The purpose of the present study is twofold: 1) to elucidate the effect of applying lower body positive pressure (LBPP) on circulatory and musculoskeletal parameters, and 2) to compare the changes in circulatory and musculoskeletal parameters during treadmill exercise in human subjects with and without LBPP. We hypothesized that application of LBPP decreases net ground reaction force (GRF), apparent body weight, and that a low level of LBPP causes minimal changes in cardiovascular parameters and produces beneficial effects on musculoskeletal parameters. Ten male and five female subjects, who gave their informed consents, participated in this study. After body weight of the subjects was measured, the pressure inside LBPP chamber was increased until it reached 15 mmHg above the atmospheric pressure, resulting in 30-35 kg decrease of the net GRF. Electrocardiogram, arterial blood pressure, skin blood flow in the lower extremity, skin temperature, electromyogram from the gastrocnemius, gait pace were monitored during treadmill exercise (4 km/h walking). Exposure to LBPP did not change significantly the heart rate, arterial blood pressure, skin blood flow in the leg, and gastrocnemius EMG during rest. On the other hand, exercise-induced increases in heart rate and EMG were reduced by exposure to LBPP. These results suggest that LBPP reduces the net GRF effectively without changing cardiovascular functions and that the reduction of net GRF may play a beneficial role in rehabilitation. [J Physiol Sci. 2007;57 Suppl:S192]

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The essential oils have been used to reduce tensions from some stressful conditions that might cause insomnia. The aim of this study was to examine whether frankincense oil affects plasma levels of corticosterone (CS) and biological antioxidant potential (BAP) as well as glutathione (GSH) concentrations of sleep deprived rats. Male SD rats (12-h light and 12-h dark) were used. Group A (control) and group B rats (sleep deprived between 9:00 to 15:00) received one drop (0.05ml) of jojoba oil as a control or frankincense oil as an experimental oil at every hour from 10:00 to 15:00 under sleep monitoring. Blood samples were collected to measure the CS by ELISA and the reactive oxygen metabolites and BAP by FRAS, respectively. The GSH levels in CSF were measured by HPLC. Blood CS levels were higher in sleep-deprived rats than non-sleep deprived rats. Frankincense induced dose-dependent suppression of CS comparing with the jojoba treated rats. Frankincense treated rats also demonstrated dose dependent suppression of BAP comparing with the jojoba treated rats. Body weight decreased in the frankincense group. The amount of wakefulness was increased, and slow wave sleep was decreased by frankincense oil comparing to the jojoba oil, while paradoxical sleep did not show any change. These results suggest that frankincense oil more likely improve the anti-stress effect, anti-dote effect and sleep compensation effect. [J Physiol Sci. 2007;57 Suppl:S192]

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Purpose: The university student chiefly takes an examination of the national exam for clinical laboratory technician at the end of the university. It is difficult to look for the student who suffers from the academic (=mental) stress because there is no index with a good academic stress. Then, it examined it by using some stress indices aiming to know the method that was able to be passed by a lot of students in this research.Methods: Subjects: 57 healthy students were given informed written consent. Chemical stress index: Levels of salivary cortisol and chromogranin A. Psychological evaluation: Affect grid developed by Russell JR, Weiss A and Mendelsohn GA. Measurement time: Ten months ago and before two days examining.Results: The saliva Chromogranin A concentration increased significantly from 2.26pg/dl ten months ago to 6.01pg/dl two days before though the saliva cortisol did not change significantly. In Affect grid, the standard of Pleasantness decreased from 5.96 to 4.78 in significant though the standard of Awake did not change. Moreover, there was an opposite correlation between the result of the examination (score) and the saliva chromogranin A concentration (r=-0.302).Conclusions: The result of the person with high chromogranin A concentration examination before two days tended to be low. It was suggested that the measurement of the saliva chromogranin A concentration immediately before the examination for making a lot of students pass the examination and caring mentally be effective. [J Physiol Sci. 2007;57 Suppl:S192]

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On Earth, signals from otolith organs can be interpreted either as linear motion or as tilt with respect to gravity. In microgravity, static tilt will no longer give rise to changes in otolith activity. However, linear acceleration as well as angular acceleration stimulates the otolith organ. Therefore, during adaptation to microgravity, otolith-mediated response such as eye movements would alter. Flatfish provide a natural model for the study of adaptive changes in the vestibulo-ocular reflex. During metamorphosis, vestibular and oculomotor coordinate of flatfish displaced 90 degrees about the longitudinal body axis. Therefore, it is expected that microgravity induce the sensory mismatch in adult flatfish. In this study, we analyzed the eye movements of flatfish for body tilting. The eye movements for body tilting along the different body axis were video-recorded. The vertical and torsional eye rotations were analyzed frame by frame. In normal flatfish, tilting the fish body to the left produced an upward rotation in the left eye and a downward rotation in the right eye. For head up or head down tilting, both eyes rotated downward or upward respectively. Furthermore, the properties of torsional eye movements for body tilt were almost the same as that in goldfish. These results suggested that flatfish perfectly adapted gravity environment and sacculus and/or lagena were important for otolith-ocular reflex. [J Physiol Sci. 2007;57 Suppl:S192]

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Estrogen receptors (ERs) are widely expressed in the brain, where estrogen modulates central nervous functions. In the present study, we examined whether estrogen alters the stress-induced c-Fos expression through ER α in the brain of ovariectomized (OVX) rats. Female rats aged 9wk were divided randomly into three groups: sham-operated (S), OVX plus placebo-treated (P) and OVX plus estrogen-treated (E) groups. The rats in the P or E group were implanted with pellets containing either placebo or 17 b-estradiol. After 4wk, each group of rats underwent cage-switch for 30 min and was sacrificed at 60 min after the cessation of the stress. The rats were perfused, and the brains were immunohistochemically processed for c-Fos and ER α. The cage-switch stress significantly increased the number of c-Fos positive cells in the paraventricular hypothalamic nucleus (PVN), arcuate hypothalamic nucleus, medial amygdaloid nucleus, dorsomedial hypothalamic nucleus (DMD), locus coeruleus (LC) in the P and E groups, but not in the S group. However, the numbers of c-Fos positive cells in LC, PVN and DMD were lower in the E group than the P group. Moreover, ER α positive cells were observed in PVN and LC in the both groups, though the number of these cells were higher in the P group. These findings suggest that estrogen would affect stress-induced neuronal activation at least partly through ER α. [J Physiol Sci. 2007;57 Suppl:S193]

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Activation of nicotinic acetylcholine receptors is thought to enhance activity-dependent synaptic plasticity processes. The purpose of this study was to assess the effect of systemic administration of nicotine on higher brain function and other physiological system in conscious rats. At least 3 days before study, Wistar male rats (n=12) were chronically implanted with a probe to measure brain blood flow, and catheters to measure systemic arterial pressure and to intravenously infuse nicotine. In addition, electrodes were also placed for measurements of electroencephalogram (EEG), renal and lumbar sympathetic nerve activities (RSNA and LSNA), trapezius electromyogram (EMG) and electrocardiogram (ECG). The EEG was analyzed by Fast Fourier Transform in 10-s epochs. Either 50 or 25 µg of nicotine was administrated through venous catheter after an hour observation, in which rats kept quiet. The response of brain blood flow after nicotine injection was characterized by a rapid elevation and followed by a gradual decrease. The power spectrum of EEG was broadly inhibited after the nicotine injection. Similarly the RSNA, LSNA, EMG and heart rate were rapidly increased, and then steeply decreased. These variables were inhibited more than an hour, while the response of mean arterial pressure was increased and decreased but remained elevated. These data shows that intravenous administration of nicotine causes biphasic responses, such as a rapid elevation and followed by suppression, in both central and sympathetic nervous system. [J Physiol Sci. 2007;57 Suppl:S193]

