KCC4 and H⁺,K⁺-ATPase are functionally associated in SA. KCC4 may be involved in the basal gastric acid secretion of gastric parietal cells. [J Physiol Sci. 2008;58 Suppl:S54]

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Most smooth muscles including intestinal muscles and systemic vessels except pulmonary artery relax to hypoxia. We showed that at high stimulus conditions with high potassium guinea pig taenia caeci and porcine coronary artery were relaxed by hypoxia without a change in [Ca²⁺]_i. The level of myosin regulatory light chain phosphorylation (p-MRLC) was not reduced by hypoxia in the taenia (Obara et al. 1997), but significantly reduced in the coronary (Gu et al. 2005). Thus, the mechanisms of relaxation to hypoxia are different as the p-MRLC-dissociating hypoxic relaxation in the taenia and the Ca²⁺-desensitizing hypoxic relaxation in the coronary. This Ca²⁺-desensitizing hypoxic relaxation was observed even in the skinned coronary muscle under the fixed [Ca²⁺]. Hypoxia relaxed the skinned coronary muscle pretreated with GTP-γS, but not with ATP-γS. The protection protocol for myosin light chain kinase and Rho kinase using ML7 and Y27632 revealed that hypoxia directly inhibits Rho kinase-dependent phosphorylation of myosin phosphatase (MYPT1), thus leading to the Ca²⁺-desensitizing hypoxic relaxation of the skinned coronary via decreases in both p-MYPT1 and p-MRLC. The different mechanisms between intestinal and arterial muscles may be dependent on the different property of contractile apparatus; rapid contractile activity in the intestine and slow activity in the artery. [J Physiol Sci. 2008;58 Suppl:S55]

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Evidence now being accumulated suggests that a subpopulation of interstitial cells in the myenteric plexus generates basal pacemaker electrical activity in the gut, including the ileum. These special pacemaker cells are referred to as interstitial cells of Cajal (ICC) from the histological resemblanceones. In this study, we employed an 8x8 MEA (with 150 μm in interpolar distance) to perform a spatio-temporal analysis of electrical activities in the ileal smooth muscle of BALB/c and W/W^V mice lacking myenteric ICC. Smooth muscle layer containing the myenteric plexus was isolated from the ileum, and mounted in a recording chamber. To differentiate ICC pacemaker electrical activity from smooth muscle and neural activities, dihydropyridine (DHP) Ca²⁺ antagonists and TTX were added to the medium. Field potentials were recorded after high-pass filtering at 0.1 Hz. In analyses, digital band pass filter (0.05-0.6Hz) was applied. In normal (BALB/c) mice, field potentials of several tens of μV were observed over the recording area, whereas in W/W^V mice the amplitude was much smaller and the occurrence was irregular. In power spectrum analysis, only normal mice showed a prominent peak corresponding to the ICC pacemaking frequency. Furthermore, potential mapping clearly demonstrated that ileal smooth muscle from normal mice generates propagating spontaneous electrical potentials, but that from W/W^V mice does not. These results suggest that network-forming ICC in the myenteric plexus play a crucial role in coordinating electrical activities of the ileum as well as generating pacemaker potentials. [J Physiol Sci. 2008;58 Suppl:S55]

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The effects of K channel blockers on slow waves and pacemaker potentials recorded from mouse small intestine were investigated intracellulary. Application of iberiotoxin, a blocker of large conductance Ca-activated K channel, or charybdotoxin, a blocker of intermediate conductance Ca-activated K channel, had no effect on the generation of slow waves recorded from circular smooth muscles, while the blocking of small conductance Ca-activated K channel (SK channel) by apamin depolarized the membrane and inhibited the early, rapid repolarization of slow waves. 4-Aminopyridine (4-AP), a blocker of transient outward K channel, depolarized the membrane and increased the amplitude and maximum rate of rise (dV/dt_max) of the primary component of slow waves. On the other hand, the amplitude and dV/dt_max of pacemaker potentials recorded from in situ interstitial cells of Cajal distributed in the myenteric region (ICC-MY) were decreased by application of apamin or 4-AP. These observations indicate that both apamin-sensitive K conductance and 4-AP-sensitive K conductance may contribute to the resting membrane potential of circular smooth muscles. The observations also indicate that 4-AP-sensitive K conductance is likely to be activated during the generation of the primary component of slow waves. The early, rapid repolarization seems to be a specific response of smooth muscle cells resulting from the activation of apamin-sensitive K conductance. [J Physiol Sci. 2008;58 Suppl:S55]

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Sensory nerves play a significant role in regulating spontaneous contractions of the renal pelvis, while autonomic motor nerves have a relatively small role. The major inhibitory neurotransmitter is calcitonin gene-related peptide (CGRP), but the cellular mechanisms of its action are not well understood. Changes in the intracellular Ca concentrations were recorded from fluo-4 loaded smooth muscle bundles of the mouse renal pelvis. Electrical and mechanical responses were recorded separately. The effects of stimulating capsaicin (CAP)-sensitive sensory nerves on spontaneous activity were compared with the effects of applying human CGRP (hCGRP).Renal pelvis preparations exhibited spontaneous action potentials, Ca transients and contractions. CAP (10 μM) caused a long-lasting inhibition of spontaneous activity which was often preceded by a transient excitation. Similarly, hCGRP (0.1 μM), isoproterenol (10 μM), forskolin (1 μM) and 8 Bromo-cAMP (1 mM) all suppressed spontaneous activity. These inhibitory effects were reduced in preparations previously treated with the ATP-sensitive K channel blocker, glybenclamide (10 μM). In mouse renal pelvis, stimulation of cyclic AMP production in smooth muscle may activate ATP-sensitive K channels to hyperpolarize the membrane and reduce the frequency of spontaneous activity. This pathway may be involved in sensory nerve-mediated regulation of spontaneous activity by releasing CGRP. [J Physiol Sci. 2008;58 Suppl:S55]

