The high concentration of Mg (high Mg) during hypoxia shows the protective effects on the cardiac hypoxia-reoxygenation injury. We examined the effects of high Mg on mitochondria. Male SD rat hearts were excised and perfused with Langendorff manner. After stabilization, heart was made hypoxic 30 min and reoxygenated 30 min. In control group, the Mg concentration is 1.2 mM and in high Mg group was 12 mM. The left ventricular developed pressure, heart rate and coronary perfusion pressure were monitored throughout the experiments. At the various timing of the experiment, perfusion was stopped and heart tissue was homogenized. The cytosol fraction was obtained by ultracentrifuge of homogenate and its cytochrome c and caspase-3 content were quantitated by Western blot analysis. In addition, mitochondrial ultrastructure was examined by electron microscopy. In high Mg group, the recovery level of pressure rate product by reoxygenation (30min; 80.3%±4.5 p<0.01 vs. control group) was significantly higher than that of control group (54.3%±4.5). Cytochrome c and caspase-3 content of cytosol were increased by reoxygenation, but the extent of the increase was significantly inhibited by high Mg during hypoxia. Mitochondria were swollen by hypoxia and incompletely recovered by reoxygenation. However, in high Mg group, the structure recovered to almost normal. These results suggest that high Mg during hypoxia protects mitochondria from cardiac hypoxia-reoxygenation injury, restrains apoptosis of the myocardium and facilitates the functional recovery of the hearts. [J Physiol Sci. 2007;57 Suppl:S204]

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Lymph nodes are known to exchange hydrophilic substances between the lymph and the blood compartments. Usually small molecular substances are absorbed to blood vessels and consequently lymph protein concentration increases in the lymph nodes. In this study, we focused on exchange of water through the lymph nodes in vivo. Male Japan White rabbits were anesthetized with 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. Physiological saline solution was infused through the upstream cannula. Lymph outflow rate was determined at the constant outflow pressure of -5 cmH₂O. Lymph outflow rate increased monotonically with protein concentration of infused solution (0, 2.6 and 10% bovine serum albumin) and with venous pressure of the hind leg (control, 20, 30 and 40 mmHg). No apparent change was observed after decrease of arterial pressure of the hind leg. From these results, colloid osmotic pressure and hydrostatic pressure may have important role in penetration of hydrophilic substances in the lymph nodes. [J Physiol Sci. 2007;57 Suppl:S205]

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We examined whether pressor responses to isometric biting were partially caused by sensory receptors in periodontal tissue through increased MSNA and attenuated baroreflex gains in humans. First, we measured muscle sympathetic nervous activity from the peroneal nerve (MSNA, microneurography), arterial pressures (AP, finometer), heart rate (HR), electromyogram of masseter muscle (EMG), biting force (BF), and finger skin blood flow (FSBF, laser-Doppler flowmetry) during 2-min isometric biting at 50% maximal voluntary contraction (MVC) before (No-Blk) and after pharmacological alveolar nerve block (Blk) in 8 young men. Second, baroreflex gain was determined by the Oxford method during 5-min periods of rest without biting (No-BT) and biting at 30%MVC (BT) in Blk and No-Blk, respectively. In the 1^st study, biting increased AP, HR, and total MSNA (burst amplitude x frequency) and decreased finger cutaneous vascular conductance (FCVC, FSBF/mean AP) in Blk and No-Blk (P<0.05). However, the increases in AP, total MSNA, and the decrease in FCVC were markedly less in Blk than in No-Blk (P<0.05) with no differences in EMG, BF, and HR response between the trials. In the 2^nd study, baroreflex gains from HR (HR/systolic AP) and MSNA (total MSNA/diastolic AP) responses to pharmacologically altered AP were lower in BT than in No-BT (P<0.05) for no-Blk, while the differences disappeared in Blk. Thus, the sensory receptors in periodontal tissue were involved in pressor response to isometric biting through increased MSNA and reduced baroreflex gains. [J Physiol Sci. 2007;57 Suppl:S205]

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By analyzing the short-term mechanical restitution after twitches of various amplitudes in rat heart muscle, we have hypothesized that Ca²⁺ repletion in the junctional sarcoplasmic reticulum (JSR) occurs with a common Ca²⁺ content vs. time relationship after various degrees of JSR Ca²⁺ release and deduced a time course of Ca²⁺ repletion and a ratio of Ca²⁺ release/content in the JSR (T.Tameyasu et al, Jpn J Physiol,2004).Based on this hypothesis,we reported slower JSR Ca²⁺ repletion and smaller ratio of JSR Ca²⁺ release/content in heart muscle of adult spontaneously hypertensive rat(SHR) than control (WKY) in previous physiological meetings.We examined short-term mechanical restitution with ventricular papillary muscle of infant (3 wks-old) SHR and control to determine how and when functional impairment occurs in the SR of SHR heart muscle in the present study.There were little differences in the rate of JSR Ca²⁺ repletion nor the ratio of JSR Ca²⁺ release/content between the infant SHR and control.The result,taken together with previous ones, suggests that the slowing of JSR Ca²⁺ repletion and decrease of the ratio of JSR Ca²⁺ release/content initiates between ages of 3 to 7 wks in SHR and that the latter continues to decrease at least until 20 wks of age. [J Physiol Sci. 2007;57 Suppl:S205]

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By finding a suggestive evidence that Ca repletion in the junctional sarcoplasmic reticulum(JSR) occurs with a common JSR Ca content vs. time relationship after various degrees of JSR Ca release, we developed a method to estimate a time course of JSR Ca repletion after JSR Ca release and JSR Ca release/content relation by analyzing short-term mechanical restitution in papillary muscle of murine hearts (Tameyasu et al, Jpn J Physiol, 2004). Using this method, we could characterize the SR function in heart muscle of hypertensive rat and diabetic mouse, as reported recently. Applicability and limitation of our method was further tested in the present study. We show here that (1) there are little differences in a time course of JSR Ca repletion nor relation between the JSR Ca release and content as examined with papillary muscles among several different strains of mice, (2) the Ca handling property of the JSR obtained with whole heart preparation is similar to that with papillary muscle in mouse, (3) our method is applicable to analyze the JSR function under a low external Ca concentration, and (4) addition of caffeine (0.5 mM) violates a basic assumption that the JSR Ca repletion occurs with a common JSR Ca content vs. time relationship after various degrees of Ca release. The result ascertains that our method is quite useful to assess the JSR function at least in murine hearts. Analysis of the JSR function in rabbit heart with our method is in progress. [J Physiol Sci. 2007;57 Suppl:S205]

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Molecular mechanism on leukocyte transendothelial migration (TEM) in a variety of pathogenesis such as atherosclerosis is mostly unclear. We have previously reported that oxidized LDL, one of the essential players in atherogenesis, induced the expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) on endothelial junctions, and specifically promoted monocyte TEM. In the present study, to shed light on the specific role of the transmembrane protein PECAM-1 in monocyte TEM, we constructed the recombinant vector expressing red fluorescent protein DsRed fused to the C-terminus (intracellular side) of PECAM-1. This mode of fusion could keep the extracellular domain of the molecule intact, allowing interaction with other cells during TEM. The vector, which was constructed with general procedure (primer design, RNA extraction, reverse transcription, PCR amplification, vector ligation, clone selection, plasmid purification, and DNA sequence) was transfected into human umbilical vein endothelial cells (HUVECs) using nucleofection technique. The expressed fusion protein was confirmed to migrate as a single molecular mass with the expected size of 160 kDa (western blotting), and localize at cell-cell contact, which is the same distribution with endogenous PECAM-1 (Immunofluorescence). We are now studying the live-cell dual imaging of endothelial (DsRed) and monocyte (GFP) PECAM-1 during TEM with two-photon excitation microscopy. [J Physiol Sci. 2007;57 Suppl:S206]

