We have clarified in our previous papers that the bound thrombin (B-th), from crushed fibrin clots, enhanced SMemb mRNA and protein expression in vascular smooth muscle cells (VSMCs). However, it was not known whether B-th contributed to restenosis of blood vessel through the proliferation of VSMCs. If B-th could contribute to restenosis, the B-th might pass through endothelial cells (ECs). To investigate the passage of B-th through ECs, we used the dual chamber with the rat aortic endothelial cells (RAECs). After the application of B-th to RAECs in upper chamber, the medium of lower chamber were electrophoretically analyzed by the SDS-PAGE. In addition, the analysis on the western blot using the polyclonal antibody against fibrinogen (pFbg), two types of monoclonal antibodies against thrombin; CC2 and GE12, which have been made in our laboratory. From Western blot analysis of the medium of the lower chamber, it was possible for us to measure separately the quantity of B-th and native thrombin (N-th). ELISA to measure the quantity of B-th, or that of N-th was developed. The rates of passage of B-th from upper to lower chamber were 7.02 ± 3.9% and that of N-th was 10.16 ± 4.9%, respectively. In the present study, we demonstrated that B-th, composed of α-thrombin and fibrin derivatives, passed through the RAECs, similarly to N-th. It, therefore, was shown that B-th might occur the proliferation of VSMCs and contribute to restenosis of blood vessel. [Jpn J Physiol 55 Suppl:S109 (2005)]

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Thrombin is a multifunctional serine protease in haemostasis and is a glycoprotein containing one carbohydrate chain, consisting of lactosamins and N-acetylneuraminic acid (NeuAc). The glycosylated site at Asn53 is located near the hydrophobic pocket flanked by the catalytic triad in an active site cleft of the thrombin molecule. Although the carbohydrate chain is binding near the important domain involved in thrombin protease activity, the function remains unknown. In this report, the enzymatic property of the carbohydrate chain moiety-modified thrombin was compared to that of intact thrombin in order to clarify the function of the carbohydrate chain. The NeuAc at the non-reductive terminal of the carbohydrate chain was removed by neuraminidase. Release of fibrinopeptides was measured by HPLC. The intact thrombin ([M+H]⁺=37,680, measured by a MALDI/TOF mass spectrometer) released fibrinopeptides A (FPA) and B (FPB) during fibrin clot formation. The NeuAc-lacking thrombin ([M+H]⁺=36,976) formed fibrin clot, however, FPA was primarily released while little FPB was released. The result suggests that access of the fibrinogen Bβ chain to the active site cleft was restricted by removing NeuAc. The modification of the carbohydrate chain thus may cause the structural alteration of the active site cleft of the thrombin molecule, and the carbohydrate chain may play an important role in thrombin protease activity, especially the recognition of a macromolecular substrate such as fibrinogen. [Jpn J Physiol 55 Suppl:S109 (2005)]

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We have reported that the electrical shock stress reduced blood fluidity and the reduced blood fluidity was restored by α-blocker in rats. In the present study we investigated the effects of adrenergic α- and β- agonists and antagonists on the blood fluidly. Used drugs were as followed: Phenylephrine as an α-agonist, phentolamime as an α-antagonist, isoproterenole as a β-agonist, propranolol as a β-antagonist were used respectively. The blood fluidity was measured by micro channel array flow analyzer (MC-FAN) which mimicked capillary vessel. Phentolamine and isoproterenole accelerated the blood fluidity. On the other hand phenylephrine and propranolol reduced the blood fluidity. These results indicate that stressor reduced the blood fluidity through noradrenaline system but adrenaline might accelerate the blood fluidity preferably. [Jpn J Physiol 55 Suppl:S110 (2005)]

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Nonspecific effects of stress during suspension are known to produce glucocorticoid-mediated muscle metabolism. However, the in vivo effects of hypokinesia/hypodynamia on the numbers of white blood cells, neutrophil, monocyte, lymphocyte, eosinophil and basophil are still unknown. It is necessary to elucidate systematically the physiological responses obtained by various hypokinesia/hypodynamia models such as restricted physical activity and whole body suspension. In addition, β₂-agonist, clenbuterol (CLE) is known to be useful as doping drugs. Therefore, the effects of immobilization (IMM), whole body suspension (WBS) and CLE-administration (CLE) on the numbers of white blood cells were studied in rats. 7 weeks old male SD rats (n=59) were used, and divided into the control (CON) groups, and IMM, WBS and CLE groups in each experiment. IMM, WBS and CLE-administration were maintained for 10 days. IMM, WBS and CLE-administration nonspecifically increased numbers of neutrophil and monocyte. Although IMM and WBS significantly increased numbers of total white blood cells, neutrophil, monocyte, eosinophil and basophil, WBS increased numbers of monocyte, eosinophil and basophil more significantly than IMM. IMM and WBS did not change numbers of lymphocyte. CLE-administration decreased numbers of lymphocyte and eosinophil. In conclusion, IMM and WBS increase the numbers of natural immunity cells without changing acquired immunity cells. However, different responses of natural immunity cells are found in three conditions. [Jpn J Physiol 55 Suppl:S110 (2005)]

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A novel continuous cell separation method is developed for application to transfusion medicine. The performance of this method was examined on separation of human buffy coat and peripheral blood containing nucleated cells (>10⁸) among a large number of erythrocytes (>10¹⁰). Differential leukocyte counts revealed that lymphocytes were concentrated in fractions 3 and 4 and neutrophils in fraction 6, while rare basophils were often observed in fraction 5 and some cells present in fraction 2 were difficult to identify microscopically. These cells had nucleoli like immature cells. Flow cytometry analysis revealed that CD34-positive cells, which are considered to be hematopoietic stem cells, were relatively concentrated in fraction 2 (density: 1.060). Recently, the present method was successfully applied for the separation of human mast cells in a cultured dish. When the co-cultured cell suspension containing >10⁶ cells (approximately 10% of mast cells and 90% of fibroblasts) was separated, fibroblasts were concentrated in fraction 2 (density: 1.065) with a few young mast cells while densely granulated mast cells were collected in fraction 6 (density: 1.085). Because this method separates the cells according to their densities, it may be efficiently applied for isolation of stem cells, including both CD34-positive and CD34-negative species, from human cord blood. We also propose that the method might be used to detect minute changes in cell density under various physiological and pathological conditions. [Jpn J Physiol 55 Suppl:S110 (2005)]

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The liver regeneration after injury is regulated by various growth factors and cytokines. Release of these growth factors is deeply related to degradation of extracellular matrix (ECM). This ECM degradation is regulated by the activation of plasminogen (PIg) and matrix metalloproteinase (MMP) systems. The liver regenerations in knockout mice (KO) for fibrinolytic factors were examined by using CCl₄ injection modeI. The ability of liver regeneration was significantly increased in the α₂-antiplasmin (α₂-AP) KO as compared to the wild-type mice (WT), but was significantly decreased in the PlgKO. The proteolytic activity in liver tissue extract from PlgKO after liver injury was lower than that α₂-APKO and WT. These results suggest that the plasmin/α₂-AP system playes an important role in the hepatic repair. Further, we analyzed the liver regeneration by using the hepatocytes isolated from mouse liver. The proliferation ability of the hepatocytes from mice of 5 days after CCl₄ injection was significantly increased in the α₂-APKO as compared to the WT but was decreased in the PlgKO. The Plg bound to the isolated hepatocytes. The activation of Plg was significantly increased in the presence of mouse hepatocytes. The plasmin inhibition by α₂-AP in the presence of mouse hepatocytes was weaker than that in the absence of mouse hepatocytes. These results indicate that the plasmin/α₂-AP system on the surfaces of mouse hepatocytes plays important roles in liver regeneration. [Jpn J Physiol 55 Suppl:S110 (2005)]