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A whole-cell patch-clamp study was carried out using brain slice preparations of the rats (P21-38) to elucidate the developmental changes in calcium channel subtypes involved in non-NMDA glutamatergic transmission onto cholinergic neurons in the basal forebrain (BF) and D1-like receptor-mediated presynaptic inhibition. Pharmacologically-isolated non-NMDA EPSCs were evoked by focal stimulation. The EPSC fraction suppressed by ω-CgTX (3 μM), an N-type calcium channel blocker, decreased with age, whereas that suppressed by ω-Aga-TK (200 nM), a P/Q-type channel blocker, increased. Inhibition of EPSCs by SKF 81297, a D1-like receptor agonist, also increased with age in parallel with the increase in ω-Aga-TK-induced suppression. SKF 81297 showed no further inhibition of EPSCs after the effect of ω-Aga-TK had reached steady state throughout the developmental stages examined. These results suggest a tight functional linkage between presynaptic D1-like receptors and P/Q type channels for the glutamate release throughout development. [J Physiol Sci. 2008;58 Suppl:S56]

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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 1-3 weeks old GFAP-GFP mice expressed GFP in astrocytes were used. We can discriminate whether astrocytes or neurons by observation of GFP fluorescence. The slices were stained with Fura-PE3-AM to measure intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the ratiometric fluorescence measurement was conducted. Long-lasting spontaneous [Ca²⁺]_i transients, which lasted up to about 300 s, were observed in both GFP positive cells (astrocytes) and GFP negative cells (putative neurons). Distributions of the duration, interval and peak amplitude of the spontaneous [Ca²⁺]_i transients in putative neurons were slightly different from those of astrocytes (p < 0.05, Kormogorov-Smirnov test). Average values were followings; duration (s): 17.3 ± 20.8 and 16.2 ± 20.5; interval (s): 105 ± 141 and 104 ± 158; peak amplitude (ΔR): 0.0206 ± 0.0115 and 0.0202 ± 0.0114 (mean ± SD, data from 178 putative neurons and 113 astrocytes, respectively). Administration of CNQX + AP5 did not block the [Ca²⁺]_i transients in the both types of cells (in 4 slices). In contrast, the numbers of the active cells, which exhibited the [Ca²⁺]_i transients, were greatly reduced by the intracellular Ca²⁺ store depletor, thapsigargin. These results suggested that both neurons and astrocytes exhibited the long-lasting spontaneous [Ca²⁺]_i transients, which were caused mainly by Ca²⁺ release from the intracellular Ca²⁺ store. [J Physiol Sci. 2008;58 Suppl:S56]

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Stimulation of D1 type of dopamine (DA) receptor induces a slow Na⁺ current response in the identified neurons of Aplysia ganglia under voltage clamp. We previously reported that the Na⁺ current response to DA is facilitated by the activation of small GTP-binding protein RhoB or C but not RhoA, and subsequent phospholipase D (PLD). In the present study, we investigated the regulatory mechanism of the DA-induced Na⁺ current response by Rho and PLD. The Na⁺ current response to DA was significantly augmented by extracellular application of monodancylcadaverine, an inhibitor of clathrin-mediated endocytosis. In contrast, extracellular application of monensin, an inhibitor for recycling of endocytosed protein to the cytoplasmic membrane, significantly depressed the DA-induced current response. Furthermore, intracellular application of botulinum toxin type B, which cleaves the SNARE protein SNAP-25, also inhibited the Na⁺ current response to DA. These results suggest that Rho and subsequent PLD may regulate the DA-induced Na⁺ current response through recycling of the D1 receptor. [J Physiol Sci. 2008;58 Suppl:S56]

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Recent lines of evidence suggest that affective disorders such as depression are based on synaptic dysfunction in the brain. Bilateral olfactory bulbectomy (OBX) is an experimental model for depression in rodents. Altered emotional state and increased locomotor activity gradually become remarkable after OBX. It has also been known that change in several neurotransmitter systems and alteration of neuronal networks in the limbic system after OBX. To identify the molecular nature of the synaptic alteration in the OBX brain, we analyzed by quantitative RT-PCR change in the expression level of several synaptic function-related genes, including synapsin I, NMDA receptor subunit 1 (NR1), drebrin A, PSD-95, BDNF and c-fos, in the hippocampus 2 and 4 weeks after OBX. As reported previously in rat, we observed transient increase in the level of BDNF mRNA 2 weeks after OBX. The levels of NR1 and synapsin I mRNAs were also slightly increased. The levels of these mRNAs went back to the baseline by next 2 weeks. In this time point we found the decrease in c-fos and drebrin A mRNAs. Furthermore, we observed that increase in nocturnal activity measured 4 weeks after OBX was greater in drebrin A-knockout mice (n = 6) than that in wild-type mice (n = 6). Loss of drebrin A may lead to a subsequent synaptic dysfunction in the hippocampus of OBX mice. Thus, our data suggest that drebrin A is a candidate vulnerability molecule in a certain type of affective disorders. [J Physiol Sci. 2008;58 Suppl:S56]