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Aging, obesity and underexercise are one of the risk factors for atherosclerosis. We evaluated the effect of these risk factors to atherosclerosis with the oxidative stress index in endothelial cells. We randomly assigned 39 rats to two experimental diet groups, taking food ad libitum (AD) and taking food 70% of the average food consumption (70%). 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 superoxide and NO 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. Dietary restriction and chronic swimming exercise independently decreased the physiological risks for atherosclerosis, growth rate of body weight, blood pressure and serum triglyceride, and more decreased in concert. There was no difference in the oxidative stress index between arterial portions, among dietary restriction and exercise conditions. There were no difference in the superoxide dismutase activity of heart, kidney, liver, abdominal aorta and serum from dietary restriction and exercise conditions. These data indicate that dietary restriction and exercise conditions improve the risk for atherosclerosis through the mechanism which is not connected with oxidative stress. [J Physiol Sci. 2007;57 Suppl:S206]

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Blockade of I_Kr prolongs action potential duration (APD) and may cause lethal arrhythmia such as torsades de pointes. The risk of this arrhythmia depends on voltage- and time-dependence of I_Kr blockade. In the present study, we introduced a novel method that can assess the risk of I_Kr blockers with variation of I_Ks channel density in the ventricular myocardium. In this method, the Luo-Rudy phase II model was adopted to calculate drug-induced prolongation of APD and the effects of I_Kr blockers (such as dofetilide, quinidine and vesnarinone) under the various magnitudes of I_Ks were shown in a two-dimensional contour map. For the tachycardia condition, the slope of APD-isoline in the I_Kr (horizontal axis)–I_Ks (vertical axis) map was large. This corresponds to the weak effect of the I_Kr blockade due to the accumulation of the I_Ks. The voltage- and time-dependence of I_Kr blockers, such as quinidine and vesnarinone, curved isolines in the map and the curvature for vesnarinone was increased under bradycardia. Because the I_Kr block accumulates at short cycle length due to the long time constant of the block by vesnarinone. These results suggest that the two-dimensional mapping is a useful method to estimate arrhythmogenic risk of I_Kr blockers. [J Physiol Sci. 2007;57 Suppl:S206]

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Electric heart activity of fetuses can be measured noninvasively not with electrocardiography but with magnetocardiography, which can measure faint magnetic fields generated by the fetal heart. Recently, intervals between peaks on magnetocardiograms have been mainly investigated, especially for the diagnosis of fetal arrhythmia; however, the amplitudes of each peak (P, QRS, and T waves) of the magnetocardiogram have not yet fully studied. In this study, the amplitude of the P wave on a fetal magnetocardiogram was investigated to examine whether P wave amplitude may directly relate to atrial load and fetal prognosis. Magnetocardiograms of 36 fetuses (14 normal fetuses and 22 fetuses with congenital heart diseases) were recorded using 64-channel SQUID (superconducting quantum interference device) magnetocardiography. By averaging the magnetocardiogram triggering R peaks, we obtained an averaged magnetocardiogram and measured the peak amplitudes of the P. In cases of monophasic P waves, the absolute amplitude of the P wave was measured. In cases of biphasic P wave, the amplitude between positive and negative peaks was measured. The peak amplitudes of P waves with atrial load such as pulmonary atresia, pulmonary stenosis, Ebstein disease were significantly larger than those in normal fetuses and were related to poor prognosis, indicating that excess atrial load resulted in a large P wave even on a fetal magnetocardiogram. [J Physiol Sci. 2007;57 Suppl:S206]

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Background: Increasing alcohol intake is known to link to increased risk of atrial fibrillation that has possibly been associated with apoptosis. Recent studies have revealed that ethanol induces apoptosis of various tissues. However, apoptotic effects of ethanol on atrium are unclear. Therefore, we hypothesized that ethanol induces apoptosis of atrium, and prove the hypothesis using HL-1 mouse atrial myocytes. Methods and Results: The cultured HL-1 cells were incubated for 18 hours with 0.25, 0.50, 1.0, and 2.0% of ethanol. To determine apoptosis in HL-1 cells, mitochondrial transmembrane potential (ΔΨ_m), the percentage of the cells with hypodiploid DNA, and the activity of caspase-3 were measured quantitatively by flow cytometric assay with DiOC₆(3), propidium iodide, and PhiPhiLux G1D2, respectively. Ethanol at 1.0% or more induced loss of ΔΨ_m, the occurrence of hypodiploid cells, and activation of caspase-3. Conclusions: Ethanol induced apoptosis of HL-1 cells, suggesting that ethanol intake leads to structural remodeling of atrial myocytes through the induction of apoptosis, followed by the development of the arrhythmogenic substrate, which tends to initiate and perpetuate atrial fibrillation. [J Physiol Sci. 2007;57 Suppl:S207]

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Background: Intracellular and extracellular magnesium (Mg²⁺) plays a important role to control the excitability in cardiac myocytes. Objective: To assess the effects of low magnesium diet on rat heart associated with action potentials (AP), ionic currents and expression of mRNA focusing on the K⁺ channels in cardiac myocytes. Methods and Result: Serum and intracellular Mg²⁺ concentrations were measured from normal (control) and the magnesium-deficient food feeded rats (hypomagnesium rat). ECG in hypomagnesium rat shows significantly shorter R-R intervals (control, 180.3 ± 6.1 ms vs hypomagnesium 146.8 ± 6.8 ms, p<0.05). Action potential duration (APD) in ventricular myocytes were recorded by intracellular microelectrode. APD₉₀ in myocytes obtained from hypomagnesium rats were significantly prolonged (control, 101.2 ± 3.2 ms vs hypomagnesium, 314 ± 11.8 ms, p<0.05). Inward-rectifier K⁺ current (I_K1) obtained by whole-cell patch clamp method indicates a decrease in I_K1 density and it's conductance in myocytes from hypomagnesium rat. Expression of Kir2.1 mRNA was significantly downregulated by 38.5 ± 4.2% in myocytes derived from hypomagnesium rat, while expression of Kir2.2 mRNA was unchanged. Conclusion: These results suggest that intracellular and extracellular magnesium ion modifies the inward-rectifier K⁺ current, Kir2.1 in particular, by means of channel kinetics and expression. [J Physiol Sci. 2007;57 Suppl:S207]

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Neurons in the rostral ventrolateral medulla (RVLM neurons) receive inputs from various sources, including baroreceptors, and then regulate activity of sympathetic preganglionic neurons. Though the RVLM neurons are assumed to mediate the viscero-sympathetic reflex, it has not been clarified yet. In the present study, we investigated the role of the RVLM neurons in the viscero-renal sympathetic reflex in anesthetized and vagotomized rabbits. Electrical stimulation of the greater splanchnic nerve (SPN) evoked reflex responses in the renal sympathetic nerve activity that were composed with inhibitory and/or excitatory components. Bilateral microinjection of muscimol, a GABA receptor agonist, into the RVLM, considerably blocked the reflex responses. Bilateral microinjection of bicuculline, a GABA receptor antagonist, attenuated largely inhibitory component, on the other hand, excitatory component was eliminated by kynurenic acid, the glutamate receptor antagonist. Activities of 21 RVLM neurons were recorded. Twenty neurons among them responded to the SPN stimulation. Responses of neurons also consisted of inhibitory and/or excitatory components. Excitatory component of these neurons preceded that of renal nerve activity by about 100 ms. This latency difference was almost same as that of the inhibitory responses evoked by the aortic nerve stimulation. In conclusion, the renal sympathetic reflex responses evoked by the SPN stimulation are mediated by the RVLM neurons, and GABA and glutamate are used as transmitters in this reflex at the RVLM. [J Physiol Sci. 2007;57 Suppl:S207]

