Alterations in arterial properties detected by pulse wave velocity and pulse wave analyses in patients with hypercholesterolemia are controversial. Therefore, we used an animal model of atherosclerosis to test the potential of arterial mechanical properties indexed by aortic input impedance (Z_in) as markers for early detection of atheromatous lesions in the aorta. In 12-month-old anesthetized Kurosawa and Kusanagi-Hypercholesterolemic Rabbits (KHC; n=15), pressure and flow were measured at the ascending aorta during sinus rhythm and random pacing. We confirmed histologically in previous studies that early-stage atheromas developed in the aorta by this age. Age-matched Japanese White rabbits (JW; n=13) had no overt atheroma. High-resolution Z_in derived from random beating data was characterized by a lower ratio of the first local maximum to minimum |Z_in| (1.36±0.26 vs. 2.08±0.33; p<10⁻⁵) accompanied by more delayed phases over a range between 6 and 8 Hz (-0.35±0.21 vs. -0.05±0.15 radian; p=0.0002); and a lower characteristic impedance (2.30±0.67 vs. 2.92±0.59 mmHg·s·kg BW/ml; p=0.016) in KHC than in JW rabbits. We concluded that frequency-specific changes in Z_in are sensitive hemodynamic markers that can detect early-stage atheromatous lesions in the aorta. High-resolution Z_in analysis is essential and useful for detecting such frequency-dependent findings. [Jpn J Physiol 54 Suppl:S105 (2004)]

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Cholesterol is a main structural component in the lipid raft in caveolae that are involved in cAMP-dependent increase of voltage-gated Na⁺ current in cardiac myocytes. We showed previously that intracellular methyl-beta-cyclodextrin (MβCD) that deprives cholesterol from the membrane accelerated run-down of L-type calcium current (I_CaL) and suppressed isoproterenol (ISO)-induced increase of I_CaL in isolated rabbit ventricular myocytes. Here we examined further the effects of MβCD on I_CaL increase produced by forskolin (Forsk) or cAMP analogue. Whole-cell I_CaL were recorded by applying double pulses in the presence of Na⁺, Cs⁺ and 1.8 mM Ca²⁺ in the superfusate and CsCl and BAPTA in the pipette solutions. Pipette solutions that contained no (control) or 30 mM MβCD were dialyzed 10 minutes before the challenge of following test drugs. The ratios of the maximal I_CaL amplitude before and after the challenge of the drug in control and in the presence of MβCD were: ISO (1 μM) 2.9±0.1 (n=21) in control and 1.0±0.2 (n=12) in MβCD; Forsk (10 μM) 3.0±0.4 (n=8) in control and 1.3±0.2 (n=8) in MβCD; dibutyl-cAMP (3 mM) 3.1±0.4 (n=9) in control and 1.4±0.2 (n=9) in MβCD. These results indicated unequivocally that cAMP-induced increase of cardiac I_CaL was suppressed by intracellular MβCD. It suggests that cholesterol is indispensable for the cAMP-mediated augmentation of cardiac I_CaL either in the phosphorylation process or in the normal function of the phosphorylated channels. [Jpn J Physiol 54 Suppl:S106 (2004)]

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Left ventricular (LV) mouse myocardial slices of the 300-μm thick freely shortened by 1-Hz electrical field stimulation. Mean myocardial slice oxygen consumption per minute (VO2) without stimulation was 0.97 ± 0.14 ml O2.min-1.100 g LV-1 and mean VO2 with stimulation increased to 1.80 ± 0.17 ml O2.min-1. 100 g LV-1 in normal Tyrode solution. Mean delta VO2 was 0.83 ± 0.12 ml O2.min-1.100 g LV-1. Cyclopiazonic acid 30 μM significantly reduced the delta VO2 to 33% of control. The increases in extracellular Ca concentrations up to 14.4 mM significantly increased the delta VO2 to 190% of control. BDM 3-5 mM decreased the delta VO2 by 33-37% of control not in a dose-dependent manner, though BDM 3-5 mM dose-dependently decreased the slice free shortening. Present results indicate the possibility that the VO2 of mouse LV slices during shortening under mechanically unloaded conditions consists of energy expenditure for total Ca handling in the excitation-contraction coupling, basal metabolism and possibly definite residual crossbridge cycling (maximally 33% of control). [Jpn J Physiol 54 Suppl:S106 (2004)]

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Aiming at clarifying the physiological and pathophysiological roles of Na⁺-Ca²⁺ exchanger (NCX) in cardiac EC coupling, we studied the ventricular myocytes of homozygous mice over-expressing NCX1.1 (NCX1.1(Tg), Iwamoto et al., J. Pharmacol. Sci. 92 (2004) Suppl.). In ventricular myocytes of NCX1.1(Tg), the protein content of NCX and NCX currents were enhanced to two fold of those of wild type (wt). NCX1.1(Tg) displayed modest cardiac hypertrophy at 8-11 weeks of age (Iwamoto et al., 2004). The fractional shortening of single ventricular myocytes was not different between NCX1.1(Tg) and wt when stimulated at 0.33 Hz. However, in contrast to wt, NCX1.1(Tg) showed frequency-dependent reduction of shortening at frequencies higher than 0.5 Hz. Partial blockade of the forward mode NCX activity by replacing 50% of the extracellular Na⁺ by Li⁺ abolished the frequency-dependent contractile dysfunction. The severely impaired cardiac function of NCX1.1(Tg) may have been masked by compensatory mechanisms. We therefore measured the plasma norepinephrine level, and found it to be significantly higher in NCX1.1(Tg) than in wt. These results indicate that, in NCX1.1(Tg), the augmented forward mode NCX activity impairs the cardiac E-C coupling, which is masked by compensatory up-regulation of sympathetic tone that may be partly responsible for the cardiac hypertrophy observed in NCX1.1(Tg). [Jpn J Physiol 54 Suppl:S106 (2004)]