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Striosome (or patch)/matrix compartments are one of the candidates of functional module in the striatum. We previously reported that activation of μ opioid receptor (MOR) suppressed GABAergic inhibitory postsynaptic currents (IPSCs) in medium-spiny projection neurons in compartment-specific manner. Interestingly, the inhibition of IPSCs was also observed in cholinergic interneurons. In this study, we investigated the MOR-mediated effects on the excitability of cholinergic interneurons. To identify the striosomes, we used a TH-GFP transgenic mouse strain expressing eGFP under the control of promoter of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis. Because dopaminergic neurons densely innervate the striosomes in early postnatal stage, a striosome is identified as a bright area under a fluorescence microscope. Using corticostriatal slices obtained from TH-GFP mice, we made whole-cell recordings from cholinergic interneurons. GABAergic IPSCs were elicited in the presence of CNQX and AP5. Intrastriatal stimulation evoked multiphasic GABAergic IPSCs consisted of monosynaptic early and polysynaptic, nicotinic acetylcholine receptor (nAchR) mediated late components. DAMGO (1 microM), an agonist of MOR, suppressed only the early-IPSCs in the striosomes. Dihydro-β-erythroidine, an antagonist of non-α7 nAchR, suppressed only late-IPSCs, suggesting nAchR-mediated activation of GABAergic neurons. These studies would help to explain the MOR-mediated effects on the activity of local neural circuits in striosome/matrix compartments. [J Physiol Sci. 2008;58 Suppl:S57]

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We sought to identify novel gene(s) expressed specifically in the LC and to elucidate its function. The tyrosine hydroxylase (TH)-green fluorescence protein (GFP) transgenic mouse was used. The pontine region containing the LC, obtained from E.14.5 or E.18.5 mice, was dissected out. Dispersed cells underwent sorting by flow cytometry (FACS), and GFP-labeled NA neurons were collected with high precision. The transcripts expressed in the LC-NA neurons were compared with those expressed in controls by GeneChip (Affymetrix). The categorically different transcripts, expressed selectively in the LC, included transcription factors, transporters, ion channels, enzymes, and miscellaneous proteins. Selected species of transcripts was localized to the LC by in situ hybridization. We focused on the transcription factors, expressed in the LC neurons with high selectivity, and examined whether these may modulate the gene expression of catecholamine synthesizing enzymes. Expression assay was done in a cell line which was co-transfected with the ORF of the respective transcription factor and a construct with each one of the promoter of catecholamine synthesizing enzymes plus luciferase. As a result, we identified transcription factors which enhanced gene expression of the enzymes. [J Physiol Sci. 2008;58 Suppl:S57]

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We reported a novel mouse model in which the LC noradrenergic (NA) neurons were ablated selectively, leaving other NA cell groups intact. Transgenic mouse was used that expressed human interleukin-2 receptors (hIL2R) under the promoter of dopamine-β hydroxylase. The hIL2R was colocalized with tyrosine hydroxylase (TH) immunoreactivity in anatomically defined NA neurons. The hIL2R was identified in the cell bodies and dendrites but not in axons. The immunotoxins (conjugates of antibody against hIL2R and Pseudomonas exotoxin) was injected into bilateral LC. The LC cells disappeared at day 7 following the immunotoxin injection, and did not regenerate at day 28. The TH-immunoreactive axon terminals decreased remarkably in brain regions receiving NA inputs exclusively from the LC. Ambulatory behaviors (elevated plus-maze and open field exploration) were analyzed by the software developed in our laboratory. The number of marbles buried, as well as the light/dark transition, was assessed. Overall, the levels of anxiety decreased in the LC-ablated mice in comparison with the controls. However, plasma ACTH levels were not affected following the restraint stress or LPS-induced immune challenge. The results suggest the involvement of the LC in anxiety-like behavior but not in neuroendocrine stress responses in mice. [J Physiol Sci. 2008;58 Suppl:S57]

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Disturbances in social skills are the most pervasive aspects of schizophrenic patients. In rodents, both phencyclidine (PCP) and methamphetamine (MAP) induced schizophrenia-like abnormal behaviors. However, there is no animal model of schizophrenia with deficits of social behaviors in primates. To establish a primate animal model of schizophrenia by using PCP and MAP treatment, 6 monkeys were used and divided into 2 groups; control (n=3) and PCP groups (n=3). PCP group received chronic PCP treatment (0.3 mg/kg/day, i.m.) for more than 7 months. The PCP monkeys sometimes received additional acute MAP treatment (2 mg/kg, i.m.) (PCP+MAP group, n=3). Monkey behaviors were recorded by CCD cameras and analyzed automatically by computer software and manually by visual inspection. Social behaviors were analyzed while each of 2 monkeys was put in both side cages that were connected to a center cage. The results indicated that social behaviors (e.g., approaching to, leaving from, following, grooming with, and mounting on other monkey) were less observed in the PCP than the control groups. The PCP group also spent less time in close proximity to other monkey than the control group. Acute treatment of MAP with the PCP monkeys further decreased social behaviors and time spent in close proximity to other monkey. These results indicated that chronic PCP and acute MAP treatment induced human schizophrenic-like abnormal social behaviors, and suggest that these monkeys can be used for a primate animal model of schizophrenia. [J Physiol Sci. 2008;58 Suppl:S57]