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Cardiac contraction is elicited by the release of stored Ca²⁺, following the opening of L-type Ca channels (L-Ch). We observe properties of Ca²⁺-induced Ca²⁺-release (CICR) as a result of averaged behavior of individual Ca-release unit (local control theory). Mathematical modeling of CICR thus requires stochastic simulations of numerous dyadic spaces, and is computationally highly demanding. The possible simplest formulation of local control is to treat Ca-release units as a distribution of four distinct states, where ryanodine receptors (RyR) open/close and L-Ch open/close. Based on the rapid equilibrium approximation of [Ca²⁺] between dyadic space and cytosol (Hinch, 2004), we developed a model of CICR where the distribution of Ca-release units over four states was estimated using macroscopic and/or single channel gating properties of L-Ch and RyR. We then incorporate it into a human ventricular action potential model (tenTusscher, 2004) with minor modifications. We tried several types of macroscopic L-Ch gating scheme obtained in a variety of experimental settings. Data on physiological RyR gating, in contrast, is scarce. We used a simple C-O-I Markov model and a popular four-states scheme (Stern, 1999; Bers' lab. 2004,2005). After tuning of RyR gating constants, models with different sets of RyR/L-Ch properties consistently reproduced essential features of CICR: (1)graded release, (2)experimentally observed voltage-dependence of EC-coupling gain. Being extensible to tissue levels, our model provides basis to investigate intracellular Ca²⁺ dynamics in wide physiological/pathological conditions. [J Physiol Sci. 2007;57 Suppl:S207]

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We investigated whether the increase in Ca²⁺ uptake rate of sarcoplasmic reticulum (SR) would influence Ca²⁺ content and Ca²⁺ leakage in SR. To investigate SR functions at different Ca²⁺ uptake rates, we used transgenic mouse myocardium overexpressed SR-Ca²⁺ ATPase (SERCA2a-TG). We evaluated Ca²⁺ uptake rate, Ca²⁺ release and Ca²⁺ leakage using saponin-treated thin trabeculae obtained from the TG and non-TG mice hearts. Each SR function was estimated by measuring the remaining Ca²⁺ in SR which was released by caffeine (50 mM) after SR was loaded with Ca²⁺ at various concentrations (pCa 8-5.6) for different periods (5-120 sec). The released Ca²⁺ was measured with fluo-3. In saponin-treated preparation, the Ca²⁺ handling proteins (SERCA2a, phospholamban and ryanodine receptor) were well preserved, confirmed with Western immunoblotting. The structures of transverse tubules and SR were intact, confirmed by electron microscopy. At short Ca²⁺ loading time, Ca²⁺ content was larger in TG myocardium (113.8%) than that in non-TG myocardium. The maximal Ca²⁺ content and Ca²⁺ leakage were similar in both myocardium. In conclusion, the modulation of Ca²⁺ uptake rate would influence Ca²⁺ handling in each heartbeat but does not alter the maximal Ca²⁺ content and Ca²⁺ leakage at a steady state. [J Physiol Sci. 2007;57 Suppl:S208]

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We studied the effect of doxapram HCl on the cardiac conduction system in the adult Wister rats under urethane anesthesia. ECG and respiratory movements were recorded by Biopac MP-30 system. The doxapram HCl was intravenously injected and blood samples were obtained at 5, 15, 30 and 45 min. after the injection. The concentration of the drug was measured with the high performance liquid chromatography. The effect of the drug on RR, PR and QT intervals of ECG was evaluated in two groups of rats with and without preadministration of atropine. The cardiac conduction velocity was analyzed with Fluclet program. The concentration of doxapram HCl in the blood showed the maximum value at 5 min. after injection of the drug and decreased gradually in progress of time. Administration of the atropine sulfate reduced RR interval and PR interval without any change in QT interval. On the other hand administration of the doxapram HCl prolonged QT and PR interval but reduced RR interval. The effect of the drug depended on concentration. The results suggest that the doxapram HCl affect the repolarization of the ventricle as well as atrioventricular conduction. [J Physiol Sci. 2007;57 Suppl:S208]

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The purpose of this study was to measure blood flow velocities in common carotid, brachial and femoral arteries simultaneously during head-up-tilt (HUT) using our developed telemetry measurement system. This system was implemented with simultaneous measurement of electrocardiogram (ECG). The velocity waveforms of 3 arteries, heart rate (HR), and blood pressure (BP) were assessed for 20 s at the stages of postural change. As a result, HUT-induced increase in HR was more pronounced in the young subjects. There was significant increase in diastolic BP, but a non significant change in systolic BP to tilt. The decreased of the blood flow index in the carotid artery was expected to be the effect of cerebral autoregulation (CA) to control blood flow to the brain. In conclusion, HUT produced larger changes in the 3 arteries, particularly in brachial and femoral, and also in HR and diastolic BP. This study demonstrated that simultaneous measurement of blood flow velocity waveforms in carotid, brachial, and femoral in HUT postural change using our telemetry measurement device was attainable. [J Physiol Sci. 2007;57 Suppl:S208]

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Recombinant adenoviruses are widely used as gene transfer vectors because of their flexible packaging constraints and their ability to transduce nonreplicating cells. The aim of this study was to examine the acute and chronic toxic effects of adenoviral vector on cardiac function in terms of left ventricular (LV) mechanoenergetics. E1-deleted recombinant adenoviral vector carrying β-galactosidase (Ad.βgal.) cDNA was used. Cardiac function was examined on the cross-circulated rat heart preparations, where end-systolic/diastolic pressure-volume relation (ESPVR/EDPVR), systolic pressure-volume area (PVA) and myocardial oxygen consumption (VO₂) were measured. Data were obtained before and after coronary infusion of Ad.βgal solution to examine the acute toxic effects of the virus, and further obtained 3 days after global cardiac gene transfer of Ad.βgal to examine its chronic toxic effects. There was no significant difference between pre- and post-virus data concerning ESPVR/EDPVR, LV relaxation rate, basal metabolic VO₂, VO₂-PVA relation and oxygen cost of contractility. Likewise, there was no difference in all data between Ad.βgal-transferred and sham-operated hearts. We conclude that adenoviral vector does not have acute and chronic toxic effects on rat cardiac function in terms of LV mechanoenergetics. Further testing of adenoassociated virus with longer term expression will be necessary to evaluate the safety of these agents in therapeutic gene transfer. [J Physiol Sci. 2007;57 Suppl:S209]

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We investigated left ventricular (LV) mechanical work and energetics in the cross-circulated (blood-perfused) isoproterenol (Iso 1.2 mg/kg/day for 3 or 7 days)-induced hypertrophied rat heart preparation under isovolumic contraction-relaxation. [Methods] We evaluated pressure-time curves per beat, end-systolic pressure-volume and end-diastolic pressure-volume relations, and myocardial O₂ consumption per beat (VO₂)-systolic pressure-volume area (PVA; a total mechanical energy per beat) linear relations. [Results] The LV relaxation rate at 240 bpm in Iso-induced hypertrophied hearts was significantly slower than that in control hearts (saline 24 μl/day for 3 or 7 days) with unchanged contraction rate. Interestingly, the relaxation half-time (T_1/2) was not prolonged by increasing LV volume in Iso-induced hypertrophied hearts, although it was prolonged in control hearts. Although the expression of Ca²⁺ cycling proteins, the ratio of sarcoplasmic reticulum Ca²⁺-ATPase versus phospholamban (SERCA2A/PLB) expression and Na⁺-Ca²⁺ exchanger (NCX1) expression, and the VO₂-intercept (summation of basal metabolism and Ca²⁺ cycling energy consumption) of VO₂-PVA linear relation was unchanged, the collagen production (type I and III) was markedly enhanced in Iso-induced hypertrophied hearts. [Conclusion] The present results suggested the possibility that LV lusitropic dysfunction of isoproterenol-induced hypertrophied rat heart is due to marked collagen production but not due to Ca²⁺ cycling impairment. [J Physiol Sci. 2007;57 Suppl:S209]

