The KCNE proteins (KCNE1-5) are single transmembrane peptides that function as ancillary β-subunits of voltage-gated K⁺ channels. Functional coupling of KCNE1 (E1) with KCNQ1 (Q1) to form cardiac I_Ks channel is well documented. However, Q1 is also found to have an affinity for all other members of KCNEs. The present study tested the possibility for the formation of functional channels consisting of Q1, E1 and KCNE2 (E2). We constructed a plasmid encoding a fusion protein in which the N terminus of Q1 was fused to the C terminus of either E1 or E2, and characterized the function of heteromeric channels by measuring whole-cell currents from COS7 cells transiently transfected with the plasmids. It was comfirmed that E1-Q1 fusion channels produced voltage- and time-dependent currents, similar to those obtained by coexpression of E1 together with Q1, while E2-Q1 fusion channels evoked constitutively active time-independent currents, again similar to those induced by coexpression of E2 and Q1. Coexpression of the E1-Q1 with E2 results in time-dependent currents which had a faster deactivation time course and a more depolarized half-activation voltage, compared to the E1-Q1 current. On the other hand, coexpression of E2-Q1 and E1 exclusively produced voltage- and time-dependent current without any constitutively active component, providing a functional evidence to show that all E2-Q1 coassembles with E1. These results suggest that E1 and E2 can be couple to a Q1 channel molecule forming complexes with unique current properties. [J Physiol Sci. 2006;56 Suppl:S128]

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[Background] Regulation of intracellular and extracellular free magnesium (Mg²⁺) concentration is important to control the excitability in cardiac myocytes. [Objective] To assess the role of intracellular and extracellular Mg²⁺ imbalance in cardiac myocytes associated with membrane potentials and ionic currents derangement. [Methods and Results] Serum and intracellular Mg²⁺ concentrations were measured from normal (control) and the Mg²⁺ -deficient food feeded rats. The serum [Mg²⁺]e and intracellular Mg²⁺ concentrations [Mg²⁺]i were significantly decreased from 6.9 ± 0.3 mg/dl and 2.3 ± 0.2 mg/dl to 4.4 ± 0.2 mg/dl and 0.7 ± 0.1 mg/dl on Mg²⁺ -deficient diet on day 28, respectively. We obtained ECG signals by a radio transmitter from control and the Mg²⁺-deficient rats. Low [Mg²⁺]e significantly prolonged RR duration (control, 181 ± 17 ms versus low [Mg²⁺]e, 158 ± 18 ms, p < 0.05) in accordance with various types of cardiac arrhythmias such as supraventricular extrasystole, atrioventricular blocks and so on. Action potentials (AP) from rat ventricle myocytes were recorded by using intracellular microelectrode technique. AP durations (APD₉₀) in myocytes obtained from Mg²⁺ -deficient rats were significantly prolonged (control, 101.2 ± 3.2 ms versus low-Mg²⁺, 314.0 ± 11.8 ms, p < 0.01). [Conclusion] Decreases in intracellular and extracellular Mg²⁺ concentrations cooperatively contribute toward arrhythmogenecity presumably affecting intracellular Ca²⁺ concentrations. [J Physiol Sci. 2006;56 Suppl:S128]

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In recent years, some of biological actions of lysophosphotidylcholine (LPC) have been found to be mediated through a class of G protein-coupled receptors, termed G2A. The present study was designed to examine the possible regulation of the muscarinic K⁺ current (I_K,ACh) by LPC and its associated signaling pathways in guinea-pig atrial myocytes, using whole-cell patch-clamp method. Bath application of LPC (2 μM) reversibly and almost completely (93.3%; n = 9) inhibited I_K,ACh preactivated by acetylcholine (ACh). On the other hand, LPC almost irreversibly inhibited I_K,ACh preactivated by intracellular loading of non-hydrolyzable GTP analogue GTPγS (84.2%; n = 6), suggesting an involvement of G protein. The inhibitory action of LPC was partially, but significantly, attenuated by pretreating myocytes with an anti-G2A antibody and phospholipase C (PLC) blockers (compound 48/80 and neomycin). Furthermore, the inhibitory effect of LPC was still significantly reduced by exogenously adding phosphatidylinositol 4,5-bisphosphate (PIP₂, 50 μM) to the cell inside, and became nearly irreversible when atrial myocytes were continuously exposed to wortmannin (10 μM), which suppresses the resynthesis of PIP₂. As expected, LPC greatly reversed the shortening of action potential duration evoked by ACh. Based on these results, we conclude that LPC markedly inhibits I_K,ACh through a mechanism involving an activation of G protein-coupled G2A receptor causing a depletion of membrane PIP₂ via PLC activation. [J Physiol Sci. 2006;56 Suppl:S128]

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In general, animal cells tend to swell if Na/K pump is blocked. However, the volume of guinea-pig ventricular myocytes remained almost constant during 3 hours under 40 μM ouabain. The Na/K pump block simulation using the Kyoto model showed that the cell swelling is retarded in parallel to a decrease in the background membrane conductances for Cl⁻ and Na⁺, suggesting very low conductances for these ions in actual ventricular cells. In addition, it is suggested by the simulation that the active Ca²⁺ extrusion by sarcomlemmal Ca²⁺ pump (PMCA) has an important role in the delayed cell swelling. The transmembrane [Ca²⁺] gradient is partially maintained by PMCA, and this makes the Na/Ca exchanger extrudes Na⁺ in reverse mode, compensating for Na/K pump. In the experiment where the Cl⁻ leak was increased beyond these compensatory mechanisms by applying 5 μM isopreterenol, cell swelling could be induced, but only after a delay of about 55 min after ouabain application. The simulation revealed a gradual membrane depolarization during the delay, and finally it reached a voltage range of continuous opening of a significant fraction of L-type Ca²⁺ channels (window current). A rapid accumulation of Ca²⁺ subsequently activates Ca²⁺-dependent Na⁺ conductance, which finally initiates the rapid cell swelling. [J Physiol Sci. 2006;56 Suppl:S128]

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Spiral-wave reentry is a principal mechanism of life-threatening ventricular tachyarrhythmias. We investigated the effects of regional cooling (RC) on the dynamics of spiral-waves. Methods and Results: Optical action potential signals were recorded from 2-dimensional ventricular myocardium (1 mm thick) of Langendorff-perfused rabbit hearts. Ventricular tachycardias (VTs, >2 min) were induced by burst stimulation. RC (by 5-7ºC) was applied to the left ventricular free wall using a transparent cooling device (diameter, 10 mm). RC caused a marked prolongation of action potential (by 25±4%) and a slight reduction of conduction velocity (by 16±5%) in the affected region under constant pacing (2.5Hz, n=10). Spiral-wave dynamics during VTs were transformed by RC from stationary to chaotic meandering types. During RC, the rotors moved irregularly around the cooled region, and often collided with the anatomical boundary (atrioventricular groove) resulting in spontaneous termination. The incidence of termination of VTs within 15 s was increased from 2/18 (11%) without RC to 14/18 (78%) with RC. Conclusion: RC facilitates spontaneous termination of spiral-wave reentry through the unpinning of rotors. [J Physiol Sci. 2006;56 Suppl:S129]

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The cardiac biological pacemaker (BP) has been created by suppression of the inward-rectifier K⁺ current (I_K1) or overexpression of the hyperpolarization-activated current (I_h) in ventricular (Purkinje) or atrial myocytes, suggesting possible development of the functional BP as a therapeutic alternative to the electronic pacemaker. In this study, we investigated the effects of incorporating regulatory inward currents (pacemaker currents) such as I_h, sustained inward current (I_st), and low voltage-activated L-type Ca²⁺ channel current (I_Ca,LD) on 1) creation of BP cells, 2) robustness of BP activity to electrotonic loads of adjacent non-pacemaker cells, and 3) BP cell ability to drive the surrounding non-pacemaker cells. Bifurcation structures of single BP cell and coupled-cell models for human ventricular myocytes (HVMs) were explored during changes in conductance of I_K1, the regulatory inward currents, and gap junction. Our findings suggest that 1) incorporating I_h and I_st (or I_h and I_Ca,LD) facilitates BP generation during I_K1 suppression, although it does not lead to BP oscillation without I_K1 inhibition, and that 2) incorporating I_st or I_Ca,LD significantly improve the structural stability of BP cells to electrotonic loads and BP cell ability to drive adjacent HVMs. [J Physiol Sci. 2006;56 Suppl:S129]

