It has been known that atrial natriuretic peptide (ANP) inhibits Na⁺ reabsorption in renal tubules of the mammalian kidney. We, however, recently reported that ANP and cGMP stimulate amiloride-blockable Na⁺ absorption through PKA-dependent pathway in the urinary bladder of the Japanese tree frog, Hyla japonica. We also suggested that there is a possibility of expression of cyclic nucleotide-gated channels in the frog urinary bladder using Ussing chamber voltage-clamp and whole cell patch-clamp techniques (Yamada et al., 2005). In the present study, we examined to identify characteristics of the channels activated with ANP and cGMP by using a single channel recording in epithelial cells of urinary bladder. In the recordings of cell-attached patch with NaCl in a patch pipette, a variety of ion channels were revealed the activity with conductance ranging from 5 to 30 pS. Ion channels with conductance for inward current of 5-7 pS were examined because the channels have the same properties, such as conductance and slow opening and closing kinetics with the epithelial Na channels in the toad urinary bladder. When Cl⁻ in the pipette solution was replaced by gluconate, the conductance of the channel did not change. This shows the channel carries cations but anions. Addition of 10⁻⁴ M 8-Br-cGMP to the bath solution significantly stimulated the inward current in the cell-attached patches. These results suggest that ANP- and cGMP-dependent increases in Na⁺ absorption are mediated by 5-7 pS cation channels in the epitherlial cells of the frog urinary bladder. [J Physiol Sci. 2006;56 Suppl:S141]

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Potent appetite-stimulating effects of neuropeptide Y (NPY) are well known. The present study undertook to demonstrate that the role of NPY in gastric relaxation of the proximal stomach was examined in anaesthetized rats. Intragastric pressure (IGP) was measured using a balloon situated in the proximal part of the stomach. The administration of NPY into the fourth ventricle induced relaxation of the proximal stomach in a dose-dependent manner. The administration of an Y1 receptor agonist [Leu³¹, Pro³⁴] NPY induced a larger relaxation than NPY. The administration of an Y2 receptor agonist (NPY13-36) did not induce significant changes in motility. Microinjection of [Leu³¹, Pro³⁴] NPY into the caudal part of the dorsal vagal complex (DVC) induced relaxation of the proximal stomach. In contrast, similar injections into the intermediate part of the DVC increased IGP of the proximal stomach. The administration of NPY into the fourth ventricle did not induce relaxation after bilateral injections of the Y1 receptor antagonist (1229U91) into the caudal DVC. Bilateral vagotomy below the diaphragm abolished the relaxation induced by the administration of NPY into the fourth ventricle. The intravenous injection of atropine methyl nitrate reduced the magnitude of relaxation induced by the administration of NPY. These results indicate that NPY induces relaxation in the proximal stomach via Y1 receptors situated in the DVC. This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science to MK. [J Physiol Sci. 2006;56 Suppl:S142]

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Aim: Mosapride, selective 5-HT₄ receptor agonist, and itopride, selective D₂ receptor antagonist, were known to enhance motility of the distal stomach and facilitate gastric empting. On the other hand, sumatriptan, selective 5-HT₁ receptor agonist, and paroxetine, 5-HT reuptake inhibitor, were reported to induce a relaxation in the proximal stomach. In this study, effects of these four drugs on proximal gastric tone were researched using a barostat in conscious dogs with a gastric fistula. Methods: The effects of the drugs and those vehicles on the proximal gastric volume at a constant intragastric tone were studied using a barostat. Simultaneously, proximal and distal gastric contractility, and duodenal contractility was measured using three force transducers. Furthermore, stepwise isobaric gastric distensions were performed before and after the administration of the drugs. The same experiments were done in fasting phase and in postprandial phase. Results & Conclusion: Effects of the drugs on proximal gastric tone were more clearly seen in postprandial phase than fasting phase, and sometimes differed from those on distal gastric and duodenal contractility. Such a measurement of proximal gastric tone using a barostat in animal experiments seems to be useful for estimation of effectiveness of some drug on gastrointestinal disorders. [J Physiol Sci. 2006;56 Suppl:S142]

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The gastrointestinal (GI) tract demonstrates a number of changes that accompany advanced age. In the present study, we investigated gastric acid secretion in young and old mice in response to chemical stimulation and mechanical stimulation. Gastric acid secretion was measured in anesthetized mice. Proton pump (H⁺,K⁺ ATP ase) is a good maker of the parietal cell function, therefore, protein expression of proton pump was determined by Western blotting. After 60 min basal collection, acid secretion was stimulated by histamine (500 and 1000 mg/kg) or carbachol (10 and 20 mg/kg). To investigate the response to mechanical stimulation, the stomach was distended by an intragastric injection of isotonic saline (0.5, 1.0, 1.5 and 2.0 ml). 1) Administration of 2 doses of histamine produced a dose-dependent increase in acid secretion in young mice, whereas a higher dose of histamine could not produce a significant increase in acid secretion in old mice. 2) Stimulatory effect of carbachol on gastric acid secretion did not differ between young and old mice, although the mean values tended to be lower in old animals than in the young. 3) The response to mechanical stimulation tended to be lower in old animals. 4) Protein expression of H⁺, K⁺ ATPase was significantly lower in old mice than the young. The present study showed that gastric acid secretion was different between young and old mice concerning to various kinds of stimulations. The decrease in the secretory function of the stomach in the old mice is partly associated with a decrease in parietal cells (H⁺,K⁺ ATPase ). [J Physiol Sci. 2006;56 Suppl:S142]

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Thirteen young male subjects first lied in the supine position on bed with eyes opened for 10 min, followed by 10 min exercise of a cycle ergometer with a load randomly chosen among three different ones inducible heart rates as high as 15, 30, or 60% of the predicted maximal heart rate, and again took a supine position as during the first rest for 40 min. ECG and EGG were measured by using bio-amplifiers during all the experimental period, AD converted at the rate of 1kHz and stored into PC. RR-intervals obtained from ECG were Fourier transformed by every 64, serially from the top to the end of the file with doubling about its 50%. Cardiac sympathetic (CS) as well as parasympathetic nerve activities (CPS) were assumed from the power in high-frequency band (HF), and the relative magnitude of the power in low-frequency band (LF) to HF, respectively. EGG re-sampled by 10 Hz from the row EGG datum was Fourier transformed by every 512, serially from the top to the end, with doubling about its 50%. The power summed in the range between 2.4 and 3.6 cpm was assumed to be gastric parasympathetic nerve activities (GPS). CPS tended to increase only during 10-20 min district after 30% exercise (ex30). GPS was significantly increased during 10-15 min district (p<0.05) in the post-ex30 period. Either CPS or GPS after 60% exercise was significantly depressed. These results suggested that moderate exercise could augment parasympathetic nerve activities, particularly in the gastric region. [J Physiol Sci. 2006;56 Suppl:S143]

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In vascular endothelial cells acetylcholine (ACh) increases [Ca²⁺]_i which in turn activates charybdotoxin-sensitive IK_Ca, apamin-sensitive SK_Ca and Cl_Ca channels. As a result, ACh induces a membrane hyperpolarization, but the response is sometimes transient and followed by the membrane depolarization. To investigate the possible mechanism for this variation, a sheet of endothelial cells was isolated from a guinea-pig mesenteric artery and perforated whole-cell clamp experiments using amphotericin B were performed. The intracellular Cl⁻ concentration was modified by using either low-Cl⁻ (20 mM) or high-Cl⁻ (150 mM) pipette solutions. In the current clamp mode, application of ACh (0.5 μM) induced a large and sustained hyperpolarization when [Cl⁻]_i was low, while the response was small and transient when [Cl⁻]_i was high. In the voltage clamp mode in low [Cl⁻]_i condition, the reversal potential of ACh-induced current was -54.1 mV and it was changed to -27.8 mV after the K conductance was blocked by charybdotoxin and apamin. On the other hand, the reversal potential was changed from -57.8 mV to -76.0 mV by blocking Cl conductance with DIDS. When [Cl⁻]_i was high, the reversal potential was -22.0 mV and it was changed to -0.6 mV after the K channels were blocked. These results indicate that enhanced Ca²⁺-activated Cl⁻ current modifies ACh-induced hyperpolarization to be small and transient when the [Cl⁻]_i is increased in some pathological conditions. [J Physiol Sci. 2006;56 Suppl:S143]