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In a model of early-stage type II diabetes, we investigated whether angiogenic factors increase the percentage of smaller longitudinal capillaries and interconnecting anastomoses in skeletal muscle, thereby "derecruiting" capillaries through which red blood cells (RBCs) pass, thus accounting for the development of insulin resistance in muscle. The OLETF rat model of type II diabetes was used, with LETO rats as controls. By 16 weeks of age, OLETF rats had developed higher body weights and insulin levels. The RBC velocity (V_RBC) in soleus muscle capillaries, measured by intravital microscopy, was significantly higher in OLETF rats. The three-dimensional architecture of the soleus muscle microvessels, visualized by confocal laser-scanning microscopy, showed a higher percentage of longitudinal capillaries and interconnecting anastomoses with luminal diameters < 3 μm in OLETF rats. Real-time PCR showed that OLETF rats had higher messenger RNA levels for the angiogenic factors, VEGF, KDR, Flk-1, angiopoietin-1, and Tie-2. They also had higher levels of VEGF protein, as measured by an enzyme-linked immunosorbent assay. In the presence of increased levels of angiogenic factors, the skeletal muscle microcirculation developed greater numbers of small capillaries and anastomoses during the early stages of diabetes; this reduced the number of capillaries carrying RBCs and increased the velocity of flow in those that continued to transport these cells. [J Physiol Sci. 2007;57 Suppl:S209]

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The values of b/a and d/a of 2nd derivative photoplethysmogram (PTG) have been shown to reflect distensibility of the arterial wall and peripheral circulation, respectively. Pulse wave velocity (PWV) and augmentation index (AIx) of pulse waves have often been employed as indices of arterial rigidity and reflected wave from the peripheral sites, respectively. We investigated relations of b/a and d/a to wall distensibility, PWV and AIx in the normal and Kurosawa and Kusanagi-Hypercholesterolemic (KHC) rabbits aged 22-24 months under pentobarbital anesthesia. Pulse waves were measured at different aortic sites using two catheter-tip transducers. PTG was recorded in the left forearm. Diameter of the aorta was measured at different aortic sites using intravascular ultrasonic system. PWV was determined as a velocity from the ascending to distal abdominal aortas. The values of b/a and d/a were significantly higher and lower in the KHC rabbits than in the control rabbits, respectively. Pressure-strain elastic modulus (Ep) was significantly higher in the KHC rabbits than in the normal rabbits in the proximal thoracic and abdominal aortas. The value of b/a showed significant correlation with Ep and PWV in the KHC rabbit group and d/a correlated significantly to AIx of pulse waves at the ascending aorta in the two rabbit group. These suggest that b/a and d/a are as useful indices which reflect PWV and AIx in the KHC rabbit group, respectively. [J Physiol Sci. 2007;57 Suppl:S209]

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The sinoatrial node (SAN) locates in the right atrium at the junction of the crista terminals and generates the pacemaker activity. Little is known about structural correlates of complex behaviors of SAN such as pacemaker shift. We visually analyzed the spatial distribution of action potential properties in the neonatal rat SAN using a voltage sensitive dye, di-4-ANEPPS. Two parameters, upstroke time and action potential duration at 30% repolarization level (APD30) were calculated. Although these parameters showed roughly downward gradients from the leading pacemaker site, pseudocolor images of both upstroke time and APD30 exhibited patchy appearances due to the existence of multiple local maxima. The leading pacemaker activity site occurred at one of these local maxima. Further, pacemaker activity was affected by internal conditions such as gap junction conductance. A gap junction blocker, 100 μM carbenoxolone, reduced conduction velocity from 134±8 to 62±11 μm/s without affecting beating rate (n=7). Carbenoxolone changed action potential properties and shifted the leading pacemaker site in all cases. The new leading pacemaker site occurred in one of the regions where upstroke time and APD30 were at maximum levels even before the application of carbenoxolone. These results indicate that both intrinsic cellular properties and mutual interactions through gap junctions contribute to determine the action potential characteristics of SAN cells and leading pacemaker site. [J Physiol Sci. 2007;57 Suppl:S210]

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To simulate a biological relationship between arterial pressure (AP), renal sympathetic nerve activity (RSNA), heart rate (HR) and respiration rate (RR), we have built a new simulation model by a neural network (NN) algorithm (Neural Network Toolbox of MATLAB) using only measurements but without approximation equations. The learning algorithm of the layered NN was a back propagation. AP, RSNA, HR and RR were measured in conscious chronically instrumented rats. Previously, averaged values of experimental observations were used for the learning of NN model, while in this study, individual values of experimental observations were used for the leaning. The errors generated from the present simulation were smaller than those generated from the previous method. Using the present simulation model that was constructed using the data from normotensive Sprague Dawley (SD) rats, we examined whether the simulated AP values would be consistent with the measured AP values in salt-sensitive hypertensive Dahl rats, or chronic heart failure Dahl rats. The simulated AP values were significantly lower than the measured values. The results suggested that the NN modeling could indicate that the biological relationship between RSNA, HR, RR and AP in hypertensive or heart failure state was theoretically different from that of the SD rat strain. [J Physiol Sci. 2007;57 Suppl:S210]

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We previously reported that low-frequency variability (< 0.6 Hz) in arterial pressure (AP) oscillations originates from sympathetic activity and is buffered by nitric oxide (NO). The power spectral density (PSD) in the 0–0.2 Hz (VLF) band was increased by L-NAME, while the PSD in the 0.2–0.6 Hz (LF) band was decreased. In contrast, L-Arg decreased the PSD in the VLF band and increased the PSD in the LF band. This shows that NO buffers low-frequency variability of AP oscillations via two-pathways. In the present study, we investigated the mechanical pathways of NO-buffering action on AP variability to assess the role of NO in blood pressure regulation. We conducted experiments on male normotensive Wistar rats (n = 30), 10–13 wks of age in order to examine the dynamic effects of NO and K_ATP channels on AP variability and heart rate variability (measured in the conscious resting state). Findings indicate that (1) the PSD in the VLF band increases with the increase in mean AP by the NO-buffering mechanism at resistance vessels, and (2) the PSD in the LF band decreases with the increase in mean AP by the NO-buffering mechanism via the baroreflex, which is complemented by activating K_ATP channels of baroreceptors. We conclude that VLF variability is a risk factor exerting serious damage on the cardiovascular system during an increase in AP, while LF variability is a homeostatic control factor maintaining the fluctuation of AP within a narrow range via the baroreflex. [J Physiol Sci. 2007;57 Suppl:S210]

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Low values in plasma lipids and HbA1c were found in the control SHRSP group in the previous study. The continuous rise in blood pressure, which readily results in cerebral stroke, has been said to be the cause for the short life span of SHRSP. In addition to the pressure rise, the low plasma cholesterol level may produce fragile microvessel walls and the short life span of SHRSP. In a similar fashion the plasma membrane of erythrocytes may suffer from the low plasma cholesterol, which shortens the life span of erythrocytes. Measured values in SHRSP are listed here together with reported values in Wistar rats; plasma total cholesterol (TC) 57.3 vs 86.7, LDL 4.5 vs 17.1, HDL 23.0 vs 56.3, TG 45.0 vs 64.7 each in the unit of mg/dl, and HbA1c 0.2 vs 3.41%. Thus, the values in SHRSP are smaller than those in Wistar rats. The life span of erythrocytes are reported 25-40 days in SHRSP and 60 days in Wistar rats. Meanwhile high TC (200-240 mg/dl), and HbA1c (<6%) and a long life span of erythrocytes (120 days) which are larger than those in Wistar are given in human subjects. Since HbA1c represents the cumulative amount of the glycated hemoglobin over the period of 2 months, a comparison of these data suggests that the low value in HbA1c in SHRSP is reasonable because of the small life span of erythrocytes. In addition, SHRSP has genetic disorders also in lipid metabolisms. [J Physiol Sci. 2007;57 Suppl:S210]