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Cardiac hypertrophy is an adaptive response to chronic cardiac overload, and causes an abnormal electrical activity leading to arrhythmia. It is important to understand the electrophysiological mechanisms underlying the development of this condition. Therefore we analyzed the electrocardiogram (ECG) in hypertrophic heart of mice persistently pressure-overloaded by transverse aortic constriction (TAC). In pre-operated or sham-operated (SO) mice, the first upstroke in QRS-complex (designed to as "a") is followed by a fast second upstroke ("b") and sometimes a slower downstroke ("c"). In TAC mice, "b" is often followed by another slow upstroke ("b'") before "c", and a prolongation of Qc interval was revealed. These results suggest that ECG is sensitive enough to confirm development of cardiac hypertrophy in TAC mice. In addition, the whole-cell patch clamp recordings showed a prolongation of the action potential duration (APD) and a basal activation of chloride (Cl⁻) current in single myocytes freshly isolated from left ventricle of TAC mice. The Cl⁻ current may reflect a persistent activation of volume-regulated Cl⁻ current (I_Cl,vol) in isotonic condition, because of the time-dependent inactivation at higher positive potentials, the outwardly rectifier current-voltage relationships and the inhibitory effect of 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid. The basal activation of I_Cl,vol may be responsible for prolongation of APD and Qc interval in TAC mice. [J Physiol Sci. 2006;56 Suppl:S129]

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β-adrenergic receptor agonists, such as isoproterenol, activate protein kinase A (PKA) and induce positive inotropy in the ventricular myocyte. Major mechanisms underlying the positive inotropy are enhanced activities of the sarcolemmal L-type Ca²⁺ channel (I_CaL) and the sarcoplasmic reticulum Ca²⁺-ATPase via phosphorylation of phospholamban. However, role of activation of cystic fibrosis transmembrane conductance regulator channel (I_CFTR) in the inotropic effect has not been well understood.We have constructed a computer model of β-adrenergic signaling network including PKA activation of I_CaL, phospholamban and I_CFTR based on Saucerman et al., (2003, 2004) and implemented it into Kyoto model (Matsuoka et al., 2004). When 1 μM isoproterenol was applied, the amplitudes of I_CaL, Ca²⁺ transient, and cell shortening were increased, and the action potential duration was prolonged. Elimination of the activation of I_CFTR resulted in a slight rise of the plateau phase of action potential and a decrease in the amplitudes of I_CaL due to the decrease in driving force. Ca²⁺ transient was attenuated by about 20%. The results demonstrate that the activation of I_CFTR induces compensatory increase in I_CaL and facilitates indirectly the positive inotropic action of isoproterenol. [J Physiol Sci. 2006;56 Suppl:S129]

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The analytical method of cardiac energetics, developed by H.Suga, has been extensively used to evaluate the relationship between mechanical contractility and energy consumption by the left ventricle both in experimental and in clinical studies. The end-systolic maximum elastance (Emax) is measured to indicate contractility of the heart, and the mechanical energy is obtained by the pressure-volume area of a cardiac contraction cycle (PVA), which well correlates with the oxygen consumption. Here, we aim at interpreting the cardiac energetics in terms of the cellular mechanisms. We constructed a heart model of spherical shape to apply the Laplace law in converting the wall tension to pressure. The wall tension was calculated by the contraction model of Negroni and Lascano (1996), which is driven by the Ca transient of the comprehensive cardiac myocyte model, Kyoto Model. The 'Laplace heart' was connected with preload and afterload, and the pressure-volume loop was constructed. Our results of simulation under an aerobic condition show that the Negroni & Lascano model well reconstructs the linear PVA–oxygen consumption curve. The PVA-independent oxygen consumption could be well simulated by the ATP hydrolysis via the Na/K pump and the Ca pump on the sarcoplasmic reticulum. [J Physiol Sci. 2006;56 Suppl:S130]

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Though non-insulin-dependent (type 2) diabetes which accounts for over 90% of all diabetic patients leads to congestive heart failure, contractile property of cardiac muscle in type 2 diabetes has not been well characterized. We studied the contractile property of left ventricular papillary muscle of model mouse of the type 2 diabetes, db/db mouse, and effect of insulin and palmitate on it at 20 °C. There was no difference in the time course of twitch between db/db mouse and the control except its magnitude; the rested-state twitch force (Fmax) in the Krebs solution (10 mM glucose) was 21.2 mg/mm² (n=6) in the db/bd mouse and 190.3 mg/mm² (n=6) in the control. Application of either insulin (80 i.u./l) or palmitate (0.4 mM) increased Fmax in both the db/db mouse and the control. Steady-state force decreased with increasing stimulus frequency. Insulin and palmitate increased the steady-state force at a frequency > 1 Hz in the bd/bd mouse and the control except that palmitate decreased it at a frequency > 1 Hz in the db/db mouse. The rate of short-term mechanical restitution (SMR) decreased with increasing magnitude of the preceding twitch. Palmitate delayed the SMR, while insulin had variable effect on it. There was no apparent difference in the characteristics of the SMR between the db/db mouse and the control. We characterized Ca²⁺ release/content in the cardiac sarcoplasmic reticulum from the SMR with or without insulin and palmitate in the db/db mouse and the control according to the rationale described previously (Tameyasu et al, 2004). [J Physiol Sci. 2006;56 Suppl:S130]

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Excitation and contraction are the two main functions of heart cells. Therefore for studies of physiology and pathophysiology of heart cells, measurement of cell contraction is as important as that of membrane current and action potentials. In addition, measurement of cell contraction provides a simple way for assesing intracellular calcium transients. However, the measurement of single-cell contraction has not been a popular experimental technique, because it requires very specialized instruments, and because the measurement of cell contraction from patch-clamped cells was not quite trivial. In this report, we describe a simple system for video-based measurement of heart cell contraction, which can be used with patch-clamp instruments without difficulty. In this system, motion of the cell is recorded with a CCD video camera attached to the microscope, and the video images are digitized and analysed on a Windows-based PC using ScionImage public domain software. With the aid of software image manipulation using Sobel filter, cell shortening could easily be measured at 1/60 s time-resolution, from patch-clamped single heart cells. Values of the measurements are stored in text-based data files, which can easily be imported into pClamp or spreadsheets for further analysis. Contraction properties of patch-clamped mouse heart cells is also provided as an example of cell analysis using this system. [J Physiol Sci. 2006;56 Suppl:S130]

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Cardiovascular effects of sea buckthorn (Hippophae rhamnoides L.) called simply "Saji" in China were studied in SHRSP. Saji is a spiky shrub with yellow or orange berries.It has nodule rooting capability of fixing nitrogen from the air. Chemical analyses of Saji berries revealed that it contained vitamins C, B1, B2, E, F, K, P, provitamin A. The average protein content was 22% of the crude material, including polyphenols having high SOD activity. Further, it contained serotonin, cytosterol and selenium as well as zinc. In the in vivo study 5 SHRSPs were fed with rat chow containing powdered Saji fruits at the dose of 30mg/400g for 60 days. As the control group 4 SHRSPs were fed with normal rat chow. Then the arterial blood pressure and heart rate were measured on the tail. The venous blood weas sampled under anesthesia and subjected to biochemical analyses, and the heart was removed and frozen quickly in liquid nitrogen for histochemical analyses. The left ventricle was sliced horizontally in a crosssectional plane and studied by the double staining methods as described previously for the analyses of the arteriolar, intermediate and venular capillary portions. The arterial blood pressure and heart rate decreased. HbA1c, total plasma cholesterol and triglyceride decreased significantly. The arteriolar capillary portion tended to decrease, indicating the release from the stress caused by the hypertension.Thus the herbal medicine, Saji, is beneficial for cardiovascular and metabolic functions. [J Physiol Sci. 2006;56 Suppl:S130]