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Recently we showed that chronic estrogen replacement in ovariectomized rats significantly attenuates cardiovascular responses to mild psychological stress induced by cage-switch. Our hypothesis is that these estrogen effects are mediated by nitric oxide (NO) in the brain involved in decreasing sympathetic output. In this study, we investigated whether estrogen affects the stress-induced activation of nitric oxide-producing neurons in the paraventricular hypothalamic nucleus (PVN) in ovariectomized rat. Female rats aged 9 wk were ovariectomized and assigned randomly to placebo-treated (P) and estrogen-treated (E) groups. The P and E groups were implanted with pellets containing either placebo or 17 β-estradiol (1.5 mg/60-day release). After 4 wk, each group of rats underwent cage-switch for 30 min and was sacrificed at 60 min after the cessation of the stress. The rats were perfused, and the brain sections were processed for c-Fos immunohistochemistry and NADPH-diaphorase histochemistry. In the medial parvocellular PVN (mpPVN), cage-switch stress significantly increased c-Fos positive neurons with a greater extent in the P than E group. Moreover, stress-induced elevation in the percentage of double-stained neurons (c-Fos + NADPH-d positive neurons) was significantly larger in the E than P group. These findings suggest that estrogen modulates the activation of nitric oxide-producing neurons in the mpPVN. [J Physiol Sci. 2007;57 Suppl:S193]

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There are many studies reporting that enriched rearing environment has the advantages in promoting brain plasticity and learning. In the present study, we examined the effects of environmental enrichment during rearing on sexual and social behavior after maturation in male Wistar rats. Following weaning on day 25, 7 males were housed together in a large cage with several objects and equipments, and other 7 males were kept in conventional cages without objects (2 or 3 males / cage). When tested in a narrow cylinder (20 cm diameter) or in a round open field (60 cm diameter), males reared in enriched environment showed increased immobility (decrease in locomotor activity) than males reared in conventional cages. Following the activity tests, 3 successive sexual behavior tests were weekly carried out with receptive females that were ovariectomized and primed with estrogen and progesterone. The rearing environment had no effects on male sexual behavior, locomotion and anogenital investigation were significantly decreased in males reared in enriched environment. The decrease in these social activities culminated in diminished soliciting behavior (hopping and darting) shown by stimulus females. Current data suggest in combination with our earlier result that motivational states in social behavior regulated by dopamine may be affected by enriched environment in early life. [J Physiol Sci. 2007;57 Suppl:S193]

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Long-term cold exposure resulted in good effects on lipid metabolism and energy balance of obese rats. However, different effects were observed between obese rats: OLETF and obese Zucker rats, in food intake, weight-reducing effect through growth, morphology of brown adipose tissue (BAT) and amount of adipocytokines in blood and adipose tissues. In this time we analyzed changes in blood lipids and hormones, and expressions of UCP1, GLUT4, insulin receptor, leptin receptor and PGC1 of BAT by Western blot analysis in OLETF and LETO (lean) rats. Methods: Male OLETF and LETO rats aged 7 weeks were allocated into 3 groups; 25°C for 11 weeks (W group), 10°C for 11 weeks (C group) after acclimation to 10°C for 9 weeks and 25°C for 2 weeks (D group). BAT was isolated after 11 weeks acclimation and analyzed mass value and morphological change. Results: Serum lipid levels of OLETF were higher than those of LETO, similar tendency as in Zucker fatty rats but their degree was not so remarkable. They were decreased during cold acclimation. Changes in amount of UCP1 in BAT during cold acclimation and deacclimation were similar to those of Zucker rats, but amount of UCP1 in W group was relatively high in OLETF and increase during cold acclimation was not so strikingly. Expressions of GLUT4, insulin receptor and PGC1 of BAT were differentially expressed among OLETF/LETO rats and obese/lean Zucker rats during cold acclimation and deacclimation. [J Physiol Sci. 2007;57 Suppl:S194]

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We reported sinusoidal acceleration induced heart rate (HR) modulation synchronized to the acceleration frequency in case of human and rat. The synchrony may be caused by a sinusoidal fluid pressure change or by a vestibulo-cardiovascular reflex, however the detail was not well evaluated. We examined the contribution of the vestibular system using goldfish in water where the body fluid may not be directly affected by external acceleration due to the neutral buoyancy. The fish was subjected to linear and rotational accelerations with various frequencies and amplitudes in different postures. Two types of goldfish with and without vestibular function were also tested to verify the vestibular contribution. The off-centered rotation was applied in two different postures relative to the rotation vector keeping a constant maximum radial acceleration and varying the tangential acceleration. The synchrony was evaluated by cross correlation power spectra of HR. In case of linear acceleration fixing body in both horizontal and vertical positions, a synchrony was observed in the HR power spectrum at the acceleration frequency as well as the off-centered rotational stimulation. When the head was on the rotational axis, the synchrony was not observed in neither of two directions. The synchrony was diminished without vestibular function. It strongly implied that the input from vestibular system may play an important role in the heart rate variability. [J Physiol Sci. 2007;57 Suppl:S194]

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ATP is known to be released from injured or inflamed tissues, and to excite/sensitize nociceptors to heat and possibly to mechanical stimulation. We have reported that the extensor digitorum longus (EDL) muscle of rats 2 days after lengthening contraction (LC) is mechanically hyperalgesic and that the C-fiber receptors recorded from this hyperalgesic muscle have facilitated sensitivity to mechanical stimulation. To examine the possibility that this facilitated mechanical sensitivity is mediated by ATP released from the muscle, we measured ATP release from the muscle upon mechanical stimulation. Rats underwent LC of flexor muscle (mainly, EDL muscle) of the lower hindleg under anesthesia. Two days later, EDL muscle was excised under anesthesia and superfused with Krebs-Henseleit solution. The superfusate was sampled at 0.8 ml/min and its ATP concentration was measured with Luciferin-Luciferase method. At first we examined ATP release to repetitive application of 20 g force/10 s stimulation, which was used for the study of nociceptor characteristics, with a servo-controlled mechanical stimulator with intervals of 30 min. ATP release was clearly decreased on repetition of the mechanical stimulus. Then we examined stimulus response relationship by stimulating the muscle five times at 5, 10, 20 and 40 g forces at a rate of 10 g/s. ATP release was increased roughly stimulus-strength dependently in this range. Disappointingly, these results were not different from our previous results on normal unexercised muscles. [J Physiol Sci. 2007;57 Suppl:S194]

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Complaints of chronic pain patients may increase when the low-pressure system comes. We have demonstrated that artificially lowering the barometric pressure within the range of weather change intensifies pain-related behaviors in neuropathic rats. Furthermore, chemical destruction of inner ear organs inhibited this aggravating effect, suggesting that the barometric pressure sensors are located in the inner ear. In the present study, we extracellularly recorded neural activities in the vestibular nuclei with a glass microelectrode and examined the effect of lowering barometric pressure (LP, 40 hPa/ 8 min) in normal anesthetized rats. A set of stimuli (electrical stimulation of the vestibular nerve, rotation of the body or caloric test) was also applied to characterize neurons. Location of the neurons within the vestibular nuclei was histologically confirmed after the experiment. Five out of 17 recorded neurons increased their discharge frequency upon LP. There were three response patterns: 1) discharge rate increased along LP and vice versa, 2) discharge increased along LP but remained increased upon normalization of the pressure, 3) fluctuating discharge frequency was stabilized during LP. One neuron responded to the LP only, while others responded to one or more other stimuli (rotation or caloric test). Our present results indicate that the vestibular organs have an important role in detecting barometric pressure change. [J Physiol Sci. 2007;57 Suppl:S194]

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Our lab reported that LP by 10hPa aggravated pain-related behaviors in chronic pain rats, and this effect was blocked by surgical lumbar sympathectomy. LP also increased systemic blood pressure (BP) and heart rate (HR) in unanesthetized normal rats. Therefore, we hypothesize that SNA plays an important role in pain-aggravation by LP. To prove this hypothesis, I directly recorded SNA in decerebrated rats and examined its change by LP. Normal male SD rats (BW 350-450g) were decerebrated rostral to the superior colliculus under pentobarbital anesthesia. SNA was recorded from the left sympathetic nerve trunk between L3 and L5. LP exposure (30hPa / 10min) was done after removal of anesthesia and under artificial ventilation after immobilization with gallamine. LP tended to increase lumbar SNA and HR. However, these changes were not reversible during observation period (–1.5hr), and no significant change in BP was induced by LP. Increased SNA and HR during LP in decerebrated rats are consistent with the previous observation in normal conscious rats. Our preliminary experiment with decerebrated rats without abdominal surgery for SNA recording, showed reversible increases in BP and HR during LP. This might indicate that abdominal surgery might be the cause of no BP change and irreversibility in HR and SNA changes. [J Physiol Sci. 2007;57 Suppl:S195]