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The purpose of the present study is to elucidate the effects of iron-deficiency (ID) on the numbers of neutrophil, monocyte, basophil, eosinophil and lymphocyte, and the weights of perirenal and periepididymal adipose tissues in rats. Male 6 weeks old Sprague Dawley rats (n=16) were used and divided into the control (CON: iron intake=6.1mg/day) and the ID-food (IDF: iron intake=0.05mg/day) groups. The durations of ID-feeding were maintained for 23 days. Tail venous white blood cells were analyzed by a cell counter apparatus. Numbers of total white blood cells and neutrophil in the IDF increased markedly from 15th days. Eosinophil and monocyte numbers in the IDF group increased relatively in 7-21 th days of ID-feeding, as compared with the those of CON. Lymphocyte numbers decreased markedly in 15-23 th days of ID-feeding. Basophil numbers did not change with ID-feeding. These results are similar with results obtained by the administration of β₂-agonist, clenbuterol (1.0mg/kg BW/day). Further, the weights of perirenal and periepididymal adipose tissues per body weight in the IDF group were 2.12 and 1.44 times, respectively significantly higher than those of the CON. The IDF increased total white blood cells, neutrophils, eosinophil and monocyte numbers and decreased lymphocyte numbers without changing basophil numbers. These results suggest that iron-deficient food intake increases the numbers of natural immunity cells and decreases the numbers of acquired immunity cells in rats. [Jpn J Physiol 55 Suppl:S111 (2005)]

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[Objective] Protein S (PS), a vitamin K dependent anticoagulant protein functions as a cofactor for activated protein C (aPC), has recently drawn interest due to its new properties in inflammation. We examined, whether PS is able to modify cell-associated fibrinolysis and invasive potential of inflammatory cells. [Methods and Results] We analyzed tissue plasminogen activator (tPA) catalyzed activation of Glu-plasminogen in the presence of THP-1 cells, monocyte-like cells to which both plasminogen and t-PA bind. tPA catalyzed activation of Glu-plasminogen was enhanced by THP-1 cells as reported before, which was partially abolished by PS (100 nM). PS attenuated the binding of biotin-labeled plasminogen to the surface of THP-1 cells in a dose dependent manner (50-300 nM: 50% at 100 nM and 65% at 300 nM). These two phenomena were not further enhanced by aPC. PS also attenuated plasminogen-dependent invasion of THP-1 cells through matrigel in response to monocyte chemotactic protein 1, whereas it did not modify plasminogen-independent cell migration. [Conclusion] The study identifies PS as a factor to suppress plasminogen-dependent invasive potential of THP-1 cells. [Jpn J Physiol 55 Suppl:S111 (2005)]

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The present study was carried out to investigate whether angiotensin II (ANG II) peptide is induced in the rat's kidney under endotoxemic conditions. Immunohistochemical studies revealed that strong ANG II-like immunoreactivity was observed in the renal tubules of rats which received intraperitoneal (i.p.) injection of a high dose of LPS (1,000 μg/kg). Although ANG II-like immunoreactivity was slightly observed at 1 h after the injection of LPS, LPS-injected rats showed marked expression of ANG II-like immunoreactivity in the renal tubules at 3 h. There were few signals in the kidney of the saline-injected control rats. An i.p. injection of one of three doses of LPS (0.1, 10 or 1,000 μg/kg) produced dose-related increases in ANG II-like immunoreactivity in the renal tubules. Treatment with a prostaglandin-synthesis blocker, indomethacin, had no effect on the LPS-induced ANG II expression in the renal tubules. The ELISA study measuring the concentration of ANG II in the whole kidney supports the above mentioned findings. These results suggest that systemic administration of LPS results in the expression of ANG II peptide in the renal tubules, the mechanism being independent on the mediation by prostaglandins. [Jpn J Physiol 55 Suppl:S111 (2005)]

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Unstimulated saliva is thought to play an important role in oral immunity, enamel stability and in keeping the mucous membrane of the oral cavity moist. Although it is known that the rates of flow of unstimulated saliva in healthy humans show wide variation, there have been no studies about sourses of the variation. Some studies have reported a positive correlation between the rates of flow of "stimulated" saliva and the parotid or submandibular gland seizes. Therefore, variations in the salivary gland sizes may possibly explain, the individual differences of "unstimulated" saliva. In this study, we investigated three major salivary gland size by using MR imaging in 40 healthy humans and examined the relationships among the estimated salivary gland sizes and the salivary parameters of unstimulated saliva. Bilateral sizes of parotid, submandibular and sublingual glands were respectively 81.4±2.8, 31.1±1.2 and 11.2±0.5 cm³. Whole saliva secretion rate was 1.1±0.1 ml/min, and showed a significant positive correlation with the estimated sizes of the parotid (Peason r = 0.48) and submandibular (Peason r = 0.57) glands. Total protein secretion rate was 1.28±0.15 mg/min, and showed a significant positive correlation with the estimated sizes of the parotid (Peason r = 0.44) and submandibular (Peason r = 0.40)glands. These results suggest that the lager the salivary gland, the greater the rates of fluid flow and protein secretion in the unstimulated saliva. [Jpn J Physiol 55 Suppl:S112 (2005)]

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Interestingly, pilocarpine, which is used as a sialogogue for the treatment of dry mouth, induces drinking behavior though muscarinic receptors in the central nervous system when applied peripherally. Although studies from anesthetized rats suggested that the pilocarpine administration affects the central nervous system and modulates salivary secretion, the mechanism is not clear. In this experiment, pilocarpine was applied intracerebroventricularly (ICV) or intraperitoneally (IP) in conscious rats, and salivary secretion and water intake were measured. IP-injected pilocarpine increased salivary secretion at 0.4-12 μmol/mL/kg and water intake at 1.2-12 μmol/mL/kg, in dose-dependent manners. Atropine injected by ICV in advance suppressed the pilocarpine-induced water intake while it did not the salivary secretion. Further, ICV-injected pilocarpine increased water intake in a dose-dependent manner while it had no effects on salivary secretion. Atropine injected by ICV in advance blocked the water intake by ICV pilocarpine. These results suggest that while peripherally-applied pilocarpine acts centrally the thirst center and induces drinking behavior, the central action is not enough to modulate salivary secretion in conscious rats. [Jpn J Physiol 55 Suppl:S112 (2005)]

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It has been demonstrated that there are sexual differences in saliva volume and components between men and women. However, it is unclear what is based on the differences in salivary secretion. We investigated flow rate, compositions, spinberkeit of saliva and the size of the salivary glands by using MR imaging. The salivary flow rate was 1.4 ± 0.2 ml/min in men ( n = 15 ) and 0.8 ± 0.1 ml/min in women( n = 15 ) ( p = 0.01 ). Na+concentration was 7.5 ± 1.3 mM in men( n = 15 ) and 3.9 ± 0.7 mM in women( n = 15 ) ( p = 0.02 ). Total protein was 1.7 ± 0.2 mg/min in men( n = 15 ) and 0.9 ± 0.2 mg/min in women( n = 15 ) ( p = 0.02 ). There were no significant differences in the protein concentration and the spinberkeit of saliva between men and women. The size of the salivary glands were 140 ± 6 cm3 in men( n = 15 ) and 108 ± 5 cm3 in women( n = 15 ). The size of the salivary glands in men was significantly larger than in women ( p = 0.0002 ). We will discuss the sexual differences in saliva volume and components. [Jpn J Physiol 55 Suppl:S112 (2005)]

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Potassium secretion along the renal collecting duct plays a crucial role in potassium homeostasis of the body fluid. Although K channels in the apical membrane of collecting duct is a major candidate for the passage of the potassium secretion, apical K-permeable channels in cultured collecting duct cells have not precisely been known. In this study, we investigated the K-permeable channels in the apical membrane of principal cells in cultured mouse collecting duct (M1) cells. M1 cells were cultured in the presence of aldosterone before experiments for 5-7 days, and the patch-clamp technique was applied to the apical surface of the M1 principal cells of confluent monolayer. In cell-attached patches, two types of K-permeable channels were observed. One was large conductance (70-80 pS) non-selective cation channel, whose activity was usually low in cell-attached patches, and was enhanced by intracellular Ca (1 mM) in inside-out patches. The other was a low conductance (30-40 pS) inwardly rectifying K-permeable channel, which was activated by cAMP-dependent processes but not by cGMP- or PKC-dependent processes in cell-attached patches. Furthermore, the latter low conductance channel was activated by intracellular ATP (1 mM), but not by intracellular Ca, in inside-out patches. We suggest that the low conductance K-permeable channel was ROMK-like channel and was involved in K secretion in mouse kidney collecting duct in physiological conditions. [Jpn J Physiol 55 Suppl:S112 (2005)]