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Heat shock protein 20 (HSP20) has actin binding capacity and its amino acid sequence of actin binding region (residues 110-121; GFVAREFHRRYR) is highly homogenous to the inhibitory region of skeletal muscle troponin I (residues 104-115). Our previous study showed that, in Titon-X-100 skinned muscle preparations from guinea pig taenia caeci, a synthetic peptide of the actin binding region of HSP20 (HSP20_p) had both a suppressing effect on the maximal Ca²⁺ induced force and an enhancing effect on the Ca²⁺ sensitivity for the force (Yoshino et al., ). In the present study, to evaluate the contribution of each amino acid residue of HSP20_p to the dual effects of HSP20_p on the skinned taenia, we compared the effects of 11 HSP20_p analogues, which consisted of single glycine replacement, on the Ca²⁺-induced contraction. Every residue of HSP20_p was necessary to achieve maximum inhibition of the Ca²⁺-induced contraction. On the other hand, replacing F111, V112, A113, E115, F116 or R121 for glycine resulted in losing enhancing effects on the Ca²⁺ sensitivity for the force. These results suggest that, in phasic smooth muscles, the pathways through which HSP20_p modulated the maximal Ca²⁺ induced force and Ca²⁺ sensitivity for the force appears to be different. [J Physiol Sci. 2006;56 Suppl:S143]

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Contractions evoked by electrical stimulation of 50 pulses with 40 Hz to the guinea-pig vas deferens consist of L- and T-type Ca²⁺ channel-mediated adrenergic (α₁) component and L-type Ca²⁺ channel-mediated purinergic component. SK and BK channels closely located L- and T-type Ca²⁺ channels greatly affect on the contraction, especially α₁ adrenergic component. The α₁ component consisting of the early and late components was obtained by the treatment of P2 antagonist. P2 antagonist suramin (300 μM) not only antagonizes P2x purinoceptors but also activates SK channels via P2Y2 purinoceptors (Ming et al). PPADS (30 μM)-treated vas deferens revealed large early α₁ component suggesting that PPADS itself might inactivate SK channels. α,β-Methylene ATP (desensitization of P2x purinoceptors, 10 μM) did not affect directly K⁺ channels. SK channel antagonist apamin (100 nM) amplified both early- and late-α₁ components. BK channel antagonist iberiotoxin (100 nM) amplified the late-α₁ component without affecting on the early-α₁ component. On the contrary, another BK channel antagonist CTX (100 nM) amplified not only the late-α₁ component but also the early-α₁ component. CTX seemed to deprive the action of PPADS to antagonize P2x purinoceptors. The results suggest that α,β-methylene ATP was suitable for antagonizing P2x purinoceptors, and that apamin and iberiotoxin were more suitable for antagonizing SK and BK channels, respectively. [J Physiol Sci. 2006;56 Suppl:S144]

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Gastric smooth muscle cells generate slow waves spontaneously, and their activities may be originated from interstitial cells of Cajal. Experiments were carried out to investigate the effects of hypoxia on the activities of smooth muscle cells isolated from the guinea-pig stomach antrum by recording intracellular membrane potentials using conventional microelectrode methods, and also by measuring intracellular Ca²⁺concentration ([Ca²⁺]i) with fura-2 fluorescence. Hypoxic condition was produced by bubbling Krebs solution with N₂-gass containing 5% CO₂. Hypoxia increased the frequency of slow waves with no significant alteration to the membrane potential, and these were associated with the increase in frequency and decreased in amplitude of Ca²⁺-transients. In the presence of 10 μM cyclopiazonic acid (CPA, an inhibitor of Ca²⁺-ATPase at internal Ca stores), the increase in frequency of slow waves in response to hypoxia was abolished. In the presence of 1 mM KCN (a blocker of oxidative phosphorylation), the frequency of slow waves was decreased, with associated reduction of the amplitude. In the presence of KCN, hypoxia again increased the frequency of slow waves. These results suggest that hypoxia-induced increase in the frequency of slow waves is associated with the elevated release of Ca²⁺ from internal CPA-sensitive stores, possibly in the pacemaker cells. As the hypoxia-induced increase in the frequency is not altered by KCN, no significant contribution of mitochondrial factor in pacemaker cells is considered. [J Physiol Sci. 2006;56 Suppl:S144]

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TnIp, a synthetic peptide originating from an actin tropomyosin binding region of rabbit cardiac troponin I [residues 136-147; GKFKRPTLRRVR], had biphasic effects on the relaxation of skinned smooth muscle, as accelerating the initial phase and slowing the following latter phase of the relaxation, resulted from accelerating fast cross-bridge dissociation and also transformation from fast to slow (latch) cross-bridges (Watanabe et al., 2004). To evaluate the contribution of each amino acid residue of TnIp to its biphasic effects on the relaxation, we compared the effects of 11 TnIp analogues, which consisted of single glycine replacement, on the relaxation time course by lowering Ca²⁺ concentration in contracting skinned taenia caeci from guinea pig. Replacing K₁₃₇, F₁₃₈, K₁₃₉, R₁₄₀, T₁₄₂, L₁₄₃, R₁₄₄, or R₁₄₅ for glycine resulted in loss of the accelerating effect of TnIp on the fast cross-bridge dissociation, seeming that the entire residues of TnIp might be necessary for acceleration of the cross-bridge dissociation by TnIp. On the other hand, the enhancing effect of TnIp on the translation from fast cross-bridges to latch bridges was kept except only when R₁₄₇ was substituted for glycine. R₁₄₇ seems to be a key residue for regulation of latch bridge formation by TnIp. [J Physiol Sci. 2006;56 Suppl:S144]

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Muscle satellite cells have been considered to play an important role in postnatal growth and the regeneration of skeletal muscle. Recently it has been suggested that the activation of muscle satellite cells is also associated with muscle hypertrophy. Therefore, the regenerative process of injured muscles may be facilitated by the activation of muscle satellite cells induced by various hypertrophic stimuli. The purpose of this study was to investigate the effects of overloading on the regenerative process of injured skeletal muscle in mice. Male mice (C57BL/6J) were divided randomly into four groups: (1) cage control (CC), (2) cardiotoxin-injected (CX), (3) functional-overloaded (FO) and (4) CX+FO groups. In the groups of FO with and without CX, overloading on soleus of both hindlimbs was performed by cutting the distal tendons of both plantaris and gastrocnemius muscles. Two weeks after the surgery, cardiotoxin was injected into soleus muscles of both limbs in CX and CX+FO groups. Soleus muscles were dissected 14, 28 and 35 days after cardiotoxin-injection. Responses of muscular protein contents and Pax7-positive muscle satellite cells during the regeneration were analyzed. Evidences suggest that functional overloading may facilitate the regeneration of injured skeletal muscles. [J Physiol Sci. 2006;56 Suppl:S144]

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Recently, we have identified multipotent stem cell populations residing in the interstitial spaces of skeletal muscle. We characterized these cells using FACS on the basis of cell surface antigen expression, and sorted them as a CD34+/45- and CD34-/45- cell fractions from enzymatically isolated cells. Cells in the CD34+/45- fraction (designated Sk-34 cells) were >94% positive for Sca-1 and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit and FLK-1showing that Sk-34 cells are not committed endothelial progenitors. However, Sk-34 cells formed colonies in clonal cell culture, and CFU displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. We also identified cells in the CD34-/45- (designated Sk-DN cells) fraction as a putative cell population that includes further immature stem cells; that can form clonal sphere-like colonies in a collagen based cell culture with bFGF and EGF; and that exhibits the potential to differentiate into myogenic and endothelial cells. In addition, the Sk-34 and Sk-DN cells included only a few side-population (SP) cells and mostly composed by main-population cells (1:1000). These findings demonstrated that multipotent stem cells (Sk-34 and Sk-DN cells) residing in the interstitial spaces of skeletal muscle, and potentially contribute to postnatal myogenesis and vasculogenesis following muscle growth and/or muscle hypertrophy, and there were not SP cells. [J Physiol Sci. 2006;56 Suppl:S145]