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Sphingosine–1–phosphate (S1P) regulates cell migration, proliferation and apoptosis in vascular smooth muscle cells (VSMCs) and endothelial cells through the Edg family of multiple G protein–coupled receptor isoforms. The major source of S1P is activated platelets, suggesting its role in vascular remodeling. We previously demonstrated that S1P stimulated PDGF_B chain mRNA expression in newborn VSMCs ,but not in adult VSMCs, through the mechanisms mainly involving Gi–Ras–ERK. In this study we investigated which S1P receptor subtype is involved in S1P–induced PDGF_B chain mRNA expression in VSMCs. Newborn and adult VSMCs expressed comparable levels of S1P₂ and S1P₃ mRNAs. However, the expression levels of S1P₁ mRNA were quite different : it was abundant in newborn RASM, whereas it was barely detectable in adult VSMCs. We found in adult VSMCs that overexpression of S1P₁, but not S1P₂, induced a robust S1P stimulation of PDGF–B chain mRNA. To test more directly the possibility that S1P1 mediates S1P stimulation of PDGF_B chain expression, we down–regulated the expression of S1P₁ mRNA by RNA interference. Treatment of the newborn VSMCs with the siRNA specifically targeting S1P₁, but not scrambled RNA duplex, effectively reduced S1P₁ mRNA level and markedly inhibited S1P–induced expression of PDGF–B chain. These observations indicate that S1P₁ mediates S1P upregulation of PDGF–B chain. [Jpn J Physiol 54 Suppl:S106 (2004)]

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The sinoatrial (SA) node is a complex and heterogeneous tissue in terms of electrical activity: its pacemaker activity, shape of the action potential and conduction velocity vary between the center and periphery of the SA node. The aim of this study was to investigate regional heterogeneity of ion channel mRNA expression in the rabbit SA node and its surrounding atrial muscle. Small tissue samples (1.5x1.5x0.5 mm) including the center or the periphery of the SA node (C-SAN or P-SAN) as well as the right atrial appendage (RA) were dissected from the rabbit heart and mRNAs of various ion channel subunits were evaluated using a quantitative real-time PCR method. There were progressive increases in the expression of HCN1, HCN4 and Cav1.3 mRNAs from the RA to the center of the SA node (RA<P-SAN<C-SAN). In contrast, Cav1.2, Kv1.4, connexin43 and ryanodine receptor mRNAs were less abundant in the SA node than in the RA. There was no significant difference in sarcolemmal Na/K pump and connexin45 mRNAs among the three sites. These observations may provide a molecular basis for understanding the sophisticated structural and functional organization of the SA node as a dependable pacemaker of the heart. [Jpn J Physiol 54 Suppl:S107 (2004)]

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Experimental and clinical evidence suggests an important role for the intraatrial septum (IAS) in the atrial fibrillation (AF) of the vagal origin. The muscarinic K⁺ channel (I_K,ACh) represents a relevant target for the action of vagal neurotransmitter acetylcholine (ACh) and its activation should be involved in the alterations of electrical activity in atria associated with an elevation of the vagal tone. In the present study we examined the distribution of I_K,ACh in different regions of guinea-pig atria. Atrial myocytes were isolated from right atrial appendage (RAA), right atrial free wall (RA), IAS, left atrial appendage (LAA) and left atrial free wall (LA) and were voltage-clamped using the whole-cell patch-clamp technique. I_K,ACh was maximally activated by 10 μM ACh at a holding potential of –40 mV and the current density of I_K,ACh was estimated by normalizing the amplitude of the outward shift in the holding current with reference to the cell membrane capacitance. The densities of I_K,ACh in RAA, RA, IAS, LAA and LA were 29.8±6.9 (n = 4), 31.1±2.7 (n = 5), 16.0±1.9 (n = 5), 13.5±2.3 (n = 6) and 20.8±3.0 (n = 5) pA/pF respectively. Thus, the distribution of I_K,ACh in IAS is significantly smaller compared with that in RAA (P<0.05) or RA (P<0.01). These regional differences in I_K,ACh density may contribute at least partly to the inhomogeneity in repolarization process in atria involved in the vagally-induced AF. [Jpn J Physiol 54 Suppl:S107 (2004)]

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Action potential waveform is surely the most important issue for discussing electrical activities in the heart. However in the mouse, the waveform of cardiac action potentials is still controversial and large variations are observed among the reports. Here, I aim to confirm physiological waveform of ventricular action potentials of the mouse, and also to reveal the cellular mechanism that underlies those waveform variations. For this purpose, I recorded action potentials from whole-cell clamped adult mouse ventricular myocytes, with the aid of a novel concentration-jump device that rapidly switches between superfusates. When being recorded with conventional whole-cell patch, the action potentials had a plateau that peaked at around -30 mV. This waveform is considered to be physiological because the same action potentials were also recorded with nystatin-perforated patch. On the other hand, the plateau was abolished when BAPTA (2 mM) or ryanodine (20 uM) was included in the electrodes, indicating that the development of the plateau demands the presence of [Ca²⁺]_i transients. The plateau was abolished by KB-R7943 (5 uM), a blocker of the Na/Ca exchanger, or by temporarily substituting extracellular Na with Li during the action potential, indicating a mandatory involvement of the Na/Ca exchange current in the development of the plateau. These results indicate that the variation in the action potential waveform is closely related to the variation in [Ca²⁺]_i transient, via the operation of Na/Ca exchange that provides inward current during the [Ca²⁺]_i transient. [Jpn J Physiol 54 Suppl:S107 (2004)]

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It is well known that fetal types of cardiac ion channels, i.e. hyperpolarization activated cation channel (I_h) and T-type Ca²⁺ channel (I_Ca-T) are re-expressed in ventricular myocytes of hypertrophied heart, giving rise to electrophysiological remodeling. We have recently reported that a transcriptional repressor, neuron restricted silencing factor (NRSF) regulated the expression of fetal cardiac genes such as atrial naturetic factor. Transgenic mice expressing dominant negative NRSF in their heart (dnNRSF Tg), exhibited dilated cardiomyopathy and susceptibility to lethal ventricular arrhythmia. In the present study, we explored the mechanism of lethal arrhythmia in dnNRSF Tg. Among genes encoding I_h and I_Ca-T, hcn2, hcn4 and cacna1h possess NRSE, a cis-acting element of NRSF. In the ventricle of dnNRSF Tg, mRNAs of HCN2, HCN4 and CACNA1H were re-expressed. In accordance with this, I_h was activated only in the cardiac myocyte of dnNRSF Tg, but not in wild type littermate (WT). The density of I_Ca-T was higher in dnNRSF Tg than in WT. I_Ca-T in dnNRSF Tg was almost completely suppressed by, whereas I_Ca-T in WT was insensitive to, 50 μM Ni²⁺. The resting membrane potential was more depolarized, and the action potential duration was prolonged in dnNRSF Tg. Early after depolarization was consistently induced in dnNRSF Tg in the presence of 1 μM isoprotenol. These electrophysiological alterations appeared responsible for the lethal arrhythmia in dnNRSF Tg mice. [Jpn J Physiol 54 Suppl:S107 (2004)]