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We tested whether pulmonary vascular effects of propofol depend on the level of vasomotor tone. We used the isolated perfused normal rat lung and monocrotaline (MCT)-induced pulmonary hypertension (PH) rat model. Rats were subcutaneously given MCT (PH group). On day 21 after injection, the rats were anesthetized, and the lungs were isolated and perfused. Pulmonary perfusion pressure (PPP) was measured. Changes in PPP during administration of propofol were compared between both groups in the absence or presence of phenylephrine (PE). Propofol had no effect on PPP in the absence of PE. Propofol caused dose-dependent pulmonary vasoconstriction in the presence of PE. To study effects of propofol on endothelial function or myofilament Ca²⁺ sensitivity, N-nitro-L-arginine methyl ester and indomethacin or an inhibitor of PKC, bisindolylmaleimide I (BIS) and calphostin C were administrated before PE treatment. BIS and Calphostin C significantly decreased propofol-induced PPP elevation in MCT rats. In contrast, indomethacin significantly decreased propofol-induced PPP elevation in normal rats. Propofol may exert pulmonary vascular contractility through PKC activation in MCT rats and through inhibition of cyclooxygenase pathway in normal rats. [J Physiol Sci. 2006;56 Suppl:S131]

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We have been trying to calculate of arterial pressure (AP) on simulation model by a neural network (NN) algorithm without approximate equations, to develop a new approach to estimate the relationship between AP and other factors (renal sympathetic nerve activity (RSNA), heart rate (HR) and respiration rate). Previously, we confirmed the simulated AP consisted with measured AP by the NN model. In this study, we examined whether the simulated AP would consist with measured AP when measured values of hypertensive rats were input to the learned NN by those of normotensive rats. The AP simulation model was developed using Neural Network Toolbox of MATLAB (The Mathworks, Inc.). The back propagation was selected as a learning algorithm of the layered NN. AP, HR, RSNA and respiration rate were obtained from conscious chronically instrumented rats. Those were used for the learning of NN algorithm to establish the relationship between AP and other factors. When measured values obtained from hypertensive rats were input to the learned NN by those from normotensive rats, the simulated AP were lower than measured AP. We confirmed that the relationship between AP and other factors of hypertensive rats were different from those of normotensive rats by the NN model. These results suggest that AP of hypertensive rats was not only dependent on the relationship between HR, RSNA and respiration rate. [J Physiol Sci. 2006;56 Suppl:S131]

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We investigated the effects of exercise on endothelial function in Dahl salt-sensitive rats with risk factors for atherosclerosis, i.e. hypertension and/or hypercholesterolemia. We randomly assigned 174 rats to four experimental diet groups: (MF) control diet; (S) high-salt diet; (C) high-cholesterol diet; and (SC) combination of high-salt and high-cholesterol diet. The rats took these diets ad libitum. Each group was further assigned to two exercise conditions: sedentary group (SD) and exercise group (EX), swimming for 1 hour/day, 5 days/week. Chronic swimming exercise and experimental diet were started at the same time and continued for 8 weeks. After physiological examination every 2 weeks, we observed the distribution of NO and superoxide production in endothelial cells around aorto-renal bifurcation by using confocal laser-scanning microscopy. We evaluated the endothelial function with the oxidative stress index, the ratio of superoxide production to NO production. Chronic swimming exercise decreased the physiological risk for atherosclerosis, growth rate of body weight in all diet groups significantly, blood pressure in MF and C significantly, tended to decrease trigriseride, and increased HDL in all diet groups significantly. There was no difference in the oxidative stress index between artery portions, risk factors, and sedentary and exercise, but, decrease of NO production was related to atheroma prone portion. Therefore NO production may more contribute to the localization of atherosclerosis than oxidative stress. [J Physiol Sci. 2006;56 Suppl:S131]

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Colloidal particles are widely used as tracer and vehicle of drugs which target the lymph nodes. Little information, however, exists regarding mechanisms of colloidal accumulation in the lymph nodes in vivo. Thus, in this study, we have attempted to evaluate size-dependent mechanisms for accumulation of colloidal particles in the lymph nodes in vivo. Male Japan White rabbits were anesthetized with ketamine (20 mg/kg iv.) and pentobarbital (20 mg/kg iv.). Centripetal and retrograde cannulations were performed in one of the popliteal afferent and efferent lymph vessels, respectively. All other efferent lymphatics were ligated completely. Labeled latex with fluorescence microspheres with 0.5, 1.1, 1.7 and 1.9 μm in diameter were injected into the afferent lymph vessel. Two microgram of each particle was administered at one time and artificial lymph fluid was injected through the same route at constant rate of 1.47 ml/h. Then, changes in concentration of the microspheres in the efferent lymph vessel were determined by measuring the number of the particles under a fluorescent microscope. 11.0 ± 6.4% of the 0.5 μm-microsphere passed through the lymph node in 2.5 h. On the other hand, no microsphere with 1.9 μm in diameter was observed in the efferent lymph. The decreasing order of ability for accumulating in the lymph node was as follows: 1.9 μm > 1.7 μm >1.1 μm > 0.5 μm-microsphere. These results strongly suggest that size-dependent accumulating mechanisms exist in the lymph node. [J Physiol Sci. 2006;56 Suppl:S131]

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Micro- and nano-particles are known to be taken up into initial lymphatics. The particles have been used for efficient delivery of drugs and diagnostic agents to lymphatic system. Little information, however, exists regarding size-dependent mechanisms for the particle uptake. Thus, in this study, we have attempted to evaluate effects of size on uptake of the particles. Male Japan White rabbits were anesthetized with ketamine chloride (20 mg/kg iv.) and pentobarbital sodium (20 mg/kg iv.). Retrograde cannulation was performed in one of the popliteal afferent lymph vessels. Labeled latex with fluorescence microspheres with 0.5, 1.1, 2.0, 5.6 and 10.0 μm in diameter were injected subcutaneously at dorsal portion of rabbit foot. Then, concentrations of the microspheres in the efferent lymph vessel were determined by measuring the number of the particles under a fluorescent microscope. The decreasing order of particle concentration in efferent lymph was as follows: 0.5 μm > 1.1 μm > 2.0 μm-particle. No particle with 5.6 and 10.0 μm in diameter was observed in the efferent lymph. When mechanical massage was administered on the injection site, both lymph flow rate and particle concentration were markedly increased. These results strongly suggest that size- and mechanical stimulation-dependent mechanisms exist in transport of micro- and nano-particles from subcutaneous tissue into the lymphatic system. [J Physiol Sci. 2006;56 Suppl:S132]

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The lymphatic vessels and lymph nodes serve a one-way drainage system which return fluid and protein from the interstitial space to blood stream. Almost all of lymph passes through one or more lymph nodes in which water is exchanged freely between lymph and blood compartments. Little information, however, exists regarding lymphdynamics through the lymph nodes in vivo. Thus, in this study, we have attempted to evaluate pressure-flow relationships in rabbit leg lymphatic system and then estimate perfusion resistance through the lymph node. Male Japan White rabbits were anesthetized with ketamine chloride (20 mg/kg iv.) and pentobarbital sodium (20 mg/kg iv.). Retrograde cannulation was performed in one of the popliteal efferent lymph vessels at the groin. All other efferent lymphatics were ligated completely. Then, centripetal cannulation was performed in one of the popliteal afferent or efferent lymph vessels near to the lymph node. Lymph infusion pressure-outflow rate relationship was determined at the constant outflow pressure of -5 cmH₂O. Lymph flow rate increased monotonically with infusion pressure. Calculated resistance at infusion pressure of 25 cmH₂O was about 50 cmH₂O/ml/h with the lymph node and about 15 cmH₂O/ml/h without the lymph node. From these results, resistance of the lymph node was calculated as about 35 cmH₂O/ml/h. Effect of water exchange between lymph and blood compartments should be evaluated in future. [J Physiol Sci. 2006;56 Suppl:S132]