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Diabetes mellitus is a metabolic disorder associated with structural and functional alteration of various organ systems including the central nervous system. One major contributor is the Diabetes related alterations in the function of the blood-brain barrier (BBB). This study aimed to investigate whether the permeability of the BBB is affected in the brains of persons with diabetes mellitus (DM).On postmortem frozen sections of prefrontal and temporal tissue of cortex of 25 diabetic patients and 25 controls, immunohistochemical stainings were carried out using specific antibodies against three proteins (PAL-E, IgG and albumin), which are considered as markers for the vascular permeability status of the BBB.Patchy or uniform microvascular PAL-E staining was observed in the capillaries of the prefrontal and temporal cortex parenchyma, in 44% of DM patients but just 4% of controls. In the diabetic patients , PAL-E staining co-localized with microvascular staining for albumin and IgG.Increased BBB permeability was detected in patients with diabetes . Increased permeability of the BBB might account for some of the cerebral effects of type II diabetes, and so possibly also for the effect of other conditions that affect the microvasculature (like hypertension), on the brain. [J Physiol Sci. 2007;57 Suppl:S195]

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The aim of this study was to investigate sympathetic nerve responses to activation of muscle reflex in heart failure (HF), induced by myocardial infarction (MI). Ligating surgery of coronary artery was performed in rats. Echocardiogram was utilized to measure fractional shortening (FS), an index of the left ventricular function. We examined renal and sympathetic nerve activities (RSNA and LSNA, respectively) during 1-min repetitive (1- to 4-s stimulation-to-relaxation) contraction or stretch of the triceps surae muscles in decerebated rats. During these interventions, the RSNA and LSNA responded synchronously as tension was developed. The RSNA or LSNA response to contraction was significantly greater in rats with MI (n=7) with FS < 30% than in control animals (n=8) with FS > 40% (RSNA: +127±13 vs. +75±11%, P<0.05; LSNA: +75±9 vs. +36±9% P<0.05; Developed tension 475±70 vs. 487±44 g, P>0.05). In the same manner, larger RSNA or LSNA response to stretch was also observed in MIs than in controls (RSNA: +109±21 vs. +55±12%, P<0.05; LSNA: +62±9 vs. +21±4%, P<0.05; Tension: 502±46 vs. 532±43g P>0.05). The data demonstrate that HF exaggerates sympathetic nerve responses to muscle contraction as well as stretch. Muscle mechanoreflex may play a dominant role in enhancing the reflex sympathetic nerve responses in HF. We suggest that muscle afferents mediated sympathetic outflows contribute to the abnormal distribution of cardiac output seen during exercise in HF. Supported by NIH R01 HL075533 & 078866 (JL) and HL060800 (LIS). [J Physiol Sci. 2007;57 Suppl:S195]

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Sarcalumenin (SAR), a Ca²⁺-binding protein located in the longitudinal sarcoplasmic reticulum (SR) of striated muscle cells, interacts with sarcoplasmic reticulum (SR) Ca²⁺-ATPase protein (SERCA2a). Since aging is associated with a decrease in SERCA2a activity that causes cardiac dysfunction and impaired exercise capacity, SAR may play a role in the impairment. To test the hypothesis, we herein examined cardiac function by using a high-fidelity pressure transducer and exercise performance by treadmill test in aging SAR-deficient mice. As previously demonstrated in senescent human and animals, cardiac function and exercise performance were decreased in both senescent wild-type and SAR-deficient mice (over 18-month old) when compared with young adult mice (2-4-month old). However, the declines were much greater in senescent SAR-deficient mice. Our data demonstrated that systemic ablation of SAR caused a significant reduction in cardiac function and exercise ability in aging animals, indicating that SAR plays an important roles in preventing the age-related decline in striated muscle performance. [J Physiol Sci. 2007;57 Suppl:S196]

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Bathing in carbonated springs is considered to dilate skin blood vessels and increase blood flow. To improve circulation as a part of skin and health care, carbon dioxide was percutaneously supplied to skin by applying carbon dioxide sheets (containing 1000 ppm or higher carbon dioxide dissolved in water), and the microcirculatory dynamics was investigated. In healthy adult subjects, carbon dioxide sheets stored in a gas-impermeable container were applied to the flexor aspect of the forearm, and changes in the microcirculatory dynamics were investigated by direct observation of subepidermal microvessels using a Capillary Scope System equipped with a small sensor and a light source, which irradiates the skin surface with indirect light. The vascular diameter was measured, and the blood flow was measured by the venous obstruction method using infrared spectroscopy. In addition, the blood flow was measured by 2-dimensional image mapping using a laser speckle imaging system. Compared to sham sheets, the carbon dioxide sheets significantly increased the diameter of the subepidermal microvessels, and increased the skin blood flow from 5 minutes after the application. Development of commercial products using the sheets for the improvement of blood circulation for use in the skin and health care fields may be possible. [J Physiol Sci. 2007;57 Suppl:S196]

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Periodic function can be displayed as summation of sinusoids using Fourier series. In the present study, arterial pressure waveforms during infusion of vasoactive agents were analyzed with Fourier series. Abdominal aortic pressure, abdominal aortic flow in adult anesthetized rats during infusion of Angiotensin II (ANG), Phenylephrine (PHE), Nitroprusside (NP), and Nitroglycerine (NG) were measured. Peripheral vascular resistance (R) was calculated with the pressure and the flow. The pressure waveforms were analyzed with the Fourier series. Harmonic distortion (HD) and mean frequency were calculated from the amplitudes and the frequencies derived by the series. Mean frequency was normalized with the fundamental frequency, which corresponds to pulse rate per second. As results, change in R was inversely proportion to change in HD, and change in normalized mean frequency (NMF). Increase in R with vasopressor i.e., ANG and PHE infusion lowerd the HD and the NMF. Decrease in R with vasodilator i.e., NG and NP infusion made the HD and the NMF larger than those during control. That means the waveform of the arterial pressure during vasopressor infusion is getting similar to the sinusoid of the fundamental frequency, but the waveform is complicated during vasodilator infusion. Thus, changes in HDR and NMF are possibly used as indices of changes in R, but further study is needed to clarify the reason of changes in the waveform. [J Physiol Sci. 2007;57 Suppl:S196]

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Previous studies conducted in our laboratory demonstrated that the vestibular system plays a significant role in controlling arterial pressure (AP) in conscious rats during transient conditions of microgravity. The vestibular system is known to be highly plastic, and on exposure to a different gravitational environment, the sensitivity of the vestibular system-mediated AP response might be altered. To test this hypothesis, rats were maintained in 3-G or a normal 1-G environment for 2 weeks, and the AP responses to free drop-induced microgravity were determined. In 1-G rats, the microgravity increased the AP by 37 ± 3 mmHg; this fluctuation in AP was significantly attenuated by vestibular lesion (VL) (24 ± 3 mmHg) or body stabilization (29 ± 2 mmHg). Thus, the microgravity-induced pressor response was mediated by both the vestibular and nonvestibular systems; the input of the latter system was blocked by body stabilization. In the 3-G rats, the pressor responses were significantly suppressed compared to those in the corresponding 1-G rats, i.e., the AP increased by 24 ± 2 mmHg in freely moving 3-G rats, by 10 ± 4 mmHg in 3-G rats with VL, and by 13 ± 4 mmHg in stabilized 3-G rats. Furthermore, there was no difference between the 1-G and 3-G rats in terms of the pressor response induced by stressors such as a loud noise or an air jet. These results indicate that pre-exposure to 3-G for 2 weeks induces plasticity in both vestibular- and nonvestibular-mediated AP responses to microgravity. [J Physiol Sci. 2007;57 Suppl:S196]