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We have shown that stem cell populations residing in the interstitial spaces of skeletal muscle can give rise to myogenic-endothelial cell lineages, and were designated Sk-34 (CD34+/45-) and Sk-DN (CD34-/45-) cells. Potential therapeutic use of these cells, such as the functional significance of the transplanted tissue, and vasculogenesis, myogenesis was investigated in detail. For this purpose, we developed a severe-damage model of mouse tibialis anterior muscle with a large deficit of nerve fibers, muscle fibers, and blood vessels. Freshly isolated and cultured Sk-34 and Sk-DN cells were transplanted directly into damaged portion of the muscle. Results showed that, after transplantation, implanted cells give rise to myogenic, vascular (pericytes, vascular smooth muscle and endothelial cells), and neural (Schwann cells) cells, as well as contributed to the synchronized reconstitution of blood vessels, muscle fibers, and peripheral nerves, with significant recovery of both mass and contractile function after transplantation. Intrinsic plasticity of these cells was also revealed by fluorescence in situ hybridization (FISH) analysis for the transplanted muscle detecting the Y chromosome. As well, there were no donor-derived Sk-34 and Sk-DN cells in the muscle of lethally irradiated bone marrow-transplanted animals, indicating that the Sk-34 cells were not derived from bone marrow. [J Physiol Sci. 2006;56 Suppl:S145]

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We have shown that multipotent stem cell populations residing in the interstitial spaces of skeletal muscle (Sk-34 and Sk-DN cells) can give rise to myogenic, vascular (pericytes, vascular smooth muscle and endothelial cells), and neural (Schwann cells) cells, as well as contributed to the synchronized reconstitution of blood vessels, muscle fibers, and peripheral nerves, with significant recovery of both mass and contractile function after transplantation. Intrinsic plasticity of these cells when there were transplanted into non-skeletal muscle tissue environment was investigated. The renal capsule, spinal cord and heart were selected as non-skeletal muscle tissues. In the renal capsule, these cells could also give rise to muscle fibers, nerve fibers, and blood vessels as well as in the skeletal muscle tissue. In the spinal cord, however, MAP-2 positive neural cells and blood vessels were observed. Interestingly, in the cardiac muscle tissue milieu, these skeletal muscle derived stem cells could differentiate into mono-nucleated cardiac muscle cells with an apparent desmosome junctions between donor-recipient cells, and/or donor-donor cells. Donor cell derived blood vessels were also observed. These results suggest that Sk-34 and Sk-DN cells can give rise to myogenic, neural and vascular cell lineage intrinsically, and these capacities can be affected by the different tissue milieus. [J Physiol Sci. 2006;56 Suppl:S145]

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We have found multipotent stem cell populations residing in the interstitial spaces of skeletal muscle (designated Sk-34 and Sk-DN cells) can give rise to myogenic, vascular (pericytes, vascular smooth muscle and endothelial cells), and neural (Schwann cells) cells, as well as contributed to the synchronized reconstitution of blood vessels, muscle fibers, and peripheral nerves. To expecting vasculogenic capacity of these cells, we investigated the hypothesis that Sk-34 (CD34+/45-) cells may play an important role for the ischemic tissues as a "tissue specific vasculogenic cells" and may be contribute to vasculogenesis. For this purpose, the Sk-34 cells, obtained from the muscles of 3-6 week-old GFP mice, were administrated intramuscularly into the nude mice with hindlimb ischemic models (n=4 each), and same amount of physiological saline was administrated for control group. Two weeks after transplantation, the Sk-34 cells transplanted group demonstrated significantly less toe necrosis (p<0.05), and enhanced recovery of peripheral perfusion measured by Laser Doppler (p<0.05) compared to control group. Moreover, increase in donor derived (GFP positive) CD31 positive cells and/or vessels can be seen in treated animals compared to control by immunohistochemical analysis. These findings indicate that this cell population represents an accessible cell source that can be used therapeutically to improve postnatal neovascularization. [J Physiol Sci. 2006;56 Suppl:S145]

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Skeletal muscle crush injuries occur in an incident when the limb is trapped with a compressing force. Little is known about the recovering mechanisms of injured muscles. To study these, we developed a new apparatus. For induction of crush injury, a pressure of 4.25 kg/cm² was applied for 2 h on the left lower limb of the rat. We examined changes in their histological and physiological properties. We report that slow fibers play important roles in recovery of skeletal muscles from such injury. A follow-up survey was done for 8 weeks on 12 species of hind limb muscles; their wet-weight, number of fibers, diameter and area of fibers were measured. Slow fibers were identified by immunohistological staining. Muscle function was analyzed from footprint. Soon after the crush, wet-weight of muscles increased by 10-20% in 1-2 days (acute edematous phase), decreased by 30% during 2 days to 2 weeks (atrophic phase) and then increased gradually for 8 weeks (recovery phase). Diameter and area of muscle fibers, which reduced in the first 2 weeks, started to increase gradually in the recovery phase. At 8 weeks, muscle weight exceeded 100% of the control (opposite-side muscles) although their fibers were thinner than the control. Immunohistology revealed that slow fibers showed a large increment in number in the recovery phase; it indicates that they play a major role in recovery from the injury. [J Physiol Sci. 2006;56 Suppl:S146]

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We investigated the effects of hindlimb immobilization (HI) on contractile properties of single skinned soleus fibers in rats. HI (6 weeks) resulted in reduced wet weight of soleus muscle (–40%). In immobilized fibers, maximal Ca²⁺-actiavated force was reduced by ∼40% and the force-pCa curve was shifted to the lower pCa side by ∼0.15 pCa units (sarcomere length, 2.20 μm). Our EM observation revealed thinner myofbrils in immobilized muscle, with the width of Z-line similar in control and immobilized muscles during relaxation and contraction (pCa 4.5). We reconstituted thin filaments of control and immobilized fibers with the identical troponin complex (from rabbit psoas muscle). It was found that Ca²⁺ sensitivity was still lower in immobilized fibers by ∼0.15 pCa units after troponin reconstitution, suggesting that troponin isoform switches, if at all, can not account for functional changes of immobilized fibers. We then induced Ca²⁺-independent active force by lowering the MgATP concentration (= -log [MgATP], from 5 to 7) to investigate whether or not the decreases in Ca²⁺-activated force are the result of reduced cross-bridge formation. We found that in immobilized fibers, maximal Ca²⁺-independent active force was reduced by ∼40%, with a leftward shift of the force-pMgATP curve. These results suggest that cross-bridge recruitment is suppressed in immobilized muscle, via e.g., structural changes of the sarcomere, resulting in reduced active force production. [J Physiol Sci. 2006;56 Suppl:S146]

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Many reports have shown that there are sex differences in pain sensitivity, and that hormonal condition also modulates pain sensitivity. Because muscle pain sensitivity was not studied yet in Japanese, we examined sex differences and differences in different phases of menstrual cycle in muscle pain sensitivity of young Japanese. Healthy subjects (10 males, 10 females in follicular phase, and 10 females in luteal phase) were recruited from students of Aichi Gakusen University and provided informed consent. The age ranged between 18 and 22 years old. Experimental muscle pain was induced in the biceps brachii muscle by eccentric exercise (ECC). A weight was placed around the wrist of undominant arm, and the subjects were asked to flex and extend the arm. Pressure pain threshold of the biceps brachii muscle (PPT), elbow joint angles that the subjects could flex or extend without pain, blood pressure, hemoglobin and skin fold thickness were measured. The females showed a significantly lower basal PPT, maximal blood pressure, and blood hemoglobin content, and their skin fold was thicker than the males. The PPT and blood hemoglobin in luteal phase were significantly lower than the follicular phase. PPT decreased 1 day after ECC and returned to the pre-exercise value 4 days after ECC. This time course in the development of delayed onset of muscle soreness was not different among groups. Present data showed clear sex and menstrual cycle differences in muscle pain threshold. [J Physiol Sci. 2006;56 Suppl:S146]