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The mineralocorticoid receptor has been clearly implicated in the pathogenesis of cardiac hypertrophy linked to heart failure. However, the cellular and molecular mechanisms of aldosterone action on the cardiac function remain to be elucidated. We have previously shown that aldosterone increases the rate of spontaneous contraction in neonatal rat cardiomyocytes concomitantly with an increase of T-type Ca²⁺ channel expression and current amplitude.Here, we show that glucocorticoids mimicked aldosterone effects, accelerating spontaneous contraction frequency and increasing both T channel expression and current amplitude. Oxidant agents significantly sensitized the cells to the effect of aldosterone on the beating acceleration while a reducing agent decreased. Interestingly, redox agent did not affect T channel expression, however, the acute application of redox agents affected both T-type Ca²⁺ currents and spontaneous contractions in steroids treated cells. In conclusion, aldosterone and corticosterone modulate the expression of T channels and accelerate spontaneous beatings in cardiomyocytes. The chronotropic effect appears directly linked to increased T-type Ca²⁺ channel activity, modulated by the cell redox state and could contribute in vivo to the deleterious effect of an inappropriate activation of the mineralocorticoid receptor on a disrupted cardiac function and on an increased susceptibility to arrhythmias. [J Physiol Sci. 2008;58 Suppl:S58]

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It has been reported that thromboxane A₂ (TXA₂) and prostaglandin F₂ (PGF_2α) mediates inflammatory tachycardia by directly facilitating the automaticity of cardiac sinoatrial node preparations (Takayama et al, 2005). In this study, the action potential and membrane currents were recorded in isolated guinea-pig sinoatrial node cells and the ionic mechanisms underlying the positive chronotropic action of PGF_2α and TXA₂ were investigated by the patch clamp method. Both PGF_2α and TXA₂ increased the spontaneous firing frequency of isolated sinoatrial node cells, and this increase was partially blocked by 50 μM Ni²⁺. Under the voltage clamp condition, I-BOP, a selective agonist for TP receptor, increased the amplitude of low voltage-activated Ca²⁺ current with little affecting the high-threshold Ca²⁺ current, the delayed rectifier K⁺ current and the hyperpolarization-activated cation current. In the single channel recording, I-BOP increased the open probability of Ca²⁺ channels, which had tiny (-8pS) conductance and displayed rapid inactivation. The results indicate the functional role of T-type Ca²⁺ channel in the positive chronotropic action of PGF_2α and TXA₂. [J Physiol Sci. 2008;58 Suppl:S58]

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The sino-atrial node (SAN) is the natural pacemaker region of the heart. Little is known about structural correlates of the complex behaviors of the SAN. We visualized the excitation propagation in the rabbit SAN using a voltage sensitive dye, di-4-ANEPPS, and analyzed the spatial distribution of action potential properties to elucidate the mechanism of the leading pacemaker site formation. Action potential parameters, the onset and peak of activation, action potential duration at 50% repolarizaion (APD50), the slope of upstroke (phase 0 slope), and the slope of the pacemaker depolarization (phase 4 slope), were analyzed. The leading pacemaker did not necessarily emerge at the site of maximum APD50 or phase 4 slope. The changes in action potential characteristics were complex. We did not observe a gradual transition from the leading pacemaker site to the periphery. Then, we hypothesized that the leading pacemaker emerges as a consequence of mutual interactions of SAN cells, and predicted that the reduction of mutual interactions through gap junctions induces a pacemaker shift. Carbenoxolone, a gap junction blocker, changed action potential properties and caused a pacemaker shift. Model simulation, assuming a random distribution of intrinsic properties of SAN cells, reproduced the experimental results. We conclude that both intrinsic properties and mutual interactions through gap junctions of SAN cells contribute to the characterization of action potentials and the formation of the leading pacemaker site. [J Physiol Sci. 2008;58 Suppl:S58]

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Atrial myocytes express L-type calcium channel α₁ subunits, Ca_V1.2 and Ca_V1.3. Ca_V1.3 activates and inactivates at voltages lower than those of Ca_V1.2 by approximately -17mV. We have previously reported that Ca_V1.3 shows slow voltage-dependent inactivation as compared to Ca_V1.2 and that the carboxy(C)-terminal region of Ca_V1.3 plays an important role in its slow inactivation kinetics. To elucidate the contribution of Ca_V1.3 currents in pacemaker potential, we examined ion current kinetics of Ca_V1.3, Ca_V1.2 and the chimeric channel CTD in which the C-terminal region of Ca_V1.2 was replaced by that of Ca_V1.3 by use of heterologous expression system. The recovery from the inactivation of Ca_V1.3 was significantly faster than those of Ca_V1.2 and CTD. The Ca_V1.3 channel current increased at both diastolic depolarization and repolarization during an action potential (AP). Under repetitive AP clamping with AP waveform of rabbit sino-atrial node at 1Hz, Ca²⁺ current and Ba²⁺ current through the three Ca²⁺ channels were gradually decreased and reached plateau. Interestingly, the availability of Ca_V1.3 was kept at higher level than that of Ca_V1.2. The amount of charge influx through Ca_V1.3 during an AP turned out to be larger than that of Ca_V1.2. Those results suggest that the unique voltage-dependent kinetics of Ca_V1.3 increase its contribution in Ca²⁺ influx and in keeping AP duration. [J Physiol Sci. 2008;58 Suppl:S58]

