Expression of the cardiac Na⁺/Ca²⁺ exchanger1 (NCX) changes under various pathophysiological conditions but the mechanism is not known. Recently found that in H9c2 cardiomyoblasts fluvastatin (Flv), an HMG-CoA reductase (HMGR) inhibitor, decreased NCX mRNA and protein. This effect of Flv was prevented by the presence of either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP), which are isoprenoids required for small GTP-binding protein signaling. Here, we examined the role of small GTP-binding proteins in the regulation of NCX mRNA levels in H9c2 cells. Intracellular expression of C3 toxin, a Rho-GTPase inhibitor, decreased NCX mRNA. Conversely, lisophosphatidylcholine (LPC), a Rho-mediated signaling activator, increased NCX mRNA in a C3 toxin-sensitive manner. However, overexpression of constitutive active or dominant negative mutants of RhoA did not affect NCX mRNA. RT-PCR analysis demonstrated that H9c2 cells expressed not only RhoA but also RhoB, which is isoprenylated by either FPP or GGPP. Western blot analysis showed that membrane associated RhoB was decreased by Flv, but increased by LPC. When transcription was blocked by 5,6-dichlorobenzimidazole riboside, NCX mRNA stability was decreased by Flv, but increased by LPC. These results suggest that Rho-family protein, most probably RhoB, is involved in stabilizing NCX mRNA in cardiac myocytes [Jpn J Physiol 55 Suppl:S96 (2005)]

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In sympathetic nerve endings, N-, P/Q- and L-type Ca²⁺ channels are present and contribute to the release of noradrenaline. The N-type channel is composed of several subunits including a pore forming subunit, α1B, and accessory β subunits. To investigate the role of the N-type channel in positive inotropic response of cardiac muscle resulting from stimulation of the sympathetic nerves, we have analyzed β3-null (β3-/-), β3-transgenic (β3-Tg), α1B-null (α1B-/-) and wild-type (WT) mice by measuring isovolumic contraction of left ventricle using a Langendorff apparatus. The positive inotropic effect of field stimulation was significantly reduced in α1B-/- and β3-/- mice, but enhanced in β3-Tg mice, when compared to WT mice. The positive inotropic effect of field stimulation was almost completely abolished by ω-conotoxin GVIA in β3-/- and β3+/+ mice, but was only slightly inhibited in α1B-/- mice. On the other hand, ω-agatoxin IVA had no effect on the positive inotropic effect of field stimulation. Furthermore, voltage-dependent Ca²⁺ current, recorded in isolated ventricular cells of β3-/-, β3-Tg and WT mice, had essentially similar kinetic properties and response to beta-adrenergic stimulation. These results indicate that the N-type channel plays a major role in Ca²⁺ entry in sympathetic nerve terminals, and that β3 subunit constitutes N-type channels together with α1B subunit. [Jpn J Physiol 55 Suppl:S96 (2005)]

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Objective: We have reported that high Mg applied through out the period of hypoxia can protect rat hearts from hypoxia/reoxygenation injury. The purpose of the present study was to examine when and how long high Mg applied during hypoxia is effective. Methods: Langendorff-perfused rat hearts were subjected to 1) control (CT) group: 30 min hypoxia and 30 min reoxygenation. 2) Hypoxic perfusate containing 12 mM Mg was given at the first 5 min, 3) 10 min or 4) 15 min and then perfused with normal hypoxic solution for total 30 min hypoxia and then 30 min reoxygenation. 5) Hypoxic solution containing 12 mM Mg was given at the second 5 min or 6) the last 15 min during hypoxia and the others same as 2 group. Results: Recoveries of pressure-rate product (PRP) in 2, 3, 4 groups were not different from those perfused with high Mg through out hypoxia and were all higher than those of CT at the end of 30 min reoxygenation. Recoveries of PRP in 5, 6 groups were similar as those of in 2, 3, 4 groups at the first 5-10 min of reoxygenation period, but the recovery was suppressed and there was no difference from CT at the end of 30 min reoxygenation. Conclusions: High concentration of Mg applied at the beginning of hypoxia was important and necessary for protecting rat hearts from hypoxia/reoxygenation-induced injury. [Jpn J Physiol 55 Suppl:S96 (2005)]

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Dynamic computer simulations are essential for quantitative understanding of integrated mechanisms underlying various cell functions and can provide a powerful tool to create and test working hypotheses. We have been developing mathematical models of cell functions and biodynamics by integrating experimental data and implemented cardiac cell (Kyoto model from Matsuoka et al., 2003 and 2004, LRd model from Faber and Rudy, 2000, Noble et al., 1998), an epithelial cell (Strieter et al., 1990) and a pancreatic β cell model (Magnus and Keizer, 1998) using a common biological simulation tool called simBio, which is written in Java, uses XML and solves ordinary differential equations. In analogy to biological functional structures, a cell model in simBio is composed of independent functional modules called Reactors such as ion channels and sarcoplasmic reticulum, and dynamic variables called Nodes such as ion concentrations. Interactions between Reactors and Nodes are described by the graph theory and the resulting graph represents a blueprint of an intricate cellular system. Each Reactor can be composed or improved independently and can easily be reused for different models. We have designed simBio as a tool for a simple conversion of a biological system into machine code, for an easy model expansion, and for a reuse of models as a whole or in part as sub-models in different contexts. Thus, the development of a large variety of biological models will be enhanced. The simBio package is freely available from http://www.sim-bio.org/. [Jpn J Physiol 55 Suppl:S97 (2005)]

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Once cardiac troponin I is phosphorylated following β-adrenergic stimulation, the phosphorylated troponin I is resistant to dephosphorylation in cardiac papillary muscle preparations. In the present study, we examined whether troponin I could be dephosphorylated in the whole heart system, which could be effectively washed out the drugs through micro vessels. For tension recording of the whole heart from the mouse of 4-5 weeks of age, apex was clamped with a metal clip which was connected to a tension transducer via a silk thread. Before starting a series of experiments, the whole heart was gradually stretched until near maximal tension was obtained. Application of 0.2 μM isoprenaline initially increased both tension and heart rate. With time, however, tension was gradually decreased towards control levels, while heart rate remained increased. Interestingly, tension was gradually reduced below control levels after removal of the drugs. Because phosphorylation of cardiac troponin I is known to decrease myofilament Ca ²⁺ sensitivity, the results suggest that troponin I remained phosphorylated. Continuous perfusion of the drug-free solution, however, resulted in gradual recovery of tension close to control levels, which was accompanied by gradual decline in heart rate. These results support the notion that cardiac muscles do possess protein-phosphatase activity responsible for troponin-I dephosphorylation. [Jpn J Physiol 55 Suppl:S97 (2005)]

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Atrial excitation interval (PP) is shortened during dynamic exercise by extrinsic autonomic activity. On the other hand, when atrial pacing decreases PP, the subsequent atrioventricular (AV) conduction time lengthens, suggesting a cycle length dependent mechanism of the AV node. Although the slope of the relationship between PP and the subsequent change in PR interval (ΔPR) is considered as sensitivity of the cycle length dependent AV nodal function, the effect of cycle length on the sensitivity is unknown. To examine the possible effect of cycle length on the sensitivity, the paired data of PP, RR and ΔPR were analyzed by a linear regression test. The slope of the PP-ΔPR relationship was plotted against various levels of heart rate (HR) during dynamic exercise. Seven healthy subjects performed cycle ergometer exercise to increase HR (100, 120, 140, 160 beats/min [bpm]). During resting, no significant correlation was found between PP and ΔPR. The slope of the regression line was close to zero. On the contrary, we found a strong inverse relationship between them during dynamic exercise. As cycle length shortened, the slope became steeper; the slope of the regression line was -0.38 ± 0.22 at 100 bpm, -0.74 ± 0.16 at 120 bpm, -0.83 ± 0.17 at 140 bpm, and -0.93 ± 0.05 at 160 bpm, respectively. In contrast, no significant correlation was found between RR and ΔPR during dynamic exercise. We conclude that the AV nodal mechanism that operates at a higher level of HR may cancel fluctuation in PP during dynamic exercise. [Jpn J Physiol 55 Suppl:S97 (2005)]