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Spiral waves (SWs) are principal mechanisms of life-threatening ventricular tachyarrhythmias, but their dynamics are not fully understood. We investigated action potential (AP) dynamics of SWs in a 2-dimensional subepicardial layer of rabbit ventricular muscle by using high-resolution optical mapping. The preparations showed uniform anisotropy with an anistropic ratio of conduction at 2.6-3.2. SWs induced by cross-field stimulation rotated around a functional line of block (BL) with periodic meandering. The BL was composed of 2 segments: a central segment (C-BL) with a phase-shift between the two limbs of the circuit, and small terminal segments (T-BLs) with a localized conduction delay around the pivot points. The latter was always parallel to the fiber direction. APs changed gradually from the periphery to the center of the circuit: APs had progressively slower upstroke as close to the pivot points and were transformed into double potentials on the C-BL. I_Na blockade increased the cycle length (CL) of SWs and prolonged T-BLs. APs on T-BLs showed steps in the depolarization phase, longer duration and shorter electrical diastole as close to the pivot points. I_Ca,L blockade shortened the C-BL and decreased CL: SWs were more stabilized. These results suggest that depolarization efficiency of SWs depends primarily on the tissue anisotropy and Na channel availability. L-type Ca channel availability may affect the rotation period and stability of SWs through a modulation of AP repolarization. [J Physiol Sci. 2007;57 Suppl:S211]

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We have introduced multiple-site optical recording of transmembrane voltage activity, using a fast merocyanine-rhodanine voltage-sensitive dye (NK2761) and a multi-element (16 × 16) photodiode array, to monitor action potentials in the artificial circuits made of the isolated rat atrium. The atrial wall preparation including the right or left auricle was dissected from the adult rat heart and an artificial hole was made in the center of the preparation. The preparation was then stained with a voltage-sensitive dye. For suppression of optical artefacts due to contractile movements, a bathing medium containing 2,3-butanedione monoxime (BDM: 20 mM) was used. The spread of spontaneous excitation was assessed optically by timing the initiation of the action potential-related extrinsic absorption changes. The electrical stimulation applied by a bipolar electrode evoked the sustained circus movement of the excitatory wave around the artificial hole. We have optically mapped the spatiotemporal patterns of the circus movement of excitation. These maps suggest that the circus movement observed in the artificial circuit resembles the basic mechanism of the tachycardia-like excitation observed in the intact isolated right atrial preparation, that we reported previously. On the bases of these maps, we considered the circus movement in the artificial circuit as the in vitro model of atrial flutter. [J Physiol Sci. 2007;57 Suppl:S211]

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Urocortin (Ucn), a member of the corticotrophin-releasing factor (CRF) peptide family, has appetite-suppressing and cardiovascular effects. Its intravenous or subcutaneous injection of Ucn reportedly decreased the mean arterial blood pressure (MABP) and increased the heart rate (HR). We report that at 50 and 75 pmol/50 nl, Ucn, suspended in artificial cerebrospinal fluid (ACSF) and microinjected into the rat nucleus tractus solitarii (NTS), produced a significant, dose-dependent reduction in both MABP and HR. Our results suggest that Ucn may act as a neurotransmitter or neuromodulative substance in the NTS and that it may play a role in cardiovascular regulation in vivo. [J Physiol Sci. 2007;57 Suppl:S211]

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Background: In gene therapy, proteins are expressed about one week after injection and the degree of expression cannot be controlled. These disadvantages may be overcome by the in vivo delivery of biologically active proteins into cells. We investigated the possibility of targeted, direct in vivo protein transduction into cardiomyocytes by injecting β-galactosidase (β-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the left ventricular myocardium of rats.Methods: Male Wistar rats underwent left thoracotomy. We injected β-gal with HVJ-E vector (BH group), β-gal without the vector (B group), or the vector without β-gal (H group). The hearts were removed 1, 3, 6, 12 and 24 hours post-injection and each was cut in 5-μmthick slices with a cryostat. The sections were histochemically stained to detect β-gal enzymatic activity. β-gal-positive areas were present in these sections as β-gal activity, determined by computer-assisted colorimetric analysis.Results: The activity of β-gal in sections from rats injected with β-gal with HVJ-E vector was significantly stronger than those of β-gal without the vector and the vector without β-gal (p<0.01 at all time points).Conclusions: Our findings suggest that direct in vivo protein transduction into cardiomyocytes is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the heart. [J Physiol Sci. 2007;57 Suppl:S211]

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Peak expiratory flow rate (PEFR) of 350 rural women aged (20-70 years) in Edo State, Nigeria who actively used wood as a source of fuel for cooking was measured. The height, chest circumference, weight and blood pressure of the women were also measured. Respiratory symptoms of cough with sputum production, dyspnea, wheezing, chest tightness and chest pain were markedly elevated in the subjects compared to control. The mean PEFR values for the wood exposed women (289±19.6L/mm) was significantly lower than control (364±17.2L/mm), P<0.05.The predicted PEFR values for both Africans and Caucasians were significantly higher than the measured values. The PEFR decreased with increase in years of exposure to wood smoke and the fall was neither accounted for by age nor height. The increase in respiratory symptoms and the low PEFR values observed for women exposed to wood smoke, the severity of which is related to exposure time indicate that prolonged exposure to wood smoke is capable of impairing lung functions. [J Physiol Sci. 2007;57 Suppl:S212]

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Sevoflurane is a widely used volatile anesthetic in clinical practice, but its neural mechanisms of sevoflurane-induced central respiratory depression remain poorly understood. In the present study,we studied the mechanism of sevoflurane-induced central respiratory depression related to GABA_A receptors using the isolated brainstem-spinal cord of neonatal rats (0-4 day old). Rhythmic inspiratory burst activity was recorded from the C4 spinal ventral root. The activity of respiratory neurons in the ventrolateral medulla was recorded using a perforated patch-clamp technique. We found that bath-applied sevoflurane decreased C4 inspiratory burst rate and amplitude. C4 burst rate could be reversed by the administration of a GABA_A antagonist, bicuculline. Sevoflurane suppressed the firing of action potentials in preinspiratory and expiratory neurons. In contrast, sevoflurane had little effect on firing in inspiratory neurons. Our findings suggest that suppressed action potential of preinspiratory neurons, which is mediated by the GABA_A receptor, serves as the neuronal basis of sevoflurane-induced respiratory depression in the newborn rat. [J Physiol Sci. 2007;57 Suppl:S212]

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The parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) and preBotzinger complex/ventral respiratory group (preBotC/VRG) are considered to play key roles in respiratory rhythm generation. To test our hypothesis that responses of these two areas to respiratory and metabolic acidosis are different, we analyzed respiratory population activities in pFRG/RTN and preBot/VRG to respiratory and metabolic acidosis by voltage-imaging. CO2/pH changes induced respiratory phase switching in pFRG/RTN bidirectionally, i.e., from preinspiratory/inspiratory phase dominant to inspiratory/postinspiratory phase dominant pattern and vice versa in 20% of preparations. These switching patterns were unpredictable. Respiratory acidosis induced recruitment of respiratory-modulated neuronal activity, although pixels that gained and lost respiratory modulation were intermingled. Among the newly recruited pixels, the preinspiratory pattern appeared mainly in pFRG/RTN and the inspiratory pattern mainly in the caudal portion of preBotC/VRG. Metabolic acidosis changed the respiratory-modulated pattern from preinspiratory dominant to inspiratory dominant in pFRG/RTN. In pixels of pFRG/RTN and the caudal portion of preBotC/VRG, respiratory and metabolic acidosis induced deprivation of respiratory modulation. We conclude that respiratory and metabolic acidosis differently affect pFRG/RTN and preBotC/VRG, induce phase switching and phase shifts in pFRG/RTN, and even alter the mechanism of respiratory rhythm generation. [J Physiol Sci. 2007;57 Suppl:S212]