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Titin/connectin is a giant spring-like protein that is responsible for passive tension generation of striated muscle. We observed the passive tension of the bundles of myofibrils from rabbit cardiac and psoas muscle, and that from chicken breast muscle at various sarcomere lengths (SL) to compared it with the domain structures of connectin predicted from the cloned cDNAs. The bundles of the myofibrils of rabbit cardiac muscle were prepared mechanically from the homogenized muscle tissue in the relaxing solution. The specimen was held by a pair of sharp glass capillaries, and tension was estimated from their bending. SLs were observed microscopically. The passive tension of rabbit psoas and chicken breast muscle was measured with tension transducers. Moderate extension of the specimens developed a small passive tension that rapidly decayed to a steady level. Significant tension development began with a stretch beyond particular SL characteristic for each muscle type. That is, rabbit cardiac myofibrils began to develop tension at the shortest SL (around 2.0 μm), and chicken breast and rabbit psoas muscle began to develop tension at longer SLs (at around 2.3 and above 2.6 μm, respectively). On the other hand, the predicted length of PEVK region of connectin was human cardiac < chicken breast < human psoas < human soleus muscle. Assuming the connectin of rabbit to be similar to human, these data strongly suggests that the length of PEVK region correlates closely with the passive tension development at short SL. [J Physiol Sci. 2006;56 Suppl:S146]

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A mutant troponin T of which glutamate is replaced with aspartate (Glu244Asp) is one of the causes of familial hypertrophic cardiomyopathy (HCM). Incorporation of this mutant to skinned fibers has been reported to increase calcium sensitivity as well as maximal tension (Nakaura et al. 1999). However, mechanism has not been elucidated. Therefore, we constructed a model structure of this mutant troponin by introducing the mutation to the crystal structure of human cardiac troponin (TIC complex) obtained from Protein Data Bank (ID number 1J1E). Molecular dynamics simulation of the wild and the mutant structure was carried out in water at 310 K to estimate a dynamic structure of the mutant troponin and search a possible mechanism for the enhanced tension development. Dynamics was calculated by the use of software Amber ver.7. Iteration was done in TIP3 water with 0.5 or 1 fs time step in periodic condition at constant temperature (310 K). It was found that the electrostatic interaction between T244 and I111 which linked troponin T and I in the wild was lost in the mutant. Furthermore, when a terminal residue of troponin I was pulled toward an actin molecule, troponin T seemed to be easier to follow troponin I in the mutant than that in the wild. This may be involved in the enhanced tension development in the mutant myofilament. [J Physiol Sci. 2006;56 Suppl:S147]

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The intensity ratio of (1,0)/(1,1) equatorial x-ray diffraction indicates the mass transfer from myosin filament to actin filament. This ratio decreases during isometric contraction. During a very low load shortening, the amount of changes in this ratio reduced. To examine this reduced ratio carefully, the length clamp method was introduced in rat papillary muscle. When the shortening was stopped at any moment during a preload shortening, the isometric force was developed in accordance with the residual activity of crossbridges (length clamp). During the length clamp, both the shortening length and the force were located between those of preload and isometric contractions. Similarly the intensity ratio was also located between those of preload and isometric contractions. After the clamp of length, the time course of intensity ratio quickly separated from that of preload contraction. If the number of crossbridges actually reduced during the preload shortening, the time course of intensity ratio during length clamp was expected to follow that of preload shortening because of the latency in crossbridge formation. These results suggest that the number of crossbridges will not decrease during the preload shortening, and that not so small number of cross-bridges that can contribute the force development are left even under the low load shortening. [J Physiol Sci. 2006;56 Suppl:S147]

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At the basis of the Frank-Starling mechanism of the heart is the intrinsic ability of the contractile system to produce active force in response to stretch. It has been reported that length-dependent activation is, at least in part, modulated via interfilament lattice spacing reduction due to titin-based passive force (e.g., Fukuda et al., J. Physiol. 553, 147-154, 2003). In the current study, we examined whether or not length-dependent activation is modulated at the thin filament level. We used skinned porcine ventricular muscle that had been treated with 1% (w/v) Triton X-100. An increase in sarcomere length (SL) from 1.9 to 2.3 μm exerted a marked increase in Ca²⁺ sensitivity with a concomitant increase in passive force. The SL-dependent increase in Ca²⁺ sensitivity was markedly attenuated by perfusing preparations with rigor solution containing exogenous fast skeletal troponin (T-I-C complex; rabbit psoas muscle), with little or no effects on passive force. The magnitude of SL dependency was similar to what was observed in rabbit psoas muscle. Our SDS-PAGE analyses showed that endogenous cardiac troponin subunits were replaced with skeletal counterparts by ∼100% and that titin was not degraded upon troponin exchange. These results suggest that, presumably downstream of titin-based lattice spacing reduction, length-dependent activation is modulated at the thin filament level. [J Physiol Sci. 2006;56 Suppl:S147]

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We investigated cellular signaling cascades linked to estrogen-induced ER synthes is in mouse C2C12 myoblasts. By immunoblotting, ER was detected in the post-nuclear fraction with a corresponding molecular weight of 66 kDa. The amount of ER protein was dose-dependently (10⁻¹²-10⁻⁵ M increased after treatment with 17β-estradiol for 24 hours. 17α-estradiol (10⁻⁸M), the stereoisomer of 17β-estradiol, and a BSA-17β-estradiol-conjugate which is incapable of penetrating the plasma membrane, mimicked the increasing effects on ER. The level of ER expression was reduced by an ERK1/2 inhibitor, PD 98059(10μM), or a specific p38 inhibitor, SB 203580 (10μM) regardless of the presence of estradiol. Using ³⁵S-methionine for immunoprecipitation, newly-synthesized ER was increased by 17β-estradiol. Novo-synthesis of ER was further increased by the protein kinase C (PKC) activator, TPA (1μM). These results suggest that ERs located at the plasma membrane of mouse skeletal myoblast cell are a target of estrogen's actions. The estrogen-stimulated ER synthesis involves the PKC/MAPK signaling system, thereby presumably regulating the proliferation process. However, somewhat contra-intuitively, up-regulation of estrogen receptor was also observed in the mouse skeletal muscle treated with antiestrogens, such as tamoxifen and ICI182780. [J Physiol Sci. 2006;56 Suppl:S147]

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We assessed the involvement of serotonin receptors (5-HT-R) on pacemaker Ca²⁺ activity in gastrointestinal (GI). Small tissues of 100-300 mm diameter (cell cluster preparations) were prepared from the stomach and small intestine muscle (including the enteric neurones) of mice by enzymatic and mechanical treatments. After 2 to 4 days of culture, the fluo-3 AM (acetoxymethly ester of Fluo-3) was loaded to measure the intracellular Ca ²⁺ concentration ([Ca²⁺]_i). In the presence of nifedipine, we measured ([Ca²⁺]_i) oscillations in c-Kit-immunopositive pacemaker cells (= interstitial cells of Cajal: ICCs) and examined the effects of several drugs: LY-278584, 2-Methylserotonin maleatesalt, GR113808, Fluoxeline, SK&F96365, etc. RT-PCR and immunohistochemical examinations were carried out to characterize expression of serotonin receptor subtype in pacemaker cells. We conclude that endogenous 5-HT plays a crucial role in generating and maintaining pacemaker Ca²⁺ activity in ICCs via type 3 serotonin receptors (5-HT₃) under basal conditions, and a potentiates Ca²⁺ responses to electrical stimuli by facilitating cholinergic neurons. Our results may provide a new therapeutic target in diseases associated with abnormal GI motility, and add an important member (i.e. ICCs) to assess the brain-gut (The Second Brain) interaction. [J Physiol Sci. 2006;56 Suppl:S148]