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[Introduction]Chronic infusion of a beta stimulant, isoproterenol induces cardiac hypertrophy. In this study we infused isoproterenol to rats for 3 or 7 days, and compared these effects on left ventricular (LV) function. [Methods]Delivery of drug was achieved by implanting osmotic minipump subcutaneously in the neck. Either isoproterenol (1.2 mg/kg/day for 3 or 7 days: Iso 3 or Iso 7) or vehicle (saline 2.4 ml/kg for 3 days: Sa) infusion was performed. One hour after removal of the minipump in Sa, Iso3 and Iso7 groups and two days after removal of the minipump in Iso3 and Iso7 groups [Iso3 (-) and Iso7 (-)], we recorded continuous LV pressure-volume (P-V) loops of in situ ejecting hypertrophied rat hearts. We obtained systolic P-V area at midrange left ventricular volume (PVA_mLVV) from a series of LV P-V loops in Sa and Iso groups. [Result] End-systolic pressure at mLVV (ESP_mLVV) in Iso7 group was significantly smaller from Iso3 group, but there were no significant differences in PVA_mLVV between Iso3 and Iso7 groups. ESP_mLVV in Iso7 (-) group was significantly smaller from Iso3 (-) group, but there were no significant differences in PVA_mLVV between Iso3 (-) and Iso7 (-) groups. Although LV end-systolic volume was significantly larger in Iso7 (-) group than Iso3 (-), there were no significant differences in stroke volume between Iso3 and Iso7 groups and between Iso3 (-) and Iso7 (-) groups. [Conclusion] Isoproterenol infusion for longer term did not cause any further effects on hypertrophy formation and reversibility. [Jpn J Physiol 55 Suppl:S97 (2005)]

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Since it is known that molecular and functional expression of CFTR on the plasma membrane is enhanced under glucose-free hypoxic conditions in neonatal rat ventricular myocytes (Jpn J Physiol 53, 357, 2003), there is a possibility that the CFTR Cl⁻ channel is somehow involved in the ischemic process. Actually, we previously reported injurious effects of CFTR Cl⁻ channel blockers and protective effects of CFTR Cl⁻ channel activators on cell viability in neonatal rat ventricular myocytes in primary culture after oxygen and glucose deprivation which mimics ischemia in vitro. Using a myocardial ischemia-reperfusion (I-R) system in vivo, we then investigated further the protective effect of CFTR activators on myocardial injury after 30-min occlusion of the left anterior descending coronary artery and 48-h reperfusion in male C57BL/6J mice. We injected a phosphodiesterase III inhibitor and PKC activator as CFTR Cl⁻ channel activators intravenously. Application of both agents, but not either alone, decreased markedly the infarct size compared to vehicle when these agents were given for 10 min before and during 30-min ischemia. Thus, it is concluded that the CFTR Cl⁻ channel activity exerts a protective action against ischemic injury in heart. [Jpn J Physiol 55 Suppl:S98 (2005)]

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Respiration (RW) modifies heart rate (HR) of mammals and avian known as the heart rate variability (HRV). The origin and its development however are not well understood. By contrast, there are little or no HRV in aquatic species such as amphibians and reptiles. In this report with fish having a simpler cardiac system, we analyzed whether the respiratory gill movement modify the heart rate. The goldfish 8cm body length was anesthetized with MS-222, and restrained in a small chamber equipped with a thermostat. The apparatus provided a constant water flow to the fish mouth. ECG was recorded with needle electrodes inserted in the body muscle near the gills. The gill movement was measured with a laser displacement sensor. Data was acquired with 200Hz rate by using Biopac MP-100 system. The trends of HR and RW were analyzed to obtain power spectra by autocorrelation-FFT method. Average HR at 25°C varied individually but ranged 60-100/min. The gill movement was nearly twice faster than the HR. The HR power spectrum gave a unique peak around 0.4Hz showing clear HRV under both anesthetized and normal conditions. The power spectrum of gill movement provided doublet peaks at 2-3Hz with a peak separation of 0.3-0.4Hz. The power spectrum of the gill movement rate calculated from RW intervals showed a single peak at the exactly same frequency as the HR spectrum. The results indicated that HRV of goldfish was originated by differential fluctuation of the gill movement. Under anesthetized condition, HR increased to the same level as the gill movement suggested that the drug suppressed the vagal tone of the efferent nerve to heart. [Jpn J Physiol 55 Suppl:S98 (2005)]

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It is considered that the Cl⁻ flux across the cell membrane is coupled with water movements, and thereby with the cellular volume regulation. To examine this hypothesis in a quantitative manner, we incorporated osmotic water flux as well as the volume regulated Cl⁻ channel (VRCC) and c-AMP activated cystic fibrosis transmembrane conductance regulator (CFTR) channel into the "Kyoto-model", a mathematical ventricular cell model. The passive Cl⁻ movement was balanced simply by adding Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), neglecting other types of Cl⁻ transporters and the steady state Cl⁻ concentration was more than 50 mM when stimulated at 2.5 Hz. The model well reconstructed the experimental findings such as, 1) the cell volume decreases in a reversible manner when the CFTR Cl⁻ channel was activated. 2) The cell swelling induced by superfusing the cell with variable osmotic solutions showed volume changes as observed in experiments. 3) During superfusion of 70% hypotonic solution showed little regulatory volume decrease (RVD) with the experimental amplitude of the volume-activated Cl⁻ current. To our knowledge, the present cardiac cell model is the first in including the Cl⁻ homeostasis. [Jpn J Physiol 55 Suppl:S98 (2005)]

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We developed a computer model of excitation-contraction (E-C) coupling (Kyoto model: Matsuoka et al., Prog. Biophys. Mol. Biol. 85 (2004) 279), in which mitochondrial oxidative phosphorylation (Korzeniewski & Zoladz, Biophys. Chem. 92 (2001) 17) was incorporated. To further extend the Kyoto model, we improved the mitochondrial energy metabolism by adding pyruvate dehydrogenation, TCA cycle (Cortassa et al., Biophys. J. 84 (2003) 2734), fatty acid β oxidation (Yugi & Tomita, Bioinformatics 20 (2004) 1795) and the reactions of mitochondrial calcium transport (Fall & Keizer, J. theor. Biol. 210 (2001) 151). A kinetic model of the pyruvate dehydrogenase complex was constructed based on experimental kinetic constants. This integrated mitochondria model was developed on the simBio system that was developed to elucidate overall biological process and reproduce dynamic cell functions on computer. In this model, pyruvate dehydrogenase, isocitrate dehydrogenase and oxoglutarate dehydrogenase are activated by Ca²⁺ increase. Therefore, the ATP production is associated with Ca²⁺ concentration change. It is demonstrated that the elevation of the cytosolic Ca²⁺, which accompanied with the stimulated contraction, activates the ATP production. This integrated mitochondria model will provide closer insights into the crosstalk between E-C coupling and energy balance of the cardiac cell. [Jpn J Physiol 55 Suppl:S98 (2005)]

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Incidence of cardiovascular diseases is lower in premenopansal women than men. We examined whether gender difference affects tolerance of Mg deficiency mice hearts against ischemia/reperfusion injury. The mice hearts were Langendorff-perfused and subjected to 15 min ischemia /30 min reperfusion. Heart rate, ventricular tension and GOT release were monitored throughout the experiment. Recovery of tension-rate product (TRP) in female mice hearts which perfused with normal Krebs solution or Mg-free Krebs solution was 77.4±3.99% and 66.78±1.63% at 15 min reperfusion, respectively and significantly higher than those in male hearts (66.9±2.78% and 50.9±2.77%, p<0.05, respectively). In mice hearts fed 2-week Mg-deficient diet, TRP of female mice recovered earlier at the beginning of reperfusion than that of male, but there was no difference at the end of reperfusion. In hearts of female mice fed 3-week Mg-deficient diet, TRP recovered to 60.9±9.4% and was not significantly different from that of normal female hearts (p<0.05) when perfused with normal Krebs solution. However, recovery of TRP was significantly lower than that of normal female mice throughout reperfusion when perfused with Mg-free Krebs solution. These results suggest that tolerance of female hearts against ischemia is higher than that of male hearts and the Mg-deficiency can decrease this tolerance. [Jpn J Physiol 55 Suppl:S99 (2005)]