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Disturbances in the energy balance between demand and supply in cardiac muscle alters the kinetics of Na⁺/K⁺ pump because the Na⁺/K⁺ pump is powered by ATP. Although the change of the pump activity affects various cellular functions, such as membrane excitation, contraction, volume regulation, intracellular [Ca²⁺] and pH regulation, the relationship between free energy of ATP hydrolysis (dG_ATP) and pump-mediated cellular functions is not fully understood especially in quantitative aspects. In this study, we aimed at understanding this relationship quantitatively by computational approach. First, we developed the Na⁺/K⁺ pump model by adding key experimental findings to the Smith-Crampin model, in which ion transport is coupled to dG_ATP. Our model successfully reproduces a variety of electrophysiological characteristics of Na⁺/K⁺ pump, including the dependence on [Na⁺]_i and [K⁺]_o as well as the current-voltage relationship under various ionic conditions. By using ventricular cell model, in which the new Na⁺/K⁺ model was incorporated, we induced the energy shortage condition by inhibiting NADH synthesis rate of mitochondria. This resulted in an increase of [Na⁺]_i together with an increase in metabolites ([ADP] and [Pi]). When the inhibitory effect of metabolite accumulation on the Na⁺/K⁺ pump activity became more than the compensatory effect of [Na⁺]_i increase, an irreversible increase in [Na⁺]_i was observed. We will discuss the cellular mechanisms responsible for irreversible [Na⁺]_i accumulation. [J Physiol Sci. 2007;57 Suppl:S197]

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To quantitatively understand intracellular Na⁺ homeostasis during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling cascade based on a model by Saucermann et al. (2003), and implemented it into a comprehensive ventricular cell model (Kyoto model, Takeuchi et al. 2006), which can reconstruct membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, and osmotic volume change. An application of isoproterenol resulted in the shortening of action potential duration, the increases in both Ca²⁺ transient and cell shortening, and the decrease in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. It has been experimentally reported that intracellular Na⁺ concentration decreases during the β1-adrenergic stimulation. Our model reproduced the decrease in Na⁺ under the condition of 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase of Na⁺ affinity of Na⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at physiological beating rate because of larger Na⁺ influx. We concluded that dynamic change of the intracellular Na⁺ concentrations as well as Ca²⁺ significantly contribute to the inotropic effect of β1-adrenergic stimulation on cardiac excitation-contraction coupling. [J Physiol Sci. 2007;57 Suppl:S197]

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(Backgrounds) Several papers reported that HERG channel block, namely blockade of rapid component of delayed rectifier K⁺ channel (IKr-blockade) occasionally brings on triggered action potentials (TA) in ventricular cells under pathological conditions. However, these mechanisms are not fully understood. In the present study, we analyzed the relationship between the IKr-blockade and TA using the comprehensive cardiac cell model, Kyoto Model. (Methods) A steady state condition of 1) Ca²⁺ overload and 2) low K⁺ conductance were established. Under each of two states, the effects of decreasing the conductance of IKr were examined. (Results) 1) Under Ca²⁺ overload, the Ca²⁺ influx via the L-type Ca²⁺ channel was increased, while the enhancement of Na⁺/K⁺ pump activity interferes with the Ca²⁺ overload via the Na⁺/Ca²⁺ exchanger. We found that TA was induced when [Ca²⁺] in sarcoplasmic reticulum is increased beyond a critical level. 2) Under low K⁺ conductance, the mean membrane potential per one cycle was gradually shifted to positive. When the open probability of L-type Ca²⁺ channel during diastole was increased beyond a critical level, another type of TA was induced. (Conclusions) These mechanisms of abnormal excitability are in good agreement with the view proposed by the previous experimental studies, and the Kyoto model can provide hypothetical quantitative mechanisms. [J Physiol Sci. 2007;57 Suppl:S197]

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The intracellular Ca²⁺ dynamics is modulated in the cardiac myocytes according to the rate of stimulation, and this property contributes to the accelerated relaxation during diastole with increasing heart rate. The Ca²⁺/calmodulin-dependent protein kinase II (CaMKIIδ) may have a central role in adjustment of this frequency dependent modification of the intracellular Ca²⁺ dynamics, but detailed mechanisms remain unclear. Therefore, we prepared a computational CaMKII model which well reproduces the kinetics of CaMKII-Ca²⁺/calmodulin interaction and the frequency dependency of CaMKII autophosphorylation described in vitro experiments. In order to predict CaMKII dynamics and mechanism of Ca²⁺ dynamics regulation in vivo, we incorporate this model into cardiomyocyte model of guinea pig, KyotoModel. In this model, CaMKII is set to phosphorylate L-type Ca²⁺ channel, RyR channel, phospholamban, and SERCA pump. In consistency with experimental data, the amplitude of cytosolic Ca²⁺ transient becomes larger and the decay phase was accelerated in parallel with the frequency-dependent activation of CaMKII. From these simulation data, the mechanism governing the frequency dependency of contraction in cardiomyocytes will be discussed. [J Physiol Sci. 2007;57 Suppl:S197]

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In physiological experiments and clinical practices, the Pressure-Volume (PV) loop of left ventricle is important to evaluate the cardiac contractility and energetics. The end-systolic PV relation (ESPVR) is almost linear and its slope is well known as representing the maximum elastance (Emax) to evaluate the cardiac contractility. Furthermore, the pressure volume area (PVA) represents the total mechanical energy per beat and correlates with myocardial oxygen consumption. In order to study mechanical energetics in human cardiac myocyte, we developed a human circulation model which consisted of a preload, an afterload and a sphere heart. The pressure of the heart was converted from the tension of human ventricular myocyte model based on Kyoto Model according to Laplace's law. Our circulation model produced the PV loops with each afterload and showed a linear ESPVR. PVA was proportional to oxygen consumption like many experimental results. We calculated ATP consumptions per beat by the Na-K pump, SERCA and the cross-bridge. ATP consumptions by Na-K pump and SERCA was almost independent from PVA, while the ATP consumption by the cross-bridge was proportional to PVA. [J Physiol Sci. 2007;57 Suppl:S198]

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Thromboxane A ₂ (TxA₂), one of vasoconstrictive lipid mediators, is involved in systemic anaphylaxis. On the other hand, nitric oxide (NO) is known to attenuate anaphylactic venoconstriction of the presinusoids in isolated guinea pig and rat livers. However, it is not known whether NO attenuates TxA₂-induced hepatic venoconstriction. We therefore determined the effects of L-NAME, a NO synthase inhibitor, on TxA₂ mimic U-46619-induced venoconstriction in blood- and constant flow -perfused isolated livers of mice, rats and guinea pigs. The sinusoidal pressure was measured by the double occlusion pressure (Pdo), and was used to determine the pre- (Rpre) and post-sinusoidal (Rpost) resistances. U-46619 concentration-dependently caused predominant presinusoidal constriction in rat and guinea pig livers at 0.1-3 µM and in mouse livers at 1-3 µM. L-NAME pretreatment selectively increased the basal Rpre in rat and guinea pig livers. L-NAME also augmented the U-46619-induced increases in Rpre, but not in Rpost, in rat and guinea pig livers. This augmentation was stronger in rat livers than in guinea pig livers at high concentrations of 1-3 µM U-46619. However, L-NAME did not augment U-46619-induced venoconstriction in mouse livers. In conclusion, in rat and guinea pig livers, NO may be released selectively from the presinusoids in response to U-46619, and then attenuate the U-46619-induced presinusoidal constriction. In mouse liver, U-46619-induced venoconstriction is weak and not modulated by NO. [J Physiol Sci. 2007;57 Suppl:S198]

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A liver serves as a reserver of blood. Intrahepatic blood is mobilized to systemic circulation during hemorrhagic shock. Greenway et al. measured liver volume using ultrasonic crystals to estimate changes in blood volume in cat liver (Am. J. Physiol, 262: G934-39. 1992). They used a pair of crystals to detect the changes in liver thickness. Two sets of two crystals on a liver lobe gave different thickness responses. They concluded that the thickness responses at different sites on a liver are variable and the results is unreliable. Therefore we tried, using four ultrasonic crystals to form a tetrahedron, to measure the volume changes of the tetrahedron to reduce the influence of the variability in the responses at different site. Isolated rat livers were portally perfused with diluted blood (Hct 10%) at constant flow rate. The liver weight was continuously measured. Ultrasonic crystals were applied to the surface of the middle lobe of a liver. One crystal was attached on the lower surface of the lobe and the other three on the upper surface to form a triangle. Each length of six pairs of crystals was measured simultaneously to calculate the volume of the tetrahedron. When the blood flow was decreased, the liver weight and each length of the pair of crystals were decreased. Though each change in the thickness was different at each region, the volume of the tetrahedron correlated with the change in liver weight. We concluded that this method using four crystals can reliably monitor changes in liver volume. [J Physiol Sci. 2007;57 Suppl:S198]