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In the proximal tubules, water movement from tubular lumen to peritubular capillary is the passive consequence of active solute reabsorption. This vectorial transport is characterized by a combination of many carrier proteins, expressed differently in polarized membranes, apical and basolateral membranes. In the present study, we successfully reconstructed a mathematical model of proximal tubular epithelial cells, reported by Weinstein (Am J Physiol 263: F784, 1992), on our originally developed cell simulation platform "simBio". When tubular Na⁺ was replaced with impermeable molecule, apical Na⁺ flux into the cell via Na⁺-driven transporter, such as Na⁺-glucose or Na⁺-H₂PO₄⁻ cotransporter and Na⁺/H⁺ exchanger, was greatly reduced. Then intracellular Na⁺ concentration decreased gradually, resulting in the reduction of basolateral Na⁺ efflux via Na⁺-coupled transport processes such as Na⁺/K⁺ pump and Na⁺-3HCO₃⁻ cotransproter. According to the reduction of Na⁺ fluxes in each membrane, water fluxes were also greatly reduced, confirming the significance of osmosis in water reabsorption at the proximal tubules. Furthermore, known observations regarding the change of water transport in response to changes in composition of extracellular fluid, such as reduction of HCO₃⁻ and addition of cyanide and glucose, were well explained by the model. [Jpn J Physiol 55 Suppl:S113 (2005)]

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Backgroud & objects: EGGs were recorded before and during MDT in order to study the stress effects on EGG. Methods: EGGs(τ=5 sec, high cut=0.5 Hz) were recorded from 16 locations on thoraco-abdominal surface. EGG spectral groups were classified into 5 ones, 1cpm ones (0-2.4 cpm), 3 (2.5-4.9), 6 (5.0-7.4), 8 (7.5-9.9) and 10 (10.0-12.9). Peak power ratio(MDT/rest) of epigastric(ch3-6), supraumbilical(ch7-9) and infraumbilical region(ch12-14) was calculated. Anxiety and depression scores were obtained from HADS(after Zigmond & Snaith). Informed consent was obtained from the subjects(n=43) Results: Respiratory frequency and heart rate at rest significantly increased during MDT. The MDT stress induced dual excitatory(MDT/rest>1.0) or inhibitory effects(MDT/rest<1) on EGG. The mean frequencies of the inhibitory groups at rest were significantly higher than those of the excitatory ones and they became lower during MDT. Those of the excitatory ones at rest were significantly lower than those of the inhibitory ones and they became higher during MDT. Anxiety or depression scores of the excitatory groups were significantly higher than those of the inhibitory ones in epigastric 6 and 10 cpm, supraumbilical 6 cpm and infraumbilical 1, 3 and 6 cpm groups. The LF/HF during MDT of the inhibitory group was significantly higher than that of the excitatory one just in infraumbilical 3 cpm group. Discussion & conclusion: Anxiety, depression and LF/HF seemed to have some relationships to MDT stress responses of EGG. [Jpn J Physiol 55 Suppl:S113 (2005)]

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Plant cells have some specific enzymes for transformation of compounds. By selecting some appropriate kinds of plant cells, Hamada succeeded glycosylation of some spice compounds like thymol and carvacrol, and stability and solubility in water of those compounds were improved. In the present study, capsaicin was transformed to capsaicin-O-β-glucoside (capsaicin glucoside) by the cultured Phytolacca americana cells. Actions of capsaicin and capsaicin glucoside on gut functions in the rat small intestine were investigated and compared. Intestinal contraction of whole ileal segments was measured by Magnus method, and the amplitude of ileal twitch elicited by selective nerve electrical stimulation was inhibited by capsaicin (50μM ), but capsaicin glucoside (50μM ) didn't induce any changes. Capsaicin elicited remarkable decreases in transmural short-circuit currents (Isc) measured by Ussing chamber technique as a mucosal transport function when it was applied to both mucosal and serosal sides at ileum, but Isc was not changed when appliled to mucosal side at jejunum. On the other hand, capsaicin glucoside increased in Isc when applied to mucosal side at jejunum and ileum, but Isc change was less or small when applied to serosal side. The mechanism of Isc changes by capsaicin and capsaicin glucoside is not yet clear, but characteristics changes by glucosylation of capsaicin might be related to the differences of actions of both compounds on gut functions. [Jpn J Physiol 55 Suppl:S113 (2005)]

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Various physiological phenomena are under the control of an endogenous circadian rhythm. The molecular circadian clock system is thought to be based on transcriptional/translational feedback loops consisting of several “clock genes” and their products. Cell cycle progression is regulated through several different cyclin dependent kinase (Cdk) regulatory mechanisms. Cdk inhibitors play important roles in cell cycle control by coordinating internal and/or external signals and impeding proliferation at several key check points. Out of several Cdk inhibitors, p21 is an important mediator of cell cycle arrest imposed by the tumor suppressor p53 in response to DNA damage. p21 gene is regulated by clock system, because its promoter has RevErb/ROR binding elements and Bmal1 gene promoter has a same regulation site. Cholic acid has reported to regulate the cell cycle through the p21 gene expression. We still do not know how clock and cholic acid regulate the p21 gene expression in the mouse liver. In mouse liver, expression of p21 mRNA exhibits weak circadian rhythm in normal food, but cholic acid containing diet causes a robust circadian rhythm. However other organs like small intestine and heart, cholic acid did not affect p21 expression. Interestingly, in the Clock mutant mice, cholic acid strongly up-regulates the p21 gene expression through the whole day.In summary, cholic acid intake under clock mutation may strongly inhibit cell division in a liver through the up-regulation of p21. [Jpn J Physiol 55 Suppl:S114 (2005)]

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Potassium secretion in the colon involves the basolateral, bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter and the apical TEA-sensitive K⁺ channel. This study investigated whether increasing plasma K⁺ concentration directly affects the rate of colonic K⁺ secretion, in addition to activating it indirectly through aldosterone. [Methods] Isolated distal colon of the mouse fed on a normal diet or high-K⁺ diet was mounted in Ussing chamber and short-circuit current (Isc) was determined. [Results] In animals fed on control diet, serosal bumetanide or increasing K⁺ concentration in the serosal side from 3 mM to 7 mM had no effect on Isc under basal condition. The addition of isoproterenol (5 μM, serosal) caused a decrease in Isc, a magnitude of which became larger as serosal K⁺ concentration was increased. The Isc decrease that was stimulated by isoproterenol and dependent on serosal K⁺ concentration was largely inhibited by mucosal TEA (10 mM), indicating that it represent K⁺ secretion. In animals fed on high-K⁺ diet, Isc under basal condition was significantly increased by serosal bumetanide, suggesting the presence of on-going K⁺ secretion. However the increase in serosal K⁺ concentration did not affect Isc. On the other hand, When K⁺ secretion was stimulated by isoproterenol, the increase in serosal K⁺ concentration stimulated TEA-sensitive Isc decrease. [Conclusion] Under certain conditions, the colonic K⁺ secretion is enhanced as the plasma K⁺ concentration increases, thereby probably contributing to the homeostatic K⁺ regulation in the body. [Jpn J Physiol 55 Suppl:S114 (2005)]