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Our previous data obtained by cDNA microarray analysis and quantitative RT-PCR indicated that the arachidonic acid–leukotriene B4 (LTB4) may be up-regulated in the nucleus tractus solitarii (NTS), a pivotal region regulating arterial pressure, in the spontaneously hypertensive rat (SHR) compared to their progenitor strain, Wistar-Kyoto rat (WKY). In this study, we tested the functional significance of LTB4 in the NTS in regulating cardiovascular system. In radio-telemetered WKY LTB4 was microinjected into the NTS and cardiovascular parameters were monitored over one week. Although the arterial pressure (BP) did not change acutely, it increased gradually over 4 days (about 15 mmHg, n=5) demonstrating its pro-hypertensive effect. Heart rate and spontaneous baroreflex gain did not change. Power spectral analysis of BP demonstrated that three days post LTB4 injection the LF + VLF power of BP variability was increased (about 45%, n=5) indicative of raised sympathetic activity. We suggest that LTB4 in the NTS is affecting mechanisms controlling the set point of arterial pressure independent of alteration in baroreflex gain. Since LTB4 is known to be involved in inflammatory diseases, we propose a new hypothesis that inflammatory reactions in the NTS may lead to detrimental consequences for cardiovascular autonomic homeostasis resulting in neurogenic hypertension. British Heart Foundation funded research. [J Physiol Sci. 2008;58 Suppl:S59]

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We previously reported that the direct delivery of nNOS induced the infracted area after acute myocardial infarction (AMI). We investigated the hemodynamics effect of targeted, direct delivery of nNOS and the hemagglutinating virus of Japan envelope (HVJ-E) vector transduced into rat cardiomyocytes after AMI. AMI induced to ligate the left anterior descending coronary artery of Wistar rat. And then 90 minutes later, we injected nNOS with the HVJ-E vector (NV group, n=8), β-galactosidase with HVJ-E vector (BV group, n=8), nNOS alone (N group, n=8) or HVJ-E vector alone (V group, n=8) into the hearts and continued to measure left ventricular pressure in order to calculate the value of dp/dt. We removed them at 3 hours later and evaluated the infarcted area by a colormetry. The infarcted areas in sections from the NV group were significantly narrower than those from the other groups (p<0.05). The value of dp/dt at the end point in the NV group was significantly higher than those from the other groups (p<0.05). Our findings suggest that direct nNOS transduction into cardiomyocytes rapidly diminished the size of infarcted areas after AMI. This type of targeted delivery should have wide application in terms of delivering therapeutic nNOS to the heart, and provide a powerful therapeutic tool with which to treat ischemic heart disease. [J Physiol Sci. 2008;58 Suppl:S59]

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Background: While apelin and its endogenous receptor APJ have been reported to play roles in pathophysiology of the cardiovascular system, the relationship of apelin-APJ system with cardiac hypertrophy is still not well understood. Recently, Kuba et al. have reported that the degree of cardiac hypertrophy induced by pressure overload in apelin-deficient and wild type (WT) mice are not significantly different (2007, Circ Res). Since we had generated APJ-deficient (APJ KO) mice, we examined whether apelin-APJ system affected the pathogenesis of catecholamine-induced cardiac hypertrophy. Materials and Methods: APJ KO mice and WT mice at 3-month old (n=11, respectively) received chronic infusion of isoproterenol (ISO, 60mg/kg/day) for 7 days. Echocardiography was performed before and after infusion. Results: In the absence of ISO infusion, the heart of APJ KO mice was not morphologifcally different from that of WT mice. Excessive β-adrenergic stimulation developed significant cardiac hypertrophy in both strains, but to a greater extent in APJ KO mice (the ratio of heart weight to tibial length: 8.1±0.7 versus 7.5±0.7 mg/mm, p<0.05). Conclusion: The present study demonstrated that apelin-APJ system played an important role in preventing catecholamine-induced cardiac hypertrophy. [J Physiol Sci. 2008;58 Suppl:S59]

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The sarcolemmal Na⁺/H⁺ exchanger1 (NHE1) is a primary mechanism for acid extrusion and a major Na⁺-influx pathway in myocardium. Recent evidence suggests that NHE1 activation is involved in cardiac hypertrophy and heart failure (HF). However, whether NHE1 activation alone is sufficient to induce detrimental effects or what molecular pathways are involved in pathogenesis remains unknown. To directly answer these questions, we generated cardiac-specific transgenic (Tg) mice overexpressing a constitutively-active form of human NHE1 (delta 637-656). We found that Tg hearts developed hypertrophy and HF in vivo, with activation of CaMKII and p38 MAPK. In isolated myocytes, intracellular pH, Na⁺ and Ca²⁺ concentrations were significantly elevated in Tg group, accompanied by enhanced SR Ca²⁺-loading via CaMKII-dependent phosphorylation of phospholamban. Furthermore, NHE1-overexpression induced translocation of HDAC4 and NFATc, fully to the cytoplasm but partially to the nucleous, respectively in cultured neonatal myocytes, suggesting that NHE1 activation can drive the Ca²⁺-dependent prohypertrophic pathway CaMKII-HDAC and partially calcineurin-NFAT signals. All NHE1-induced effects observed in vivo and in vitro were largely prevented by the specific inhibitor cariporide. Taken together, these data indicate that NHE1 activation, which induces an elevation of intracellular Na⁺ followed by altered Ca²⁺-handling, is sufficient to initiate cardiac hypertrophy and HF through mainly activation of the CaMKII-HDAC pathway. [J Physiol Sci. 2008;58 Suppl:S59]