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As we reported in the last meeting in Sendai, we established in situ Ca²⁺ imaging technique of the rat renal pelvis using a macro zoom microscope (Olympus MVX 10). The clear view and smooth zooming operation with this technique enabled us to search upstream of Ca²⁺ transient and to identify the pacemaker region easily. With higher magnification of the region, we could observe that not only one cell but several cells increased their intracellular Ca²⁺ concentration simultaneously. Interestingly their propagating pathway were slightly different every time. Spontaneous Ca²⁺ rises in the other part of renal pelvis rarely occurred; they never propagated to the downstream. Contrary many spontaneous but asynchronous Ca²⁺ rises were observed in the presence of low concentration of heptanol, a gap junction blocker. Using the same technique we could also successfully record image of di-4-ANEPPS, a voltage-sensitive dye. The initial depolarization occurred at the exactly same place as the Ca²⁺ rise. These results might suggest that the smooth muscle cells connected via gap junctions and with synchronous Ca²⁺ rise played an important role in the pacemaker mechanisms. [J Physiol Sci. 2006;56 Suppl:S148]

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Heat shock protein 20 (HSP20) has actin binding capacity and its amino acid sequence of actin binding region (residue 110-121; GFVAREFHRRYR) is highly homogenous to the inhibitory region of skeletal muscle troponin I (residue 104-115; TnIp). Previous study by Rembold et al (2000) showed that, in carotid artery, HSP20 monomer might directly interfere with actin-myosin resulting in the smooth muscle relaxation. In phasic skinned taenia caeci, however, a synthetic peptide of HSP20_110-121 enhanced Ca²⁺-induced force development except under the condition of high concentrations of Ca²⁺, so increased in the Ca²⁺-sensitivity for the force (Yoshino et al., 2003). To determine whether HSP20p also enhance Ca²⁺-induced contraction in tonic skinned carotid artery as well as in taenia, we studied the effects of HSP20p on contractile properties of beta escin skinned muscle preparations from guinea pig carotid artery. In skinned carotid artery, HSP20p suppressed Ca²⁺-induced contraction with little effects on the Ca²⁺-sensitivity for the force. Also a synthetic peptide originating from HSP27 actin binding region (residues 131-142; GYISRCFTRKYT; HSP27p) suppressed Ca²⁺-induced contraction. These results suggest that, different from the case of phasic smooth muscles, HSP20p might mainly affect thin filament and interfere with actin-myosin interaction causing muscle relaxation in tonic carotid artery. [J Physiol Sci. 2006;56 Suppl:S148]

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We examined whether 8-bromo-cAMP (8-Br-cAMP)-induced modification of tetrodotoxin-resistant (TTX-R) sodium current (I_Na) in neonatal rat nodose ganglion neurons is mediated by the activation of protein kinase A (PKA) and/or protein kinase C (PKC). In 8-Br-cAMP applications ranging from 0.001 to 1.0 mM, 8-Br-cAMP at 0.1 mM showed a maximal increase in the peak TTX-R Na⁺ (Nav1.8) current and produced a hyperpolarizing shift in the conductance-voltage (G-V) curve. The PKC inhibitor bisindolylmaleimide Ro-31-8425 (Ro-31-8425, 0.5 μM) decreased the peak Nav 1.8 current. The Ro-31-8425-induced modulation of the G_V1/2 baseline (a percent change in G at baseline V_1/2) was not affected by additional 8-Br-cAMP application (0.1 mM). The application of a PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.1 μM), increased the Nav 1.8 current, and this increase was not significantly affected by additional 0.1 mM 8-Br-cAMP application. Intracellular application of a PKA inhibitor, protein kinase inhibitor (PKI, 0.1 mM), greatly attenuated the 8-Br-cAMP-induced increase in the peak Nav 1.8 current but caused a significant increase in the slope factor of both activation and inactivation curves. The PKI application lowered the G max to below the control level. These results suggest that the 8-Br-cAMP-induced increase in Nav 1.8 currents may be mediated by activation of PKC. [J Physiol Sci. 2006;56 Suppl:S149]

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Experimental evidence has been presented to suggest that the protein kinase Cβ inhibitor LY333531 is effective at alleviating diabetic hyperalgesia to some extent. The present study was designed to examine the acute action of LY333531 (0.01-1μM) on the tetrodotoxin (TTX)-resistant Na⁺ current (I_Na) in small (<25 mm in soma diameter) dorsal root ganglion (DRG) neurons in control and streptozocin (STZ)-induced diabetic rats, using the whole-cell patch-clamp method. The cell membrane was initially hyperpolarized from a holding potential of -70 mV to -120 mV for 20 ms and then depolarized to various test potentials ranging from -50 to +50 mV, in the presence of TTX (0.1 mM) and the appropriate blockers for Ca²⁺ and K⁺ currents. I_Na was measured as a transient inward current during depolarizing steps. The maximal density of I_Na was significantly increased in diabetic rats compared with control (50.5 pApF⁻¹ vs 32.3 ⁻¹). I_Na recorded from diabetic rats was found to exhibit a significantly higher sensitivity to inhibition by LY333531 compared with control (IC₅₀, 6 nM vs 30 nM). Thus, our results provide experimental evidence to show that the sensitivity of I_Na to LY333531 is substantially enhanced by diabetic state, which suggests that I_Na is considerably inhibited by LY333531 at clinically relevant concentrations of <100 nM in diabetic state. [J Physiol Sci. 2006;56 Suppl:S149]

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One possible mechanism that may contribute to the inflammatory hyperalgesia, an inflammation-induced sensitization of primary afferent neurons, is the modulation of tetrodotoxin-resistant (TTX-R) voltage-gated Na currents by inflammatory mediators such as prostaglandins (PGs). Evidence for this compelling idea is mainly based on the electrophysiological finding that the TTX-R Na current, which is expressed preferentially in small primary afferent neurons and likely to be involved in nociception, is augmented by inflammatory mediators. However, the detailed mechanism underlying this observation has not been fully investigated. We re-investigated the effect of PGE2 (and also PGI2) on the heterogeneous TTX-R Na currents mediated by Nav1.8 and Nav1.9 in mouse dorsal root ganglion neurons, using conventional and nystatin-perforated whole-cell patch-clamp recordings on wild-type and Nav1.8-null mutant DRG neurons. Unexpectedly, PGE2 (and also PGI2) had no detectable effect on these TTX-R Na currents, raising a question regarding the well-known modulatory role of PGs on TTX-R Na currents in inflammatory hyperalgesia. [J Physiol Sci. 2006;56 Suppl:S149]

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Cav1.2 Ca²⁺ channel is suggested to be modulated by calmodulin (CaM) and Ca²⁺/CaM-dependent protein kinase II (CaMKII), that are proposed to underlie Ca²⁺-dependent inactivation and facilitation of channel activity. To explore phosphorylation sites for CaMKII, three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2, CT-1 (amino acids number 1509-1791), CT-2 (1777-2003) and CT-3 (1944-2169) were examined in vitro. Only CT-1 was consistently phosphorylated by CaMKII. By using mutated CT-1, the phosphorylation site was suggested to be threonine residue at position 1603. In pull-down assay, CT-1 but not CT-2 nor CT-3 was found to interact with CaM at both low and high [Ca²⁺] conditions. CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with alkaline phosphatase. These results suggest that interaction between CT-1 and CaM is modulated by phosphorylation mediated by CaMKII, and that they are consistent with the hypothesis that both CaM and CaMKII are involved in maintaining basal activity of the channel and in Ca²⁺-dependent inactivation and facilitation of the channel. [J Physiol Sci. 2006;56 Suppl:S150]

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We have previously reported that calmodulin (CaM) + ATP can reverse rundown of Cav1.2 L-type Ca²⁺ channels observed in the inside-out patch mode. In this study we have examined Ca²⁺- and dose-dependent effects of CaM on activity of the Ca²⁺ channels in guinea-pig ventricular myocytes. Application of CaM (0.1-14 &muM) + ATP (3 mM) to the intracellular side of the channels within 1 min after patch excision resulted in a bell-shaped dose-dependent effect with a maximum effect at 2-4 &muM CaM at low [Ca²⁺] condition (Ca²⁺-free to 80 nM). This relation between CaM and channel activity may be related to Ca²⁺-dependent facilitation and inactivation of the channel. This bell-shaped curve for the dose-dependent effect of CaM on channel activity was significantly shifted toward left (lower concentration) by increasing [Ca²⁺]. These results suggest that CaM plays a crucial role in regulation of the Cav1.2 Ca²⁺ channels, and that not only [Ca²⁺] but also CaM concentration are important factors for functional modulation of the Ca²⁺ channels. [J Physiol Sci. 2006;56 Suppl:S150]