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As reported previously, a majority of heart rate variability (HRV) induced by human respiration showed remarkable postnatal development especially in low birth weight neonates. In this report, we compared the frequency components of HRV in mammalian neonates (human, pig and rat) and hatched white chicken. The power spectrum was obtained by autocorrelation-FFT method from the heart rate and respiration waves. A comparison of the cardiopulmonary reflex in human and pig neonates resulted in a similar developmental progression. In case of normal human neonate, respiration induced HRV was significant within a week after the birth, however it took approximately one month in low birth weight neonate (mean gestational age 33±1 weeks). In case of pig neonates, the HRV was significant a few days after birth and developed in parallel to the body weight. Rat neonates however showed negligible HRV until days 8 and it became significant at postnatal 1 month. The white chicken indicated similar HRV development as rat, and became significant after 38days-old (17days post hatching). The results indicated that the development of the respiration linked HRV was triggered by the birth in mammalian neonates and quickly grew subsequently. The results also suggested that the developmental maturity of HRV is strongly depend on the gestational period when comparing rat and the low birth weight human neonates. The trigger for the HRV formation was however unclear. We consider the gravity exposure may play an essential role of the post natal development of the cardiopulmonary reflex. [Jpn J Physiol 55 Suppl:S99 (2005)]

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We studied effects of doxapram HCl on the cardiopulmonary system using adult Wister rats under urethane anesthesia. The drug was delivered from the inguinal femoral vein. The effect of drug was evaluated by RR, PP and QaT intervals from ECG with and without blocking of cervical vagal nerves and pre-administration of atropine. ECG and respiration wave were recorded by using Biopac MP-30 system.Frequency analysis was performed from extracted time domains characterized by RR, PP and QaT intervals with fast FFT to obtain respective power spectra. When the vagal nerves were electrically stimulated, the drug mitigated the inhibition of the respiration. A blocking of the vagal nerves bilaterally, the drug did not relief the inhibition on neither respiration nor HR. When atropine sulfate was administrated prior to the doxapram HCl, the electric stimulation resulted in the inhibition of the respiration, but no effect on the HR. Regarding the cardiac conduction times, the bilateral blocking of the vagal nerves resulted in shorten PP and PR intervals and no change in QaT. The atropine sulfate administration provided essentially the same results as the vagal nerve blocking. By contrast, doxapram HCl administration significantly elongated the QaT intervals where as the PP interval was shorten. This imply that the drug affect to elongate the repolarization time of ventricles and shorten the atrium contraction and atrio-ventriclar conduction. [Jpn J Physiol 55 Suppl:S99 (2005)]

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To study the development of regulatory function of the circulation during and after spaceflight, the 9-day-old rats were raised in microgravity on the Space Shuttle (FLT) for 16 days and age-matched rats remained on the ground (1G). On the landing day (Recovery day 0, R+0) and 30th day after landing (R+30), we performed the functional and histologic experiments in the anesthetized rats (urethane, 1.2-1.8 g/kg). We reported previously that the FLT rats on R+0 indicated the fewer unmyelinated fibers (UMF) in the afferent of aortic baroreflex (aortic nerve, AN), which related with the lower index of baroreflex sensitivity (IBRS) at the peak increase in arterial pressure (phenylephrine, iv), compared to 1G rats. On R+30, though the AN in the FLT remained fewer UMF, there was no significant difference in the IBRS between the FLT and 1G. The ratio of heart weight to body weight on R+0 or R+30 was larger in the FLT than in the 1G. The basal heart rates in the FLT were higher on R+0 and significantly lower on R+30 compared to 1G, and no statistical difference in the basal blood pressure between the two groups was observed on each day. These results suggest that the regulatory function of blood circulation can develop compensatively for the modification of AN in the young rat which returns to Earth form space while growing. [Jpn J Physiol 55 Suppl:S99 (2005)]

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In angiogenesis, the development of new blood vessels from preexisting vascular bed, it is believed that newly formed blood vessels consist of capillary. In this study, when angiogenesis was initiated by cancer cells, we showed that capillary in preexisting vascular bed changed to arteriole resulting in formation of new pathway to carry large amount of blood. [Methods] Angiogenesis was observed on mouse cornea. When cancer cells were transplanted into cornea pocket, newly formed vessels extended from vascular plexus around cornea. In the vascular plexus, we targeted a capillary which situated in the same topological situation for all individuals, and time-dependent change in the preexisting vessels and the newly formed vessels were examined histologically. [Results] The newly formed vessels grew towards cancer cells followed by an increase of blood flow in the targeted capillary. As growing of newly formed vessels, the diameter of the capillary increased. At 10-15 days after the transplantation of cancer cells, the targeted capillary was surrounded by smooth muscle cells, suggesting that the capillary changed to arteriole. And also, the newly formed vessel originated from the targeted capillary was identified as arteriole. This change of capillary to arteriole was not observed for weak angiogenesis induced by chemical cautery, suggesting that reconstruction of vascular network took place for supplying large amount of blood to cancer cells. [Jpn J Physiol 55 Suppl:S100 (2005)]

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We investigated augmentation index (AIx) of pulse waves in Kurosawa and Kusanagi-Hypercholesterolemic (KHC) rabbits aged 10-12, 22-24 and 34-36 months. Pulse and flow waves were simultaneously recorded at the ascending aorta under pentobarbital anesthesia. The time at which the 2nd zero crossing from above to below in the 4th derivative of the pulse wave in each cardiac cycle was estimated as a peak time of early systolic waves. AIx was determined as an amplitude ratio of pulse pressure to early systolic waves. Systolic and pulse pressures increased significantly with aging in the KHC rabbit group. Mean arterial pressure and total peripheral vascular resistance were significantly greater in the KHC rabbit group than in the age-matched control rabbit group, respectively. AIx did not show significant change with aging in the KHC rabbit group although it was significantly high compared to that in the age-matched control rabbit group. The early systolic waves increased significantly with age in the KHC rabbit group. There were no significant differences in mean ascending aortic flow, heart rate and stroke volume between the two rabbit groups at any age. Elastic modulus of the ascending aortic wall increased significantly with progress of atherosclerosis. We conclude that the age-independency of AIx in the KHC rabbit group is mainly caused by the progressive increase in early systolic waves due to the decrease in distensibility of the wall, in addition to the age-related increase in PP. [Jpn J Physiol 55 Suppl:S100 (2005)]

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Lactate is a metabolite of anaerobic glycolysis which generates during such as high intensity exercise and ischemia, and is used as an energy substrate in slow twitch fiber and cardic myocytes. On the effect of pH on L-type Ca²⁺ current (I_Ca,L) of cardiac myocytes, there are conflicting reports, i.e. some reports that it is suppressed and others are not. In isolated rabbit myocytes, we investigated the effect of pH and lactate on I_Ca,L by using whole-cell patch clamp technique. When pH of superfusate was decreased by HCl from 7.3 to 7.0 and 6.7, I_Ca,L (normalized by value at pH=7.3) decrease to 0.80±0.04 and 0.64±0.09. Application of lactate similarly decreased I_Ca,L. However, when pH of lactate solution was adjusted to 7.3 by administration of NaOH, I_Ca,L increased by lactate dose-dependently (0.80±0.02, 0.88±0.01, 0.95±0.04 at 3, 10, 30 mM, respectively). [Jpn J Physiol 55 Suppl:S100 (2005)]