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Cholecystokinin (CCK) is a peptide hormone that has important physiological functions. Recently, CCK-A and -B receptor(R)s were cloned. In mammals including humans, CCK-AR mediates satiety effect and CCK-BR does gastric acid secretion. We generated CCK-AR gene deficient (-/-) mice and found that the daily food intake, energy expenditure, and gastric emptying of a liquid meal were not changed, compared with wild-type mice. As CCK-AR(-/-) mice showed an anxiolytic status, we examined the changes in daily food intake after the swimming stress or after the restraint stress. Five-minute swimming for 3 days did not modify the changes in body weight and daily food intake between the two genotypes. Seven-h restraint stress significantly decreased body weight as well as food intake during subsequent 3 days. Then, on the fourth day, food intake increased in both genotypes. CCK-AR(-/-) mice showed a significantly higher level than the value before restraint stress. However, the food intake was only recovered to the level before the stress in wild-type mice. At this time, gastric acid secretion and gastric emptying were measured. Both parameters did not differ significantly between the two genotypes. In conclusion the gastric functions in CCK-AR(-/-) mice were not altered by a restraint stress, which produced a greater increase in food intake compared to wild-type mice. [Jpn J Physiol 55 Suppl:S114 (2005)]

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To investigate the membrane current involved in acetylcholine (ACh)-induced hyperpolarization of the vascular endothelium, a sheet of endothelial cells was isolated from a guinea-pig mesenteric artery and placed with the luminal surface upword. The endothelial cells were electrically coupled to one another and voltage control of the whole syncytium with a patch electrode was impossible. To decrease the number of the cells to be controlled, a pipette having a tip diameter of about 100 μm was pressed down onto the preparation. After removing the pipette, circularly-aliened cells compressed by the edge of the tip were destroyed leaving small number of intact cells within the circle. A patch electrode containing amphotericin B was applied to one of the intact cells and perforated whole-cell clamp experiments were performed. As the membrane potential was usually less negative than -10 mV, the holding potential was always set to 0 mV. Application of ACh induced an outward current, the reversal potential of which varied by preparations. This current was relatively resistant to charibdotoxin and/or apamin. Although this current seemed to be potassium current, there was significant inward current at negative potentials even if the bath solution did not contain any potassium. The potassium channels involved in this current seem to be permeable to other ions than potassium. [Jpn J Physiol 55 Suppl:S115 (2005)]

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To elucidate Ca²⁺ handling by the junctional sarcoplasmic reticulum (JSR) in heart muscle of SHR, we applied our method for determination of the time course of Ca²⁺ replenishment and release of the JSR in the papillary muscle of both SHR and age-matched control(WKY; 7-weeks-old).The time course of short-term mechanical restitution after varying magnitude of twitch contraction was examined. Furthermore, Ca²⁺ content of SR was also measured using rapid cooling contracture(RCC) in both straines. The mechanical restitution consisted of a pretwitch latency period followed by a rapid and a subsequent much slower restitution of twitch force in both straines. The slope of the rapid restitution was similar and almost constant in both SHR and WKY, but it was significantly smaller in the former than the latter after the rested state twitch(SHR; 1.47 ± 0.04, n=7,WKY; 2.49 ± 0.10, n=6,P < 0.0001). Based on this finding, the functions G(t) and H(t), representing the time courses of the JSR Ca²⁺ replenishment and release, respectively, were derived graphically from a family of the mechanical restitution curves for the both strains.G(t)increased monotonically with time at a decreasing rate, while H(t) increased with time in a sigmoid manner. At rested state contraction, magnitude of RCC was significantly declined in SHR by approximately 40% of the control ones( p < 0.001).These results suggest that the JSR Ca²⁺ content immediately after the rested state twitch is much smaller in SHR than WKY. [Jpn J Physiol 55 Suppl:S115 (2005)]

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Our previous studies revealed that cardiac myofibrils are significantly stiffer in the transverse direction than skeletal myofibrils. To clarify molecular architectures responsible for this mechanical difference, myofibrils were specifically disrupted by the treatments with calpain and trypsin, and carefully investigated by use of atomic force microscope (AFM) and SDS-PAGE techniques how the transverse stiffness changes associated with decompositions of myofibrils. Single myofibrils were prepared by homogenizing glycerinated muscle fibers of the left ventricle of rat heart and the rabbit psoas muscle. Myofibrils were treated with calpain (0.15mg/ml) and trypsin (0.25μg/ml), and their transverse stiffness examined with an AFM as previously. SDS-PAGE was performed on 2.5%∼12% gradient gels. As the proteolytic digestions of myofibrils advanced under the present conditions, α-actinin and connectin/titin became gradually disrupted while actin and myosin remained undigested. In parallel, the transverse stiffness along myofibrils decreased in characteristic fashions. It was concluded that (1) Z-disc of cardiac myofibrils is ca. 3 times more rigid and thick than that of skeletal myofibrils, (2) connectin/titin is bound to thin filaments in cardiac myofibrils but not in skeletal myofibrils, and (3) the stiffness of the lattice structure of actin and myosin filaments is almost the same for cardiac and skeletal myofibrils. [Jpn J Physiol 55 Suppl:S115 (2005)]

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It is reported that heat stress activates proliferative potential and muscle hypertrophy. However, the mechanism responsible for the effects of heat stress on muscle hypertrophy remains unclear. Our hypothesis is that the up-regulation of heat shock protein (HSP) induced by heat stress, as well as mechanical stretch, may be one of the signals for muscle hypertrophy. Geranylgeranylaceton (GGA), which is an acyclic polyisoprenoid and an anti-ulcer drug developed in Japan, stimulates HSP72 expression in the gastric mucosa, small intestine, and heart in rats. Thus, the current study was carried out to investigate the effects of GGA on HSP72 expression of cultured skeletal muscle cells. Mouse skeletal muscle cells (C2C12) were plated on collagenized culture dishes. And the C2C12 cells were subjected to one of the following four kinds of conditions; 1) control, 2) GGA administration (10⁻⁷ M), 3) heating at 41°C for 60 min, or 4) GGA administration followed by heating at 41°C for 60 min. Following the above treatments, the cells were incubated for few days at 37°C. Expression of HSP72 was up-regulated by the administration of GGA. The GGA administration enhanced the heat stress-induced up-regulation of HSP72, suggesting that heat stress-associated muscle hypertrophy may be stimulated further by GGA. [Jpn J Physiol 55 Suppl:S116 (2005)]

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Contractions induced by electrical stimulation of 50 pulses at 40 Hz to the guinea-pig vas deferens consisted of purinergic and adrenergic (α₁) components. As Ca²⁺ influx mediated by P2x purinoceptors activates Ca²⁺-dependent K⁺ channels, the α₁-mediated contraction may be modified by inhibiting P2X purinoceptors. P2 antagonists, suramin (300 μM), α,β-methylene ATP (10 μM) and PPADS (30 μM) abolished the purinergic component, leaving biphasic (early and late) α₁ component. The early α₁ component can be isolated from the α₁ component by removing the late α₁ component with nifedipine (10 μM). Apamin (100 nM), a SK channel antagonist increased the early α₁ component up to 214±44% (n=5, p<0.05) and 111±4% (n=4, p<0.05) in the suramin- and α,β-methylene ATP-treated vas deferens, respectively. However, the effect of apamin on the early α₁ component was not significant (102±3%, n=4) in the PPADS-treated vas deferens. Suramin decreased the early α₁ component concentration dependently in the α,β-methylene ATP-treated vas deferens but not in the PPADS-treated vas deferens. These findings indicate that 1) α,β-methylene ATP inactivates SK channels by decreasing P2X-mediated Ca²⁺ influx, 2) PPADS may close SK channels and 3) suramin reverses the SK channels blocked by P2Y purinoceptors, as Ming et al (2000) reported in the cortical collecting duct of mouse kidney. [Jpn J Physiol 55 Suppl:S116 (2005)]