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The distribution and abundance of the calcium binding protein, calbindin D-28k immunoreactivity in the taste buds of circumvallate papillae and larynx were compared between normoxic and chronically hypoxic rats (10% O₂ for 2 months). In the normoxic control rats, calbindin D-28k immunoreactivity was observed in the cells and fibers of the intragemmal region of the taste buds in the circumvallate papillae. In contrast, in the subgemmal region of the laryngeal taste buds, fibers but not cells were immunoreactive for calbindin D-28k. In chronically hypoxic rats, calbindin D-28k immunoreactive cells and fibers in the taste buds were decreased in the circumvallate papillae. In the laryngeal taste buds, the density of the subgemmal calbindin D-28k immunoreactive fibers in the chronically hypoxic rats was greater than in normoxic rats. It is considered that function of the laryngeal taste buds is different from that of the lingual taste buds, so that laryngeal taste buds may be involved in chemosensation other than taste. The altered density of calbindin D-28k immunoreactive cells and fibers in the lingual and laryngeal taste buds is a predominant feature of hypoxic adaptation, and chronic hypoxic exposure might change the chemical sensitivity of the circumvallate papillae and larynx through the regulation of intracellular calcium ion. [J Physiol Sci. 2007;57 Suppl:S212]

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Breath contains numerous kinds of chemical compounds derived not only from metabolites but exogenous chemicals. To develop a biogas diagnostic system, such exogenous chemicals compounds as cigarette smoking, air pollutions, ingestions of tablets and/or meals and so on must be considered. In the present study, we examined the effects of cigarette smoking on the biogas with low molecular weights. Nine hundreds and eighty eight subjects aged from 20 to 88 years old volunteered for the present study. Among them, current, ex-, and non-smokers were 20, 30 and 50%, respectively. Current smokers were asked to stop smoking after getting up in the morning. After tooth brushing and gargling, fasted subjects breathed purified artificial air via a mouthpiece in a sitting position for 12 min, during which exhaled air was collected via one-way valve into the Douglas bag. Minute ventilation volume and concentrations of carbon dioxide (CO2) and oxygen (O2) in exhaled air were monitored by a respiratory metabolic analyzer with the chemical sensors. Concentrations of hydrogen (H2), methane (CH4), carbon monoxide (CO), nitric oxide (NO) and nitric dioxide (NO2 = Nox–NO) in the bag were analyzed by gas chromatography and chemiluninescence methods. Significant increases in VCO and FCO of current smokers were observed in the heavy smokers, depending upon the pieces of cigarette smoking per day. On the other hand, VNO and FENO decreased in a dose-dependent manner. The other kinds of gases analyzed in the present study were not significantly affected by smoking. [J Physiol Sci. 2007;57 Suppl:S213]

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The respiratory rhythm response to the afferent inputs from the pulmonary stretch receptors depends critically on the discharge frequency of afferent fibers. (Takano and Kato, J. Physiol., 1999, 2003). P2X receptors modulate excitatory syn-aptic transmission in the nucleus of the solitary tract (NTS) in a manner sensitive to pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (PPADS; Kato and Shige-tomi, J. Physiol., 2001). To analyze how P2X receptors in the NTS participate in the frequency-dependent switching of the effect of vagal afferent inputs, we locally injected PPADS (20 nmole/ side/rabbit) into unilateral NTS in anesthetized rabbits. In the side of PPDAS injection, the inspiratory pro-motion by low frequency vagus nerve stimulation (20 Hz) was significantly attenuated for 120 min after injection, whereas the inspiratory suppression by high-frequency stimulation (160 Hz) remained unchanged. The effects of both low- and high- frequency stimulation of the vagus nerve remained in-tact in the side in which vehicle (PBS) was injected. The ex-tent of the injected drug diffusion in the brain stem, as confirmed with Evans blue staining, was limited within a ra-dius of 1 mm, showing that the frequency-dependent influence of PPADS was mediated by its effect on the NTS network. These results suggest that the ATP-P2X receptor-mediated transmission in the NTS plays critical roles in the afferent frequency-dependent responses of the central respiratory pat-tern generator. [J Physiol Sci. 2007;57 Suppl:S213]

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Inspiratory muscle conditioning at a lower lung volume based on the principles of muscle thixotropy decreases end-expiratory chest wall volume (Vcw) during resting breathing. The aim of the present study was to determine the deflating effect of thixotropy conditioning of inspiratory muscles on dynamic increases in operating chest wall volumes (end-expiratory Vcw and end-inspiratory Vcw). We examined the deflating effect on hyperinflation induced by continuous positive airway pressure (CPAP; 10 cm H ₂ O) in 13 healthy men with respiratory inductive plethysmography. Conditioning at maximal expiratory volume, positioned 1.30 ± 0.57 L below true functional residual capacity (FRC), decreased end-expiratory Vcw by 0.16 ± 0.12 L and end-inspiratory Vcw by 0.20 ± 0.14 L at 180 s (P < 0.01). Inspiratory capacity (1.59 ± 0.45 L) and inspiratory reserve volume (1.18 ± 0.40 L) were greater than baseline values during CPAP breathing (1.44 ± 0.49 L and 0.99 ± 0.41 L, respectively; P < 0.01). Although small changes in these parameters were observed immediately after conditioning at or above true FRC, these changes disappeared by 180 s after conditioning. Thus, conditioning should occur at an expiratory position below true FRC to result in lasting decreases in operating chest wall volumes during CPAP-induced hyperinflation. [J Physiol Sci. 2007;57 Suppl:S213]

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We reported the effects of 5-hydroxytryptamine 2 (5-HT2) receptor activities in the dorsomedial medulla oblongata (DMM) on airway resistance and ventilation in mice. We compared perfusion of a selective serotonin reuptake inhibitor, fluoxetine, and the co-perfusion of fluoxetine plus a 5-HT2 receptor antagonist, LY-53857, in a double-chamber plethysmograph. The results suggested that 5-HT2 receptor activity in the DMM determines the set points for both airway dilation and ventilatory volume that are suited to the facilitated respiratory drive. We also found that the mice we placed in the double-chamber plethysmograph spent a lot of time awake. In the present study, we examined basal activity of the 5-HT2 receptors in the DMM to airway resistance without background stimulated by fluoxetine. Each mouse was anesthetized intraperitoneally with pentobarbital sodium and the medulla oblongata exposed dorsally. A microdialysis probe (CUP7; membrane length, 1 mm) was inserted into the DMM and fixed to the cranial bone. The skin incision was closed. Each mouse was placed in a double-chamber plethysmograph. After recovery from anesthesia, airway resistance obtained by single perfusion of 10⁻⁵ M LY-53857 was compared with that obtained by artificial cerebrospinal fluid. The preliminary experiments showed that a single perfusion of LY-53857 tended to increase airway resistance. As well, we discuss basal activity of 5-HT2 receptors in the DMM in respect to airway resistance. [J Physiol Sci. 2007;57 Suppl:S213]

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The circadian change of ventilatory responses to hypoxic stimuli have rarely been studied in experimental animals. We examined the ventilatory and metabolic responses to hypoxia in the light and dark periods in wild-type (WT) and histamine H1 receptors knockout (H1RKO) mice with a whole-body plethysmograph and an open-circuit system by means of magnetic-type mass spectrometry. Animals were subjected to a 20-min hypoxic gas exposure (7% O₂ and 3% CO₂ in N₂) after acclimatization to the chamber for 90 min. In both groups of mice, hypoxia initially increased minute ventilation (V_E) and then decreased it. However, we found differences in the ventilatory response to hypoxia (HVR) between WT and H1RKO mice. In WT mice, the V_E response to hypoxia was higher in the dark period than those in the light period. Hypoxia decreased metabolic rate in WT mice. O₂ consumption and CO₂ excretion (V_CO2) in WT mice were higher in the dark period than light period. However, in H1RKO mice, changes in V_E between the light and dark periods were minimal. There were no changes in V_CO2 between the light and dark periods, between normoxia and hypoxia. Thus, there was a circadian light/dark cycle in the HVR in WT mice, whereas the cycle disappeared in H1RKO mice. These results suggest that H1 receptors play a part in the light/dark cycle variations in HVR via affecting metabolic control. [J Physiol Sci. 2007;57 Suppl:S214]