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L-type Ca²⁺ channel currents have the voltage-dependent inactivation (VDI) and Ca²⁺ dependent inactivation (CDI) processes. These inactivation processes regulate the the amount of Ca²⁺ influx into cardiac myocytes. Under the β-adrenergic stimulation, the CDI dominate the decay of Ca²⁺ current since the VDI is slow (I. Findlay, J. Physiol., 2002). In this study, we have modeled the VDI and CDI under the control and the β-adrenergic stimulation (100nM isoproterenol). We have developed the VDI model by using the experimental time course of VDI estimated from the outward-going K⁺ current through the Ca²⁺ channel in the absence of extracellular Ca²⁺. The current dacay calculated by using the pure VDI was close to the experimental Ba²⁺ current decay which possessed minimal CDI. After that, we modeled the CDI satisfying the experimental current. The inactivation rate was expressed as a function of the Ca²⁺ current. In this formula, two components CDI was adopted to reproduce the strong bi-phasic decay for the β-adrenergic stimulation. The Ca²⁺ current dependent K⁺ current through the Ca²⁺ channel are introduced into this model to reproduce the experimental reversal potential. The calculated time course of the inward Ca²⁺ current was in excellent agreement with the experimental results. Therefore we have successfully established the inactivation model of the L-type Ca²⁺ channel in consistent with the experimental results. [J Physiol Sci. 2006;56 Suppl:S150]

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Store-operated Ca²⁺ entry activated by hyperpolarization in rat submandibular acinar cellsYoshida, H. and Nakahari, T.Department of Physiology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, JapanIn rat submandibular cells, ACh evoked a biphasic increase in [Ca²⁺]_i, that is, an initial transient phase followed by a sustained phase. The initial transient phase is induced by a Ca²⁺ release from the intracellular stores and the sustained phase is maintained by a Ca²⁺ influx from the extracellular fluid, which is the so-called "store-operated Ca²⁺ entry". Store-operated Ca²⁺ entry were stimulated using 4 µM thapsigargin, sarco(end)plasmic reticulum Ca²⁺ ATPase inhibitor, in Ca²⁺-free solution followed by superfusion with control (Ca²⁺-containing) solution. This protocol resulted in a [Ca²⁺]_i increase, which was inhibited in the presence of 1 µM Gd³⁺. A restoration from 150 mM K⁺ solution(Ca²⁺-containing) to control solution also evoked an [Ca²⁺]_i increase during thapsigargin stimulation, and an [Ca²⁺]_i increase following reintroduction of Ca²⁺ during thapsigargin stimulation was completely inhibited by addition of 1 µM Gd³⁺. Although a restoration from 7.5 mM K⁺ solution to control solution did not evoke an [Ca²⁺]_i increase during thapsigargin stimulation, addition of tetraethyl ammonium into 7.5 mM K⁺ solution evoked an [Ca²⁺]_i increase. Consequently, Ca²⁺ entry pathways is activated by the store-depletion and hyperpolarization. [J Physiol Sci. 2006;56 Suppl:S150]

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Ca²⁺ release from intracellular Ca²⁺ stores terminates after a rapid release of a fraction of releasable Ca²⁺ (called "quantal" release). To explain the quantal nature, it has been hypothesized that a decrease in luminal Ca²⁺ attenuates Ca²⁺ efflux. However, the mechanism remains an enigma. We show that voltage- and Ca²⁺-activated potassium channels in Ca²⁺ store control Ca²⁺ release. The potassium channel was identified as BK-type by patch-clamp recordings from an enlarged nuclear envelope in the nucleus-attached mode and by immunolabeling. The store BK channel was activated by positive shifts in luminal potential and luminal Ca²⁺ increases. The closing or blockage of store BK channels developed lumen-negative potentials and suppressed Ca²⁺ release. Ca²⁺ uptake by store Ca²⁺ pumps would reactivate the store BK channels regeneratively with K⁺ entry to allow repetitive Ca²⁺ release. Indeed, the luminal potential oscillated bistably ∼45 mV in amplitude as revealed with an organelle-specific voltage-sensitive dye [DiOC₅(3)]. Our study suggests that Ca²⁺ release-induced closings of store BK channels cause a lumen-negative potential towards the equilibrium potential for Ca²⁺ to attenuate Ca²⁺ efflux. [J Physiol Sci. 2006;56 Suppl:S151]

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Blockers of cardiac I _kr channels encoded by HERG gene is reported as the drug clinically effective for lethal ventricular arrhythmias. However the kinetic property of a HERG channel by these blockers is not yet studied in detail. We attempted to find the characteristics. By using the standard two-microelectrode voltage clamp technique on HERG channels expressed in Xenopus oocytes, HERG blocker (nifekalant, quinidine, carvedilol, E-4031, and dofetilide) blocked HERG channels in a use-dependent manner . However, some characters were found under the block effect. Nifekalant, quinidine or carvedilol increased HERG channel current at low voltages only in the presence of a previous strong depolarizing pulse, and so this pulse could separate the facilitation effect from the block effect. On the other hand, with the facilitation effect, they caused a significant negative shift in the voltage-dependence of activation. In the case of E-4031 etc, the facilitation effect was not found. Moreover, some mutant HERG channel blocked by quinidine induced stabilization of the closed state of channel. These results reveal that each HERG blocker has a different mechanism of block effect, and has not only block effect but also other effects. [J Physiol Sci. 2006;56 Suppl:S151]

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Kir2 subfamily members of inward rectifier K⁺ channel are known to co- assemble to form heteromultimers, and it is also known for Kir3. Here we examined whether Kir2.1 (IRK1) and Kir3.4 (GIRK4) belonging to different subfamilies can assemble each other or not. First, we examined the association by co-immunoprecipitation experiments using FLAG or myc tagged constructs co-transfected in HEK 293 cells. We observed "GIRK4-FLAG and IRK1-myc" as well as "IRK1-FLAG and GIRK4-myc" co-immunoprecipitated at a comparable level with a positive control pair, IRK1-FLAG/IRK1-myc. This clear co-immunoprecipitation was not observed in a negative control pair, P2X₂ receptor-FLAG/GIRK4-myc. As a next step, we anlayzed electrophysiologically using Xenopus oocyte expression system the effect of co-injection of GIRK4 or GIRK4/GIRK1 cRNA on IRK1 current. We could not record a clear emergence of current with unique features which reflects formation of functional heteromultimers, but observed a decrease in the amplitude of IRK1 current, suggesting a suppression effect possibly by hetero-multimerization. Finally, we carried out FRET analysis of IRK1-CFP/GIRK4-YFP pair and GIRK1-CFP/IRK1-YFP pair expressed in CHO cells, and obtained preliminary data supporting association of these two subunits. Taken together, these results suggest that IRK1 and GIRK4 subunits have a capability to form heteromultimers in heterologous expression systems. [J Physiol Sci. 2006;56 Suppl:S151]

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P2X receptors are ligand-gated cation channels activated by extracellular ATP. The P2X₂ channel current at the steady-state after ATP application is known to have voltage-dependence, i.e.–it shows inward rectification, and a gradual increase in the inward current is observed upon hyperpolarization. We analyzed this "activation" phase quantitatively under two-electrode voltage clamp using Xenopus oocytes expression system, and also approached its structural background by mutating a glycine residue (G344) in the 2nd-transmembrane helix (2nd-TM), a putative kink for the "gating". We observed that the inward current of G344A mutant increased instantaneously upon hyperpolarization without a gradual increase. On the contrary, G344P mutant showed a slower "activation" than that of WT. We also analyzed the conductance-voltage relationship by measuring the tail current, and observed that the half-maximal voltage of "activation" of G344P was shifted to the hyperpolarized potential in comparison with that of WT. The mutation did not affect the ATP dose-response relationships significantly. By the glycine scanning mutageneses on the G344A mutant background, we observed a recovery of the "activation" phase by introducing a glycine residue to the middle region of 2nd-TM. Taken together, we speculate that the flexibility of G344 in the 2nd-TM contributes to the voltage dependent "gating" of P2X₂ channel, which could be caused by an intrinsic or extrinsic mechanism to the channel molecule. [J Physiol Sci. 2006;56 Suppl:S151]