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We compared how hindlimb immobilization (HI) affected contractile functions of single skinned soleus fibers from the rat. HI (6 weeks) resulted in reduced wet weight of soleus muscle (∼40%). The area of single skinned fibers was ∼30% less in immobilized muscle than in control muscle. We found that in immobilized muscle, maximal Ca²⁺-actiavated force (pCa 4.5) was markedly reduced (by ∼60%) and the force-pCa curve was shifted to the lower pCa (higher Ca²⁺ concentration) side (sarcomere length, 2.20 μm), suggesting a reduction in Ca²⁺ sensitivity of force. We also induced Ca²⁺-independent active force by lowering the MgATP concentration to investigate whether the decreases in Ca²⁺-activated force result from reduced likelihood of cross-bridge formation. We found that in immobilized muscle, maximal Ca²⁺-independent active force was reduced by ∼40%, with the leftward shift of the force-pMgATP curve. In muscular fatigue, the intracellular concentration of inorganic phosphate (Pi) increases. Pi reverses the Pi release step of the cross-bridge cycle, resulting in decreases in active force. We tested the effect of Pi (up to 20 mM) on maximal Ca²⁺-activated force in control and immobilized muscles. The inhibitory effect of Pi was more pronounced in immobilized muscle, suggesting a reduced fraction of active cross-bridges. These results are consistent with the notion that cross-bridge formation is suppressed in immobilized muscle via, probably, structural changes of the sarcomere, resulting in reduced active force production. [Jpn J Physiol 55 Suppl:S116 (2005)]

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The effect of 17β-estradiol on the estrogen receptor (ER) expression and its related signal pathways were examined in the C2C12 mouse skeletal myoblast cells. Using immunoblotting methods, ER was detected in the post-nuclear fraction with the corresponding molecular weight of 66 kDa. The ER expression was increased by an administration of 17bβ-estradiol (0.1-100 nM), and also increased by 17α-estradiol (10 nM), the stereoisomer of 17β-estradiol. The amount of ER expression was more increased by an estradiol-17β-BSA (10 nM). This stimulatory effect of estradiol was not inhibited by antagonists, tamoxifen (1 nM) and ICI 182780(1-10 nM). By the immunoprecipitaiton methods using 35S-methionine, the newly-synthesized ER with 66 kDa was also detected in the post-nuclear fraction. The newly-synthesized ER was increased time dependently (1, 3, 5 hrs). The administration of 17β-estradiol (0.1-100 nM) increased the synthesis of ER dose-dependently. More detailed studies revealed the involvement of PKC/MAPK signaling system. These results suggest that ERs located at membranes are a target of estrogen action in mouse skeletal myoblast cells. And the estrogen-stimulated ER synthesis involves the PKC/MAPK signaling system. [Jpn J Physiol 55 Suppl:S116 (2005)]

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It has been considered that satellite cells are responsible for the postnatal growth and regeneration of skeletal muscle. However, the details of mechanism for skeletal muscle regeneration are still unclear. The purpose of this study was to investigate the effects of heat stress on the regeneration processes of injured skeletal muscles. Male Wistar rats (7 weeks old) were divided into control (n = 25), non-heated (n = 50), and heat-stressed groups (n = 50). To induce a necrosis-regeneration cycle, 0.1 mL of cardiotoxin (CTX, 10 μmol/L) was injected into the left tibialis anterior muscle of rats except in the control group. Rats in the heat-stressed group were exposed to environmental heat stress (41°C for 60 min) in a heat chamber 24 hours before or immediately after CTX injection without anesthesia. The tibialis anterior muscles were dissected 1, 3, 7, 14, and 28 days after CTX injection. The muscle protein contents in the heat-stressed group were significantly greater than controls 28 days after CTX injection (p<0.05). Increment in Pax7-positive nuclei, which is a specific indicator for muscle satellite cell, in the heated group was larger than in the non-heated group. These results suggest that heat stress could activate satellite cells, promote the proliferation and the differentiation of satellite cells, and facilitate the regeneration. [Jpn J Physiol 55 Suppl:S117 (2005)]

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We have devised a new cytochemical method for detection of Cu²⁺ ions penetrated into the postsynaptic subneural apparatus of the frog skeletal muscle cells, during electrical motor nerve stimulation. CuCl₂ was added (final concentration; 1.8 mM) to the physiological solution immeadiately after the motor nerves tetanic stimulation (5 Hz for 2 min). The tetanus was disappeared within 2 min after Cu²⁺ application. After washing with an isotonic NaCl solution containing EDTA, the tissue was treated with a solution containing 2.5% glutaraldehyde (tissue fixator), 10 mM potassium ferrocyanide (precipitating agent of Cu²⁺ and donor of an oxidoreductase-like catalytic activity to the copper precipitate) and 100 mM NaCl (osmotic compensator). After permeabilizing the muscle membrane, the tissues were treated with conventional DAB-H₂O₂ procedure. The catalytic activity of copper ferrocyanide was revealed as red staining in the site of the precipitate in the level of the subneural apparatus. The subneural area was diffusively stained when direct muscle stimulation was superposed. The opposite area of the subneural apparatus in the neural region and the aneural regions of the muscle cells were devoid of the staining. d-Tubocurarine applied before CuCl₂ markedly reduced the staining and the absence of CuCl₂ provided no staining. [Jpn J Physiol 55 Suppl:S117 (2005)]

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Effects of the inhibited phsphorylation of heat shock proteins (HSPs) and ribosomal protein S6 phosphorylated at serine-235/236 (p-S6) on the deafferentation-related atrophy of soleus muscle were studied in rats. Adult male Wistar rats were randomly separated into the control, functionally overloaded (FO), sham-operated, deafferentated (DA), FO+DA, and hindlimb-unloaded (U) group. The tendons of plantaris and gastrocnemius muscles were transected in the FO rats. The dorsal roots of the spinal cord at the L4-5 segmental levels were transected in the DA rats. The rats in U were tail-suspended. The sampling of the soleus muscle was performed 2 weeks after the treatments shown above. The cytoplasmic fraction of the soleus muscle homogenate was used for the quantitative analysis of the expression levels of 72 kDa (HSP72) and 27 kDa proteins (total and phosphorylated at serine-85, HSP27 and p-HSP27), and p-S6. All of theses proteins were down-regulated by U. Although the levels of the expression in these proteins were not affected by FO and sham operation, the levels of p-HSP27 and p-S6 235/236 were decreased in DA and FO+DA groups. It was known that the p-HSP27 and p-S6 play a role in the cytoskeletal organization and the initiation of the global mRNA translation, respectively. Therefore, the results suggested that the inhibition of the phosphorylation of HSP27 and S6 may be one of the major causes for the deafferentation-related soleus muscle atrophy. [Jpn J Physiol 55 Suppl:S117 (2005)]

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Protein kinase B (PKB) and calcineurin (CaN) are both postulated to play important roles in integrating intracellular signaling in skeletal muscle in response to both disuse and increased muscle loading. These experiments investigated the age-related differences in signal transduction of the downstream pathways of both PKB and CaN during a period of recovery following immobilization-induced muscle atrophy. The hindlimb muscles of one leg in young (10 weeks) and adult (40 weeks) were immobilized in plantar flexion position using a plaster cast for 10 days. At 1-10 days after the removal of plaster cast, both the atrophic and the contralateral plantaris muscles were removed. Immobilization resulted in significant muscle atrophy in both young and adult animals. Muscle mass in young animal recovered up to -90% following 10-days of recovery period, but not in adult animals. The levels of phosphorylated p70S6K were significantly higher in both animals following 1-day of recovery, with the greatest changes seen in young rats. Muscle levels of phosphorylated S6 (pS6) in young rats were higher in all of recovery period, but pS6 in adult rats returned to control levels by day 10 of recovery. CaN levels in adult animals were higher at recovery day 10, but not in young animals. These results show that the signaling pathways during recovery in atrophied plantaris muscle immobilization differ in young and adult muscles. [Jpn J Physiol 55 Suppl:S117 (2005)]