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We examined whether intrinsic orexin participates in the control of breathing in a vigilance state-dependent manner. Ventilation was recorded together with electroencephalography and electromyography for 6 hrs during the daytime in prepro-orexin knockout mice (ORX-KO) and wild-type (WT) littermates. Respiratory parameters were separately determined during quiet wakefulness (QW), slow wave sleep (SWS), or rapid eye movement (REM) sleep. Basal ventilation was normal in ORX-KO, irrespective of vigilance states. Hypercapnic ventilatory response during QW in ORX-KO was significantly smaller than that in WT mice, whereas the responses during SWS and REM in ORX-KO were comparable to those in WT mice. Hypoxic responses during wake and sleep periods were not different between the genotypes. Spontaneous but not post-sigh sleep apneas were more frequent in ORX-KO than in WT littermates during both SWS and REM sleep. We concluded that orexin plays a crucial role both in CO2-sensitivity during wakefulness and in preservation of ventilation stability during sleep. [J Physiol Sci. 2007;57 Suppl:S214]

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Respiratory long-term facilitation (LTF) is a long-lasting augmentation of respiratory motor output following intermittent hypoxia (IH), known to be serotonin-dependent. Serotonergic raphe nuclei receive dense projections from the orexin containing neurons in the hypothalamus. We have recently found that orexin modulates chemosensitivity in a vigilance state dependent manner. Moreover, a recent study has demonstrated that LTF is state-dependent. Therefore, we hypothesized that orexin might contribute to generating LTF after IH. To test this hypothesis, we examined ventilatory LTF in prepro-orexin knockout mice (ORX-KO) during sleep. Minute ventilation (VE) was measured in unanesthetized ORX-KO and wild-type littermates (WT) by body plethysmography before, during, and after exposure to IH (5× 5min 10%O₂) or sustained hypoxia (SH) (25min 10%O₂). EEG and neck EMG were recorded concomitantly. Respiratory data during slow wave sleep (SWS) were calculated. Whereas WT showed augmented VE during SWS for 2 hours following IH, ORX-KO showed no significant increase. Both types of mice showed no LTF after SH. We concluded that orexin played an important role for eliciting ventilatory LTF during SWS in mice. [J Physiol Sci. 2007;57 Suppl:S214]

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Considerable evidence has suggested that the midline region of the brainstem is involved in the control of respiration. Respiratory-modulated neuronal activities have been recorded in the raphe nuclei of rats, and some of these respiratory-related neurons were antidromically activated from the spinal cord. However, the role of the raphe neurons remains uncertain. Recently, it has been demonstrated that the medullary raphe nuclei form one of several brainstem regions of central chemoreception. Although there is abundant evidence for chemoreception within the ventrolateral medulla (VLM) in the intact animal, responsiveness of respiratory-related neurons in the medullary raphe nuclei to a ventilatory challenge has not been examined. The present study investigated whether the respiratory neurons in the medullary raphe nuclei are chemosensitive to the low range end-tidal CO2 levels that are relevant to normal physiological conditions. The experiments were conducted on decerebrated, vagotomized, paralyzed and artificially ventirated rats. Etracellular recordings were obtained from neurons showing firing patterns related to phrenic nerve discharge in the midline medullary tegmentum. The recorded respiratory-related neurons were tested for responsiveness to hypercarbic ventilatory challenge. The steady-state relationship between some inspiratory throughout neuronal activity was obtained by step-wise increases in the inspired CO2 level to various values (2.0-7.4%). These findings suggest that the midline caudal raphe nuclei are involved in central chemoreception. [J Physiol Sci. 2007;57 Suppl:S214]

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Rats have been extensively used as a model of obstructive lung disease although information is limited. We therefore analyzed lung function, in particular flow-volume <FV> curves, in rats using a body-surface pressure ventilator. Male Wistar rats were anesthetized without paralysis, intratracheally intubated, and kept in the supine position. The rats were ventilated using a cyclic extrapulmonary pressure change of -25 and 0 cmH₂O in the tightly sealed chamber. After rat's respiratory phase was locked to ventilator's cycle, FV curves were obtained using the pressure change of -25 and +60 cmH₂O in the same chamber. Then the procedures were repeated after bilateral vagotomy. The mean values of the forced vital capacity <FVC> and the peak expiratory flow rate <PEFR> were 7.9 ml, 80 ml/sec respectively. The shape of FV curve was quite different from that of man. It consists of an ascending limb <part I>, steeply descending limb <part II> followed by a mildly descending limb <part III>. They were 63%, 15% and 22% of FVC respectively. When the positive pressure increased, PEFR became higher and the part I became shorter, but the shape and length of part III were not affected, i.e., effort-independent part. Bilateral vagotomy increased the PEFR by 7%, but did not change the FRC significantly. After vagotomy part I became shorter while part III became longer. Vagally-mediated descending signals appear to regulate calibers of both large and small airways in the rat. [J Physiol Sci. 2007;57 Suppl:S215]

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Using optical imaging with voltage-sensitive dye in newborn rat cerebellum-pons-medulla (-spinal cord) block preparations, we recorded respiratory activities in the cerebellum, rostral ventrolateral medulla (RVLM), inferior olive and nucleus parabrachialis (NPB). Cycle triggering with C4 inspiratory activity revealed optical responses distributed in the lateral part of VII-VIII lobules, paraflocculus, lateral end of vermis and also the dorsal respiratory group (DRG) in the dorsal view of the medulla. There are two types of respiratory pathways in the cerebellum related to pulmonary afferent and respiratory rhythm. Vagus nerve stimulation activated the nucleus tractus solitarii (NTS), NPB and also ipsilateral hemisphere of the cerebellum. It is notable that respiratory neuronal activities in the cerebellum are mediated by the inferior olive and NPB. It was confirmed using DiI transport that the inferior olive projects to the paraflocculus and lateral hemisphere around VII-VIII lobule, while it receives input from the neurons below the ambiguus nucleus. The present results indicate that respiratory activities are major sources of both climbing fiber and mossy fiber inputs to the cerebellum in the neonatal stage. These activities may play a role in the activity-dependent formation of cerebellar neuronal circuitry. [J Physiol Sci. 2007;57 Suppl:S215]

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Because breath holding time (BHT) is influenced by a combined effect of alveolar PO2 and PCO2, it has been speculated that ventilatory responses to hypoxia and hypercapnia play an important roll to determine it. To clarify the contribution of respiratory chemosensitivities on breath holding (BH) performance, 15 healthy subjects were examined their BHT, the lowest SpO2, the lowest PETO2 and highest PETCO2 at breaking point (BP) of their BH trials with or without oxygen inhalation started from three different lung volumes, i.e., total lung capacity (TLC), functional residual capacity (FRC) and residual volume (RV). The data were compared with their isocapnic progressive hypoxic ventilatory response (HVR) and hypercapnic ventilatory response (HCVR). HVR showed significant negative correlation with PETO2 and SpO2 at BP as well as positive correlation with BHT.HCVR had not clear relationship with any parameters. We conclude that HCVR, but not HCVR, is a strong contributor to BH performance and that alveolar PO2 is a key determinant to BHT other than PCO2. [J Physiol Sci. 2007;57 Suppl:S215]

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A novel continuous-flow cell separation method can separate a large number of cells into five fractions according to their densities. It has been developed for application to the transfusion medicine. In the past the capability of the method has been demonstrated on the separation of nucleated cells from human peripheral blood. Nucleated cells (>10⁷) present among a large population of erythrocytes (>10¹⁰) were separated from 10ml of peripheral blood for about 100 minutes. At the last annual meeting, we reported that a variety of progenitor cells such as CFU-E, CFU-GM and CFU-GEMM were found among harvested cells without the loss of viability. In the batch method, low-density nucleated cells are separated by density gradient centrifugation from a much smaller volume of blood. Generally, a polysucrose solution, adjusted to a density of 1.077, is used for the preparation of cell samples for the colony-forming cell assay. Therefore, we compared the performance between the above two separation methods. Using the batch method, mononuclear cells trapped above the interface and within the polysucrose layer were collected and examined. CFU-GEMMs, multi-potential progenitor cells, were found not only at the interface, but also within the polysucrose layer which was usually discarded. This result suggested that the batch method may lose substantial amount of progenitor cells whereas a novel cell separation method could prevent the potential loss of progenitor cells. [J Physiol Sci. 2007;57 Suppl:S215]