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Seasonal variations have been reported in a wide range of physiological parameters, including components of the neuroendocrine and immune system.In the present study we investigated the seasonal changes on hormonal and autonomic nervous systems. 8 volunteers were subjected for the experiment at four times of the year–around the vernal and autumnal equinoxes and the summer and winter solstices. Seasonal changes in the thermoregulatory responses are assessed by measuring core and skin temperatures, sweat rate and blood flow during immersion of the leg in hot water (mild heat at 40^oC to 42^oC) for 30 min. The following parameters were analyzed: metabolites of catecholamine (VMA, HVA, 5-HIAA), angiotensin II, aldosterone, plasma renin activity, ADH, cortisol, ACTH, GH, leptin, IL-6. Tympanic temperature increased significantly in winter during water immersion of 42^oC compared with the same condition in spring and summer. Sweating rate was significantly higher and sweating more quickly appeared in summer season compared with winter or spring. Concentration of ADH and angiotensin II was significantly higher in summer compared with winter and spring. Concentration of IL-6 was significantly lower in spring and summer compared with winter. Concentration of ACTH and cortisol was significantly higher in summer. The results of this study demonstrated statistically significant seasonal changes in ADH, angiotensin II, VMA, IL-6, and core body temperature and sweat rate. [J Physiol Sci. 2008;58 Suppl:S60]

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In Japan, environmental temperature changes very widely. With annual climate changes, body temperature regulation as well as hormonal regulation may be changed. The purpose of this study is to investigate the seasonal changes in temperature and hormonal regulation in elder subjects (69±4.6 yrs; mean±SD) and young subjects (21±1.3 yrs; mean±SD). The experiments were performed in the climatic chamber set at 26^oC and 50% RH during spring, summer, autumn and winter. Each subject immersed his lower legs in hot water at 40^oC for 30 min in a sitting position. Core (tympanic) temperature, skin temperature and forearm sweat rates were recorded continuously. Blood was taken in pre-immersion and in post-immersion to measure the concentrations of antidiuretic hormone, plasma renin activity, angiotensin II, aldosterone, TSH, fT3 and fT4. The results obtained in spring and summer experiments showed that the rise of core body temperature and sweat rate during hot water immersion was lower in elder group than in young group. We will discuss the entire profile of the seasonal changes in thermoregulatory and hormonal function in young and elder subjects when the experiments in autumn and winter are completed. [J Physiol Sci. 2008;58 Suppl:S60]

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Introduction We have reported that estrogen has influence on thermoregulation in the heat and cold. In ovariectomized rats, in the heat, an increase in body temperature (T_b) was greater with a reduction in heat dissipation from the skin. In the cold, T_b was smaller with a decrease in metabolic heat production. However, 17β-estradiol (E₂) replacement restores these responses to normal levels. The purpose of the present study was to test the hypothesis that the estrogen fluctuation in normal estrus cycle would modulate the thermoregulary response to the cold in rats. Methods Female WKY rats were laparectomized and radiotelemeters for T_b were placed. At least 2 wks after the surgery, rats' vaginal smear was obtained to determine the estrus cycle. At 9 am, rats in proestrus or diestrus period were exposed to 5°C or 25°C environment for 2 h, and T_b was monitored. After the cold exposure, rats were perfused with phosphate buffered saline and the brains were excised. cFos immunoreactive cells in the hypothalamic area were counted. Results T_b increased in the cold in the proestrus period, but decreased in the diestrus period. Plasma E₂ was higher in the proestrus period than estrus period with a small difference in progesterone level. cFos immunoreactive cells in the medial preoptic area and dorsomedial hypothalamus were greater in the proestrus period. Conclusion Thermoregulation in the cold differs among the periods of estrus cycle in rats. The fluctuation of plasma E₂ level may be involved in the mechanism. [J Physiol Sci. 2008;58 Suppl:S60]

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In bears and squirrels, core temperature is decreased in winter. On the other hand, it has been considered that core temperature is not decreased in humans and mice. However, when energy intake is insufficient in mice, they enter torpor, in which core temperature is daily decreased. Furthermore, Swoap et al. and Zhang et al. report that intraperitoneal injection of adenosine monophosphate (AMP) reduces core temperature in mice. Therefore, we hypothesized that thermoregulatory system of mammals has a shutdown switch to stop thermoregulatory responses. The purpose of this study is to verify this hypothesis. Mice were injected with AMP (3.0-7.5 μmol/g, i.p.), and their core temperatures and autonomic and behavioral heat-gain responses were investigated. Saline was used as control. After AMP injection, core temperature was decreased. This temperature drop was dose-dependent. Core temperature of AMP-injected mice was changed with ambient temperature, indicating that mice acted like heterotherms. When ambient temperature was low, skin temperature around the brown adipose tissue (BAT) was higher than that of other skin areas. In AMP-injected mice, however, skin temperature around BAT was as low as that of other skin areas, showing that BAT activity was decreased by AMP. These results demonstrate that thermoregulatory system of mammals has a shutdown switch to stop thermoregulatory responses. [J Physiol Sci. 2008;58 Suppl:S60]

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To shed light on the specific role of the transmembrane protein PECAM-1 in monocyte diapedesis, PECAM-1-GFP fusion vector was constructed and transfected into human umbilical vein endothelial cells (HUVECs). Since GFP was fused to the C-terminus (intracellular side) of PECAM-1, interactions of the PECAM-1 extracellular domain with monocytes can be kept intact. Three-dimensional-live-cell molecular imaging revealed that endothelial PECAM-1 was dynamically recruited to surround the transmigrating monocyte locally during paracellular diapedesis. This recruitment was not observed during transcellular diapedesis, which was only 5% of total diapedesis events. Three-dimensional image analysis showed a <1.5–1.8 fold increase of local PECAM-1 fluorescent signals near transmigrating monocytes within 20 min of paracellular events. Flow cytometric analysis also indicated a <1.4-fold increase of overall PECAM-1 signals from endothelial cells in 50 min of monocyte coincubation. The recruitment was not seen in the local region which was not associated with diapedesis events, which excludes the involvement of soluble factors secreted by monocytes. Blocking of neither monocyte adhesion nor transmigration induced the recruitment. These data indicate that local transmigration events, but not adhesion, or monocyte soluble factors, could induce the dynamic recruitment of endothelial PECAM-1 to transmigrating monocytes during paracellular diapedesis. [J Physiol Sci. 2008;58 Suppl:S61]