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The mechanism controlling blood flow distribution during exercise is still unclear. We previously reported that electrical stimulation of the mesencephalic locomotor region (MLR) induces vasoconstriction in both kidney and skeletal muscle via sympathetic nervous system (SNS). In this study, we compared sympathetic and circulatory responses to stimulation of the MLR between kidney and triceps surae muscles in pre-collicular decerebrate and paralyzed rats (n=9). The MLR stimulation at 30 μA current intensity significantly (p<0.05) increased arterial pressure (+36 ± 7 mmHg) and both renal and lumbar sympathetic nerve activities (+115 ± 20 and +42 ± 10%, respectively). There was a significant difference between the magnitudes of the sympathetic activation. The stimulation significantly decreased renal blood flow (-8 ± 3%) and increased muscle blood flow (+17 ± 6%). Vascular conductance response to the stimulation, which was estimated from dividing blood flow by arterial pressure, was significantly lower in the kidney (-28 ± 5%) than in the muscles (-12 ± 4%). These results suggest that differential sympathetic outflow induces different degrees of vasoconstriction at kidney and skeletal muscle during stimulation of the MLR, and imply that a neural mechanism mediated by central command contributes to the blood flow distribution during exercise via SNS. [Jpn J Physiol 55 Suppl:S100 (2005)]

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To develop a new approach to estimate the relationship between arterial blood pressure (ABP) and other factors (renal sympathetic nerve activity (RSNA), heart rate (HR) and respiration rate), we tried to construct the ABP simulation model by a neural network (NN) algorithm without approximate equations. The ABP simulation model was developed from a layered NN algorithm using Neural Network Toolbox of MATLAB (The Mathworks, Inc.). The back propagation (i.e., supervised learning) was selected as a learning algorithm of the layered NN. RSNA, HR, and respiration rate were obtained from conscious and unrestricted Sprague-Dawley rats, and used for the NN algorithm to learn the relationship ABP and other factors. A predicted value of ABP was calculated from the learned NN algorithm. When unused values for training the NN were inputted to the learned NN, the derived ABP values consisted with measured values. This result indicates that ABP can be predicted from given RSNA, HR and respiration rate. It is possible to integrate any experimental information by applying a NN. [Jpn J Physiol 55 Suppl:S101 (2005)]

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Flow-induced dilation has been widely observed in various arterioles. Increase of intraluminal flow is known to produce vasodilation of the arterioles via release of endogenous factors, such as NO, vasodilator prostanoids (PGs), and EDHF. In this study, we attempted to investigate the mechanisms of the flow-induced vasodilation in the pressurized rabbit cerebral penetrating arterioles. The arterioles (∼100 μm in diameter) were cannulated, and suffused with MOPS solution at 37°C. The inflow and outflow cannula was connected to different reservoirs which can be moved independently. The intraluminal flow was established by changing the height of inflow and outflow reservoirs with keeping the midpoint luminal pressure constant. After an equilibration period 40min, the pressurized arteriolar preparations developed spontaneous constricted to ∼50% of their maximal diameter. Intraluminal flow produced a dilation of the arterioles. L-NAME reduced the flow-induced vasodilation. Additional administration of the K_Ca channels inhibitor charybdotoxin and apamin further reduced the dilation. In the presence of L-NAME together with charybdotoxin and apamin, the residual vasodilation was completely diminished by additional treatment with indomethacin. The removal of endothelium completely abolished the flow-induced vasodilation of the arterioles. These findings suggest that in rabbit cerebral penetrating arterioles, the intraluminal flow produced flow-induced dilation via production and release of endogenous NO, PGs, and EDHF. [Jpn J Physiol 55 Suppl:S101 (2005)]

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In the study we compared the effect of nitrous oxide mixed in volatile anesthetics and volatile anesthetics only on the L-type calcium current (I_Ca,L) of isolated rabbit ventricular myocytes and investigated the influence of phosphorilation using the whole cell configuration of the patch clamp technique. Signal myocytes were isolated using collagenase. The depolarization pulses the acquire peak current were applied every 10 seconds. The changes of I_Ca,L were measured in response to two experimental gas mixtures: 1) isoflurane (ISO) 0.5%, nitrous oxide (N₂O) 66%, and oxygen (O₂) 33%(I-N₂O); and 2) ISO 1.15%, nitrogen (N₂) 66% and O₂ 33% (I-N₂). Air was bubbled in the solution for control. Exposure to I-N₂O (n=17) and I-N₂ (n=14) reduced peak I_Ca,L to 84.4% and 85.4%, respectively. Phosphorilation by 1 micro mol isoproterenol and exposure to I-N₂O (n=13) and I-N₂ (n=12) reduced peak it to 90.8% and 87.1%, respectively. Anesthetic effect of I-N₂O and I-N₂ are 1 minimum alveolar concentration (1MAC). These gas mixtures suppressed calcium channel without phosphorilation similarly, but with phosphorolation differently. In same anesthetic level cardiac sensitivity for catecholamine seems to be higher under N₂O combined with ISO than ISO only. [Jpn J Physiol 55 Suppl:S101 (2005)]

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Aim: Chronic infusion with a beta-stimulant, isoproterenol induces cardiac hypertrophy associated with increased collagen production. The aim of the present study was to evaluate the composition of O₂ consumption of myocardial slices at mechanically unloaded conditions from rat isoproterenol-induced hypertrophied rat hearts. Method: Osmotic mini-pump containing isoproterenol was subcutaneously implanted for 3 days to induce hypertrophy. O₂ consumption per minute (mVO₂) was measured without and with 1-Hz field stimulation by oximetric system. Delta mVO2 corresponds to O₂ consumption for Ca²⁺ handling in excitation-contraction (E-C) coupling. Results: Basal metabolic mVO₂ was significantly smaller than that of the normal heart slices. Delta mVO₂ increased in an extracellular Ca²⁺ concentration-dependent manner. However, there were no significant differences in the relations of Ca²⁺ concentration-response in delta mVO₂ between normal and hypertrophied hearts. In contrast, an SR Ca²⁺ pump inhibitor, cyclopiazonic acid (CPA) significantly decreased delta mVO₂, but the decrease in delta mVO₂ was significantly smaller in hypertrophied hearts (about 45%) than in normal hearts (about 70%). Conclusion: The decrease in basal metabolic mVO₂ is due to the increased collagen production and the decrease in CPA-induced decrease of delta mVO₂ is due to changes in re-circulation fraction of intracellular Ca²⁺ in Ca²⁺ handling mechanisms in E-C coupling. [Jpn J Physiol 55 Suppl:S101 (2005)]

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A special feature of our cardiac cell model (Kyoto Model) is the implementation of a muscle contraction model, namely the NL model from Negroni and Lascano (1996; J Mol Cell Cardiol, 28, 915-29), in addition to the membrane excitation. To introduce cooperativity in crossbridge (Xb) activation, new contraction models have been developed which all include the NL model Xb mechanics part but consist of different Ca kinetics parts. In Hybrid Model (HM) 1 this is a 6 state system modified from Rice Model 3 (Rice et al., 1999; Am J Physiol, 276, H1734-54). In HM2 the kinetics part is composed of a 2 state troponin (Tn) activation, a 2 state system for tropomyosin and a 4 state myosin Xb formation with cooperativity mechanisms similar to the Sachse Model (Sachse et al., 2003; Int J Bifurc Chaos, 13, 3561-78). The simulated twitch contraction time course of HM2 is more comparable to experimental data than that of HM1. However, the force-length-Ca relation has been found in experiments to be steeper (Hill coefficient n=6-7) than in HM1 (n=3.4) and HM2 (n=1.8). In HM3 Tn activation has been extended to 3 states according to new experimental data suggesting that a conformational change of TnI needs to be considered for a full activation of myofilaments (Robinson et al., 2004; J Mol Biol, 340, 295-305). HM3 looks promising concerning the force-length-relationship, a characteristic especially important for a heart model. [Jpn J Physiol 55 Suppl:S102 (2005)]