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Gastric smooth muscle generates slow waves spontaneously, and they are regulated by autonomic nerves. Experiments were carried out to investigate the effects of hypoxia on spontaneous electrical activity and their autonomic neuronal regulation in smooth muscle of the guinea-pig stomach. Segments of smooth muscle tissues were isolated from the antrum region of the stomach, and electrical activities of smooth muscle cells were recorded using conventional microelectrode technique. Junction potentials produced by transmural nerve stimulation were also recorded. Isometric mechanical responses of smooth muscle tissue were also examined. Low oxygen condition, produced by bubbling with N₂-gasses, induced a reduction in resting tension, spontaneous rhythmic contractions and mechanical responses produced by nerve stimulation, with no significant alteration to the frequency of spontaneous contraction. These changes were not associated with change in electrical responses of the membrane (the resting membrane potential, shape of slow waves and junction potentials). Acetylcholine (Ach)-induced depolarization and contraction responses were also not changed significantly during hypoxia. The results suggest that oxygen requirement for mitochondrial activity responsible for production of rhythmic activity is relatively low. [Jpn J Physiol 55 Suppl:S118 (2005)]

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The larva of ascidians represents a minimal form of chordates. Its tail has bilateral muscle bands, each composed of about 20 cells, and whose activities are controlled by 6-10 motor neurons. This simplicity as well as the recent extensive accumulation of genomic data provides an opportunity to study a detailed mechanism for regulating the complex locomotion patterns in chordates. In order for investigating the relationship between locomotion patterns and muscle activities, we tried a combinatorial approach to tail-beating patterns of Ciona intestinalis larva, using a high-speed video camera (HSVC), extracellular suction electrode for field potential recording from muscle bands, and intracellular microelectrode for recording membrane potential from the muscle cell. This study revealed that (1) the activities of muscle bands are considerably varied with respect to the rhythm, pattern, and velocity of tail bending. (2) As larva matured, the propulsive movement became efficient, and the behavioral modes could be complicated. (3) The changes in muscle field potential during a swimming train corresponded well with the beating patterns simultaneously recorded under HSVC; which promised a correlation between the observable locomotion patterns and intrinsic physiological properties of the tail. (4) Stepwise current injections into a muscle cell did not evoke an 'all-or-none' response, but a 'graded' one. These illuminate an intrinsic mechanism of the larva to show variable locomotion patterns using a pair of single layers of apparently-uniform muscle cells. [Jpn J Physiol 55 Suppl:S118 (2005)]

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It is known that muscle sympathetic nerve activity (MSNA) increases during respiratory apnoea, cold pressor test, handgrip exercise, and mental arithmetic task in humans. We hypothesized that increased MSNA may modify the sensitivity of muscle spindle afferents. To test this, we evaluated tonic vibration reflex (TVR) in the condition with enhanced MSNA. Eight subjects with no neurologic impairment performed four maneuvers accompanying sustained activation of MSNA: 1) respiratory apnoea, 2) cold pressor test for 1 min, 3) mental arithmetic task for 2 min, and 4) static handgrip exercise at 30-40% of maximal voluntary contraction for 1.5 min followed by post-handgrip ischemia for 1 min. Arterial blood pressure (AP) and ECG were noninvasively monitored and cardiac output and total peripheral resistance (TPR) were estimated from the AP waveform using the Modelflow method. TPR was considered as an index of peripheral sympathetic nerve activity. TVR tended to increase as TPR increased during respiratory apnoea, cold pressor test, and mental arithmetic task. TVR tended to increase when AP increased during static handgrip exercise. However, TVR changed little, when MAP didn’t change during post-handgrip ischemia, mental arithmetic task, or cold pressor test in some subjects. TVR decreased in one subject, when TPR decreased during mental arithmetic task. The correlation between TVR and TPR indicates that increased sympathetic outflow may facilitate TVR in support of the concept that MSNA can influence the sensitivity of human muscle spindles. [Jpn J Physiol 55 Suppl:S118 (2005)]

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The sartorius muscles from a bullfrog were used. The X-ray patterns were recorded by an X-ray CCD detector with a time resolution of 15 msec. When the skeletal muscle is stretched slowly at the plateau of the isometric tetanus, the tension increases during the stretch. When the length change terminates, the tension starts to return to a new tension level, which is higher than the initial isometric level. In a release, the tension behaves in a similar but opposite way. The effect of these muscle length changes on the intensity of the 3rd meridional reflection (M3), which arises from the 14.3 nm repeat of the myosin heads was examined. It was found that M3 increases during stretch and decreases during release if the applied length change is smaller than 1% of the muscle length(Lo). When the applied length change is larger than 1%, both stretch and release induce the decrease in M3. When a sinusoidal length change (amplitude = 1%Lo) is applied to the tetanized muscle, M3 increases and decreases in parallel with the applied length change. On the other hand, when the length change is larger than 1%Lo (for example, 3%Lo), M3 decreases both in stretching and releasing phases. [Jpn J Physiol 55 Suppl:S118 (2005)]

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We have explored the role of the giant elastic protein titin in the Frank-Starling mechanism of the heart by measuring the sarcomere length (SL) dependence of activation in skinned cardiac muscles with different titin-based passive stiffness characteristics. We studied muscle from the bovine left ventricle (BLV), which expresses a high level of a stiff titin isoform (N2B titin), and muscle from the bovine left atrium (BLA), which expresses more compliant titin isoforms (N2BA titins). Passive tension was also varied in each muscle type by manipulating the pre-history of stretch prior to activation. We found that the SL-dependent increases in Ca²⁺ sensitivity and maximal Ca²⁺-activated tension were markedly more pronounced when titin-based passive tension was high. Control experiments suggest that the greater SL dependency in BLV is unlikely due to differences in the contractile protein isoform composition. Small-angle X-ray experiments revealed that the SL dependence of reduction of interfilament lattice spacing is greater in BLV than in BLA, and that the lattice spacing is coupled with titin-based passive tension. These results support the notion that titin-based passive tension promotes actomyosin interaction by reducing the lattice spacing. This work indicates that titin may be a factor involved in the Frank-Starling mechanism of the heart by promoting actomyosin interaction in response to stretch. [Jpn J Physiol 55 Suppl:S119 (2005)]

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Flexor carpi radialis muscle (FCR) is one of the forelimb muscles in frog used for mating posture, amplexus. We measured the change in force and stiffness of the muscle before and after amplexus of three weeks in breeding season at 4°C.It was found that the muscle before and after amplexus has characteristic ‘long-lasting relaxation (more than 30 min) after contraction’ and that the relaxation was composed of three phases, which were separated clearly by two changing points (CP) on force curve. The first phase was fast and the second and third phases were slow, the third phase being the slowest. Among these three phases, the heights of force and stiffness in the second relaxation phase were significantly higher after amplexus. Spontaneous contraction which brought about the increase in force and stiffness without stimulation was also observed after amplexus as well as before amplexus. Present results indicate that the force and stiffness level of the relaxation phase after contraction was raised after amplexus especially by increasing the second phase, suggesting that contraction changed so that it would become more suitable for amplexus. The results about cross-bridge cycle in the relaxation will be also reported. [Jpn J Physiol 55 Suppl:S119 (2005)]

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To evaluate the contribution of each amino acid residue of the cardiac troponin I inhibitory peptide (TnIp; residues 136-147, Ac-GKFKRPTLRRVR-NH₂) to the force inhibiting effects of TnIp on the skinned smooth muscle (Watanabe et al., 2003), we compared the effects of 11 TnIp analogues, which consisted of single glycine replacement, on the Ca²⁺-induced contraction of Triton X-100-skinned preparations of taenia caeci from guinea pig. Every residue of the cardiac TnIp was necessary to achieve maximum inhibition of the Ca²⁺-induced contraction. Furthermore, replacing R₁₄₀, L₁₄₃ or V₁₄₆ with glycine resulted in enhancement of Ca²⁺ sensitivity for the force. Since these analogues did not enhance myosin phosphorylation independent contraction induced by 30 mM Mg²⁺, they may accelerate phosphorylation of myosin light chain as well as a synthetic peptide of acting binding region of heat shock protein 20 (HSP20p, residues 110-121). [Jpn J Physiol 55 Suppl:S119 (2005)]