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Fluorescence imaging is a powerful method to visualize and analyze spatiotemporal dynamics of molecules in a living cell. Here we have developed a prism-based total internal reflection fluorescence microscopy (TIRFM ) system to simultaneously visualize and analyze three 2nd messengers, Ca²⁺, cAMP and diacylglycerol (DAG), that involve many cellular functions. Employed were three fluorescent indicators: 1) fura-2 AM for Ca²⁺, 2) Epac1-camp, a CFP-YFP FRET-based cAMP indicator, for cAMP and 3) C1-mRFP, a tandem DAG binding domain of PKC gamma, for DAG. These indicators were transfected into Cos 7 cells. DAG signal was taken by TIRFM, and Ca²⁺ and cAMP signals were taken by epifluorescence microscopy. When three 2nd messengers were evoked in Cos7 cell stimulated with 100uM ATP, each signal was successfully dissected without significant overlap of emission wave length among three signals. Unmixing of overlapping signals was also discussed in this study. [J Physiol Sci. 2008;58 Suppl:S61]

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Rabphilin is generally thought to be involved in the regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells. The N-terminal part of rabphilin has recently been shown to function as an effector domain for Rab3A/Rab27A in PC12 cells, but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and exocytotic proteins and found that the C2B domain of rabphilin directly interacts with a plasma membrane protein, SNAP-25. Furthermore, vesicle dynamics were imaged by total internal reflection fluorescent microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-deltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25. [J Physiol Sci. 2008;58 Suppl:S61]

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Sperm are highly differentiated cells adapted to perform one specific role: to fertilize the egg. Despite their small size, sperm have the ability to precisely recognize physicochemical signals from the environment and the female reproductive organs and respond in a specific and regulated manner. Therefore, sperm are often considered to be comparable to the sensory neurons. Motility is one of the most important sperm functions and its defect directly leads to infertility. To study sperm motility regulation in detail, we developed a high-speed fluorescence imaging system based around stroboscopic illumination with a light-emitting diode (LED). We also developed several caged compounds (ligands and second messengers) to rapidly stimulate sperm without causing any turbulence in the surrounding medium, a major limitation of classical bath application protocols. Using these techniques, we induced a chemotaxis-like motility response in sea urchin sperm and confirmed that sperm flagella become more asymmetric when the flagellar intracellular Ca²⁺ concentration increases. The advantages of our imaging system and the insights it has revealed regarding sperm motility regulation will be summarized. [J Physiol Sci. 2008;58 Suppl:S61]

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One of the forelimb flexor muscles, flexor carpi radialis muscle (FCR), in male frogs is used for long-lasting mating posture, so-called amplexus, and its mechanical properties are known to be much different from those in other skeletal muscles, e.g. the hindlimb muscles used for jumping. It was reported previously that both the rates of increase and decrease in contractile force of FCR at the beginning of and after the end of the electrical repetitive stimulation were much slower than those in hindlimb. In the present experiment, we measured histochemical activities of FCR and hindlimb muscles to know the difference in muscle fiber types in frog.We stained FCR muscle and two hindlimb muscles, iliofibularis muscle (IFM) and glutaeus magnus muscle (GMM), in breeding season (Feb.- Mar.) for adenosine triphosphatase (ATPase) with the preincubation in pH 4.9 and 10.1. In addition, these muscles were also stained for succinate dehydrogenase (SDH). With these three histochemical methods, it was demonstrated that FCR in forelimb consisted of various fiber types including slow types, while GMM and IFM in hindlimb consisted of one fast type and of one fast and one slow types respectively. [J Physiol Sci. 2008;58 Suppl:S64]

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X-ray diffraction is a unique method which enables us to measure structural changes of contractile and regulatory proteins in live muscles. When a skeletal muscle is electrically stimulated, calcium is released from sarcoplasmic reticulum and binds to troponin in the thin filament. The calcium binding can be monitored by measuring the intensity of the troponin meridional reflection in the x-ray diffraction pattern. Using high flux x-rays from SPring-8, we measured the time course of this intensity change at the time resolution of up to 0.5 ms in a frog skeletal muscle. At 8°C, the onset of the intensity change was about 4 ms after the stimulus, much earlier than intensity changes of myosin-related reflections and the tension development. It also proceeded the latency relaxation in tension. The intensity change of the troponin reflection showed a marked delay to the rise in the intracellular calcium concentration, suggesting a cooperative nature of the structural change in the thin filament. Results on overstretched muscle and double-pulse stimulation will be also presented and discussed. [J Physiol Sci. 2008;58 Suppl:S64]

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As an extension of our previous NMR studies of myofibrils, both the diameter and transverse stiffness of single skeletal myofibrils were examined under various physiological conditions by AFM. Single myofibrils were prepared by homogenizing glycerinated muscle fibers of rabbit psoas muscle. AFM measurements were made as previously for myofibrils fixed on the surface of cover slip coated with aminosilane. Modified AFM cantilever having a micro-bead tip were used for measurements. The diameter of single myofibrils was 1.12, 1.01, 1.01 μm in a relaxed, contracting and rigor states, and Their transverse stiffness 0.5, 1.0 and 1.5 pN/nm respectively. By increasingly adding Dextran up to 16%, the diameter of myofibrils graduary decreased by 54% in a relaxed state and by 34% in a rigor state. On the other hand, the transverse stiffness linearly increased 2-3 times in a rigor state, and 1.5-2 times in relaxed and contracting states up to 2% Dextran, and, by further increase of Dextran, leveled off at 7-8 pN/nm for all of these states. Interestingly, in the presence of BDM, the diameter and the transverse stiffness changes of myofibrils took place as those in a contracting state at the low Dextran concentrations while they took place as those in a relaxed state at the high Dextran concentrations. The results were analyzed in the relation with the cross-bridge structure and the force production in the actomyosin filaments lattice in myofibrils. [J Physiol Sci. 2008;58 Suppl:S64]