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We investigated effects of swimming exercise on endothelial function in Dahl salt-sensitive rats with risk factors for atherosclerosis, i.e. hypertension and/or hypercholesterolemia. 359 rats were randomly assigned 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 groups: sedentary group (SD) and swimming exercise group (ED), 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 production and superoxide production in endothelial cells around aorto-renal bifurcation by using confocal laser-scanning microscopy. The ratio of NO production to superoxide production of diet group S was the smallest quantity, followed by SC, C, MF among swimming exercise groups. Chronic swimming exercise decreased growth rate of body weight in all diet groups significantly, blood pressure in MF and C significantly, tended to decrease trigriseride in all diet groups, and increased HDL in all diet groups significantly. In conclusion, chronic swimming exercise decreased the physiological risk of atherosclerosis in the rats with or without risk factors for atherosclerosis, and hypertension is the highest factor for superoxide production in endothelial cells around aorto-renal bifurcation in the exercising experimental rats. [Jpn J Physiol 55 Suppl:S102 (2005)]

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During development of a new drug, it is essential to check effects of the drug on the electrical activity as well as the mechanical function of human heart. However, it is difficult to obtain human cardiac cells. Therefore, developing a mathematical model of excitation-contraction (E-C) of human ventricular cell is awaited to complement wet experiments and also to targeting specific mechanisms for drug development. So far, several types of human cardiac cell models are available for membrane excitation, and only one E-C coupling model was published by Sachse et al. (2003), which is based on the excitation model provided by Priebe and Beuckelmann (1998). The electrical model developed by ten Tusscher et al. (2004) is based on recent data obtained from human ventricular myocytes and human cardiac channels expressed in cultured cells. Thus, we develop a human E-C coupling model combining the model from ten Tusscher et al. (2004) with cardiac contraction models from Negroni and Lascano (1996) and Sachse et al. (2003). We improved intracellular calcium handling model to reproduce calcium transient measurements obtained from human cardiac tissue. Parameters of the combined contraction model were optimized to fit human experimental results, and a frequent-dependency of contraction was tested. [Jpn J Physiol 55 Suppl:S102 (2005)]

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A global system model of the coronary circulation is implemented with a lumped parameter approach to simulate circulation characteristics in an ischemic heart due to a stenosed coronary artery. In the lumped model, the hemodynamic elements of the coronary system are represented as a series of equivalent elements in an electric circuit. The increase in the coronary collateral vasculature in response to the cardiac tissue demands for blood flow is a major long-term regulation process of the coronary circulation in an ischemic heart. We use a simple control theory to simulate this long-term regulatory process of the coronary circulation and combine it with the hemodynamic elements represented in a lumped parameter approach. The severity of coronary artery stenosis is approximated by varying the stenosis resistance in the electric circuit. For code verification, the computational results for a normal coronary circulation are compared with the experimental results. To assess the effect of collateral circulation on the blood supply to an ischemic heart, we conduct parametric studies varying the stenotic severity. Numerical results show that the coronary blood flow and stenotic severity are strongly coupled. It is observed that collateral blood flow also increases as coronary stenosis severity increases. [Jpn J Physiol 55 Suppl:S102 (2005)]

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Tissue regeneration by stem/progenitor cells has been recognized as a maintenance or recovery system of many organs in the adult, and analysis of therapeutic applications for the damaged organs have used ischemic hindlimb or myocardium mostly. Here we examined for new approach that the potential impact of reduced oxygen tension in vivo using a new ischemia model. Direct measurement of oxygen tension allowed its to define three discrete tissue segments with increasingly ischemic microenvironments in nude mice. In this model, a peninsular shaped incision was made dividing epidermis, papillary dermis, and skeletal muscle from the systemic circulation. Following 1 week ex-vivo expansion of endothelial progenitor cells (EPCs) isolated from adult human, EPCs were administrated intravenously into animals for in vivo cell transplantation (n=6). The animal model determined by direct measurements at 4 points were successfully established demonstrating increasingly oxygen tension in ischemic area. The treated animals demonstrated significantly less toe necrosis compared to controls in peripheral perfusion measured by Laser Doppler at day 14. In the measurement of oxygen tension level, the treated animals induce significantly compared to controls in ischemic tissue. Moreover, the ischemic area transplanted EPCs were higher uptake VEGF after cell transplantation compared to controls. In summary, this animal model may be used convenience for analysis of tissue regeneration. [Jpn J Physiol 55 Suppl:S103 (2005)]

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Purpose: To reveal the cause of the transmural difference in crossbridge (CB) dynamics and the effect of the preload on that. Methods: We recorded X-ray diffraction images of the isolated and perfused rat left ventricle (LV) at SPring-8 with LV pressure (LVP) and analyzed transmural CB duration (CBD) and myofilament lattice spacing (MLS) using transmural variations of epi-myocardial (CBD_E and MLS_E) and mid-myocardial (CBD_M and MLS_M) myofilament orientations at the end-diastolic pressure of 0 and 20 mmHg (EDP₀ and EDP₂₀). Results : During contraction, normalized LVP and transmural CBDs temporally harmonized well each other. However, at both EDP₀ and EDP₂₀, CBDEs were significantly longer than CBD_Ms (EDP₀, CBD_E; 195±16 msec, CBD_M; 172±19 msec, EDP₂₀, CBD_E; 226±27 msec, CBD_M; 205±23 msec) and also MLS_Es were significantly smaller than MLS_Ms (EDP₀, MLS_E; 36.6±1.3 nm, MLS_M 37.2±1.4 nm, EDP₂₀, MLS_E; 35.7±1.4 nm, MLS_M; 36.9±1.5 nm). MLS is though to be reciprocal proportion to sarcomere length and it decreased with increasing EDP. Conclusion: The transmural difference in CB dynamics may be partially due to the difference in the amplitude and duration of twitch contraction of regional myocardium at regionally different sarcomere length. [Jpn J Physiol 55 Suppl:S103 (2005)]

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Stress can be classified into mental and physical ones. We previously reported that mental stress might be evaluated by the heart rate (HR) deviations from the regression line between HR and physical activity (PA). In the present study, we studied relationships between subjective stress and HR obtained from a portable monitor and answer-sheet during free moving. Subjects were healthy adults. Continuous recordings of HR and PA were performed for 24 hours or more by using a portable monitor. Their PAs were assessed by the number of counts beyond an acceleration of 0.02G. Physical and mental stresses were marked on the sheets of the Visual Analog Scales when their physical behaviors were briefly described. In most cases, HRs were well correlated with physical accelerations during exercise. However, physical stress did not show statistically significant correlations with HR and PA, possibly depending upon tolerance to variable kinds of PA. On the other hands, in a half of the subjects, mental stress exhibited significant correlations with their physical stress. We could not find any significant relationships between mental/physical stresses and parameters derived from spectrum analysis of HR. In addition to HR, another biomarkers may be needed to quantify mental and physical stresses. The other possible factors relating to the stress will be discussed by using several kinds of questionnaires. [Jpn J Physiol 55 Suppl:S103 (2005)]

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Background: Hypertrophied RV (HRV) due to PH results in RV heart failure. We hypothesized that mechanical stress of HRV impairs coronary microcirculation with increased superoxide. Methods: Male SD rats (8 weeks old) were divided into two groups (Normal: n=40 and HRV: n=49). In HRV group, Monocrotaline (MCT) was administered at 5-week age to induce PH. RV coronary arterioles (<100μm) were visualized in vivo with our intravital videomicroscope. After cyclooxigenase blockade, vascular responses to ACh were examined under 4 conditions, Control, L-NAME, L-NAME + tetraethylammonium (TEA) and SOD. Difference of%dilation between Control and L-NAME was used as index for NO-contribution, and that between L-NAME and L-NAME + TEA as EDHF. Superoxide in RV was measured by lucigenin chemiluminescence. Results: RV systolic pressure was greatly increased in HRV (75±12 vs 33±3 mmHg, p<.05). In HRV, ACh-induced vasodilation was significantly reduced (by 51%, p<.05). NO-mediated vasodilation in HRV was greatly decreased (by 74%). EDHF-mediated vasodilation in HRV was robust, but decreased (by 43%). Eventually, relative vasodilatory contribution of EDHF increased (59 vs 51%). Superoxide was significantly elevated in HRV (330±31 vs 255±88 cpm/mg, P<.05). Decreased vasodilation in HRV was improved after SOD (by 33%, p<.05). Conclusion: NO-mediated vasodilation of coronary arterioles was impaired in HRV, while vasodilatory ability of EDHF was relatively robust. Increased superoxide in HRV may be involved crucially in NO dysfunction. [Jpn J Physiol 55 Suppl:S103 (2005)]