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In vivo measurement of oxygen concentration in various tissues is crucial for understanding the adaptation of mitochondrial oxidative metabolism in hypoxia. In these oxygen measurements, a submicron spatial resolution is required because significant heterogeneities in oxygen concentration may be established within a cell. Now, we propose that GFP can be used as an in situ oxygen probe if it is efficiently expressed in cells. In cultured COS7 cell transiently expressing GFP (enhanced GFP), a brief exposure of the cell to 470-490 nm light elicited a red fluorescence (excitation and emission wavelengths, 520-550 nm and >580 nm, respectively) when the cell was exposed to anoxic gas. This red shift was quickly abolished upon reoxygenation. Then, using this technique, we examined whether hypoxia/anoxia can be detected in single cardiomyocytes in a submicron spatial resolution. We used transgenic mice in which GFP was stably expressed (green mouse). All the ventricular myocyte isolated from the green mice showed significant green fluorescence although its intensity was ~1/200 of the transiently GFP expressing COS7 cells. Photoactivation in anoxia certainly produced the red fluorescence in these cells but the magnitude of the increase was much smaller than expected. In summary, GFP can be used as an in situ probe for hypoxia/anoxia. In GFP expressing transgenic animals, in vivo imaging of anoxic loci with a submicron spatial resolution may be possible.
Supported by JSPS KAKENHI 15390061. [Jpn J Physiol 54 Suppl:S108 (2004)]

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Na⁺/Ca²⁺ exchangers of ionic transporters expel the intracellular Ca²⁺ to the extracellular side by utilising the concentration gradient between the cell membrane of only Na⁺ in the cardiac-type K⁺-independent family (NCX1) or both Na⁺ and K⁺ in the cone/brain neuron-type K⁺-dependent one (NCKX2). The structural and functional properties on the bindings of the substrate and the other cations to the exchanger molecule requires to examine to clarify the physiological mechanisms of the ion transports of the exchangers. The MTSET susceptibility test demonstrated that the exchanger protein molecule is consisted of multiple transmembranal segments, two membrane α-loops and one cytoplasmic loop. A variety of extracellular cations affected the reverse-mode exchange activity in both families of the exchangers in a dose-dependent manner. Any cations were considered to bind to a single site of the exchanger molecule from the Hill analysis on the dose response curves. The results obtained from the site-directed mutagenesis of NCX1 and NCKX2 suggested that the cation binding sites of the both exchangers are located in the α-loop domains. [Jpn J Physiol 54 Suppl:S108 (2004)]

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Expulsive phase of cough is produced by a large contraction of abdominal muscles. To understand neuronal mechanisms controlling this phase, the activity of augmenting expiratory (aug-E) neurons were investigated during fictive cough in decerebrate and paralyzed cats. Repetitive stimulation of the superior laryngeal nerve produced fictive cough characterized by an increased augmenting discharge in the phrenic nerve (stage 1 of cough; SC1) and large decrementing discharge in the iliohypogastric nerve (stage 2 of cough; SC2). Aug-E neurons showed 3 types of responses during fictive cough. A half (14/28) of aug-E neurons gradually depolarized during the SC1 phase and abruptly depolarized with high frequency discharges during the SC2 phase (D-D type). Ten other neurons showed no detectable change during the SC1 phase and a large decrementing depolarization during the SC2 phase (N-D type). The remaining 4 neurons hyperpolarized during the SC1 and SC2 phases (H-H type). In contrast, postinspiratory (post-I) and augmenting inspiratory (aug-I) neurons showed uniform responses during fictive cough. Post-I neurons (n=13) depolarized during both SC1 and SC2 phases. Aug-I (n=6) showed an increased depolarization during the SC1 phase and large hyperpolarization during the SC2 phase. The present results demonstrated heterogeneity of aug-E neurons in their responses during fictive cough and suggest a possible source of the N-D type of neuron for abdominal muscles during cough. [Jpn J Physiol 54 Suppl:S108 (2004)]

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We intended to elucidate the mechanism how hypoxia and ischemia affect the neural function of the spinal cord. Transverse slices of the juvenile rat cervical spinal cord were stained with a voltage-sensitive dye. Preparations were superfused with a control mock cerebrospinal fluid (mock CSF) (O₂=95%, CO₂=5%, glucose 11mM). We recorded processes of electrical stimulation induced neural excitation propagation on the transverse plain of the spinal cord using a high-speed optical recording system. We, then, analyzed the effect of hypoxia by superfusing the preparations with a hypoxic mock CSF (N₂=95%, CO₂=5%, glucose 11mM) as well as the effect of ischemia with an ischemic mock CSF (N₂=95%, CO₂=5%, glucose free). Under the control condition, two components were identified in the depolarizing optical responses to dorsal root stimulation: a fast component of short duration and a slow component of long duration. Hypoxic condition as well as short term ischemic exposure reversibly reduced the slow component without affecting the fast component. However, long term ischemic exposure irreversibly reduced not only the slow component but the fast component. Because the fast component represents pre-synaptic, and the slow component represents post-synaptic neural activity, it was suggested that hypoxia and ischemia disturb synaptic transmission in the neuronal network of the spinal cord. [Jpn J Physiol 54 Suppl:S109 (2004)]