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In sitting position, combined effects of movement restriction and hydrostatic pressure difference between the heart and the lower limb might enhance venous pooling/stasis, and then enhance development of deep venous thrombosis. However, our understanding of the actual hemodynamic effects generated by sitting position in the venous circulation of the lower limb remains surprisingly incomplete. Accordingly, the purpose of the present study was to examine this and the effect of electrical muscle stimulation (EMS) on the popliteal venous flow was further examined. Fifteen healthy adult volunteers, none with a history of lower limb surgery or thromboembolism, were recruited. One lower limb was randomly selected for stimulation with the other serving as a control. The sitting position significantly decreased maximum velocity (Vmax) from 30.2 ± 5.7 to 6.5 ± 0.5 cm/s at 30 min and continuously suppressed for 120 min. In contrast to the Vmax, cross sectional area was significantly increased from 9.6 ± 1.2 to 53.1 ± 6.7 mm². Due to the combined effect of the decreased Vmax and the increased cross sectional area, the flow volume was not affected by the sitting position (from 116.8 ± 20.7 to 115.9 ± 21.1 ml/min). The Vmax of the EMS leg was significantly higher than that in the non-EMS leg throughout the 120 min sitting period. There was no difference in the cross sectional area between the EMS leg and the non-EMS leg, while the flow volume was significantly higher in the EMS leg compared in the non-EMS leg. Thus, lower limb venous stasis elicited by sitting position was improved by the EMS. [J Physiol Sci. 2006;56 Suppl:S132]

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One of the major functions of the lymphatic system is to return plasma proteins from the interstitial space to the blood stream. Previously, we have demonstrated that small molecular FITC-dextran are permeable from the intraluminal to extraluminal space of isolated lymph vessels and that the endothelial cell surface structure plays a barrier role in effective permeability of medium size of FITC-dexran through the wall of the lymph vessels (Am. J. Physiol. 289: H1676-H1682, 2005). In the present study, we further investigated the effects of trypsin, which is useful enzyme for removal of lymphatic endothelial cells in vitro, on effective permeability of large size of FITC-dextran (77 KDa) in isolated lymph vessels. Afferent lymph vessels were isolated from iliac lymph nodes of Wistar rats. The isolated lymph vessels were cannulated with glass-micropipettes and perfused with Krebs-bicarbonate solution with or without 77 KDa FITC-dextran. Changes in the intensity of 77 KDa FITC-dextran in an intraluminal space of the lymph vessels were measured by a video-microscope system and the concentration of 77 KDa FITC-dextran were calculated before and after treatment with trypsin. 77 KDa FITC-dextran did not permeate the wall of lymph vessels before and after the treatment with trypsin. These results suggest that the non-endothelial wall structure of lymph vessels may be a barrier for effective permeability of a large size of FITC-dextran in vitro. [J Physiol Sci. 2006;56 Suppl:S132]

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One of the important functions of lymph vessels and lymph nodes is to return plasma proteins from the interstitial space in tissues. There exists few or no report, except for one (Ono, et al: AJP H1676-H1682, 2005), regarding for evaluation of hydrophilic permeability through the walls of lymph vessels. No experiment in vitro model using cultured lymphatic endothelial cells has been reported to elucidate macromolecular permeability through the cell layer. Therefore, we have attempted to develop firstly in vitro assay system to study hydrophilic permeability and then investigated the effects of TNF-α.Rat lymphatic endothelial cells cultured from the thoracic ducts or afferent lymphatic vessels were seeded onto the collagen-coated inserts of double chambers in-vitro vascular permeability assay kit (CHENICON). The cell monolayers were treated with or without TNF-α (10ng/ml) over night, and then fluorescent-labeled dextrans (4, 12, and 77KD) were added on the top of the cells coated on upper chamber to evaluate the hydrophilic permeability. The extent of permeability was determined by measuring fluorescent activity of the solution dropped down the lower chamber. The pretreatment with TNF-α caused a significant increase of the permeability of all FITC-dextran through the cultured endothelial cell layer.These findings suggest that the in vitro assay system may be suitable for hydrophilic permeability through cultured lymphatic endothelial cell layer. Inflammatory cytokine, TNF-α, caused a significant increase of permeability with FITC-dextran. [J Physiol Sci. 2006;56 Suppl:S133]

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According to Windkessel theory, diastolic pressure waveform is a function of the total peripheral resistance and the compliance of the artery. Higher compliance makes the decrescence gentler, and the waveform would be further different from the input pressure waveform made by the cardiac contraction. Frequency analysis of the arterial pressure waveform shows fundamental frequency (F) derived from the heart rate and harmonic frequency which is the integral multiple of the F. In the present study, we measured abdominal aortic pressure, abdominal aortic flow, and the diameter during infusion of Nitroprusside with the speed of 1, 0.6, 0.3 μg/kg/h. The compliance was calculated from the pulse pressure and volume change calculated by the diameter and the length of the aorta. The compliance was also calculated with first order of the Windkessel theory. High frequency centroid (HFC), which is amplitude-weighted mean frequency of the third to seventh peaks of the harmonic waveform was analyzed. HFC was normalized by F. Total harmonic distortion (THD), which is an index of distortion from a pure sine wave was also analyzed. Measured compliance and the calculated compliance of the aorta were significantly correlated. Normalized HFC and both measured compliance and the calculated compliance also showed significant linear correlation. THD did not show significant correlation between the compliance. These results suggest HFC can be an index of the compliance or the aorta, and the compliance may be analyzed from the aortic pressure waveform only. [J Physiol Sci. 2006;56 Suppl:S133]

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August Krogh first reported that the local application of vasodilators could stimulate extensive vasodilatation which spreads rapidly to distant sites. Conducted responses of this type are referred to as spreading dilatation, a response which cannot be explained simply on the basis of diffusion of the locally applied dilator agent. The present study was designed to test the hypothesis that local stimulation of hyperpolarization can be conducted longitudinally over significant distances to spread dilatation in small mesenteric arteries. A segment of mouse mesenteric artery was cannulated at each end with a glass pipette and then pressurized. In the presence of the NO synthase inhibitor, L-NAME, focal application of the PAR2 agonist, SLIGRL induced dilatation at the site of application. This local dilatation was associated with simultaneous dilatation along the entire artery segment. Measurement of smooth muscle membrane potential revealed local hyperpolarization to SLIGRL which was also observed at distant sites. In contrast, focal application of forskolin did not induced any hyperpolarization, and although it evoked local dilatation, this was not associated with any coordinated dilatation along the isolated mesenteric artery. These results indicate that spreading dilatation responses in mesenteric resistance arteries are only evoked with vasodilators which hyperpolarize the artery. [J Physiol Sci. 2006;56 Suppl:S133]

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Previous studies from our laboratory demonstrated that the vestibular system has a significant role in controlling arterial pressure during gravitational stress. The vestibular system is thought to be stimulated not only by gravitational change but also by postural change. Thus, the vestibular system might have a significant role in controlling arterial pressure during postural change. To examine this, arterial pressure was measured during 60°head-up tilt in conscious rats with or without intact vestibular system. Rats were divided into 3 groups: intact, sinoaortic denervation (SAD), and SAD+vestibular lesion (VL). The posture change did not alter arterial pressure in intact group. In SAD group, however, arterial pressure was increased by the posture change. This increase in arterial pressure was completely abolished by additional VL. Thus, the vestibular system has a significant role in an increase in arterial pressure during posture change in SAD group. The increased arterial pressure induced by the vestibular system is buffered by baroreflex, thus arterial pressure is well maintained during posture change in intact group. [J Physiol Sci. 2006;56 Suppl:S133]

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We investigated effect of deep ocean water on hemodynamics in Kurosawa and Kusanagi-Hypercholesterolemic (KHC) rabbits. The refined deep ocean water (RDOW) at a degree of hardness of 1,000 was freely accessed to the KHC rabbits aged 4 months old for 6 months. Aortic pressure and flow waves were simultaneously recorded under pentobarbital anesthesia. There were no significant differences in body weight and food and water intakes. Systolic, diastolic, mean and pulse pressures, augmentation index of pressure waves and total peripheral vascular resistance were significantly lower in the RDOW group than in the control group, whereas no significant differences were observed in mean aortic flow and total percent lesioned area of the aorta between the two groups. Though serum lipid levels, plasma renin and ACE activities and angiotensin 1 and 2 levels tended to decrease after the feeding of RDOW in the two groups, the difference in these parameters before and after the feeding of RDOW was almost the same between the two groups. Serum electrolyte levels except Mg²⁺ changed little during the intake of RDOW. We can conclude that RDOW improved mild hypertension in KHC rabbits, which might be partly related to the slight but significant increase in serum Mg²⁺ due to the intake of RDOW. [J Physiol Sci. 2006;56 Suppl:S134]