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Using the gas environmental chamber,with which electron microscopic images of living muscle thick filaments can be recorded on the imaging plate, we have attempted to record ATP-induced cross-bridge movement at both sides of the thick filament bare region, across which the cross-bridge polarity is reversed. Large bipolar thick filaments were prepared by slowly polymerizing rabbit skeletal muscle myosin. Without ATP application, the position of gold particles, attached to the junctional peptide between 50 and 23kDa fragments of myosin heavy chain with a site-directed antibody, did not change appreciably with time, indicating that the time-averaged cross-bridge position remained almost unchanged despite thermal motion. On application of ATP to myosin filaments, the cross-bridges were found to move by 5-8nm in the direction away from, but not towards, the filament bare region. Since thin filaments are absent in the experimental system, the ATP-induced cross-bridge movement is associated with the formation of intermediate, M-ADP-Pi, and is therefore regarded as the cross-bridge preparatory stroke, having the amplitude identical with, and the direction opposite to, the cross-bridge power stroke. We thank Japan Electron Optics Laboratory for providing facilities to carry out our work. [J Physiol Sci. 2008;58 Suppl:S64]

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To study the effects of bite-opening treatment (BO) on the daily duty time determined as the total duration of the activity bursts relative to the experimental periods (i.e.,% of 24 h period) and the fiber-type composition in rat jaw muscles, we measured both the electromyographic activity (EMG) by using a radio-telemetry system and the myosin heavy chain (MHC) composition by using a SDS-PAGE analysis in masseter and digastric muscles of the control and bite-opened rats. The daily duty time of the digastric muscle was significantly (P < 0.05) higher than that of the masseter muscle at the activity levels exceeding 5 and 20% of the day's peak activity, whereas the opposite was true at the activity levels exceeding 50 and 80% of the day'peak activity (P < 0.05) in the control state. The MHCs in the digastric muscle consisted of a mixture of fast and slow types, while mostly fast type in the masseter muscle. BO produced not only a significant increase (P < 0.05) in the daily duty time at any activity levels but also a MHC transition toward slower isoforms in both the masseter and digastric muscles, though the opposite mechanical load were imposed to the two different muscles by BO. These results suggest that in rat the increase in the duty time associated with BO induces a fiber-type transition toward slower phenotypes both in masseter and digastric muscles. [J Physiol Sci. 2008;58 Suppl:S65]

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Mechanical response and structural changes of contractile filaments in skinned smooth muscles from guinea pig taenia caeci were simultaneously measured using X-ray diffraction technique. Cell membrane of the intact taenia was chemically permeabilized with β-escin. The diffraction pattern showed the 14.4-nm myosin reflection, the 5.9-nm actin layer-line and the equatorial peak at 1/11.4 nm⁻¹ originated from packed array of thin filaments as previously observed in intact preparatios from taenia caeci (Watanabe et al., 1993). During activation with Ca²⁺, the skinned preparations contracted and the intensity of 14.4-nm reflection significantly increased. When the contraction was inhibited by application of blebbistatin, a potent inhibitor of myosin II ATPase, the 14.4-nm reflection was significantly decreased. Since blebbistatin is known to disrupt myosin filament organization, X-ray diffraction may be a useful tool to evaluate dynamics of myosin filament organization in smooth muscle cells. [J Physiol Sci. 2008;58 Suppl:S65]

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Polyethylenglycol narrows the lattice spacing of skinned skeletal muscle sarcomere. Since the size of polyethylenglycol ( M.W. 3350 ) seems around few nanometers, the lattice spacing of around 40 nanometers was seemed to be large enough for penetration of polyethylenglycol. To reveal whether the polyethylenglycol penetrates into the sarcomere or not, I measured the specific gravity of myofibril suspensions from rabbit psoas muscle in the presence or absence of polyethylenglycol. If polyethylenglycol dose not penetrate into the sarcomere, the specific gravity of the supernatant after centrifugation of myofibril suspension is larger than the specific gravity of myofibril suspension. [J Physiol Sci. 2008;58 Suppl:S65]

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Water in skeletal muscle is inhomogeneous from various aspects. This is the source of the power of MRI to distinguish various tissues of various patho-physiological states. The states of water would naturally affect biochemical reactions taking place in vivo, and therefore significantly affect our sound understanding of living organisms. We have been studied the osmotic activity of myowater in intact and skinned skeletal muscle, whose liquid-crystalline structure is advantageous in defining water states in the tissues. Our results revealed that water in skeletal muscle tissue can be grouped into four characteristic components. In the present study, considering the water vapor pressure in equilibrium with each water components, we found that each water components can be characterized by the activity profile of water molecules which is one of the basic physicochemical characteristics of molecules. To extend our fining in skeletal muscle to general biological tissues, we tried to find relationships between water components and water activity in several tissues of characteristic histological features (such as brain and testis). The results suggested an apparent discrepancy form the general view of water states developed by ordinary radiologists to evaluate MRI. [J Physiol Sci. 2008;58 Suppl:S65]

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