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We have previously reported a novel functional respiratory neuron group in the limited region of the rostral ventrolateral medulla; 'para-facial respiratory group (pFRG)', ventrolateral to the facial nucleus and close to the ventral surface. Since neuronal activity in the pFRG was demonstrated to precede the inspiratory burst by the optical recordings using voltage sensitive dyes, pre-inspiratory (Pre-I) neurons are a dominant subtype of respiratory neurons in this group. However, we had experience that the electrophysiological recordings of the neurons were rather difficult compared with those in more caudal medulla. The present study was carried out to reveal the more detailed feature of the pFRG in the brainstem-spinal cord preparation from newborn rat. We recorded mainly Pre-I neurons in the pFRG by whole-cell patch clamp and stained them with Lucifer Yellow for subsequent histological analysis. We found that Pre-I neurons were located in the area immediately beneath the ventral pia mater. These neurons had fusiform or spherical cell bodies and the dendrites extending medially and laterally along the ventral surface. The most neurons examined responded with membrane depolarization to substance P or high CO2 (8%) application in the presence of TTX. We concluded that burst activity of such neurons in the superficial ventral region could be a signal source for fluorescence changes detected by the optical recordings, and the characteristic distribution may be a reason for the technical difficulty of whole-cell recordings by the ventral approach. [Jpn J Physiol 54 Suppl:S109 (2004)]

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A number of studies have shown that stimulation of the medullary raphe nuclei, consisting of the raphe obscurus, raphe pallidus and raphe magnus, produces inhibitory or excitatory effects on respiration. However, details of the role of the raphe nuclei in controlling of respiration have not been clarified. Previously, we showed that respiratory neurons existed in the medullary raphe nuclei of cats and rats, and that electrical stimulation of the recording sites of these neurons caused a change in respiratory activity. The present study was undertaken to investigate the firing patterns, localization and spinal projection of respiratory neurons in the medullary raphe nuclei of rabbits. The extracellular spikes of single respiratory neurons in the midline medullary tegmentum of urethane-anesthetized and spontaneously breathing rabbits were explored. Expiratory (E), inspiratory (I), late-I, I-frequency modulated, I-E phase-spanning and E-I phase-spanning neurons, based on the relation of their firing to the phase of respiration were recorded. They were located in the raphe magnus, obscurus and pallidus. They were tested for projections to the spinal cord by antidromic electrical stimulation. Some of E neurons were projecting to the spinal cord. These results suggest that medullary raphe nuclei may take part in control of respiration in rabbits. [Jpn J Physiol 54 Suppl:S109 (2004)]

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The larynx plays critical roles in respiration, vocalization, and several airway defensive reflexes. Functional roles of the vocal cord tensor in these behaviors are not well known. Intracellular recordings of cricothyroid muscle motoneurons (CTMs) were performed in 11 decerebrated and paralyzed cats during fictive respiration, vocalization, coughing, and swallowing. Intracellular application of continuous negative currents was attempted to confirm the presence of inhibitory postsynaptic potentials. All CTMs depolarized during the inspiratory phase of breathing; one half depolarized during the expiratory phase as well. During vocalization, CTMs rapidly depolarized at the onset of the vocal phase and retained strong depolarization throughout the vocal phase. During coughing, CTMs depolarized during the inspiratory phase, repolarized prior to the onset of the abdominal nerve burst, which was confirmed to exist inhibitory postsynaptic potentials, and then depolarized during the abdominal burst. During swallowing, most CTMs exhibited a hyperpolarization, sometimes followed by a weak depolarization, whereas some CTMs exhibited a slight depolarization. This study suggests that the main role of the vocal cord tensor is vocalization rather than airway defensive reflexes and that CTM activities are primarily generated by excitatory inputs during breathing and vocalization, while inhibitory inputs play an important role to control CTM activities during coughing and swallowing. [Jpn J Physiol 54 Suppl:S109 (2004)]

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We have previously demonstrated that the medullary raphe nuclei played important roles in respiratory control in cats. Electrical stimulation of the medullary raphe complex [raphe magnus (RM), raphe pallidus (RP) and raphe obscurus (RO)] exerts different effects on respiration. The present study aimed to clarify the effects of raphe stimulation on respiration in rats, and to identify the putative neurotransmitters (GABA and serotonin (5-HT)) involved, using electrophysiological and immunohistochemical methods. Experiments were performed on adult Wistar rats anesthetized with ketamine and xylazine. Train pulse stimulation (100 Hz, 100 pulses, 10-30 μA) was applied at the raphe nucleus regions. Electrical stimulation of RM depressed respiratory movements measured by means of abdominal pneumograph, while stimulation of RP facilitated inspiratory movements. Stimulation of RO evoked mixed (inhibitory and facilitatory) effects depending on stimulus sites and intensities. After microinjection of Texas red into phrenic motor nucleus regions of the C4-5 segments, some neurons in the RM, RP and RO were retrogradely labeled. 5-HT positive neurons were observed predominantly in RP and sparsely in RM, while GAD positive neurons were predominantly observed in RM. The results suggest that RM inhibits respiration thorough a GABAergic system, while RP facilitates respiration through a serotonergic system. [Jpn J Physiol 54 Suppl:S110 (2004)]

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In the rhythmic brainstem slice preparation, spontaneous respiratory activity of pre-Botzinger complex (PBC) is generated endogenously and can be recorded as output activity from hypoglossal XII nerve rootlets. We used reduced medullary slice preparations and analyzed changes of spontaneous respiratory activity to elucidate the mechanisms for respiratory rhythm generation. The transverse medullary slices (700-1500 μm) was prepared from neonatal rats and/or mice (1-5 days old Wistar rats and ICR mice). Rhythmic respiratory bursts were recorded from hypoglossal XII nerve rootlets with glass suction electrodes. During recordings, slices were continuously superfused (3-4 ml/min) with mock cerebrospinal fluid with elevated extracellular K⁺ concentration (5-10 mM) equilibrated with carbogen (95% O₂ and 5% CO₂), pH 7.4 at room temperature (~26°C). When rhythmic respiratory bursts (2-7 bursts/min) were observed, transverse slice preparations were cut in half along the mid-line (mid-sagittal transections) and reduced to half slices. In half slice preparations, rhythmic respiratory bursts (1-5 bursts/min) could be recorded. To determine the respiratory bursting frequency generated in hypoglossal nerve rootlets, consecutive respiratory cycles were measured and compared before and after mid-sagittal transections. Similar respiratory bursts with slightly decreased frequency were observed after mid-sagittal transection. These results demonstrate that PBC neurons in one half slice are capable of generating rhythmic respiratory bursts. [Jpn J Physiol 54 Suppl:S110 (2004)]