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The ability to genetically engineer specific gene knock-out mice has provided a powerful tool for investigating the pathogenesis of pulmonary arterial hypertension (PAH). Yet, there have been no reports describing the measurement of pulmonary arterial pressure (PAP) in conscious mice; an essential requirement for monitoring dynamic changes associated with PAH. In this study we describe a new technique for long-term measurement of PAP in conscious mice using telemetry. In five male C57BL/6 mice (B.W. 25-30 g) the sensing catheter of a telemetric transmitter was inserted into the right ventricle and advanced into the pulmonary artery. The transmitter body was positioned within the abdominal cavity or subcutaneously on the back. During recovery from surgery mean PAP was recorded daily for one week. Subsequently, the PAP responses to acute hypoxia (8% O₂ for 10 min) and L-NAME (50 mg/kg, s.c.) were tested in three mice. By one-week post surgery, all mice had fully recovered from surgery and baseline MPAP was stable at 14.9 ± 0.7 mmHg. Additionally, acute hypoxia and L-NAME provoked a 63% and 86% increase MPAP, respectively. In summary, this study has demonstrated the ability to accurately measure PAP by telemetry in conscious mice. One important application of this technique for future studies is the possibility to assess the contribution of specific genes (i.e. knock-out mice) for the development of pulmonary pathological conditions. [J Physiol Sci. 2006;56 Suppl:S134]

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We determined the changes in hepatic sinusoidal pressure and liver weight, and the effects of a NO synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME) in ischemia-reperfusion (I/R) injury of isolated mouse livers perfused portally with diluted blood (Hct 3%). Following pretreatment with L-NAME (100 μM) or D-NAME (100 μM), Ischemia was induced at room temperature by occlusion of the inflow line of the portal vein for 1 hour, followed by 1-hour reperfusion in a recirculating manner. The sinusoidal pressure was assessed by the double vascular occlusion pressure (Pdo). In the D-NAME group, immediately after reperfusion, the portal pressure increased by 2.8±0.1(SE) mmHg, which was accompanied by an increase in Pdo by 1.5±0.1 mmHg, suggesting increases in pre- and post-sinusoidal resistances in a similar degree. Liver weight increased 0.14±0.04 g after reperfusion followed by a gradual return towards baseline. Liver injury, assessed by perfusate levels of hepatic enzymes was observed at 60 min after reperfusion. There were no significant differences in changes in any variables between the D- and L-NAME groups. In conclusion, the increases in both the hepatic vascular resistances and the sinusoidal pressure were small in magnitude, resulting in absence of edematous changes in mouse hepatic I/R, and nitric oxide does not play any significant roles in this injury. [J Physiol Sci. 2006;56 Suppl:S134]

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We determined the roles of liver and splanchnic vascular bed in anaphylactic hypotension in anesthetized mice. In anesthetized mice sensitized with ovalbumin (0.01 mg), an intravenous injection of 0.01 mg ovalbumin caused not only a decrease in systemic arterial pressure (Psa) from 92±1.6 (SE) to 39±2.6 mmHg but also an increase in portal venous pressure (Ppv) which persisted for 40 min after the antigen injection. The elimination of the splanchnic vascular beds, by the occlusions of the celiac and mesenteric arteries, combined with total hepatectomy attenuated anaphylactic hypotension. In addition, a head-down tilt maneuver, which could facilitate venous return in the splanchnic organs, significantly attenuated the decrease in Psa induced by an antigen injection. These results suggest that liver and splanchnic vascular beds are involved in anaphylactic hypotension presumably due to anaphylactic contraction-induced portal hypertension, which induced splanchnic congestion resulting in a decrease in circulating blood volume and thus systemic arterial hypotension. [J Physiol Sci. 2006;56 Suppl:S134]

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Effects of a NO synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), on anaphylaxis-induced venoconstriction were examined in isolated rat liver perfused with blood of various hematocrit (Hct) to clarify the role of nitric oxide (NO) in anaphylactic venoconstriction in rat livers.The rats were sensitized with ovalbumin (1 mg), and two weeks later, the liver was excised and perfused portally and recirculatingly at a constant flow with blood at Hct of 0, 5, 16, and 22%. We measured the viscosity of perfusing blood. Using the double occlusion technique to estimate the hepatic sinusoidal pressure (Pdo), presinusoidal resistance (Rpre) and postsinusoidal resistance (Rhv) were calculated. The antigen, ovalbumin (0.1 mg), was injected into the reservoir 10 min after pretreatment with L-NAME (100 μM) or D-NAME (100 μM). The viscosity, a determinant of vascular resistance and shear-stress, increased in a Hct-dependent manner. L-NAME pretreatment increased exclusively basal Rpre in liver perfused at Hct 22%. The antigen caused hepatic venoconstriction as characterized by predominant presinusoidal constriction in all antigen administered livers. L-NAME pretreatment potentiated the antigen-induced venoconstriction, as compared with the D-NAME pretreatment, by increasing Rpre, but not Rpost. These finding suggests that hepatic anaphylaxis increased production of NO, which consequently attenuated anaphylactic presinusoidal venoconstriction in isolated perfused rat livers. [J Physiol Sci. 2006;56 Suppl:S135]

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Objective Ventriculoseptal defect;VSD and atrial septal defect;ASD exhibit increased pulmonary blood flow through the left-to-right shunt. In patients with these pathological states arising from volume and/or pressure overload in the right heart can result in sympathovagal imbalance. We investigated the correlation between the HRV and respiratory frequency, and shunt ratio (Qp/Qs) measured by the Doppler echocardiography.Subjective and methodsWe enrolled 66 patients. Diagnoses were: VSD (n=21) and ASD (n=45). Mean age was 5.6 ± 3.3 years. ECG and respiratory waves were recorded during the Doppler UCG test. We performed HRV analysis to calculate LF, HF, TF, respiratory frequency (RF), LF/HF, RF/TF, and LF/RF. Qp/Qs was also calculated and quantified by multivariate analysis with HRV parameters. Results1) Qp/Qs and LF/HF exhibited correlation in both ASD and VSD groups. 2) The ASD group showed a positive correlation between Qp/Qs and LF/RF. However, in the VSD group the correlation was negative. 3) Qp/Qs correlated negatively with RF/TF in the ASD group, however, the correlation was positive in the VSD group.ConclusionsASD and VSD showed different effects of respiratory vagal activity on HRV. We believe that a reason is that volume and low pressure overload to the right atrium in ASD inhibits respiratory vagal innervation of the sinus node. [J Physiol Sci. 2006;56 Suppl:S135]

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Carbon monoxide is formed in the process of degradating heme from biliverdin by heme oxygenase (HO) in the various tissues, including central nervous system (CNS). Previous studies suggested that inhibition of HO activity increased arterial pressure (AP) mediated by the autonomic nervous system. The present study was designed to investigate the sympathetic nerve response to inhibition of HO activity by a direct renal nerve recording in conscious rats. Zinc deuteroporphyrin 2, 4-bis glycol (ZnDPBG), an inhibitor of HO activity, was administered ip in chronically instrumented Wistar rats: 8 intact, 8 atropine-treated, and 7 sinoaortic denervated (SAD). ZnDPBG induced significant increases in mean AP (MAP) from 95.9 ± 1.6 to 116.9 ± 4.7 mmHg and renal sympathetic nerve activity (RSNA) from 100.0 to 186.7 ± 19.2%, but no significant change in heart rate (HR) in intact rats. In atropine-treated rats, ZnDPBG also induced significant increases in MAP from 96.7 ± 1.5 to 110.6 ± 2.0 mmHg and RSNA from 100.0 to 184.7 ± 22.5%, but induced no change in HR. In SAD rats, ZnDPBG induced significant increases in MAP from 91.4 ± 5.7 to 118.8 ± 8.5 mmHg, HR from 356.8 ± 10.4 to 409.1 ± 14.4 beats/min, and RSNA from 100.0 to 211.6 ± 18.9%. The present study suggested that inhibition of HO activity caused sympatho-excitation via a direct action on CNS, resulted in an increase in AP. Further study is required to investigate the underlying mechanism of HR response to HO inhibition. [J Physiol Sci. 2006;56 Suppl:S135]