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We previously reported that the response of the portal venous pressure (Ppv) of isolated perfused mouse livers to vasoconstrictors such as norepinephrine was much weak as compared with that of other mammals of rat, rabbit, and guinea pigs. The purpose of this study to determine the responsiveness of mouse hepatic vessels to various vasoconstrictors such as norepinephrine (Nor), Angiotensin II (Ang II), Endotheln-1 (ET-1) and vasopressin (VP) in anesthetized spontaneously breathing BAB/c mice weighing 25g. The systemic arterial pressure (Psa), Ppv and central venous pressure were directly and continuously measured, and the above-mentioned vasoconstrictors were injected into the portal vein as a bolus repetitively at the doses ranging 0.01-100 nmole/kg. In response to each vasoconstrictor studied, Ppv increased dose-dependently, but the peak levels did not reach higher than 10 cmH₂O. Psa increased dose-dependently after Nor and Ang II, while tachyphylaxis was observed at highest dose (100 nmole/kg) of VP. The response of either Ppv or Psa to ET-1was the weakest among the vasoconstrictors studied. In conclusion, portal pressor response of was limited in magnitude, as compared with the systemic pressor response in anesthetized BALB/c mice. [J Physiol Sci. 2007;57 Suppl:S198]

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To elucidate the dynamical mechanisms of sinoatrial node pacemaking, we investigated the bifurcation structures of mathematical models for the rabbit sinoatrial node cells. Equilibrium points (EPs), periodic orbits, stability of EPs, and bifurcation points were calculated for central and peripheral cell models during changes in bifurcation parameters. Structural stability to applications of constant bias currents (I_bias) or hyperpolarizing loads was also evaluated. Blocking L-type Ca²⁺ channel current (I_Ca,L) caused stabilization of an EP and cessation of pacemaking via a Hopf bifurcation in both central and peripheral cells, whereas blocking Na⁺ channel current (I_Na) did not. The unstable EP region determined with I_bias applications shrunk and finally disappeared as I_Ca,L diminished in the central cell; however, it did not disappear in the peripheral cell even when I_Ca,L was completely blocked. Instability of the I_Ca,L-removed peripheral cell was eliminated by blocking I_Na. The I_Ca,L-removed cell exhibited pacemaking when hyperpolarized by acetylcholine applications or electrotonic loads. These results suggest that 1) dynamical mechanisms of basal pacemaking are essentially the same in both central and peripheral cells, 2) I_Na improves the structural stability to hyperpolarizing loads, and 3) I_Na-dependent pacemaking may occur in hyperpolarized peripheral cells. [J Physiol Sci. 2007;57 Suppl:S199]

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Bioflavonoid mixture containing oligomeric procyanidins (OPCs) extracted from pine bark is drawing attention and considered to improve microcirculation via antioxidative, antiplatelet and anti-inflammatory actions. To confirm their rheologic actions, we investigated the effects of OPCs on erythrocyte filterability in two different clinical situations using nickel mesh filtration method. OPCs (40 mg/day) were administered every day for 2 weeks to 10 males with metabolic syndrome and 47 females with cold intolerance (so-called 'Hie-sho'). Because microcirculation is supposed to be impaired in these clinical situations. Mean erythrocyte filterability in the males with metabolic syndrome (64.6±14.5%) was significantly (p<0.02) improved after the intake of OPCs (77.3±5.6%). Furthermore, we found a good correlation between the improvement of erythrocyte filterability and microcirculation evaluated by laser Doppler flowmeter (r=0.92). On the other hand, no favorable effects of OPCs on erythrocyte filterability were found in the females with cold intolerance. Thus, effectiveness of OPCs is considered to be variable depending on the clinical situations. Erythrocyte filterability in metabolic syndrome is impaired probably by oxidative stress on erythrocyte membrane, whereas that in cold intolerance is impaired by some other mechanisms. [J Physiol Sci. 2007;57 Suppl:S199]

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Mental task causes a pressor response with concomitant increase in vascular resistance (VR) in the superior mesenteric artery (SMA), whereas meal intake decreases VR in the SMA. To investigate whether this decrease in VR affects the pressor response to mental task, we compared cardiovascular responses to mental task in the fasting and post-prandial state. Ten healthy subjects (5 males and 5 females, 24 ± 1 yr, 56 ± 2 kg, mean ± SEM) performed color word test (CWT) for 5 min after 5 min of baseline measurement in the over night fasting state. On another day, the subjects ingested 300 kcal solid food within 5 min immediately before the CWT. Mean arterial pressure (MAP), blood velocity (BV) in the SMA, and blood flow (BF) in the finger skin and forearm were measured throughout the protocols. MAP was divided by BV in the SMA, and BF in the skin and forearm to yield an index of VR. The baseline variables did not differ significantly between the fasting and post-prandial state. In the fasting state, CWT significantly increased MAP (13 ± 3%), and VR in SMA (8 ± 3%), skin (129 ± 48%) and forearm (64 ± 25%) from the baseline (P < 0.05). In the post-prandial state, the CWT significantly increased MAP (14 ± 4%) despite the absence of a significant change in VR in SMA, and it significantly increased VR in the skin (178 ± 64%) and forearm (124 ± 45%). These findings suggest that VR in SMA decreased by meal intake does not affect the pressor response to CWT. (2006 Danone Institute of Japan Research Grant) [J Physiol Sci. 2007;57 Suppl:S199]

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Recapitulation of the fetal cardiac gene program has been regarded as a feature of hypertrophied and failing hearts and considered to underlie electrical remodeling in diseased heart (Yasui et al. 2005). We have previously modeled developmental changes in action potential of rodent ventricular cells on the basis of the Kyoto model (Matsuoka et al. 2003), an electrophysiological model of the guinea pig cardiomyocyte; the simulation with our models for three representative developmental stages were able to reproduce action potentials that were consistent with the reported characteristics of the cells in vitro (Itoh et al. 2006). The present work further expanded the integrated mathematical model to reflect one of the contended issues in cardiomyocyte development: role of sarcoplasmic reticulum (SR) in Ca²⁺ transient. Abolishment of the gating property of the ryanodine receptor (RyR) channel in SR, which represents blockade of the channel to its open state, significantly reduced the simulated Ca²⁺ transient in neonatal model, while that in early embryonic model was not affected and still accounted for predominant fraction in the Ca²⁺ transient. To further assess the role transition of SR in Ca²⁺ transient during development, relative changes in SR volume as well as activities of ionic components in SR were shifted from one developmental stage to the next and showed that the role of SR in Ca²⁺ transient is altered between embryonic and postnatal development. [J Physiol Sci. 2007;57 Suppl:S199]

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We set up a working hypothesis that the sinoatrial (SA) node is more stable than biological pacemakers against physiological perturbations, because the physical pacemaker has assuredly increased the stability through evolutionary natural selection. In order to assess the working hypothesis, the external Na⁺ and K⁺ concentrations were varied to simulate the physiological perturbations to the following pacemakers: 1) SA node pacemaker, 2) biological pacemaker created by suppression of inward rectifier potassium current (I_K1) (Miake et al., Nature. 419 (2002) 1529), and 3) biological pacemaker created by over expression of the hyperpolarization activated cyclic-nucleotide (HCN) gated channels, which introduces HCN "pacemaker" current (I_f) (Tse et al., Circulation. 114 (2006) 1000). In addition, we varied a time constant (tau) of gate to represent two types of I_f-induced pacemaker. Simulated results suggested that SA node pacemaker is most stable against physiological perturbations, followed by biological pacemaker created via suppression of I_K1. No significant difference was observed between stability of two I_f-induced pacemakers against given physiological perturbations. These results supported our hypothesis. [J Physiol Sci. 2007;57 Suppl:S200]