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The sense of position is signaled mainly by muscle spindles. Isometric muscle contraction at a shorter (hold-short conditioning) length causes an increase in Ia afferent discharge after the muscle returns to its intermediate length by means of intrafusal muscle thixotropy, and hold-short conditioning in the biceps has been shown to produce limb position-sense errors. The contraction of muscles in the human upper body reinforces tendon jerk in the lower limbs, which is referred to as Jendrassik maneuver (JM). JM increases stretch responses of muscle spindles preconditioned with hold-short conditioning. Consequently, these findings suggest that JM would enhance the history-dependent position-sense errors that result from intrafusal muscle thixotropy. We studied the effects of quadriceps contraction (QC) on upper-limb position-sense errors induced by hold-short conditioning in the biceps of 12 healthy men. QC enhanced tonic vibration reflex of the biceps, suggesting it also has a reinforcement effect on stretch responses of muscle spindles similar to JM. After hold-short conditioning, subjects perceived that the conditioned forearm was placed in a more extended position than occurred in reality. This position error was more obvious after hold-short conditioning combined with QC. These results suggest that QC and hold-short conditioning work together efficiently to develop intrafusal muscle thixotropy. [Jpn J Physiol 55 Suppl:S119 (2005)]

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We made a preparation of the rat renal calix, pelvis, and ureter en bloc to determine and investigate the pacemaker region of its perstalsis. The origin and frequency of the movement were maintained for more than 3 hours in our experimental condition. In observations using a stereo microscope, peristalsis originated from 25.0% in superior visceral, 37.5% in superior drosal, 16.1% in inferior visceral, 12.5% in inferior drosal region of the pelvis and 8.9% in pelviureteric region. Stained with fluo-3 periodic rise in intracellular Ca²⁺ concentration was successfully observed In situ. It originated from the exactly same region as its peristalsis and propagated along muscle bundles. Interestingly the periodic Ca²⁺ rises which could propagate always occurred in the several cells synchronously. In the originating region a few cells existed showing spontaneous Ca²⁺ rise but not synchronous to surrounding cells. These asynchronous Ca²⁺ rises never propagated to the downstream. In the presence of low concentration of heptanol, the periodic and synchronous Ca²⁺ rise still persisted, but its propagation was inhibited. In the higher concentration of the drug, the synchronous Ca²⁺ rise was disappeared: all Ca²⁺ rises observed in the region were asynchronous. These results might suggest that the cells connected via gap junctions and with synchronous Ca²⁺ rise played an important role in the pacemaker mechanisms. [Jpn J Physiol 55 Suppl:S120 (2005)]

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The physiological roles of two components (primary and plateau components) of pacemaker potentials recorded in situ from myenteric interstitial cells of Cajal (ICC-MY) distributed in the mouse small intestine were studied using intracellular recording techniques. Mibefradil, a T type Ca²⁺ channel blocker, reduced the amplitude, frequency and dV/dt_max of pacemaker potentials by inhibiting the generation of the primary component in a dose-dependent manner (1-30 μM). Exposure of the tissues with 30 μM mibefradil caused a change in pacemaker potentials from the rapidly rising, high frequency-typed potential changes to the noisy, gradually depolarized slow frequency-typed potential changes. Application of caffeine (3mM) abolished pacemaker potentials in the presence of 30 μM mibefradil. Pinacidil, a well-known ATP-sensitive K⁺ channel opener, hyperpolarized the membrane, increased the amplitude and dV/dt_max and decreased the duration of pacemaker potentials without alteration to the frequency. The effects of pinacidil were blocked by glybenclamide (10μM). These results suggest that the primary component of pacemaker potentials plays a fundamental role in generating the high frequency spontaneous activity characteristic to mouse small intestine. The primary component of pacemaker potentials is due to activation of dihydropyridine-resistant Ca²⁺ channels, and this depolarization entrains pacemaker activity to create the plateau potential. [Jpn J Physiol 55 Suppl:S120 (2005)]

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Purpose: To examine the effects of ischemia on the hardness of the gastrocnemius (GS) muscle. Methods: SD rats deeply anesthetized with urethane were used. In the first experiment, the muscular blood flow was completely occluded by a tourniquet compression at the thigh. The ischemia was confirmed by Laser Doppler flow metery of the muscle. Then muscle hardness was repeatedly measured by a special device during the ischemia (2 h) and reperfusion (2 h) periods at intervals of 5 min. The contralateral hind legs were served as controls. In the second experiment, the hardness of the GS muscle under tetanic contractions induced by direct muscular electrical stimulation (50Hz, 100μs, 2min) was measured repeatedly during the reperfusion period. Simultaneously, the produced muscular tension was monitored by a push-pull gauge. Results: During the ischemic condition, the blood flow was rapidly decreased to 2.5 +/- 0.2% of the resting control value. The muscle hardness was clearly increased from 66 to 84 (A.U) accompanied with the reduction of blood flow. Reperfusion resulted in the moderate recovery of the hardness and blood flow to the control value. The hardness and tension induced by tetanic contractions reached to their maximum at 5 sec after the onset of stimulation. However their peak values in the ischemia group were less than those of the controls. Conclusions: The ischemic condition strongly affected on the muscle hardness both the resting and contracting state. These results suggest that muscular blood flow dynamics might be one important factor for the muscle hardness. [Jpn J Physiol 55 Suppl:S120 (2005)]

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The active tension generated by cardiac muscle is not merely a function of its length but related on the manner of contractions. The tension versus length curve obtained by afterload contractions lies below that of isometric contractions, which is known as the shortening deactivation. When the length of muscle is clamped at any time during a preload shortening, the muscle is fixed at any length and starts to generate tension on an isometric manner. In this way, another length-tension curve is drawn by this length clamp method. Even when the muscle shortens with the same amount of length as during afterload contraction preceded an isometric contraction, the active tension is much larger than that of afterload contraction. The length-tension curve obtained by the length clamp method lies between those curves by isometric and afterload contractions. If the amount of length change by the length clamp method is the same as that of afterload contraction, little difference is expected in shortening deactivation. However in these contractions, the curve by the length clamp method is clearly located upward of the curve by afterload contraction. Compared with this structural aspect, the mechanical difference among these three ways of contractions is how much the external work existed. However there is no external work output in these contractions, some external work should exist because it shortens with some load. During shortening some amount of work is produced and then absorbed during relaxation. The larger the work is produced, the lower the length-tension curve appears. [Jpn J Physiol 55 Suppl:S120 (2005)]

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Background: Hypoxia inducible factor (HIF)-1alpha is a bHLH-Per-Arnt-Sim transcription factor that senses low oxygen availability and enhances activation of hypoxia-inducible genes. Recently, several investigators have demonstrated that HIF-1alpha activity is also crucial for normal tissue development and functional organ assembly in murine tissue. However, HIF-1alpha function appears to be cell- or organ-type specific. Skeletal muscle is a highly plastic tissue frequently exposed to varying oxygen tension. We therefore assumed that HIF-1alpha may control the process of myogenic differentiation. Objective: To clarify the role of HIF-1alpha in myoblasts differentiation. Methods: Expression and localization of HIF-1alpha during myogenesis was detected using RT-PCR, Western blot and immunostain. Influence of HIF-1alpha knockdown by RNA interference on myoblasts differentiation was also observed. Results & Conclusion: Although HIF-1alpha mRNA was constantly expressed even under growth phase without differentiation, HIF-1alpha protein increased during myogenesis. At early stage of myogenesis, HIF-1alpha accumulated in the nucleus of myogenin positive myoblasts. HIF-1alpha was expressed both in the nucleus and the cytoplasm at matured stage. Knockdown of HIF-1alpha effectively blocked myogenesis. HIF-1alpha expression was also confirmed in the regenerative muscle tissue of mice after eccentric exercise. In addition, expression of HIF-2alpha during myogenesis was similar to expression of HIF-1alpha. We show that HIF-1alpha may be one of the crucial transcriptional factors in the regulation of C2C12 myogenesis. [Jpn J Physiol 55 Suppl:S121 (2005)]