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It has been proven that noxious articular stimulation of hindlimb produce reflex increase in heart rate, in anesthetized animals, to be a reflex response whose reflex center is in the brain and efferent arc is a cardiac sympathetic nerve. Using central nervous system (CNS)-intact and acutely spinalized anesthetized rats, the present study aimed to examine the possibility of whether articular stimulation could influence heart rate via sympathetic nerve at the spinal level. In CNS-intact rats, noxious articular movement of both knee and elbow joint produced an increase in the cardiac sympathetic nerve activity and heart rate. In acutely spinalized rats, noxious movement of elbow joint produced large increases in the cardiac sympathetic nerve activity and heart rate, while noxious movement of knee joint induced no increase in the cardiac sympathetic nerve activity and only a marginal increase in heart rate. As the marginal heart rate response following knee joint stimulation in spinalized rats was abolished after adrenalectomy, the responses were suggested to be induced by catecholamine secreted from the adrenal grand. It is concluded that the spinal cord is capable of producing propriospinally the reflex increases in heart rate via reflex activation of the cardiac sympathetic efferent nerve following stimulation to the elbow joint stimulation whose afferent information enters the spinal cord at the same segments or segments overlapping the cardiac sympathetic outflow. [J Physiol Sci. 2006;56 Suppl:S135]

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Previous studies have reported that carotid arterial compliance increased by habitual exercise, so that the cardiovagal baroreflex sensitivity increased. This concept is based on the anatomical fact that the arterial baroreceptor is a stretch sensitive receptor, a part of which is located in the carotid sinus. However, we previously reported that neural component of baroreflex sensitivity estimated by R-R interval corresponding end-systolic lumen diameter was greater in physically active young men than that in sedentary, but mechanical component of baroreflex sensitivity estimated by end-systolic lumen diameter corresponding systolic blood pressure was not different between active and sedentary group. We hypothesized that short term exercise training increase arterial baroreflex sensitivity due to the neural alteration but not increase arterial compliance because alteration in neural property will occur faster than that in mechanical property of blood vessel wall. To examine this hypothesis, we estimated baroreflex sensitivity and carotid arterial compliance in elderly subjects before exercise training, and at 2, 6, and 12 weeks after training. Arterial baroreflex sensitivity increased at 2 weeks after training but arterial compliance did not increase at this time point. This result suggests that arterial baroreflex sensitivity increase by short term exercise training, which is probably due to alteration in "neural" arc of the arterial baroreflex. [J Physiol Sci. 2006;56 Suppl:S136]

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Morphological characteristics in the carotid bodies of spontaneously hypertensive rats (SHR) and those of age-matched normotensive Wistar rats (NWR) as well as age-matched genetically comparable Wistar rats (WKY) were examined. The distribution and abundance of four different regulatory neuropeptides, substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in the carotid bodies of three strains of rat were also examined. The carotid bodies of SHR were greater than those of NWR and WKY. The values in the long axis of the carotid bodies of SHR were significantly 1.3 times larger than those of NWR and WKY. In the carotid bodies of SHR, the percentage of relatively large vessels were similar to that in the carotid bodies of WKY, although the carotid bodies themselves were significantly larger than in WKY. In the carotid bodies of NWR and WKY, the density of NPY-immunoreactive varicose fibers were more numerous than that of VIP, SP, and CGRP fibers. These immunoreactive fibers were mainly associated with the vasculature and the clusters of glomus cells. The density of VIP varicose fibers in the carotid bodies of SHR were smaller than that in the carotid bodies of WKY, although that of SP, CGRP, and NPY fibers was similar to that in the carotid bodies of NWR and WKY. The present results suggest that the mechanisms of carotid body enlargement in hypertensive rats are different from those in hypoxic rats. [J Physiol Sci. 2006;56 Suppl:S136]

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The area around the parabrachial nucleus (PB) including the Kolliker-Fuse (KF) nucles is deeply involved in respiratory control, but not enough information is available about the respiratory neurons of this area. In Nembutal-anesthetized, paralyzed, and artificially ventilated rats with intact vagal nerves, we recorded from more than 300 respiratory neurons in the PB and KF areas. The respiratory neurons were densely distributed in the KF nucleus and sparsely in the medial parabrachial nucleus (PBM) and its vicinity. Only few neurons were purely inspiratory (I) and most "inspiratory" neurons had more or less expiratory (E) activity, exhibiting the property of E-I phase-spanning neurons. By far the less number of I-E phase-spanning neurons were recorded, and their firing was labile and weak. The E neurons exhibited augmenting, decrementing or whole-phase expiratory firing patterns. Activity of the respiratory neurons was variously modulated by lung volume, but we could not find non-respiratory neurons whose activity was modulated thoroughly by lung volume. The majority of the respiratory neurons examined by antidromic stimulation had medullary axons, some having axons descending the midline medulla toward the spinal cord. Many E and "inspiratory" neurons projected to the nucleus tractus solitarii (NTS). Some "inspiratory" neurons projected to the hypoglossal nucleus. It was suggested that PB and KF neurons project to motor output nuclei, such as the nucleus ambiguus and the hypoglossal nucleus, as well as to the rhythm-related structures, such as the Botzinger complex and the NTS. [J Physiol Sci. 2006;56 Suppl:S136]

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The effects of the circadian light/dark cycle on ventilatory responses to chemical stimuli have rarely been studied in experimental animals, despite evidence that the cycle may be a factor in respiratory results. We measured the ventilatory response to hypoxia (HVR) in unanesthetized wild-type and histamine type-1 receptors knockout (H1RKO) mice in the light and dark periods with a whole-body, single-chamber plethysmograph. Animals were subjected to a 10-min hypoxic exposure (7% O₂ and 3% CO₂ in N₂) after acclimatization to the chamber for 90 min. In both groups of mice, minute ventilation increased in response to the hypoxia and declined gradually after the peak response regardless of when HVR was determined. However, we found differences in the HVR between wild-type and H1RKO mice. In wild-type mice, the minute ventilation response was higher in the dark period than in the light period, which was due to differences in the tidal volume response rather than the respiratory frequency response. Conversely, in H1RKO mice, minute ventilation responses did not differ between the two periods, which were similar to the response of wild-type mice determined in the dark period. In summary, the circadian light/dark cycle altered the HVR in wild-type mice, whereas, in H1RKO mice, the cycle difference in the HVR disappeared. These results suggest that H1R contributes to the circadian light/dark cycle differences in the HVR in unanesthetized mice. [J Physiol Sci. 2006;56 Suppl:S137]

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Breathing activity during sleep is closely related to sleep stage. Indeed, breathing rate (BR) during rapid eye movement (REM) sleep show much larger fluctuation than that during deep sleep. In the present study, breathing movement of freely moving C57B6/J mice were analyzed during sleep by piezoelectric device placed on the floor of the cage in a non-invasive manner (PCT/2003/001109), while animal behavior was monitored by an infrared camera. We found that mice momentarily exhibited an extraordinary large rise in BR by up to 14 breaths/sec during sleep. Such rise in BR was detected in all four mice tested, especially after atonia and shortly before awakening (mean BR = 10.5±1.2 breaths/sec, which is 4.3±1.3 times the BR in stable deep sleep; mean duration = 0.4±0.1 sec). In humans, interestingly, a similar momentary increase in BR by up to 116 breaths/min (2.4-3.7 times the BR in deep sleep) was detected during REM sleep in normal volunteers tested. No such increase was detected in stages 3 and 4. In addition, more prolonged increase in BR also was observed during REM/light sleep in human subjects. Further studies may clarify the correlation between the central regulation mechanisms of respiration and sleep stages. [J Physiol Sci. 2006;56 Suppl:S137]