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The axon initial segment plays important roles in spike-initiation and invasion of axonal spikes into the soma. Among PSNs, those in the MTN can exceptionally initiate spikes in response to synaptic inputs, consequently displaying two kinds of spikes, one caused by invasion of an axonal spike arising from the sensory receptor and the other initiated by somatic inputs. We addressed where spikes are initiated in MTN neurons and whether there are any differences between initiated and invaded soma spikes (S-spikes). Simultaneous patch-clamp recordings from the soma and axon hillock revealed a spike-backpropagation from the initiation site in the stem axon to the soma in response to somatic current pulse, which brought about the delayed emergence of S-spikes after the offset of the current pulse. These initiated S-spikes were smaller in amplitude than invaded ones generated by stimulation of the stem axon; however, 4AP (<=0.5mM) eliminated the amplitude difference. Furthermore, 4AP markedly shortened the delay in spike-initiation without affecting the latency to spike-invasion, whereas it prolonged the refractory period of invaded S-spikes without affecting that of presumed axonal spikes markedly. These observations suggest that 4AP-sensitive K⁺ currents exert two opposing effects on S-spikes depending on their origins; suppression of spike-initiation and facilitation of spike-invasion at higher frequencies. [J Physiol Sci. 2006;56 Suppl:S152]

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The Kir2.1 channel currents show a strong inward rectification under the whole-cell recordings because of a voltage-dependent block of the channel by intracellular polyamines and Mg²⁺. Here we examined the effects of the intracellular pH on the Kir2.1 channel current using a 293T cell expression system. When the inside-out patch membrane was exposed to the intracellular solution of pH 7.2 containing no polyamines and Mg²⁺, the currents still showed a relatively strong inward rectification, but the closing of the channel in response to depolarizing voltage steps usually required several tens of seconds. Acidification of the intracellular solution from pH 7.2 to 6.8 and 6.4 induced a voltage-dependent decay of the outward currents during depolarizing voltage steps and increased the slow time-dependent component of the inward tail currents on hyperpolarization. Lowering of the intracellular pH from 7.2 to 6.0 and 5.6 also decreased the amplitude of the inward currents. In the presence of 0.1-5 μM spermine or 1-10 μM spermidine, a decrease in pH changed the amplitude of the outward currents with a complex voltage-dependence. Analyses of the inward tail currents suggested that the contribution of the pH-induced gating to the decrease of the outward currents increased as the intracellular pH was decreased. [J Physiol Sci. 2006;56 Suppl:S152]

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The molecular background for CaMKII-mediated regulation of a murine transient receptor potential protein homologue TRPC6 was explored by mutation analysis of CAMKII phosphorylation motifs (RXX(S/T)), which were found on the N-terminal (NT) and transmembrane (TM) (but not C-terminal) regions of wild-type TRPC6 and its chimera T776 with the NT and TM domains of TRPC7. Substitution of the last serine or threonine in these motifs with alanine revealed that, out of eight and five candidate sequences, only the mutations T487A in wild type TRPC6 and T433A in T776 respectively can strongly attenuate Ba²⁺ influx or inward cationic current evoked by muscarinic receptor stimulation with 100μM carbachol. Assuming a smilar membrane topology suggested by a recent structural analysis of TRPC1, T487 in TRPC6 and T433 in T776 may be located on a long intracellular stretch between the second and third TM domains (II-III loop) of each channel. Thus, considering the absolute requirement of a putative calmodulin/IP3 receptor binding site (CIRB) for TRPC6 channel activation, it is conceivable that close spatial arrangements of CIRB and the II-III loop might allow effective phosphorylation of T487 via the actions of CAMKII, thereby increasing the availability of TRPC6 channel for opening in response to subsequent receptor stimulation. [J Physiol Sci. 2006;56 Suppl:S152]

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TRPV family, identified as thermosensitive, Ca²⁺-permeable channels, consists of six subtypes. TRPV3 and 4 were described as heat transducers operative at moderately warm temperatures (>34 °C), whereas TRPV1 is activated by temperature above 42 °C. In natural environment, infrared light is detected as thermal radiation through skin. TRPV3 and 4 were reported to express in keratinocytes. They might, therefore, respond to infrared radiation and transfer the thermal signal to CNS. In this study, we examined the role of TRPV4 in regulation of body temperature by using transgenic mice defecting in TRPV4 protein. As thermal stimulus, infrared laser irradiation (λ=830 nm, 150 or 300 mW, 15 min) was applied to the back skin of the mouse, and temperatures of both skin surface and rectum were monitored. In wild type mouse, laser radiation which caused the increase in skin temperature up to 55 °C did not induce the change in body temperature. In TRPV4-knockout mice, however, moderate thermal stimulus, which increased the skin temperature less than 43 °C, resulted in the increase in the body temperature during the laser irradiation suggesting the loss of autonomic temperature regulation. The processing of moderate thermal radiation may partly depend on the TRPV4 expressed in skin cells. [J Physiol Sci. 2006;56 Suppl:S152]

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Mechanical stress activates TRPM7 channels expressed in HEK293T cells Numata, Tomohiro; Shimizu, Takahiro; Okada, Yasunobu (Dept. Cell Physiol., Natl. Inst. Physiol. Sci., Okazaki, Japan)Stretch-activated cation channels play an essential role in sensing and transducing external mechanical stresses in living cells. In the previous meeting we reported that TRPM7 channels endogenously expressed in human epithelial HeLa cells are activated by membrane stretch or osmotic cell swelling. However, it has not been known whether TRPM7 shows mechanosensitivity when heterologously expressed. HEK293T cells overexpressed with TRPM7 exhibited whole-cell currents typical of TRPM7, such as outward rectification, conductivity to Ca²⁺, and sensitivity to Mg²⁺ and ruthenium red. In addition, TRPM7 currents were augmented by following three kinds of mechanical stimuli: shear stress imposed by perfusion of extracellular solution, membrane stretch produced by patch membrane suction, cell swelling due to hypotonic stimulation. We thus conclude that the TRPM7 channel can be activated by mechanical stress in the heterologous expression system. [J Physiol Sci. 2006;56 Suppl:S153]

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TRPV4, a member of TRPV subfamily, is a nonselective cation channel that is activated by hypotonic stimulus, warm temperatures (about 25-34°C) or chemical compounds such as 4α-PDD, and is expressed in various tissues. To investigate TRPV4 function in the epithelial tissue, we screened a cDNA library from epithelial cells to identify TRPV4 interacting protein using a yeast two-hybrid system. Sequence analysis revealed that one of the positive clones encodes a protein containing PDZ and LIM domains. We found that the N terminal region of TRPV4 bound to LIM domains of the PDZ-LIM protein. When both TRPV4 and the PDZ-LIM protein were co-expressed in HEK293 cells, patch-clamp analysis showed that 4α-PDD-evoked currents were larger than those observed in cells expressing TRPV4 alone. Increase in the 4α-PDD-evoked currents was not observed in the cells expressing TRPV4 and the PDZ-LIM protein lacking LIM domains. These results suggest that the PDZ-LIM protein regulates TRPV4 activity by physical binding through LIM domains. [J Physiol Sci. 2006;56 Suppl:S153]

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TRPM8 was cloned as the family of thermally activated TRP channels. TRPM8 is cooling activated channel involved in cold sensation. However, it is unclear which amino acid residues are critical for ion permeation as the pore. We hypothesized that the pore is made by three consecutive hydrophobic amino acid residues between TM5 and TM6, highly conserved among different species or related TRP channels. WIF (898-900) were residues consistent with above hypothesis. We examined the involvement of this region on ion permeation by constructing TRPM8 mutant WIF898AAA, in which corresponding amino acid residues were replaced by alanine. Ion permeation was investigated with Fura-2 calcium imaging method. Menthol or cooling (from 30 °C to 15 °C) elevated intracellular calcium ion concentration in wild type TRPM8 -expressing cells but not in WIF898AAA -expressing cells. We conclude that the conserved three consecutive hydrophobic amino acid residues between TM5 and TM6 (WIF) are critical for ion permeation of TRPM8. [J Physiol Sci. 2006;56 Suppl:S153]