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The effects of carbon dioxide on cutaneous vessels have been studied in the isolated vessels and tissue blood flow of the human skin. Although carbon dioxide has vasodilator action, the direct evidence for microvascular dilatation of the skin and its mechanism has been reported a little. Then, we studied direct effects of carbon dioxide on in vivo microvasculature and blood flow rate by using an intra-vital video-microscope system. Brown-Norway rats were anesthetized with alpha-chroralose and urethane intra-peritoneously. In order to measure inner diameter and red blood cell velocity (Vrbc) of a microvessel, the dorsal skin window was draped on an observation box placed inside a bath. For recording of micro-vascular images of the subcutaneous tissue, the bath was on the stage of a microscopic system equipped with a color CCD camera. Changes in inner diameter of microvasculature were manually measured on digitalized video images. Vrbc was derived from the cross-correlation function of a paired-segment of dual window in vessel images. We measured pH in subcutaneous tissue by making a dorsal skin tube. After a topical application of carbon dioxide solution via skin of rat, we observed both vasodilatation and increase in blood flow of the microvessel. The pH of subcutaneous tissue decreased after 10 minutes of carbon dioxide application. There was no change in mean systemic blood pressure during the topical application of carbon dioxide. [J Physiol Sci. 2007;57 Suppl:S200]

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Skin sympathetic nerve activity controls blood flow and sweating. Stimulation of skin sympathetic nerve activity causes vasoconstriction, which in turn decreases in skin blood flow and finger volume. We hypothesized that the hemodynamic change in skin might alter viscoelastic property of skin and modify sensitivity of mechanosensitive receptors accordingly. To test this hypothesis, we measured skin blood flow (SBF), skin spatial resolution threshold (SSRT), heart rate (HR), and arterial blood pressure (MAP) in two ambient temperature conditions (26°C vs. 19°C). A two-dimensional image of SBF was continuously measured using a laser Doppler perfusion imaging equipment. SSRT was determined with a two-point discrimination method applied to the second fingertip, the third fingertip, and the hypothenar eminence. SBF significantly decreased by 59-62% in the fingers and palm in the cold condition. Skin spatial resolution was significantly improved in all skin areas tested. SSRT of the third fingertip significantly correlated with SBF. We conclude that decreased skin bloodflow can improve skin spatial resolution. This may be attributed to a change in mechanical property of the skin induced by a decrease of blood flow and/or blood volume. [J Physiol Sci. 2007;57 Suppl:S200]

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It has been thought that a mechanoreflex from exercising muscles as well as central command descending from higher brain centers plays an important role in cardiovascular adaptation during dynamic exercise. To examine a contribution of the muscle mechanoreflex to the cardiovascular adaptation during dynamic exercise, we measured the responses in heart rate (HR), stroke volume (SV), cardiac output (CO), arterial pressure (AP), and total peripheral resistance (TPR) during passive cycling in 8 subjects. AP was continuously measured with a Finometer device. Beat-to-beat changes in SV, CO, and TPR were calculated using Modelflow®. SV and CO increased and TPR decreased during passive cycling with ergometer, whereas HR slightly increased only at the initial period of passive cycling. Because electromyogram of the leg muscles did not appear during passive movement, active contraction was excluded. To eliminate the effect of venous return on the increase in SV, we performed the passive cycling either with or without venous occlusion of the legs (bilateral thigh cuffs inflated to 100 mmHg). Venous occlusion reduced the increase in SV and thereby attenuated the increase in CO during passive cycling. These data suggests that the increases in SV and CO during passive dynamic exercise are chiefly mediated by increased venous return but not by a muscle mechanoreflex. In conclusion, a muscle mechanoreflex may play a minor role in the cardiovascular adaptation during dynamic exercise with a lower intensity. [J Physiol Sci. 2007;57 Suppl:S200]

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Feedback regulation by activation of group III and IV thin fiber afferents in exercising muscle causes the cardiovascular and sympathetic nerve responses (termed exercise pressor reflex). Mechanical stimuli activate mainly group III afferents, some of which are sensitive to a change in temperature. The mechanical response of group III afferents might be modulated by thermal stimuli. However, the effect of intramuscular temperature on the exercise pressor reflex remained little known. To test the hypothesis that the mechanoreflex might be altered by decreasing temperature of the exercising muscle, we studied the effect of local muscle cooling on the increases in cardiac sympathetic nerve activity (CSNA), heart rate (HR), and mean arterial blood pressure (MAP) during evoked static contraction and passive stretch of the triceps surae muscle in anesthetized cats. Gadolinium, a blocker of mechanosensitive ion channels, blunted the increases in CSNA, HR, and MAP during static contraction as well as passive stretch, suggesting the significant contribution of muscle mechanoreceptors to the exercise pressor reflex. Intramuscular temperature of the triceps surae muscle decreased from 35±1 °C to 19±1 °C by muscle cooling. The baseline HR and MAP were reduced by muscle cooling, while the baseline CSNA was increased. Local muscle cooling blunted the increases in CSNA, HR, and MAP during static contraction, despite the same tension development. We suggest that the exercise pressor reflex is influenced by muscle cooling, probably due to decreasing discharge of muscle mechanosensitive afferents. [J Physiol Sci. 2007;57 Suppl:S201]

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Vagal control of heart rate (HR) consists of direct and indirect actions of ACh. Direct action of ACh activates the muscarinic potassium (K_ACh) channels in sinoatrial nodes, whereas indirect action inhibits the adenylyl cyclase. The role of K_ACh channels in total picture of vagal HR control remains to be elucidated in vivo. The purpose of this study is to quantitatively examine the transfer characteristics of the K_ACh channels in the HR response to vagal stimulation (VS). In anesthetized rabbits (n=10) with sinoaortic denervation and vagotomy, the cardiac end of the sectioned right vagus was stimulated using a binary white-noise signal (0-10Hz) to examine dynamic transfer characteristic, and a stepwise signal (from 5 to 20 Hz with increments of 5Hz every minute) to examine static transfer characteristic. The dynamic transfer function from VS to HR approximated a first-order, low-pass filter with pure time delay. Tertiapin, a selective K_ACh channel blocker (30 nM/kg iv) decreased the dynamic gain (5.2±0.4 to 2.3±0.2 beats min⁻¹ Hz⁻¹) and the corner frequency (0.23±0.01 to 0.06±0.00 Hz), and increased the pure time delay (0.35±0.01 to 0.45±0.02 sec); K_ACh channels blocker significantly delayed the 90% rise time of the step response from 1.6±0.1 to 4.6±0.5 sec. Tertiapin decreased the static reductions of HR by 58±6, 46±5, 39±5 and 36±4% at 5, 10, 15 and 20 Hz of stimulus frequency, respectively. These results suggest that K_ACh channels accelerate the dynamic HR response to VS and double the static HR response to VS in vivo. [J Physiol Sci. 2007;57 Suppl:S201]

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Automated drug delivery system to optimize hemodynamics is worth developing, since management of decompensated heart failure is often time-consuming and difficult. We have succeeded in our prototype to control left ventricle pumping ability (SL) by dobutamine, stressed blood volume (V) by dextran and arterial resistance (R) by nitroprusside in open-chest dogs, so as to restore normal arterial pressure (AP), left atrial pressure (PLA) and cardiac output (CO) (J Appl Physiol 2006). However, requirement of continuous measurement of SL and PLA make it difficult to apply for clinical settings. To overcome the limitation, we controlled right ventricle pumping ability (SR), by assuming nearly parallel changes in left and right pumping functions. We estimated PLA by linear regression of it over pulmonary arterial pressure (PPA), that was calibrated every 30 min. In anesthetized dogs (n=10) with acute decompensated heart failure, the new system accurately estimated PLA from PPA (RMS=1.3±0.3 (SD) mmHg), restored all mechanical properties (SR, V, R) within 20 min, and normalized AP (85.6±18.6 to 94.4±13.1 mmHg), CO (69.6±9.8 to 102.3±8.0 mlmin-1kg-1) and PLA (19.3±2.8 to 13.5±1.2 mmHg). The restored hemodynamics was maintained for 90 min with small steady-state deviations from respective targets (AP, 4.4±3.7 mmHg; CO, 3.0±2.6 mlmin-1kg-1; PLA, 0.6±0.5 mmHg). In conclusion, the new system with SR control and PLA estimation successfully restored normal hemodynamics in decompensated heart failure in clinical settings. [J Physiol Sci. 2007;57 Suppl:S201]