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Respiratory modulation of the heart rate (i.e.,respiratory sinus arrhythmia:RSA) and phase synchronization of respiratory and cardiac rhythms are manifestation of cardiorespiratory coordination. However, the question of whether they represent two competing entities of interaction remains unresolved. The present study was designed to characterize the synchronization and modulation between patterns of breathing and RSA during normoxia and hypoxia under resting condition. For this purpose, we recorded ventilation and electrocardiogram in 9 subjects while breathing was paced either 6,8,10, or 12 breaths/min for 5 min each. The instantaneous phases were calculated for RSA curve and respiratory patterns using Hilbert transform and then relative phase differences were obtained. The cardiorespiratory synchronization and modulation were quantified by the index based on conditional probability of the phase difference and by the amplitude of RSA power calculated using a fast Fourier transform, respectively. The synchronization index decreased significantly at 12 breath/min compared with that observed at lower breathing frequency, while the modulation index (i.e., RSA power) decreased almost linearly with increasing breathing frequency. There was a significant positive correlation between the indexes of the synchronization and modulation (p<0.01). The observed relation was not altered by hypoxia, suggesting that the coordination may not depend on functional modulations. Our analysis indicates that the cardiorespiratory synchronization can coexist with the modulation. [J Physiol Sci. 2006;56 Suppl:S137]

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Numerous studies have suggested that caudal raphe nuclei may play some roles in respiration. However, details of the role of the raphe nuclei in controlling of respiration have not been clarified. We have previously reported that respiratory neurons are present in medullary raphe nuclei. The present study was undertaken to examine the effects of hypercapnia on the respiratory neurons of medullary raphe nuclei. The experiments were performed on decerebrate, paralyzed, vagotomized and artificial ventirated rat. Extracellular recordings were made from neurons having firing patterns related to phrenic nerve discharge in the midline medullary tegmentum. We recorded mainly Inspiratory (I) throughout neurons which firerd throughout the I phase in the raphe obscurus and pallidus. They were tested changes in neuronal discharges when the ventilator was stopped (during 8-24 sec; end-tidal CO2 levels were raised from 5±0.4 to 8±0.3%). Most of the neurons examined responded to hypercapnea in the neuronal discharge. But some neurons examined did not respond. These results suggest that the midline caudal raphe nuclei are involved in central chemoreception. [J Physiol Sci. 2006;56 Suppl:S137]

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We have studied the fine anatomical architecture of the central respiratory chemoreceptor using electrophysiological, optical and histological techniques. We have identified medullary chemosensitive sites in the surface parts of the rostral ventrolateral medulla, raphe pallidus and parapyramidal regions. Pontine regions (locus coeruleus and A5 region) were also chemosensitive. We found two types of CO2-excitable cells (Type I and II cells) in the superficial ventral medulla. A Type I cell is smaller in size, located within the marginal glial layer, and intrinsically CO2-excitable. A cluster of Type I cells surround fine penetrating vessels, and a large surface vessel covers this region. A Type II cell is larger, located at a depth of a few hundred micrometer from the medullary surface, and excited transsynaptically by CO2. A single Type II cell forms dendro-somatic synapses with multiple Type I cells within the marginal glial layer, and sends axonal branches to multiple neurons of the ventral respiratory group (VRG). We propose a cell-vessel architecture model for the central respiratory chemoreceptor. Type I cells are primary chemosensors. In response to increased CO2 in perivascular tissue, Type I cells secrete neurotransmitter, and excite Type II cells. Excited Type II cells relay and amplify this information by innervating multiple VRG neurons. [J Physiol Sci. 2006;56 Suppl:S138]

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We have reported that serotonin (5-HT) type 2 receptors in the dorsomedial medulla oblongata (DMM) influence ventilation and airway resistance in mice. In the present study, the role of 5-HT2 receptors in the DMM in respiration to hypoxia was investigated. Each male mouse was anesthetized with pentobarbital sodium i.p. for insertion of a microdialysis probe into the DMM. The mouse was placed into a double chamber plethysmograph to obtain two respiratory flow curves to calculate respiratory variables, while extracellular fluid was collected at 1.2 microL/minute every 5 minutes. Extracellular fluid via the microdialysis probe was analyzed with an ECD-HPLC. Artificial cerebrospinal fluid (aCSF) or 1 x 10⁻⁵M LY-53857 (a 5-HT2 receptor antagonist) was perfused via the microdialysis probe in the DMM with air or hypoxic gas (7% O₂ in N₂) inhalation. Changes in respiratory variables and the 5-HT concentration in the DMM were analyzed every 5 minutes. Respiratory variables during hypoxia in a LY-53857-perfused group were not different from those in an aCSF-perfused group. Post-hypoxia frequency in the LY-53857-perfused group was decreased compared to that in the aCSF-perfused group, while the 5-HT concentration in the DMM was significantly increased under hypoxia in both the aCSF-perfused group and the LY-53857-perfussed group. We discuss whether or not the post-hypoxia frequency decline with LY-53857 perfusion in the DMM is a result of respiratory hypoxic excitation with a more detailed analysis of respiration variables. [J Physiol Sci. 2006;56 Suppl:S138]

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We investigated electrophysiological and morphological characteristics of GABAergic neurons in the pre-Boetzinger complex (PBC, the caudal part of the rostral ventrolateral medulla), the putative kernel site for respiratory rhythm generation. We used GAD67-GFP knock-in neonatal mice, which enabled us to identify GABAergic neurons in a living condition. We recorded respiratory activity from the hypoglossal nerve in medullary transverse slices that contained the PBC and whole-cell recorded activities of GFP-positive neurons in the PBC. Six out of 32 GFP-positive neurons were inspiratory. All of the remaining neurons were non-respiratory. In addition, 6 GFP-negative inspiratory neurons were recorded in the PBC. The electrophysiological properties of GFP-positive inspiratory neurons included high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. The somata of GFP-positive inspiratory neurons were smaller than those of GFP-negative inspiratory neurons. These results indicate that GABAergic inhibition by inspiratory neurons with particular properties in the PBC is involved in neural respiratory control. [J Physiol Sci. 2006;56 Suppl:S138]

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Here we report that unique 'switching' roles of dopamine in regulating the respiratory rhythm generation. We employed medulla-spinal cord block preparation which contains intact respiratory rhythm generator. In this preparation, respiratory rhythm generator consists mainly Pre-Inspiratory (Pre-I) neurons and inspiratory (I) neurons. I neurons are pre-motor neurons and Pre-I neurons trigger I neuronal firing as a pacemaker. Optical imaging with voltage-sensitive dye revealed that application of dopamine selectively disrupted phasic Pre-I neuronal firing and slightly enhanced I neuronal firing. This dopaminergic effect for Pre-I neuron was mimicked by dopamine D4 receptor agonist, PD168077 application and the enhanced effect of I neuronal firing was mediated with D1 receptor. Dopamine depolarized Pre-I neuronal membrane potential significantly but not on I neurons under TTX perfusion. Finally, dopamine depressed I neuronal PSPs which are probably originated from Pre-I neurons. Dopamine selectively disrupts synchronized phasic Pre-I neuronal firing through dopamine D4 receptor, and enhances I neuronal network through dopamine D1 receptor. Dopamine has opposite effects for respiratory network though different type of dopamine receptors. It seems to be the switching involuntary respiration to voluntary respiration. [J Physiol Sci. 2006;56 Suppl:S138]

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Ventilation changes depending on circadian light-dark cycle. Histaminergic neurons are involved in the circadian regulation in the brain, but a role of circadian pattern of ventilation is unknown. We examined ventilation and metabolism for 24 h in histamine type 1 receptor knockout (H1RKO) and wild-type mice. Mice were measured for ventilation by a whole-body plethysmograph and metabolism by a magnetic-type mass spectrometry, and consciousness was characterized by electroencephalogram. In both genotypes, minute ventilation (V_E) and metabolic rate were greater during the dark period (20:00 to 8:00) than during the light period (8:00 to 20:00). H1RKO mice during the light period were greater in V_E with increased tidal volume and greater in VCO₂ without changes in VO₂ than wild-type mice. The increased V_E during the light period was responsible for the increased CO₂ production with an identical ratio of V_E to VCO₂. However, H1RKO mice decreased the ratio compared to that of wild-type mice during the dark period, which was responsible for an increase of delta/theta ratio calculated from power spectrum density of electroencephalogram. Thus, deletion of H1 receptor affects patterns of ventilation accompanied with changes in metabolic substrates and arousal state of the dark period. Results suggest that histamine modulates ventilatory pattern during the light and the dark periods via H1 receptors. [J Physiol Sci. 2006;56 Suppl:S139]