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Nitric oxide (NO) is produced by NO synthase (NOS) in the endothelial cells and acts as a signaling molecule for vasodilator in the blood vessels. Recently, growing experimental and clinical studies demonstrated that the decrease of the bioavailability of NO causes the initiation and progress of vascular dysfunction associated with diseases such as hypertension and diabetes mellitus. Thus, intra-aortic measurement of NO would provide valuable insights into the bioavailability of NO in the arterial vessels and pathophysiology of the vascular diseases. In the present study, using a newly-developed catheter-type NO sensor, we measured intra-aortic plasma NO concentration in vivo. An NO sensor was encased and fixed in a 4-Fr catheter. The sensor was then located in the thoracic aorta via the femoral artery through a 7-Fr catheter in anesthetized dogs. Infusion of acetylcholine (10 μg/kg) increased base-to-peak plasma NO level in the aorta by 2.4±0.4 nM (n=7). After 20-min infusion of N-methyl-L-arginine (NOS inhibitor), changes in plasma NO concentration in response to acetylcholine were attenuated significantly (1.8±0.4 nM, P<0.003, n=7). In conclusion, the newly-developed catheter-type NO sensor successfully measured acetylcholine-induced changes in intra-aortic plasma concentration of endothelium-derived NO in vivo. [Jpn J Physiol 55 Suppl:S104 (2005)]

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We hypothesize that it is critical factor for controlling red blood cell velocity (V_RBC) in longitudinal capillary running parallel to muscle fiber whether intercapillary anastomoses are non-functional, i.e. no flow of red blood cells, or functional capillaries. We examined the role of capillary network, especially, a number of anastomoses and its lumen diameter on V_RBC in atrophied rat muscle with 2-wk hindlimb suspension using a pencil-lens intravital microscopy and a confocal laser scanning microscope (CLSM). V_RBC in capillary and anastomosis of soleus was significantly increased by atrophy, while V_RBC in capillary and anastomosis of gastrocnemius was not significantly changed. CLSM images demonstrated lumen diameter of anastomoses in atrophied gastrocnemius was not changed rather increased in spite of significant reduction of lumen diameter of longitudinal capillaries, suggesting the anastomoses are functional capillaries. The lumen diameter of longitudinal capillaries and anastomoses in atrophied soleus was significantly decreased, suggesting the anastomoses are non-functional capillaries. The present results suggest that intercapillary anastomoses seem to affect V_RBC due to plasma skimming. [Jpn J Physiol 55 Suppl:S104 (2005)]

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We have developed a method, based on the oscillometric method for indirectly measuring blood pressure, that determines an arterial stiffness index (ASI). The method quantifies the stiffness of the intima and tunica media of the arterial segment. We provide a theory for the ASI measurement with the experimental data. The mechanism of occurrence of the change in the amplitude of pulsation during the blood pressure measurement using oscillometric method can be elucidated on the non-linear Pressure-Volume (P-V) curve of the artery. The properties of the tunica media and the outer membrane makes a non-linear P-V relation. Thus, the amplitude of arterial volume pulsation is varied with transmural pressure which is the difference between arterial pressure and cuff pressure. The property of the artery with stiff tunica media makes a flat-topped graphical pattern of amplitudes of arterial volume variations and that for the normal artery produce a sharp-peaked pattern. The width of a flat-topped pattern was calculated so as to produce the ASI. We have experimentally evaluated this theory using rabbit artery. The right carotid artery was subjected to pressure sufficient to cause occlusion several times, damaging the endothelial cells, while the left carotid artery was left intact. After feeding the rabbits a diet including 5% cholesterol for 6 weeks, the P-V relations in both segments were measured. The results strongly supported the theory for the indirect measurement of ASI. [Jpn J Physiol 55 Suppl:S104 (2005)]

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Recent study from our laboratory demonstrated that the vestibulo-sympathetic reflex plays an important role in controlling arterial pressure during gravitational change in conscious rats. Gravity acts on the circulatory system to reduce arterial pressure by reducing venous return, and vestibular system senses the gravitational change and increases sympathetic nerve activity to prevent hypotension. This type of circulatory stress occurs in human daily life upon postural change from spine to upright. However, the role of vestibular system in controlling arterial pressure in human is not clear. Thus the goal of the present study was to examine this. Postural change and gravitational change was produced by tilt-bed and parabolic-flight, respectively. To disturb the normal vestibular sensation, random galvanic vestibular stimulation was applied, and arterial pressure response to postural or gravitational change was compared with or without galvanic vestibular stimulation. [Jpn J Physiol 55 Suppl:S104 (2005)]

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The two mechanisms can be candidates for ventricular arrhythmia in Ca²⁺-overloaded heart: (1) oscillation of membrane potential induced by frequent Ca²⁺ waves, resulting in triggered activity; (2) inhibition of gap junction channels which disturbs conductivity of action potentials (AP), resulting in increased probability of re-entry. In this study, we examined if propagation of APs are affected in situ in Ca²⁺ overloaded myocytes. Papillary muscles dissected from guinea pig ventricle were loaded with fluo-3 or rhod-2. Sequential fluorescence images of surface cells were obtained using the laser-scanning confocal microscope as reported previously (Kurebayashi et al. Am. J. Physiol. 287. C1646, 2004). In intact muscles, APs propagated without delay over a field of view of 300x300μm. In Ca²⁺-overloaded muscles which had been stimulated at a high frequency under anoxic condition, cells themselves were already less responsive to electrical stimulation and often showed AP alternans. Surprisingly, they sometimes showed a delay of ∼100ms in cell-to-cell propagation of APs. In the presence of a high concentration of heptanol, similar delay was observed. These results mean that conduction delays in several cells within a small area (∼0.5 x 0.5 mm) are enough to allow re-entry in Ca²⁺-overloaded heart. Inactivation of Na⁺ channels and/or inhibition of gap junctions may underlie the delayed conductivity. [Jpn J Physiol 55 Suppl:S105 (2005)]

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The symptoms of hypertension and/or hypercholesterolemia were improved by continuous exercise. In these diseases, the activity of the reactive oxygen species, that is oxidative stress, rises remarkably. In this reserch, the changes of superoxide generation via heart, kidney, liver and aorta abdominals were measured and the controlling effect of continuous exercise was evaluated. Dhal salt-sensitive rat (16 weeks) inducted hypertension and/or hypercholesterolemia with continuous exercise, that was swimming, were used. Samples were obtained from delivered organs by punch biopsy and superoxide generation was measured by chemiluminescense of Lucigenin. As results, superoxide generation via heart of hypertension rat was controlled to the same level of control rats. It was suggested that the rise of superoxide generation promoted by hypertension was inhibited by continuous exercise. [Jpn J Physiol 55 Suppl:S105 (2005)]

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Although it has been reported that habitual exercise increase cardiovagal baroreflex sensitivity, the precise mechanism responsible for its modulation remains unknown. Some previous studies indicate a relation between arterial compliance and cardiovagal baroreflex sensitivity. Also, it has been reported that habitual exercise increase arterial compliance, so that the baroreflex sensitivity increse. However, it is unclear vascular stiffness fully explain the modulation of the baroreflex sensitivity. We hypothesized that habitual exercise may also modulate the neural component of the arterial baroreflex. The aim of this study is to examine mechanical and neural component of the arterial baroreflex between sedentary and exercise trained men. Mchanical component of the arterial baroreflex was estimated by the mechanical transduction of pressure into barosensory stretch (Δcarotid vessel diameter/ΔAP). Neural component of the arterial baroreflex was estimated by the neural transduction of stretch into vagal outflow (ΔR-R interval/Δcarotid vessel diameter). Integrated cardiovagal baroreflex sensitivity was assessed by carotid and radial AP and HR. [Jpn J Physiol 55 Suppl:S105 (2005)]