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Mice reduce ventilation and metabolism under hyperthermia, which indicates a reduction of thermogenesis. In order to examine the contribution of histamine (HA) H1 receptors to this process, we examined ventilation, aerobic metabolism and arterial blood gasses at normothermia and hyperthermia in HA H1 receptor knock out (HIRKO) and wild type (WT) mice. Measurements were performed in the unrestrained and conscious state. At hyperthermia, VE decreased in proportion to the decrease of O₂ consumption (VO₂) and CO₂ excretion (VCO₂) in WT mice. The VE/VO₂ ratio remained constant and blood gas showed normocapnia at both body temperatures (BTs) in WT mice. H1RKO mice increased VE and decreased VO₂ and VCO₂ compared to WT mice at both BTs. The VE/VO₂ ratios were constant at both BTs, whereas the ratio was higher and PaCO₂ was lower in H1RKO mice than in WT mice at both BTs, suggesting hyperventilation. That hyperthermia reduces VE and VO₂ in H1RKO and WT mice suggests that the HA H1 receptor does not contribute to the thermal control of respiration in mice. However, H1RKO mice showed hyperventilation induced by a decrease of VO₂ and an increase of VE, indicating that the HA H1 receptor contributes to the maintenance of metabolism. It is known that central HA reduces feeding and increases energy consumption through sympathetic pathway via H1 receptors. Therefore, the hypometabolism in H1RKO mice is reasonable, but relatively high VE with respect to the decreased VO₂ in H1RKO mice warrants further study. [Jpn J Physiol 54 Suppl:S110 (2004)]

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We found that a perfusion of fluoxetine in the dorsomedial medulla oblongata (DMM) (includes the hypoglossal nucleus, the dorsal motor nucleus of the vagus nerve, and the solitary tract nucleus) increased the 5-HT concentration and airway resistance with an occasional increase of respiratory rate, may be due to a difference in probe sites. In the present study, the role of 5-HT at the caudal DMM in respiration was examined. The role of 5-HT₂ receptors in respiration was also investigated. Male mice (C57BL/6N) were anesthetized with pentobarbital sodium i.p. to insert a microdialysis probe into the DMM and placed in a double chamber plethysmograph. After waiting over 1 hour to recover from the anesthesia and to acclimatize to the chamber, extracellular fluid was collected at 1.2 μl/min of artificial cerebrospinal fluid (aCSF). Two respiratory flow curves from the nasal and the body were recorded. A perfusion of aCSF in two groups was changed with 1x10⁻⁵M fluoxetine perfusion and a co-perfusion of 1x10⁻⁵M fluoxetine and 10⁻⁵M LY-53857, a 5-HT₂ receptor antagonist, respectively. The 5-HT concentration was measured using an ECD-HPLC. Respiratory rate, tidal volume and specific airway resistance (sRaw) were measured using PowerLab. The results of the preliminary experiment suggest that the respiratory rate and sRaw were increased by a perfusion of fluoxetine in the caudal DMM, with an increase of 5-HT concentration. We discuss the relationship between the respiratory response and the probe sites in the DMM, including the role of 5-HT₂ receptors in the respiratory response. [Jpn J Physiol 54 Suppl:S110 (2004)]

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In order to clarify whether central histamine affects breathing pattern formation, we examined breathing patterns during hypercapnia in wild type (WT) and histamine H1 receptor knock-out (H1RKO) mice. Male WT and H1RKO mice were used at 9-10 weeks of age. Breathing pattern was measured with whole body plethysmograph during stepwise CO₂ gas exposures from 3% to 9% in an unaesthetized state, which was also performed in cervical vagotomized mice after recovery of pentobarbital anesthesia. PaCO₂ was determined by blood gas analysis during 5% and 9% CO₂ gas exposures in a conscious state. Minute ventilation increased in proportion with a PaCO₂-increase similarly in both mice; however, respiratory rate (RR) was lower and tidal volume (VT) was higher in H1RKO mice than in WT mice. The VT-inspiratory time (TI) relationship during CO₂ gas exposure showed a hyperbolic curve in both mice, whereas the relationship in H1RKO mice shifted above to the right than those in WT mice. After vagotomy, the VT-TI relationship changed to a vertical line in both mice, meaning that VT increased without a change of TI. The VT-TI lines of the H1RKO mice shifted to the right of those of the WT mice. These results suggest that H1RKO mice elevated the threshold level terminating inspiration; therefore, decreased RR and increased VT were observed at the same PaCO₂, which indicates that the inspiratory off-switch mechanism was altered. It is conceivable that histamine centrally affects breathing pattern formation via H1 receptors. [Jpn J Physiol 54 Suppl:S111 (2004)]

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We studied effects of Doxapram HCl on the heart rate variability known as a major source of the cardiopulmonary modulation. The experiments were carried out using adult Wister rats under urethane anesthesia. The electric stimulation was applied to right cervical vagal nerves with and without blocking of afferent conduction. The drug was delivered from the inguinal femoral vein. Frequency analysis, autocorrelation-FFT method was applied to extracted time domains characterized by RR, PP, QT and PR intervals to obtain respective power spectra. The respiration induced component in the RR spectrum nearly identical to that of PP spectra, whereas much small amplitude in QT and PR spectra. When the vagal nerves were blocked bilaterally, the respiration became slow and lowered PP and PR intervals. The variability was less correlated to the respiration, in some cases provided double peak in PR and QT spectra. When the drug was administrated, the respiration amplitude was significantly increased and restored the variability. It further lowered the PP intervals, but significantly increased PR intervals. Elevated respiratory amplitude by Doxapram HCl administration was not affected by electric stimulation after vagal blocking. It also increased the heart rate variability component synchronized to respiratory rhythm. The present findings strongly suggested that Doxapram HCl may act in the respiration center stimulating cardiopulmonary reflex to balance the pulmonary and body circulations. [Jpn J Physiol 54 Suppl:S111 (2004)]