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Although diabetes mellitus (DM) is recognized as one of the important risk factors for the development of heart diseases, little is known about the impact of DM on cardiac cell-volume regulation. We attempted to assess this point using an animal model. Streptozotocin (STZ, 250 mg/kg body-weight) was injected to adult male mice, and many of the so-treated mice developed Type-I DM in a few days after STZ. The cell-volume regulation and the volume-regulated chloride (Cl⁻) current (I_Cl,vol) were examined in single ventricular myocytes obtained from the DM mice, using video-image analysis and the whole-cell voltage-clamp. DM mice showed a loss of body-weight and an increase in blood-glucose level, but there was no change in the heart- to body-weight ratio. Video-image analysis showed that regulatory volume decrease (RVD) was almost lost in myocytes from DM mice, even though myocytes from both age-matched normal and DM mice underwent a similar cell swelling in response to hypotonic challenge. Voltage-clamp study revealed an approximately 50% reduction of the density of I_Cl,vol in myocytes from DM mice. The density of the basal Cl⁻ current was similar in control and DM mice. Neither the loss of RVD nor the reduction of I_Cl,vol was observed in myocytes obtained from the mice which had been given STZ only 12 hr before, suggesting that STZ does not directly knock down the I_Cl,vol channels. Our results indicate that cardiac cellular RVD is attenuated in DM mice, and that the loss of RVD is due at least partly to symptomatic suppression of I_Cl,vol. [J Physiol Sci. 2007;57 Suppl:S201]

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Single heart cells from genetically-engineered mouse models are of great importance for studying physiology and pathophysiology of the heart. With single-cell measurement techniques such as patch-clamp and calcium imaging, they provide powerful tools for unveiling molecular mechanisms underlying heart functions. Doing single-cell experiments with these techniques demand a consistent supply of quality heart cells; however, it is not easy to isolate such quality cells from mouse models. This has long been a major problem for utilizing mouse models, obstructing their use in heart research. With the aim to overcome this problem, we developed a simple method for isolating single heart cells from mouse models. By the use of simple tools to facilitate handling of their tiny hearts and a refined protocol to minimize their ischemic damages, this method isolates single heart cells of mouse models with great ease and consistency. Cell isolation is done under physiological condition, without using "KB" medium or BDM that has long been used for relieving cell damages. Thus isolated heart cells are rod-shaped, quiescent, and relaxed in Tyrode solution containing 1.8 mM Ca, while they have a consistent 75 +/- 5% (mean +/- SD, n = 5) viability under this condition. Lifetime of these cells is long enough for doing same-day experiments, since they retain 45 +/- 12% viability after being stored for 8 hours in Tyrode solution at 37'C. The cells have normal morphology, electrophysiological and contraction properties. They are expected to facilitate the use of genetically-engineered mouse models in heart research. [J Physiol Sci. 2007;57 Suppl:S202]

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It is inevitable to examine drug effects on electrical activities as well as mechanical functions of human heart during drug development. Generally, drug assessments in human can only be executed at a late stage of drug development, and we predict drug effects on human by extrapolating data from animal experiments. In this process, a mathematical model of human cardiac cell, which enables us to conduct virtual experiments using computer, will be a useful tool. So far, several types of human cardiac cell models are available for membrane excitation, and only one Excitation-Contraction (E-C) coupling model was published by Sachse et al (2003). Therefore, we started developing a comprehensive model of human cardiac cell. We developed a human models of fast Na, L-type Ca, transient outward, delayed rectifier K currents and a contraction model based on the experimental data from intact human cardiac myocytes, although which was limited in comparison with animal data. Then, we incorporated the human models into a comprehensive cell model of guinea-pig heart, the Kyoto model (Takeuchi et al., 2006) to compose a cell model of human heart. We succeeded in construction of the human cardiac cell model which well reproduces E-C coupling. This novel human cardiac cell model will illustrate how we can extrapolate data from animal experiments to human response. [J Physiol Sci. 2007;57 Suppl:S202]

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The objective of this study was to investigate the effect of the mental stress on the synchronization between breathing patterns and respiratory sinus arrhythmia (RSA) of heart rate. Thirteen healthy volunteers performed a computerized color word conflict test (CWT) for 15 min after 10 min of resting control followed by 10 min of recovery. Breathing patterns, RSA and systolic blood pressure (SBP) were derived from respiratory flow meter attached to the facemask, electrocardiograms, and Finapres, respectively, which were recorded simultaneously using FM DAT tape. The analytic signals of breathing pattern and RSA were obtained by Hilbert transform. The synchronization was then quantified by the phase coherence index λ. The magnitude of RSA as a measure of parasympathetic activity, was evaluated using a short-term Fourier transform. During CWT, SBP and breathing frequency increased significantly (p<0.01) for all subjects compared with baseline (p<0.01) but heart rate increased for 8 subjects investigated. λ decreased significantly during CWT, indicating that mental stress desynchronizes the oscillations of heart rate and respiration. The magnitude of RSA also decreased in response to CWT but did not differ significantly between baseline and CWT. The results suggest that mental stress exerts an influence on the interaction of respiration and heart rate by increasing breathing frequency, reducing the strength of the cardiorespiratory phase synchrony. [J Physiol Sci. 2007;57 Suppl:S202]

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The midbrain periaqueductal grey matter (PAG) is involved in defense reaction accompanied with an increase in heart rate (HR) and arterial blood pressure (AP). This simultaneous increase in HR and AP is partly explained by the inhibition of the arterial baroreflex due to PAG activation because previous studies demonstrated that the baroreflex response, which is induced by stimulation of aortic depressor nerve (ADN), was attenuated during PAG stimulation. In this study, we examined whether the baroreflex inhibition caused by PAG activation is mediated with nitric oxide. To solve this, we studied the effect of intravenous injection of NO synthesis inhibitor (L-NAME, 10 mg/kg) on the ADN stimulation-induced baroreflex bradycardia and depressor response during PAG stimulation in anaesthetized rats. Before L-NAME, ADN stimulation provoked bradycardia and depressor response as a baroreflex response. The baroreflex bradycardia induced by ADN stimulation was inhibited during PAG stimulation while the depressor response was not inhibited. After L-NAME, ADN stimulation induced bradycardia was not inhibited during PAG stimulation. These results suggest that the neural outflow from PAG interact with the cardiac limb, but not the vasomotor limb, of the baroreflex arc, which is mediated with nitric oxide. [J Physiol Sci. 2007;57 Suppl:S202]

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The I_Kr is a rapid component of cardiac delayed rectifier potassium current responsible for cardiac repolarization. HL-1 murine cardiomyocytes exhibit large amplitude of I_Kr with minimal contamination of other time-dependent outward currents, providing useful cardiac cell model for studying many features of I_Kr channels under cardiac specific environment. In the present study, the gene expression profiles underlying I_Kr in HL-1 cells were investigated. RT-PCR analysis revealed the expression of three splicing variants of mERG1; mERG1a, mERG1a' and mERG1b. On the other hand, significant amplification was also observed for mKCNE1 but not for mKCNE2. To examine functional relevance of mERG1 expression in HL-1 cells, knockdown experiments were performed using RNA interference technique. Small interfering RNA (siRNA) duplex oligonucleotides against all isoforms of mERG1 and negative control RNA (scRNA) were individually transfected into HL-1 cells together with a plasmid vector encoding GFP. Two days after transfection, whole-cell membrane currents were recorded from GFP-positive cells (–1%). Compared to recordings in cells transfected with GFP alone (19.1 pA/pF), the I_Kr amplitude was significantly smaller in cells transfected with siRNA (1.9 pA/pF). The effect of siRNA was attributable to specific knockdown of mERG1 expression, because cells transfected with scRNA displayed currents with comparable amplitude to those recorded from cells transfected with GFP alone. Thus, gene-specific RNA interference may be an effective tool for elucidating molecular entity of I_Kr channel in HL-1 cells. [J Physiol Sci. 2007;57 Suppl:S203]