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Objective–Chronic thiamine deficiency is believed to cause changes in the myocardium that could provoke arrhythmias. This study was undertaken to explore whether thiamine deprivation has a role in cardiac arrhythmogenesis. Methods–Isolated Langendorff-perfused rat heart preparations were used to determine whether or not thiamine-deprivation is associated with changes in cardiac function. We examined hearts isolated from thiamine-deprived, rescued and control rats. We measured heart rate, diastolic and systolic tension, contraction and relaxation rates, and coronary flow. Whole-cell voltage clamp was performed in isolated cells to measure L-type Ca²⁺ current. Results–The hearts from thiamine deficient rats did not degenerate into ventricular fibrillation during 30 minutes of reperfusion. The anti-arrhythmogenic effects were characterized by the arrhythmia severity index. The values for control, thiamine-deficient and rescued were 11 ± 1.0, 2 ± 0, and 10 ± 1.6 respectively. Ca²⁺ current density was smaller in thiamine-deprived cardiac myocytes. Our results suggest that hearts from thiamine-deprived rats did not experience irreversible arrhythmias. Conclusion–Thiamine deficiency seems to exert opposing effects on myocardial viability during ischemia-reperfusion insults with cardioprotection predominant over myocardial damaging. [Jpn J Physiol 55 Suppl:S105 (2005)]

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Morphological changes and those in the peptidergic innervation were compared between the carotid bodies of the rats exposed to hypercapnic hypoxia (10% O₂ and 6-7% CO₂ for 8 weeks) and those exposed to normoxic hypercapnia (16% O₂ and 6% CO₂ for 8 weeks). In the sections stained with hematoxylin eosin,the carotid body was found to be enlarged several-fold in hypercapnic hypoxia, although the rate of vascular enlargement of carotid body was smaller in hypercapnic hypoxia than those in the isocapnic and hypocapnic hypoxia previously reported. In the chronically hypercapnic hypoxia, the density of parenchymal NPY fibers was significantly increased, that of VIP fibers was unchanged, and that of SP and CGRP fibers was decreased. In the normoxic hypercapnic carotid body, there were no morphological changes in comparison with the normoxic control carotid body. In addition, there were no distinvtive changes in the peptidergic innervation. These results suggest that the different levels of CO₂ do not cause the morphological changes in the rat carotid body and the changes in the peptidergic innervation within the carotid body in normoxic conditions. Considered together with our recent findings, CO₂ may have some additive effects on the chemoreceptor organs in hypoxic conditions. [Jpn J Physiol 55 Suppl:S106 (2005)]

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We examined whether the chemoreceptor reflex in prepro-orexin gene knockout mice was blunted or not, and if so, whether supplementation of exogenous orexin restored the abnormality. A cannula for intracerebroventricular injection to the lateral ventricle was implanted to the isoflurane-anesthetized mice together with electrodes for recording electroencephalogram and electromyogram. Ventilation was recorded by whole body plethysmography after recovery period of at least 7 days. After recording of baseline breathing for 20 min, either orexin or vehicle was intracerebroventriculary injected and hypercapnic (5% or 10% CO₂, 21% O₂, residual N₂) or hypoxic (15% or 10% O₂) gas mixture was introduced into the recording chamber for 5 min. Data were examined for only awake period because sleeping is known to distort chemoreflex sensitivity. Hypercapnic ventilatory responses but not hypoxic responses were attenuated in orexin knockout mice as compared to those in the wild-type littermates. Intracerebroventricular injection of orexin partially restored the hypercapnic chemoreflex in the mutant mice. Our findings suggest that orexin plays a crucial role for CO₂-sensitivity at least during awake periods. [Jpn J Physiol 55 Suppl:S106 (2005)]

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Previous evidence suggests that medullary raphe nuclei may be involved in the regulation of breathing. This study was undertaken to examine the effects of change in low range end-tidal CO₂ levels on the discharges of respiratory related neurons recorded in the medullary midline. The experiments were performed on pentobarbitone-anesthetized, paralysed, vagotomized and artifically ventilated rats. Extracellular recordings were made from neurons showing respiratory related activities in the midline medullary tegmentum. A total of 25 respiratory neurons were classified into Inspiratory (I) throughout (n=16), I-frequency modulated (n=3), Pre-I (n=2), Post-I (n=2), Expiratory (E) (n=1) and E-frequency modulated (n=1) neurons based on their firing patterns in relation to the phase of respiration. They were located in the raphe magnus, obscurus and pallidus. Changes in neuronal discharge of these respiratory neurons were assessed by recording ongoing activities of the neurons during times when the ventilator was stopped. When end-tidal CO₂ concentration was raised (from 5±0.5% to 8±0.5%), 3 types of response of raphe respiratory neurons were shown to the change in end-tidal CO₂ levels:firing rate 1) increased (n=18), 2) decreased (n=5) and 3) no response (n=2). These results suggest that the midline caudal raphe nuclei are involved in central chemoreception. [Jpn J Physiol 55 Suppl:S106 (2005)]

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Gasping is a robust respiration pattern with slow rhythm that animals of various species show when subjected to ischemia/hypoxia, hypercapnea, or decapitation. Spontaneous recovery from coma due to stroke or cardio/pulmonary failure can occur as a result of gasping. Gasping is characterized by a rapid activation of phrenic nerve activity and inspiratory muscle, but little is known of the central mechanisms of its initiation and maintenance. Interestingly, decapitation-induced gasping was significantly impaired in the absence of the Kir6.2 subunit of the ATP-sensitive potassium (K_ATP) channel in KO mice: only 2.9±0.8 gasps of shorter duration being observed in Kir6.2-deficient (KO) mice (n = 23) while wild type mice exhibited 11.6±0.8 gasps (n = 23, p < 0.0001). In hypoxia (5.3-5.8%O₂) under urethane anesthesia, the respiratory interval of wild-type mice increased rapidly to above 0.8/s in 10.0±2.9 s (n = 5) after a tachypnea while KO mice showed a much slower increase of 39.2±7.0 s (n = 5, p < 0.005). In addition, phrenic discharge of KO mice during hypoxia failed to show the rapid activation characteristic of gasping, and resembled normal respiration except for the last 2 to 3 gasps just before cessation. Hypercapnea (80%CO₂)-induced gasping also was significantly impaired in KO mice. These results suggest that K_ATP channels may be critically involved in the central regulation of gasping. [Jpn J Physiol 55 Suppl:S107 (2005)]

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Cells are sensitive to a change of the extracellular pH and respond to it through detection of the H⁺/HCO₃⁻ level in extracellular fluid. However, little is known about molecular details induced by acidosis such as intracellular pathways and gene expression. Here we describe properties of gene expression, protein interaction and DNA binding activity of basic region leucine zipper (bZIP) transcription factor Maf and FosB during extracellular acidification. When cells were incubated with low pH medium, the expressions of small Maf proteins and FosB were clearly increased in an extracellular pH-dependent manner and expressed transiently with a peak after 1-2 h after stimulation. Immunofluorescence and protein binding studies indicated that MafG was partially co-localized with FosB in the nucleus and MafG can form heterodimers with FosB. Moreover, we found that MafG-FosB complexes are able to bind to AP-1 consensus sequence, TGACTCA. To investigate whether extracellular acidification influences to dimerization and DNA binding activity of MafG and FosB, extracellular pH of cultured cells was decreased from 7.40 to 6.80. The decrease in extracellular pH led to enhanced dimerization of MafG with FosB leading to augmentation of the DNA binding activity of the heterodimer to AP-1 consensus sequence. These results suggest that MafG-FosB complexes are involved in transcriptional regulation in response to extracellular acidification. [Jpn J Physiol 55 Suppl:S107 (2005)]