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In TRP (Transient Receptor Potential) cation channels family, cooling activated channels, TRPM8 and TRPA1, were identified in peripheral sensory neurons. However, it is still controversial whether TRPA1 is activated by cooling. Story et al [Cell,2003] reported that TRPA1 is activated by cooling below 17^oC. In contrast, Jordt et al [Nature,2004] reported that cooling did not activate TRPA1. Here, we investigated thermal sensitivity of TRPA1 by Fura-2 microfulorimetry and patch-clamp recordings in TRPA1-expressing HEK293 cells. 1: Cooling increased intracellular calcium levels. 2: In whole-cell voltage-clamp recording (-60mV), cooling below threshold evoked inward current. Threshold temperature was 17.5 ± 2.7 ^oC. 3: In inside-out single-channel recording, cooling induced activities of ion channels with non-selective cation channel properties. This indicated that channel activities occurred without intracellular soluble component. Single channel currents showed inward rectification. Single channel conductance was 74.1 ±18.8pS We conclude that TRPA1 is non-selective cation channel, activated by cooling. [J Physiol Sci. 2006;56 Suppl:S153]

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KCNQ channels have two putative coiled-coil domains in the cytoplasmic C-terminal region. These two domains, also known as A-domain and B-domain, are well conserved among KCNQ channel family and are recognized as subunit interaction domains. We have previously shown that serine residues in A-domain may be phosphorylated when PKC shifts the voltage-dependence of KCNQ channels. Therefore, we hypothesized that the stability of coiled-coil domain might determine the voltage-dependence of KCNQ channels. The amino acid sequence of the coiled-coil domain is characterized by heptad repeats (a-b-c-d-e-f-g) in which positions a and d are occupied by hydrophobic amino acid residues. To decrease the stability of the coiled-coil domain, we introduced double glutamine (Q) mutations at positions a and d of each heptad repeat. We analyzed electrophysiological properties of four double glutamine mutants for A-domain (I551Q/Y554Q, H558Q/M561Q, L565Q/I568Q, V572Q/I575Q) and three for B-domain (L606Q/V609Q, V613Q/M616Q, L620Q/L623Q). Three of them (I551Q/Y554Q, L565Q/I568Q, L620Q/L623Q) did not express detectable current. Among mutants showing functional expression, V572Q/I575Q showed drastic changes in its voltage-dependence: V_1/2 of the G-V curve was negatively shifted (-65.1 ± 1.1 mV vs. -39.0 ± 0.7 mV) and the apparent valence of charge movement (z) became smaller (1.54 ± 0.04 vs. 4.08 ± 0.07). The results of V572Q/I575Q suggest A-domain may also be responsible for the voltage-dependence other than the subunit interaction. [J Physiol Sci. 2006;56 Suppl:S154]

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MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and has been proposed to play an important role in osmoregulation. Although the MscS gating is regulated by membrane-stretch and voltage, little is known how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-74, and Arg-88) in the transmembrane domain have been proposed to serve as voltage sensors. To examine whether some of these three residues constitute voltage sensors, we neutralized the charge of each residue by substitution with asparagine (R46N and R74N) or glutamine (R88Q). Mechanical threshold for the opening of the expressed wild-type MscS did not change with voltage in the range from -40 to +100 mV. Replacement of the arginine residues with asparagine or glutamine did not alter the threshold. By contrast, inactivation process of wild-type MscS was strongly affected by voltage. At the pipette potential of -40 to -80 mV the current of the wild-type MscS rapidly declined whereas the current at -20 to +60 mV sustained for a longer time, as reported previously (Akitake et al., 2005). The voltage dependence of the inactivation rate of all mutants tested, was almost indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of Arg-46, Arg-74, and Arg-88. [J Physiol Sci. 2006;56 Suppl:S154]

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A protein called Ci-VSP (Voltage Sensor containing Phosphatase) has recently been reported (Murata et al, Nature 2005). VSPs have ion channel like transmembrane segments from S1 to S4 as the voltage sensor domain and the C-terminal cytoplasmic domain. It exhibits phosphatase activity in a voltage dependent manner. We have previously compared zebrafish ortholog of VSP (Z-VSP) with Ci-VSP and found that Z-VSP shows more robust charge movements in mammalian heterologous expression than Ci-VSP. To understand voltage-sensing mechanisms of VSP, we focused on the double mutant of Z-VSP in which two arginine residues were inserted into the S4 (DM). This shows negative shift of the Q-V curve (threshold was around -40 mV). Furthermore, DM showed biphasic profile of the Q-V curve that could not be fitted by a single Boltzmann equation: the movement of the voltage senor saturates around at 100 mV but then it increases as the membrane potential is more depolarized. Such biphasic profile of the Q-V curve did not depend on the phosphatase activity as examined from C302S mutant. This exhibited the threshold of charge movement even more negative (around -60 mV) and the simple Q-V curve that can be fitted with the single Boltzmann equation. These results suggest that the C-terminal domain but not its enzyme activity affects charge movement probably through exerting some constraint on the movement of the VS domain. [J Physiol Sci. 2006;56 Suppl:S154]

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ATP conductive maxi-anion channels are functionally expressed in rat cardiomyocytes and activated in hypotonic, hypoxic or ischemic conditions. In the present study, using a newly developed Scanning Ion Conductance Microscopy (SICM) technique combined with patch-clamp, we studied the spatial distribution of maxi-anion channels over the surface of sarcolemma of primary cultured neonatal rat cardiomyocytes and isolated adult cardiomyocytes. In primary cultured rat cardiomyocytes, maxi-anion channels were found to be predominantly expressed around the center of the cell body compared to the cell extensions. Using a P2X-receptor-based biosensor technique, it was found that the local concentration of ATP released in response to hypotonic stress was higher around the cell center compared to that at cell extensions. In isolated adult rat cardiomyocytes, maxi-anion channel activity was observed when a fine-tipped patch pipettes were giga-sealed at the grooved areas of sarcolemma using a conventional patch-clamp method. After taking a 3-D image of isolated cell by SICM technique, we specifically patched T-tubules, Z-grooves and scallop crest areas. We found that maxi-anion channels are predominantly expressed in T-tubules and Z-groove areas, but not in scallop crests, in adult cardiomyocytes. The results obtained indicate that maxi-anion channels are heterogeneously expressed over the surface of the rat cardiac sarcolemma. [J Physiol Sci. 2006;56 Suppl:S154]

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The profile of opioid- and cannabinoid receptors in neurons of the nucleus tractus solitarius (NTS) have been studied using the whole cell configuration of the patch clamp technique. Experiments with selective agonists and antagonists of opioid, opioid-receptor-like-1 (ORL-1) receptor and cannabinoid receptors indicated that μ-opioid, κ-opioid, ORL-1 and CB1, but not δ-opioid, receptors inhibit VDCCs currents in NTS. Application of DAMGO (μ-opioid receptor agonist), Orphanin FQ (ORL-1 receptor agonist) and WIN55,122 (CB1 receptor agonist) caused inhibition of VDCCs currents in a concentration-dependent manner with an IC50 of 390 nM, 220 nM and 2.2 μM, respectively. Intracellular dialysis of the Gα_i-protein antibody attenuated DAMGO-, Orphanin FQ- and WIN55,122-induced inhibition of I_Ba. Both pretreatment with adenylate cyclase inhibitor and intracellular dialysis of the protein kinase A (PKA) inhibitor attenuated WIN55,122-induced inhibition of I_Ba, but not DAMGO- and Orphanin FQ-induced inhibition. Mainly N- and P/Q-type VDCCs were inhibited by both DAMGO and Orphanin FQ, while L-type VDCCs were inhibited by WIN55,122. These results suggest that μ- and κ-opioid receptors and ORL-1 receptor inhibit N- and P/Q-type VDCCs via Gα_i-proteins β γsubunits, whereas CB1 receptors inhibit L-type VDCCs via Gα_i-proteins involving PKA in NTS. [J Physiol Sci. 2006;56 Suppl:S155]

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