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The hyperpolarization-activated cation channel (HCN4) shows differential expression patterns during the embryonic development and hypertrophy of hearts. Briefly, HCN4 expression is maximally activated in embryonic hearts and quickly diminishes after birth. However, its expression is reactivated during cardiac hypertrophy. The sequence analysis of the HCN4 gene revealed the presence of a conserved neuron-restrictive silencer element (NRSE). NRSE is known to bind neuron-restrictive silencing factor (NRSF). We initiated the characterization of the 5' promoter region of the HCN4 gene. A promoter truncation analysis with cardiac myocytes revealed the -446/+400 promoter region that induced a basal transcriptional activity. This region drove a high transcriptional activity in embryonic myocytes, but not in neonatal myocytes treated with hypertrophic agents. After confirming that NRSF binds to the intronic NRSE and represses HCN4 promoter, transcriptional activities modified by wild-type or mutated NRSE were evaluated. With wild-type NRSE, HCN4 promoter activity correlated well with the expression patterns, whereas mutant constructs failed to induce such transcriptional activity. These findings demonstrate that the release of transcriptional repression mediated by NRSF is required for the upregulation of HCN4 expression with cardiac myocyte hypertrophy. We conclude that the NRSE-NRSF regulatory system was implicated in HCN4 expression during the development and hypertrophy of cardiac myocytes. [J Physiol Sci. 2007;57 Suppl:S203]

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In the healthy heart, the rhythm is moderately fluctuating (1 over f fluctuation, by Peng et al.). In unhealthy hearts, a perturbation occurs prior to an initiation of a cardiac disease. Thus, the beat to beat variations in a cardiac cycle length are carrying important information about the heart. So, the detrended fluctuation analysis (DFA) has been invented to utilize the characteristics of rhythm for an early prognosis of a cardiovascular disease. It detects a hidden sign of a cardiovascular disease. However, more experiments are still necessary before using it as a practical tool. We tested the DFA in animal models, and proved that the DFA distinguished the state of heart under or out of the control of regulatory center. We are not aware of whether the center is working healthy or not, thereby the silent angina or in a much worse case scenario, the silent heart attack can still happen in unlucky individuals (43 percent of heart attacks went unrecognized, Feb14 2006, BBC). We made a DFA like program and got results: Category 1) Hearts of dying animal exhibit a scaling exponent as low as from 0.6 to 0.7. Life never recovered if the exponent once drops down. Category 2) A heart-injured animal exhibits a high exponent, as high as up to 1.2 to 1.5. There was no significant sign of death in EKG recordings to be expected in the near future. The moment of death arrived relatively sudden compared to Category 1. Category 3) Healthy hearts exhibit exclusively the exponent 1.0, in both animal models and humans. [J Physiol Sci. 2007;57 Suppl:S203]

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Cardioprotection by anesthetic preconditioning (APC) has been reported. However the mechanism of cardic APC is not clear. In the present study, we studied the mechanisms of APC through examining the characteristics of sevo- and N₂O- induced cardiac suppression and cardioprotection. Methods: Male SD rat hearts were excised and perfused with Langendorff manner. For hypoxia-reoxygenation experiment, hearts were made hypoxic for 45 min and then reoxygenated for 40 min (control: CT group). Preconditioning was attained by 3 cycles of application of 4% sevo (sevo-pre group) or 50% N₂O (N₂O-pre group) for 5 min and 5 min of washout. Results: 1) sevo and N₂O decreased LVDP to the similar extent. Sevo elevated LVEDP, while N₂O did not 2) in sevo-pre group, the recovery of pressure-rate product (PRP) was significantly better than that of CT group, and GOT release was significantly lower than that of CT group. However, in N₂O-pre group, recovery of PRP was not different from CT group. 3) N₂O applied during hypoxia (N₂O-hyp group) improved recovery of PRP, and GOT release was lower than that of CT group. However, sevo applied during hypoxia (sevo-hyp group) did not show this cardioprotection. Conclusions: N₂O inhibit myocardial contraction, at least in part, by decreasing calcium current without metabolic inhibition. On the other hand, sevo causes not only contractile depression but also metabolic inhibition. [J Physiol Sci. 2007;57 Suppl:S203]

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It is widely known that neonatal hearts show a greater resistance against hypoxia than adult hearts, but its mechanism is not clear yet. We examined the effects and kinetics of magnesium which ion is known to have myocardial protective effect. The rat hearts of adult (10weeks old) and neonate (1week old) were Langendorff-perfused and subjected to 45 min hypoxia /30 min reoxygenation. Heart rate, ventricular tension and GOT release were monitored throughout the experiment. The magnesium content of myocardium after perfusion was measured by atomic absorption spectrophotometry. The heart rate and ventricular tension, as an index of recovery of cardiac function, was recovered by reoxygenation faster in neonatal hearts than adults. The magnesium content of adult and neonatal heart after perfusion without hypoxia was 811.6 ± 37.2 and 770.0 ± 121.6 μg/g dry respectively. After hypoxia-reoxygenation, the magnesium content of adult hearts decreased to 539.4 ± 33.1 μ/g dry, significantly lower than that after perfusion without hypoxia. However, in neonatal hearts the magnesium content was not significantly difference from that without hypoxia (830.9 ± 88.1 μg/g dry). We suggest that the high resistance against hypoxia in neonatal hearts may be caused by ability of maintaining magnesium in the myocardium. [J Physiol Sci. 2007;57 Suppl:S204]

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Caveolae are involved in various signal transductions across the membrane in cardiac myocytes. Methyl-β-cyclodextrin (MβCD) deprives the cholesterol, an important structural component of caveolae, from plasma membrane. In patch clamp experiments, we found that application of MβCD inhibit the activation of L-type Ca²⁺ current via β-stimulation in cardiac myocytes. We examined the effects of MβCD application on β-stimulant-induced elevation of cardiac function in Langendorr perfused rat heart. Application of MβCD (1 mM, 30 min) did not affect the basal cardiac function. Following the MβCD treatment (15 min), application of isoproterenol (iso; 10 nM, 15 min) in the presence of MβCD, increased HR from 196.9 ± 17.3 to 336.8 ± 13.0 beats/min, but failed to increase LVDP (75.4 ± 9.1 vs 124.4 ± 12.3%; iso application with and without treatment of MβCD, respectively). MβCD did not affect the inotropic effect of elevated extracellular Ca concentration. These results suggest that treatment of MβCD does not affect L-type Ca²⁺ channels, but damages the β-stimulation pathway to attenuate activation of L-type Ca²⁺ currents. It is considered that the relationship of L-type Ca²⁺ channel, protein kinase A and A kinase-anchoring protein (AKAP) may play important roles in the β-stimulation pathway. MβCD may cause membrane fragility and which is followed by AKAP injury in the membrane and/or damages the linkage between L-type Ca²⁺ channel and AKAP. [J Physiol Sci. 2007;57 Suppl:S204]

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In Langendorff perfused rat heart, pressure development decrease to the infra-basal level by the washout of isoproterenol (iso). Since it has been reported that iso decreases intracellular Mg concentration, we hypothesized that iso washout-induced decrease in cardiac contractility is induced by the decrease of intracellular Mg concentration. We examined the relationship between cardiac contractility and Mg content in the hearts by using Langendorff perfused rat heart model and atomic absorption spectrophotometry. Application of iso (10 nM, 15 min) caused positive chronotropic and inotropic effects. After washout of iso (30 min), heart rate (HR) recovered to basal level, but left ventricular developed pressure (LVDP) decreased to significantly lower level than that of time matched control (70.7 ± 3.9 vs 88.9 ± 7.1%, respectively; p<0.05). When iso was applied with high Mg (12 mM; iso + high Mg group), iso washout-induced deterioration of LVDP was significantly inhibited (86.9 ± 4.0%, p<0.05 vs iso group). When extracellular Ca concentration was decreased, to mimic the Ca blocking effect of Mg, iso washout-induced decrease in contractility was not affected. Intracellular Mg content decreased from 739.2 ± 15.2 (control group) to 721.6 ± 25.5 μg/g dry (iso group), but in iso + high Mg group, Mg content was kept at high level (762.3 ± 36.2 μg/g dry). These results suggest that intracellular Mg may play an important role to maintain the cardiac contractility. [J Physiol Sci. 2007;57 Suppl:S204]

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