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Purpose: We have investigated structural alterations of the capillary network in the rat soleus muscle after 2-wk hindlimb unloading with and without pre-conditioning exercise. Methods: To clarify three-dimensional architecture of capillary network, contrast medium-injected sections were visualized clearly with a confocal laser scanning microscope. Fifteen male Wistar rats were used in this study. They were randomly divided into a control group, a hindlimb unloading without pre-conditioning exercise and with pre-conditioning exercise. Pre-conditioning exercise was performed treadmill running (20 m/min, 20°grade, 25min) before hindlimb unloading. Results: After hindlimb unloading, the cross-sectional area of muscle fiber and muscle mass were significantly decreased in with and without exercise. The mean capillary density, the number of anastomotic capillaries, the tortuosity of capillaries and capillary luminal diameter were significantly smaller in hindlimb unloading than in age-matched control. However, in hindlimb unloading with pre-conditioning exercise, these were significantly higher compared with hindlimb unloading without exercise. Concluding Remarks: Although hindlimb unloading conditions resulted in a significant decrease the capillary network, results from the present investigation indicate that the pre-conditioning exercise attenuated the regression of muscle capillary in hindlimb unloading rats. [Jpn J Physiol 55 Suppl:S121 (2005)]

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Masticatory (superfast) myosin is expressed in jaw muscles of eutherian and marsupial mammals including carnivores. It develops high maximal force with high ATPase cycling rate and exhibits moderately fast contraction velocity. These characteristics were hypothesized to be due to the protrusion of its head from the thick filament backbone toward thin filaments so as to increase attachment rate to actin. In this study, we carried out an X-ray diffraction experiment at SPring8 (BL45XU) on relaxed skinned fibers of dog masseter expressing the masticatory myosin. Compared with fast type muscle (tibialis anterior), masseter exhibited significantly wider myofilament spacing, roughly comparable 1,1/1,0 ratio, and weaker myosin layer lines. These suggested that the heads of masticatory myosin are reaching close to the thin filaments relieved from restriction of the backbone as expected from the hypothesis. As a functional test of the hypothesis, we performed tension and stiffness measurements. In the absence of calcium, slight decrease in ionic strength induced tension development in the masseter fibers. It suggested that their myosin readily interact to actin consistent with the hypothesis. [Jpn J Physiol 55 Suppl:S121 (2005)]

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We observed spin-spin relaxation process of ¹H-NMR signals from suspension of myofibrils prepared from rabbit psoas muscle. As was the case in tissue skeletal muscle, decomposition analysis of the relaxation process could be well represented by the summation of several exponentials indicating that water molecules in the suspension could be conveniently grouped into several components based on the relaxation time constant (T₂). The slowest two components dominated over faster relaxation components at the myofibril concentration ranges studied. With increase in the concentration of myofibrils, water component that relaxed with T₂ around 0.15 s progressively replaced the slowest component of T₂ > 0.4 s. An equivolumic point for these two components was found at 12 mg/ml myofibril concentration at 20°C in the absence of MgATP. Water components that relaxed more rapidly existed at small fractions. Since the average separation between the myofibrils is estimated to be 1.72 μm at the myofibril concentration of 10 mg/ml, myofibril affects water molecules within a significant distance from its surface differently from water molecules in the bulk solution. [Jpn J Physiol 55 Suppl:S121 (2005)]

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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 on the wild and the mutant structure was carried out in water sphere at 310 K to estimate dynamic structure 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 sphere with 0.001 ps time step in non periodic condition at constant temperature (310 K). It was found that 600-ps run closed N and C termini of H1-H2 fragment of troponin I to narrower separation in mutant structure than in wild one. This conformational change of troponin I would correspond to a decrease in the distance between H3 region of troponin I and its binding site on the regulatory domain of troponin C so as to accelerate their binding which is considered to activate actomyosin interaction (Takeda et al., 2003). This may explain, at least partly, the enhanced tension development in the mutant myofilament. [Jpn J Physiol 55 Suppl:S122 (2005)]

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Striated muscle has highly ordered periodic structure called sarcomere, which consist of thick and thin filaments. Recent finding indicate that spontaneous Ca²⁺ transient is important for sarcomere development, but precise mechanism is still unknown. Using C2C12 cells which differentiate into muscle like morphology, we have developed a method to rapidly accelerate sarcomere assembly by applying electric pulse stimulation, which will be useful to study the molecular mechanism of sarcomere development. C2C12 cells were differentiated and electric pulse of 40 V/60 mm, 1 Hz, and 24 ms pulse width was applied at 37°C and 5% CO₂, and contraction of myotubes were observed. Myotubes initially did not contract by electric pulse stimulation, but gradually started to contract and the movement reached maximum after 2 hours of stimulation. Application of low frequency stimulation (0.1 Hz) significantly delayed the development of myotubes compared to 1 Hz. Addition of verapamil completely blocked the movement of myotubes. Sarcomere structure observed under confocal microscope also increased in accord with contraction of myotubes although amount of muscle protein did not change significantly. These results indicate that Ca²⁺ oscillation is important for sarcomere assembly and acceleration of Ca²⁺ transient enhance muscle development. [Jpn J Physiol 55 Suppl:S122 (2005)]

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Ryanodine receptor type 2 (RyR2) is thought to be the most general Ca²⁺-induced Ca²⁺ release (CICR) channel. In smooth muscle cells (SMCs), RyR2 also regulates the activity of large conductance Ca²⁺ activated K⁺ (BK) channel via CICR or spontaneous local Ca²⁺ release (Ca²⁺ spark). The contribution of RyR2 to excitation-contraction coupling and muscle tone in SM was examined using urinary bladder SMCs (UBSMCs) from RyR2 heterozygous KO mice (RyR2^+/−), in which RyR2 mRNA expression decreased by ∼50%. In resting conditions, Ca²⁺ sparks activated BK channels to elicit spontaneous transient outward currents (STOCs), and STOCs regulated resting membrane potential in UBSMs. In RyR2^+/−, the frequency of STOCs and the membrane depolarization by paxilline, a specific BK channel blocker, were significantly reduced, while BK channel expression was comparable to that in RyR2^+/+. When cells were depolarized to 0 mV, early local Ca²⁺ transients (hot spots) were observed in peripheral areas and spread into other areas of myocytes. The elevation of [Ca²⁺]i at 0 mV was significantly smaller in RyR2^+/−. The force development in tissue segments by direct electrical stimulation was also smaller in RyR2^+/−. These results strongly suggest that RyR2 play a crucial role in regulation of E-C coupling and resting tone in UBSMs. [Jpn J Physiol 55 Suppl:S122 (2005)]

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Macromolecular solutes such as Dextran T500 (Mw 500,000), are known to compress the myofilament lattice of skinned skeletal muscle. Since large molecules would be physically excluded from the lattice, increase in the accessible water for the macromolecules is considered to favor the lattice shrinkage. We found that small molecules of polyethylene glycols (PEGs; Mw200 and 900) also compress the skinned fiber. To elucidate the mechanism, we pursued the effects of relevant small molecules on the fiber volume. The optical cross section of mechanically skinned fibers of frog sartorius muscle was observed. PEGs, ethylene glycol, butanols (butanol, butanediol, butanetriol), pentanols (pentanol, pentanediol) efficiently compressed the fibers, while glycerol, threitol, ribitol and mammitol did not. The number of hydrophobic groups and their distribution on the molecule would be the important factors. The molecules with compressing efficiency exerted diverse effects on fiber function. PEGs decreased Ca-sensitivity for activation, while butanol and pentanol increased it. All the tested molecules suppressed the maximal force with varied efficiency. No correlation was found between the efficiency for fiber compression, Ca-sensitization and maximum force suppression, indicating that the molecules selectively affect multiple sites on the myofilament. This suggested that the tested molecules could, at least partly, penetrate into the myofilament lattice. We infer that the network of hydration water in the myofilament lattice filters the hydrophobic molecules forming a heterogeneous field around the myofilaments. [Jpn J Physiol 55 Suppl:S122 (2005)]

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