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Recently, we developed a fictive cough model of the guinea pig to investigate the neuronal mechanisms underlying cough reflex and to assess the site of action of antitussive agents (Brain Res., Ohi et al., 2004). To clarify the functional importance of the nucleus tractus solitarius (NTS) in cough reflex, projection of the superior laryngeal nerve (SLN) afferents to the brainstem and the response induced by microstimulation of the NTS were studied. Strong fluorescence was detected ipsilaterally in the commissural NTS and the rostral NTS. At coronal slices, the fluorescent granules, lines and patches were located in the interstitial, ventromedial and dorsal areas of the NTS. Some activity was found in the contralateral dorsal area of the commissural NTS. Microstimulation of the rostral NTS, at which the strong fluorescence was detected, induced an increase in the inspiratory discharge of the phrenic nerve that was immediately followed by a large burst discharge of the iliohypogastric nerve. This serial response was identical to that induced by electrical stimulation of the SLN. The response induced by either NTS or SLN stimulation was suppressed by intravenous injection of codeine. Furthermore, the SLN-induced response was inhibited by microinjection of codeine into the ipsilateral NTS but not into the contralateral NTS. Ipsilateral lesion of the rostral NTS abolished both NTS and SLN-induced responses. These results suggest that the NTS plays essential roles in production of cough reflex and is possible sites of action of central antitussive agents. [Jpn J Physiol 55 Suppl:S107 (2005)]

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In order to clarify functional roles of central histamine during hypoxic responses, we examined breathing pattern and metabolism under hypoxic gas inhalation in unanaesthetized wild type (WT) and H1 receptor knockout (H1RKO) mice. Male mice were measured for breathing pattern with a whole body plethysmograph, and metabolism with an open-circuit system. Hypoxic gas (7% O₂, 3%CO₂) inhalation increased respiratory rate (RR), tidal volume, and minute ventilation (VE), reached a peak in 2-3 min, and then decreased in both genotypes, indicating hypoxic depression. H1RKO mice, maintained higher levels in RR and VE during hypoxic depression than WT mice. VO₂ and VCO₂ were decreased by hypoxic gas inhalation in both mice, but VO₂ during hypoxic depression was significantly higher in H1RKO mice than WT mice. The VE/VO₂ ratio during hypoxic depression was equal in both genotypes, indicating no difference of genotypes in control of ventilation and metabolism. The effects of brief hyperoxia exposure (100% O₂) after hypoxia (7% O₂) on ventilation were examined in anesthetized, spontaneously breathing mice. No significant difference was found in ventilatory responses to brief hyperoxia between genotypes, showing possible involvement of central mechanisms in altered hypoxic responses in H1RKO mice. Thus, H1RKO mice are blunted for hypoxic depression characterized by a decrease in the peak ventilation and hypometabolism, which results from alteration of central mechanism including pons, medulla, hypothalamus and other higher brain. [Jpn J Physiol 55 Suppl:S107 (2005)]

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Spontaneous augmented breaths (SABs) occur sporadically during normal breathing and increase the episodes (f_SAB) in hypoxia. We examined the effects of MK-801, a N-methyl-D-aspartate (NMDA) antagonist, on the SABs in unanesthetized rats in normoxia and hypoxia. The rat, which had been implanted diaphragm electromyogram (EMG_dia) electrodes, was measured ventilation (by the barometric technique) and the SABs (by EMG_dia) before and after an intravenous MK-801 injection (3 mg/kg), in normoxia and hypoxia (12%O₂). MK-801 increased respiratory frequency (f_R) of normal breathing due to a shortened expiratory time (T_E) in normoxia, but decreased the f_R due to a prolonged inspiratory time (T_I) in hypoxia. On the SABs, by MK-801 injection, although inspiratory and expiratory phases in each SAB were modified as seen in normal breathing, the f_SAB was not modified as seen in the f_R in both gaseous conditions. In conclusion, NMDA receptor-mediated processes may determine cycle timing for individual normal breath and SAB, but may not crucial for determining the f_SAB in unanesthetized rats. [Jpn J Physiol 55 Suppl:S108 (2005)]

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Regulators of G protein signaling (RGS) proteins are a family of proteins, which accelerate intrinsic GTP-hydrolysis on Gα subunit and play crucial roles in the physiological regulation of G-protein mediated cell signaling. If RGS proteins were active unrestrictedly, it would completely suppress G protein-mediated signalings. Thus it is important to understand how the actions of RGS proteins are regulated in various physiological conditions. In our recent successive studies, we have shown that the action of RGS protein is inhibited by binding of a kind of membrane phospholipid, phosphatidylinositol-3,4,5,-trisphosphate (PI(3,4,5)P₃), and this inhibition is cancelled by alternate binding of Ca²⁺/calmodulin (CaM). Here we show the physiological interaction of RGS protein and calmodulin in a living cell successfully detected by fluorescence resonance energy transfer (FRET) techniques. RGS4 and CaM are fused with two distinct fluorophore, i.e., Venus (yellow fluorophore) and ECFP (cyan), respectively, and expressed in HEK293 cell lines. FRET signals from ECFP to Venus were greatly increased upon the elevation of intracellular Ca²⁺, suggesting the association between RGS4 and CaM. The time-course of the alteration of FRET signals upon [Ca²⁺]_i elevation is compatible with that of the relaxation of cardiac G-protein-activated K⁺ channel current, which we have shown to be attributable to the CaM/PI(3,4,5)P₃-dependent regulation of RGS protein. This study clearly presents the evidence that RGS protein and CaM physiologically interact each other in living cells [Jpn J Physiol 55 Suppl:S108 (2005)]

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5-HT in the hypoglossal motor nucleus stimulates the activity of the genioglossus muscle, which is mediated via 5-HT₂ receptors. In the present study, the role of 5-HT₂ receptors in the dorsomedial medulla oblongata (DMM) in airway resistance to CO₂ was studied. Each mouse was anesthetized with pentobarbital sodium i.p., for insertion of a microdialysis probe in the DMM, and placed in a double chamber plethysmograph for at least 1.5 hour to recover from anesthesia and to acclimatize to the chamber. Two respiratory flow curves were recorded from the body and head chambers to obtain specific airway resistance, while extracellular fluid was collected at 1.2 μl/minute every 25 minutes. Extracellular 5-HT concentrations were analyzed with ECD-HPLC. 1 x 10⁻⁵M fluoxetine and LY-53857 were dialyzed via a microdialysis probe in the DMM. CO₂ in O₂ was changed every 8 minutes in a stepwise series of 5, 7 and 9%. Positions of the microdialysis probes were identified by coronal sections stained with neutral red. The 5-HT concentration in the DMM during 100% O₂ inhalation with fluoxetine plus LY-53857 co-perfusion was similar to that with fluoxetine perfusion. However, specific airway resistance during 100% O₂ inhalation with fluoxetine plus LY-53857 co-perfusion was significantly higher than that with fluoxetine perfusion, which was suppressed by CO₂ gas inhalation. These results suggest that 5-HT in the DMM increases airway patency via 5-HT₂ receptors, and that interference of 5-HT₂ receptors in the DMM causes accumulation of CO₂ and stimulates airway patency through other pathways. [Jpn J Physiol 55 Suppl:S108 (2005)]

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Previous study showed that orexin-A and orexin-B, which are known as feeding and sleep-wakefulness regulators, activated a calcium-dependent potassium current in mouse peritoneal macrophages. To extend the previous observations, we examined effects of orexins on the phagocytic activity, which is one of the main functions of macrophages. To define the effects of orexins in immunocompetent cells, the effects of orexin-A and orexin-B on phagocytosis in mouse peritoneal macrophages were examined. Orexin-B induced an enhancement of phagocytosis of latex particles in a dose-dependent manner. Orexin-A is less effective than orexin-B. Even in Ca²⁺-free solutions, phagocytosis was enhanced by orexin-B. The potassium channel blocker quinine inhibited the enhanced phagocytosis by orexin-B, and 4-aminopyridine and tetraethylammonium suppressed the enhanced phagocytosis less effectively. These present results suggest that orexins can enhance the phagocytosis of macrophages mediated by potassium channels. The results showed that orexins could modulate some physiological functions of mouse peritoneal macrophages, which may be mediated by the activation of potassium channels. Furthermore, ion channel activation may be related to phagocytosis and other physiological functions such as proliferation and differentiation of the macrophages. In addition, the present study also suggests that orexins, feeding and sleep-wakefulness regulators, are also immunoregulators for macrophages. [Jpn J Physiol 55 Suppl:S109 (2005)]

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