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Low frequency stimulation of the vagal pulmonary stretch receptor (PSR) afferents promotes central inspiratory activity (vagal inspration-promoting (VIP) reflex, Takano and Kato, J. Physiol., 1999, 2003). P2X receptors modulate excitatory synaptic transmission in the nucleus of the solitary tract (NTS), where the PSR afferents terminate, in a manner sensitive to pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; Kato and Shigetomi, J. Physiol., 2001). To elucidate the role of P2X receptors in the VIP reflex, PPADS was microinjected into the NTS in anesthetized rabbits. The vehicle was injected into the NTS contralateral to the PPADS injection. Effects of afferent stimulation of the vagus nerve ipsilateral to PPADS injection (il-stim) and contralateral (cl-stim) on the phrenic nerve rhythm were compared. Unilateral microinjection of PPADS (20 nmole/side/rabbit) significantly reduced the shortening effects of il-stim at a low fre-quency (5-40 Hz) on respiratory cycle (T_TOT) and expiratory time (T_E) for 60-120 min, whereas the effects of cl-stim and the expiratory prolonging effects of il-stim and cl-stim at a higher frequency (100-160 Hz) were not affected. These results suggest that the ATP-P2X receptor-mediated signaling in the NTS is involved in the respiratory responses to low frequency afferent inputs via vagus afferents. [Jpn J Physiol 54 Suppl:S111 (2004)]

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To identify muscarinic acetylcholine receptor (mAChR) subtypes being involved in the respiratory control system, we analyzed respiratory function of each subtype (M1-M5) of mAChRs gene knockout mice. Ventilatory parameters, i.e. respiratory frequency, tidal volume and minute ventilation, were recorded by whole body plethysmography in normoxic, hyperoxic, hypoxic, and hypercapnic conditions. In a single subtype of mAChR knockout mice, respiratory parameters in each condition did not differ from those of wild type mice. In contrast, respiratory frequency (138 ± 24 breaths/min) of M1/M3-compound knockout mice was significantly lower than that (180 ± 33 breaths/min) of wild type mice under normoxia. This slow rhythm of breathing in the compound knockout mice was observed under hyperoxia and hypercapnia but not under hypoxia. Tidal volume of M1/M3-compound knockout mice in each condition was slightly smaller than that of wild type mice, although the difference was not statistically significant We conclude that M1/M3-compound subtypes of mAChRs play a significant role in the respiratory neural network.
This work was supported by Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization of Japan (M.M.). [Jpn J Physiol 54 Suppl:S111 (2004)]

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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 54 Suppl:S112 (2004)]

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[0bjective] To visualize in vivo perialveolar microcirculation in pulmomary hypertension rats with our novel CCD videomicroscopy to evaluate their vasoreactivity together with their morphological damage. [Method] We visualized in vivo perialveolar microcirculations in monocrotaline(MCT) treated rats with our novel pencil-lens CCD videomicroscopy and evaluated their possible altered morphology and vasoreactivity by endothelium-dependent vasodilator, acetylcholine(2μg/kg/min). The morphology of arterioles were studied by the injection of metacrylate resin into the pulmonary artery and visualized by scanning electron microscopy. [Results] Pulmonary arterial pressure were 22±3 under control, 37±8 at 1w, 42±4 at 2w, 64±22 mmHg at 3w after MCT administration. The morphology of arterioles exhibited a serpentine configuration with rough and uneven inner surface. Capillaries of PH also exhibited deformed morphology with apparently less vascular volume. The endothelial dependent vascular reactivity of pulmonary arterioles was reduced at 3w after MCT administration(10.6±11.0, 9.0±7.5, 3.0±7.0mmHg). [Conclusion] The pulmonary arterioles reactivity to Ach was reduced in MCT rats with arteriolar morphological changes, indicating endotherial dysfunction with remodeling occurs when PH exists. [Jpn J Physiol 54 Suppl:S112 (2004)]

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OBJECTIVE:To analyze in vivo mechanical interaction between perialveolar microcirculation and individual alveolar respirations in rats with our pencil-lens probe CCD videomicroscope.METHOD:Perialveolar microcirculation and alveolar respiration were observed via pleural visceralis. We compared the behavior of the microvessels and individual alveoli during mechanical ventilation with different tidal volumes, PEEP of 20 cmH2O, and exposure to hypoxia (10% O2). RESULTS:We observed the semi-collapsed sheet-like perialveolar capillaries at end-inspiration without cessation of flow, indicating existence of"waterfall phenomenon". When tidal volume was increased from 2 to 5 ml, the alveolar diameter was increased, and the velocity of perialveolar capillary erythrocytes was significantly decreased by about 90%, but the flow still continued through semi-collapsed capillaries during a respiratory cycle. When tidal volume was 5 ml with PEEP, the flows of arterioles and venules stopped at end-inspiration in various parts, indicating discontinuance of waterfall. We also demonstrated that precapillary arterioles clearly constricted by 18% in response to hypoxia (n=9, p<0.01).CONCLUSION:We succeeded in the visualization of in vivo perialveolar microcirculation and individual alveolar respirations. The mechanical interaction is crucial for understanding pathophysiology of pulmonary microcirculation. [Jpn J Physiol 54 Suppl:S112 (2004)]

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The purpose of this study was to test whether chronically enhanced O₂ delivery to tissues, without arterial hyperoxia, can change acute ventilatory responses to hypercapnia and hypoxia. The effects of decreased hemoglobin (Hb)-O₂ affinity on ventilatory responses during hypercapnia (0%, 5%, 7%, and 9% CO₂ in O₂) and hypoxia (10% and 15% O₂ in N₂) were assessed in mutant mice expressing Hb Presbyterian (mutation in the β-globin gene, β108 Asn→Lys). O₂ consumption during normoxia was significantly higher in the mutant mice than in wild-type mice. Respiratory measurements were conducted with a whole-body, unrestrained, single-chamber plethysmograph under conscious conditions. During hypercapnia, there was no difference between the slopes of the hypercapnic ventilatory responses, whereas minute ventilation at the same levels of PaCO₂ was lower in the Presbyterian mice than in the wild-type mice. During both hypoxic exposures, ventilatory responses were blunted in the mutant mice compared with responses in the wild-type mice. Chronically enhanced O₂ delivery to peripheral tissues can reduce ventilation during acute hypercapnic and hypoxic exposures. [Jpn J Physiol 54 Suppl:S112 (2004)]