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Physical exercise is used for improvement of hypertension and hyperglycemia, and for the rehabilitation of ischemic heart disease. However the effect of physical exercise on the blood fluidity is not well known. In the present study, we studied the effect of exercise at different intensity on the blood fluidity using micro channel array flow analyzer (MC-FAN) method. MC-FAN mimics the blood flow in the capillary vessel. Physical exercise at HRmax60% by bicycle ergometer for 30 minutes did not affect the blood fluidity in human volunteer. Water intake immediately after the physical exercise improved blood fluidity but the effect retuned to the control level 30 minute after exercise. However, by Wingate anaerobic test for 30 seconds, blood fluidity decreased 5 and 30 minutes after exercise in using heparin as an anticoagulant but not in using EDTA. EDTA blocks both blood coagulation and platelet agglutination. On the other hand, heparin only blocks blood coagulation and does not affect to the platelet agglutination. Therefore physical exercise at high intensity may impair the blood fluidity through platelet agglutination mechanism. These results indicate that high intense exercise may unsuitable for rehabilitation and water intake is effective for improvement of blood fluidity. [J Physiol Sci. 2007;57 Suppl:S216]

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Placenta contains many kinds of cell growth factors and cytokines. The extracts from placenta are used for several kinds of therapy including skin injury. The extracts promote the recovery of skin from injury. As a mechanism, through which the placenta extract promote skin recovery, the cell growth factors system is one of the candidates. In the process of the skin recovery, the blood supply to the injured skin area is important. The effect of placenta extract on blood supply, especially in blood fluidity, is not known. Therefore we investigated the effect of placenta extract on blood fluidity using micro channel array flow analyzer (MC-FAN) method, which mimics blood flow in the capillary vessel. The intra-peritoneal administration of human placenta extract for 4 weeks accelerated the blood fluidity in the case of using heparin as an anticoagulant, but not changed when EDTA was used for the anticoagulant. EDTA blocks both blood coagulation and platelet agglutination. On the other hand, heparin only blocks blood coagulation and does not affect to the platelet agglutination. Therefore placenta extract may improve the blood fluidity through platelet agglutination mechanism. The improvement of blood fluidity might be one of the mechanisms that placenta extract promote recovery from skin injury. [J Physiol Sci. 2007;57 Suppl:S216]

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In Japanese Oriental medicine, the concept of "oketsu" is one of the most important pathological state in diagnosis. Oketsu is considered as a stagnation of blood flow and acupuncture stimulation is often used the therapy of oketsu. However, the basic studies to elucidate the mechanism of acupuncture stimulation on blood fluidity were very few. We demonstrated that some stressors impaired blood fluidity but acupuncture stimulation (1Hz, 1 hr) increased blood fluidity in rats using MC-FAN method. We also reported the effects of acupuncture stimulation on blood fluidity differ depending on acupuncture points. It is known that opioid system is enralled in acupuncture analgesia. In this study we tested the effects of shorter stimulation period, 15 min, of acupuncture stimulation and the effect of naloxone in blood fluidity. Acupuncture stimulations were applied to ZuSanli(S36), and applied at intensity to cause light muscle twitch. The blood was collected from inferior vena cava under pentobarbital anesthesia. Acupuncture stimulation for 15 min augmented blood fluidity but naloxone did not affect the augmented blood fluidity induced by acupuncture stimulation. These results indicate that the effects of acupuncture stimulation will be achieved in a short time on blood fluidity and the effect may be caused through other mechanism which causes acupuncture analgesic. [J Physiol Sci. 2007;57 Suppl:S216]

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It is supposed that the factor affecting blood fluidity may be deformation of red blood cells, agglutination of platelets and leukocyte adhesion. We already demonstrated that some stressors impaired blood fluidity but the effect of acupuncture stimulation (1Hz, 1 hr) increased blood fluidity in rats.In this study, we attempted to investigate the influence of different stimulating frequency (1Hz, 100Hz, 60 min) on blood fluidity in rats using MC-FAN method. All the acupuncture stimulations were applied to ZuSanli (S36), and the stimulating intensity was controlled to cause light muscle twitch. The blood was collected from inferior vena cava under pentobarbital anesthesia.One Hz of Acupuncture stimulation augmented blood fluidity but 100Hz did not. These results indicate that the effect of acupuncture stimulation on blood fluidity is different depending on the frequency of acupuncture stimulation. [J Physiol Sci. 2007;57 Suppl:S216]

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Background: VECs express and secrete various anti-thrombotic molecules to maintain vascular patency. tPA, a key enzyme in fibrinolysis, is one of them and secreted as an active form. Employing total internal reflection fluorescence microscopy (TIRF-M), we clarified a unique dynamics of slow disappearance of tPA-GFP (half time of peak fluorescence: T_F1/2=9.86s) from plasma membrane (PM) after opening of its containing granules. Here we discuss possible role of PA inhibitor-1 (PAI-1) on both exocytotic dynamics of tPA and fibrinolytic activity on VECs. Method: An established cell-line of VECs was cultured and transfected with tPA-GFP, tPA(S478A)-GFP (Ser478 at active center is replaced by Ala, so as not to form complex with PAI-1) and tPA(CD)-GFP (deleted in FEK-domains). The exocytotic dynamics of these tPAs-GFP near PM and the associated changes in their activities were analyzed by TIRF-M and chromogenic assay, respectively. Results: 1) Supplemented PAI-1 shortened T_F1/2 of tPA-GFP and suppressed membrane-bound tPA activity. tPA-PAI-1 complex but not free tPA increased in cultured medium after PAI-1 addition. 2) TF1/2 of tPA(S478A)-GFP was slower than that of tPA-GFP and was not modified by the addition of PAI-1. 3) T_F1/2 of tPA(CD)-GFP was faster than that of tPA-GFP. Conclusion: tPA stays on PM after exocytosis by binding through FEK domains. PAI-1 facilitates tPA dissociation from PM by forming an inactive complex, which suppresses cellular fibrinolytic activity on VECs. [J Physiol Sci. 2007;57 Suppl:S217]

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In previous study, we demonstrated that habutobin, thrombin-like enzyme, bound to β₃subunit of integrin α_IIbβ₃on rabbit washed platelet, and then habutobin inhibited the collagen-induced aggregation. FAK tyrosine-phosphorylation (pTyr) was inhibited at an early stage of collagen-induced aggregation in the presence of habutobin. FAK contains six tyrosine (Tyr)-residues, which are phosphorylated in response to diverse stimuli. However, it was not cleared that habutobin was concerned in which tyrosine residues. In order to investigate the signal transduction pathway being concerned with habutobin-induced inhibition of platelet aggregation, the pTyr³⁹⁷and pTyr⁸⁶¹of FAK were studied from Western blotting using the antibodies against pTyr³⁹⁷and pTyr⁸⁶¹of FAK. From results of Western blot, pTyr³⁹⁷and pTyr⁸⁶¹of FAK were prominently observed on collagen-induced aggregation in the absence of habutobin. The pTyr³⁹⁷of FAK, however, was decreased in a concentration dependent manner by habutobin, and it was not detectable in the presence of high concentration of habutobin (final 5 μg/ml) on an early stage of collagen-induced aggregation. While, the pTyr⁸⁶¹of FAK was detectable in the presence of high concentration of habutobin. From these results, it was clarified that habutobin inhibited the phosphorylation of Tyr³⁹⁷of FAK. pTyr³⁹⁷of FAK included the activation of adaptor protein Shc and the p85 subunit of PI3K. It was suggested that habutobin inhibited the phosphorylation of PI3K. [J Physiol Sci. 2007;57 Suppl:S217]

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