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Habutobin, thrombin-like enzyme that converts rabbit fibrinogen to fibrin, was purified from T. flavoviridis . Habutobin bound to β₃ subunit of integrin α_IIbβ₃, and then habutobin inhibited the collagen-induced aggregation, in previously study. We investigated the effect of habutobin for intracellular signaling through tyrosine(Tyr)-phosphorylation of protein occurring in the collagen-induced aggregation. Rabbit platelet was washed with ACD-A and HEPES-Tyrode's sol (Ca²⁺, Mg²⁺ free) and the washed platelet was suspended in the Tyrode's sol containing Mg²⁺ (1 mM). From results of Western blot with anti-phosphotyrosine antibody(4G10), Tyr-phosphorylation of several intracellular proteins increased dramatically during collagen-induced aggregation. Tyr-phosphorylated proteins were observed in 55-60, 72, 85 and 116 kD at 90 sec after the addition of collagen. In the presence of habutobin, although Tyr-phosphorylated proteins was not observed at 90 sec after the addition of collagen, they were observed in 60, 72 and 116 kD at 3 min after the addition of collagen. In addition, in the presence of habutobin, FAK Tyr-phosphorylation was inhibited at an early stage of collagen-induced aggregation, but it was not inhibited at a late stage of collagen-induced aggregation. From the present study, it was suggested that the binding of habutobin to β₃ subunit of integrin α_IIbβ₃ resulted to inhibit FAK Tyr-phosphorylation, and habutobin inhibits the β₃ signaling, that is, outside-in signaling. [J Physiol Sci. 2006;56 Suppl:S139]

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The subfornical organ (SFO) plays important roles for drinking behavior and cardiovascular regulation. It is well known that excitation of SFO neurons by angiotensin II (ANG) induces the two behavioral responses, but that by nicotine (NIC) induces only cardiovascular responses. Therefore, there may be present subpopulations of SFO neurons showing different sensitivity between ANG and NIC. In the present study, we verified that intracerebroventricular (i.c.v.) injections of ANG at 400 ng induced large water intake in rats, but that of NIC at 50 μg induced small water intake. The i.c.v. injection of NIC induced cardiovascular responses increased c-Fos expression in the SFO. In slice preparation, we compared with the responses of ANG and NIC on 101 SFO neurons by using an extracellular recording system. ANG and NIC respectively increased spontaneous firing frequencies in 67 and 62 SFO neurons. Although 45 ANG-sensitive neurons had NIC-sensitivity, the remaining 22 neurons did not. Recently, we have reported cell classification of dissociated SFO neurons, F- and S-types, and that a half of F-type and all S-type neurons have ANG-sensitivity. A patch-clamp recording showed that although almost F-type had NIC-sensitivity (n = 19/21), S-type did not (n = 7/8). From these results, we hypothesize that classified S-type cells have ANG-sensitivity without NIC-sensitivity and are related to drinking behavior, but not to cardiovascular responses, and their function is different from that of F-type cells. [J Physiol Sci. 2006;56 Suppl:S140]

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Kidney macula densa cells sense the chloride concentration of the fluid in the adjacent lumen and control glomerular filtration rate. We have used a functionally intact macula densa cell line (NE-MD) established from immortalized renal cells in culture. NE-MD cells specifically express neuronal nitric oxide synthase (nNOS) regulated by either low NaCl intake or furosemide (an inhibitor of Na⁺-K⁺-2Cl⁻ symporter). We have examined whether L-arginine-induced NO production is pH dependent or not in NE-MD cells, by using a NO-sensitive electrode. The NO production was low in control, but increased when NE-MD cells were treated with furosemide (12 µM) for 2 hrs. When furosemide-treated NE-MD cells were incubated in the presence of 100 μM amiloride (an inhibitor of Na⁺/H⁺ exchanger), L-arginine-induced NO production was unaffected. However, the NO production significantly decreased by 42% when the cells were placed in a solution containing amiloride. Similar results were obtained when NE-MD cells were incubated in the low pH solution (pH=7.1). These results strongly suggest that furosemide-induced expression of nNOS protein is not sensitive to acidosis, but its activity is pH sensitive. This may partly account for polyuria in abnormalities of electrolyte and acid-base balance. [J Physiol Sci. 2006;56 Suppl:S140]

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We investigated the effect of basolateral CO₂ on the cytosolic pH (pH_i) regulation in the proximal tubule of perfused bullfrog kidney by using H⁺-selective microelectrode. Furthermore, in sliced kidney, we monitored the changes in acridine orange fluorescence in acid vesicles of proximal tubule cells during the elevation of basolateral CO₂ with or without H⁺-pump inhibitors. Elevating baolateral CO₂ from 1.5 to 5% at constant HCO₃⁻ concentration induced an initial slight decrease followed by a sustained increase in pH_i with 10 mV hyperpolarization of basolateral membrane. In the presence of 10⁻⁶ M bafilomycin A1 (BAFA) or 10⁻⁷ M concanamycin A (CNCA), a specific H⁺-pump inhibitor, elevating basolateral CO₂ produced no increase in pH_i, but a decrease in pH_i with a depolarization of basolateral membrane. The increase in acridine orange intensity in acid vesicles was inhibited by the perfusion of 10⁻⁶ M BAFA during the elevation of CO₂ in the perfusion fluid. The peritubular perfusion of 10⁻⁶ M BAFA suppressed the alkalinaization of pH_i with 20 mM NH₃/NH₄⁺ application to peritubular perfusion fluid. These results indicate that intracellular H⁺ transport in the acid vesicles as well as Na⁺/H⁺ exchanger or Na⁺-HCO₃⁻ cotransporter in the cell membrane play an important role of the pH_i regulation in the proximal tubule of bullfrog kidney. [J Physiol Sci. 2006;56 Suppl:S140]

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Activation by angiontensin II (ANGII) of subfornical organ (SFO) neurons induces water intake and vasopressin release. On the other hand, central and peripheral administrations of galanin (GAL) have been reported to inhibit water intake and vasopressin release in recent in vivo studies. We anatomically, molecularbiologically and electrophysiologically investigated existences of GAL-receptors, and GAL-induced responses in SFO neurons of rats. Immuno-electron microscopic observation revealed existence of GAL-containing synaptic vesicles in the SFO. Conventional RT-PCR analysis demonstrated that there were present all mRNAs of three known GAL receptor subtypes, GALR1, GALR2 and GALR3, in the SFO tissues. In the extracellular recordings, application of GAL inhibited spontaneous firing rate in more than half of SFO neurons, even after synaptic blockade by a low [Ca²⁺] and high [Mg²⁺] solution. The inhibitory responses were dose-dependent. Seventy-two percent of GAL-sensitive neurons were also responsive to ANGII. ANGII increased the firing rate. These results suggest that GAL functions as an inhibitory neurotransmitter or neuromodulater in the SFO. Furthermore, the electrophysiological results offer a plausible explanation for the in vivo-observed opposite effects of ANGII and GAL on water intake and vasopressin release. [J Physiol Sci. 2006;56 Suppl:S141]

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Ion channels in the apical membrane of cultured mouse renal collecting tubule cells were investigated using the patch-clamp technique. At least two types of ion channels were observed with cell-attached patches under control conditions. One was an inwardly rectifying small conductance (20-30 pS) K channel (SK channel) and the other was an intermediate conductance (40-50 pS) non-selective cation channel (NC channel). Activity of the SK channel was relatively high, whereas that of NC channels was usually low in the control condition. Addition of membrane-permeant cAMP analogue, 8Br-cAMP (100 μM), stimulated the activity of SK channels but did not affect that of NC channels in inside-out patches. In the presence of 1mM Ca²⁺, addition of Ca²⁺-ionophore, ionomycin (1 μM), resulted in marked enhancement of NC channel activity and reduction of SK channel activity. In inside-out patches, SK channels required cytosolic ATP (1 mM) to maintain their activity, which was not affected by cytosolic Ca²⁺. On the other hand, NC channel was activated directly by elevation of cytosolic Ca²⁺, but not by ATP in inside-out patches. These results suggest that regulatory mechanisms of these channels are quite different, which may play a role in the electrolyte transport along the collecting duct under various conditions. [J Physiol Sci. 2006;56 Suppl:S141]

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