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Cells respond to physical and chemical stimulations mediated by pH, osmolarity, and oxidative and mechanical stresses. Various signal transduction pathways cooperate and participate in these responses. Here we describe the role of c-Jun NH₂-terminal kinase (JNK) in regulation of gene transcription after an increase in extracellular H⁺. When cells were incubated in low pH medium, the promotion of JNK phosphorylation and c-Jun expression was clearly observed in cells in an extracellular pH- and time-dependent manner. Activation of p38 and extracellular signal-regulated kinase 1/2 was extremely weak compared with that of JNK. An increase in extracellular H⁺ led to enhanced nuclear translocation of phosphorylated JNK leading to augmentation of the transcriptional activity of c-Jun. Nimodipine, a blocker of voltage-gated Ca²⁺ ion channels, prevented the phosphorylation of JNK and expression of c-Jun in a dose-dependent manner. These results suggest a novel intracellular signalling pathway for H⁺-induced c-Jun expression : an increase of extracellular H⁺ induces JNK phosphorylation and c-Jun expression via partly extracellular Ca²⁺ influx through voltage-gated Ca²⁺ channels. [Jpn J Physiol 54 Suppl:S113 (2004)]

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Atmospheric pressure ionization mass spectrometers (APIMS) can detect very low levels of gaseous compounds. The combined use of a clean gas-trap system and APIMS has the potential of detecting low levels of metabolites in the body. In the present study, we developed a new gas-trap system for the analysis of skin gas which may contain numerous kind of volatile compounds. The developed system consists mainly of mass-flow controllers, gas cleaners and a thermo-controlled chamber in which the cleaned gases pass. Heat sensors were attached to the surfaces of the chamber and connecting-tube, which were surrounded by a heater. The surface of the skin was completely covered with the chamber. While maintaining a constant mixing ratio, the skin gas was diluted with the same carrier gas and introduced into the APIMS. The mass spectrum of the skin gas from the palm was analyzed using both negative and positive charge modes of the APIMS. In both ionization states the spectrums were well reproduced. We next compared mass spectrums of the skin gas with those of exhaled air in the same healthy subjects. There was a significant linear relationship between ion intensities of skin gas and exhaled air. In conclusion, a new type of gas-trap system was developed for biological application of an APIMS with high sensitivity. The system successfully detected numerous kinds of volatile compounds with low mass numbers released from the skin. [Jpn J Physiol 54 Suppl:S113 (2004)]

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We previously reported that a novel continuous cell separation method using a seal-less continuous flow centrifuge was capable of separating a large number of nucleated cells with minimum damage during only a few hours of operation. Under a centrifugal force field, the introduction of cell suspension into the proximal portion of the channel resulted in continuous separation of cells according to their densities. The performance of the apparatus was demonstrated on the separation of human buffy coat. As for the differential leukocyte count on smear preparation with May-Grünwald-Giemsa stain, Lymphocytes were mainly present in fraction 4 (d=1.070) at over 90% without red blood cells. Neutrophils were observed in fraction 6 (d=1.080) at more than 90%, but red blood cells were included. Lymphocytes and neutrophils were mostly well separated. In fraction 5 (d=1.075), basophils were concentrated at near 15%. On a result analyzed by flow cytometry, lymphocytes (CD45+, CD14-) were mostly collected in fraction 3 and 4, granulocytes (CD45±, CD14-), which mainly consisted of neutrophils, were collected in fraction 5 and 6. Monocytes (CD45+, CD14+) and CD34 positive cells were collected in fraction 2. These results indicated that the present method was capable of concentrating rare cells such as basophils or CD34 positive cells in a proper fraction. The method may be applied to concentrate hematopoietic stem cells. [Jpn J Physiol 54 Suppl:S113 (2004)]

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Thrombin is a multifunctional serine protease in haemostasis. It is a two-chain glycoprotein containing an Asn53-binding carbohydrate chain, consisting of lactosamin and N-acetyl neuraminic acid (NeuAc). The glycosylated site is located in the insertion loop Lue45-Asn57 near the catalytic site cleft. However, the function of the carbohydrate chain remains unknown. In this report, the enzymatic property of the carbohydrate chain moiety-modified thrombin was compared to that of intact thrombin in order to clarify the function of the carbohydrate chain. The carbohydrate chain, especially NeuAc at its non-reductive terminal was chemically modified by PDBA-hydrazide. The intact thrombin ([M+H]⁺=37,494, measured by MALDI/TOF mass spectrometer) released fibrinopeptide A and B during fibrin clot formation. The PDBA-modified thrombin ([M+H]⁺=38,012) formed fibrin clot, however, fibrinopeptide A was primarily released while little fibrinopeptide B was released. The result led us to propose that the access of the fibrinogen Bβ chain to the catalytic site cleft was restricted by the modification of the carbohydrate chain. This chain played an important role in the protease activity, especially the interaction between thrombin and macromolecular substrate such as fibrinogen. The chemical modification of NeuAc of the carbohydrate chain may cause the structural alteration of the catalytic site cleft of thrombin. [Jpn J Physiol 54 Suppl:S114 (2004)]

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A phospholipid vesicle encapsulating a concentrated hemoglobin solution and pyridoxal 5'-phosphate as an allosteric effector (Hb-vesicle: diameter, 250 nm) has been developed as an oxygen carrier. The oxygen release from flowing Hb-vesicle was examined using an oxygen-permeable fluorinated ethylenepropylene copolymer tube (inner diameter, 0.028 mm) under deoxygenated environment. The oxygen release was measured using a scanning-grating spectrophotometer constructed on an inverted microscope. The rate of oxygen release was determined based on the visible absorption spectrum. Hb-vesicles and fresh human RBCs were mixed in various ratios at [Hb] = 100 g/L in isotonic saline containing 50 g/L albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The Hb-vesicles were homogeneously dispersed in the isotonic saline. With increasing the amount of the Hb-vesicles resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of oxygen release from the Hb-vesicle-RBC mixtures was similar with that from RBC alone. However, the addition of 50 vol% Hb solution to RBCs enhanced the release of oxygen. In conclusion, the difference in the rate of oxygen release between Hb-vesicles and Hb should mainly be due to the difference in the particle size (250 vs. 8 nm), namely in the oxygen diffusion distance. [Jpn J Physiol 54 Suppl:S114 (2004)]

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The liver regeneration after injury is is regulated by various growth factors and cytokines. Release of these growth factors is deeply related to degradation of extracellular matrix (ECM). The ECM degradation is regulated by the activation of plasminogen (Plg) and matrix metalloproteinase (MMP) systems. The liver regenerations on knockout mice (KO) for fibrinolytic factors were examined by using CCl₄ injection model. Then, the relationship between the liver regeneration and plasmin/α₂-AP system was analyzed by using the hepatocytes isolated from mouse liver. The proliferation ability of the hepatocytes isolated from mice without CCl₄ injection showed similar in all genotype mice. On the other hand, the proliferation ability of the hepatocytes from mice of 5 days after CCl₄ injection was significantly increased in the α₂-APKO as compared to the WT, but decreased in the PlgKO, and Plg-α₂-APKO. The hepatocytes isolated from all genotype mice similarly bound to the immobilized Plg. The binding ability of hepatocytes, which were isolated from mouse after CCl₄ injection, to Plg was significantly decreased in the PlgKO, and Plg-α₂-APKO as compared to the WT, or α₂-APKO. The activation of Plg was significantly increased in the presence of mouse hepatocytes. The plasmin inhibition by α₂-AP in the presence of mouse hepatocytes was weaker than that in the absence of mouse hepatocytes. These results indicate that the plasmin/α₂-AP system on the surfaces of mouse hepatocytes plays important roles in liver regeneration. [Jpn J Physiol 54 Suppl:S114 (2004)]

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Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of tissue-type and urokinase-type plasminogen activators (tPA, uPA), which convert plasminogen to plasmin. As plasmin plays a vital role in the regulation of intra- and extra-vascular fibrinolysis, the precise regulation of plasmin formation could provide a critical element of control in various processes such as fibrinolysis, tissue remodeling, inflammation and fibrous formation. We previously reported that prostaglandin I₂ (PGI₂) enhanced the expression of uPA in cultured human fibroblasts. In the present study, we examined the effect of PGI₂ on PAI-1 expression by TIG 3-20 fibroblast cells. Northern analyses revealed that beraprost sodium, a stable PGI₂ analog, inhibited PAI-1 mRNA expression induced by fetal calf serum in dose- and time-dependent manners. This inhibition by PGI₂ was abolished in the presence of DRB, an inhibitor of transcription, suggesting that transcriptional mechanism was involved in the PAI-1 regulation. Cycloheximide, an inhibitor of translation, did not affect the inhibition of PGI₂, suggesting that de novo protein synthesis was not required for the regulation. PGI₂ also inhibited the PAI-1 mRNA expression induced by cytokines such as interleukin-1β, transforming growth factor-β and tumor necrosis factor-α. Although the precise mechanism of the inhibition by PGI₂ has not been resolved in this study, it is likely that PGI₂ can modulate the fibrinolytic activity of the cells through the inhibition of PAI-1 expression. [Jpn J Physiol 54 Suppl:S114 (2004)]

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We reported that restraint or electrical shock stress reduced blood fluidity of rats estimated by MC-FAN(micro channel flow analyzer), which mimics capillary vessel and low frequency magnetic stimulation augmented the blood fluidity. Therefore we attempt to study the restoration effect of low frequency magnetic stimulation on reduced blood fluidity by stress. The restoration from reduction of blood fluidity by electrical shock stress (1Hz, duration: 10nsec, 70-110V, for 1 hour) was accelerated by magnetic stimulation (50Hz, about 45mT). Adrenergic α agonist reduced the blood fluidity but magnetic stimulation restored the reduction of blood fluidity by α agonist. These results suggest that electrical shock stress may reduce the blood fluidity through adrenergic α mechanism and magnetic stimulation may restore the blood fluidity although through adrenergic α mechanism. [Jpn J Physiol 54 Suppl:S115 (2004)]

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In oriental medicine the conception of "oketsu" is known widely. This conception means stagnation of blood flow. Some herb medicines, which eliminate "oketsu" syndrome, are known as "ku-oketsu-zai". We attempt to study the effects of "ku-oketsu-zai" on the blood fluidity of rats by MC-FAN (micro channel array flow analyzer). Repeated oral administration of tokishakuyakusan or kangenkaryu as "ku-oketsu-zai" once a day for 1 week accelerated the blood fluidity of rats but single administration did not. It was experimentally demonstrated that "ku-oketsu-zai" actually accelerated the blood fluidity of rats. We are now studying whether "ku-oketsu-zai" may restore the reduction of blood fluidity by some stressors. [Jpn J Physiol 54 Suppl:S115 (2004)]

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We have previously reported that the K⁺ channel with an inward conductance of 90 pS in opossum kidney proximal tubule (OKP) cells was inactivated by protein kinase C-mediated phosphorylation. In this study, we further examined the role of Ca²⁺/calmodulin kinase II (CaMK II)-mediated phosphorylation process on this channel activity. 1) In cell-attached patches, the channel activity was inhibited by a decrease in cytosolic Ca²⁺ by the perfusion of low Ca²⁺ (10⁻⁹ M) or 100 μM La³⁺ containing solution, or by the addition of 100 μM EGTA/AM to the bath. The application of KN-62 (10 μM) or KN-93 (5 μM), inhibitors of CaMK II, inhibited the channel activity, whereas L-NAME (5 μM) or L-NNA (10 μM), cNOS inhibitors, failed to inhibit the channel activity. These results suggest that Ca²⁺/CaMK II-mediated phosphorylation process is involved in the regulation of channel activity. 2) Membrane potential measured with nystatin-perforated patches was significantly decreased by a fall in cytosolic Ca²⁺ with low Ca²⁺ (10⁻⁹ M), La³⁺ (100 μM), or EGTA/AM (100 μM) containing solution in the bath. In addition, the application of KN-62 (10 μM) or KN-93 (5 μM) to the bath significantly decreased the membrane potential. 3) In inside-out patches with 10^−6.5 M Ca²⁺ in the bath, the channel activity was dramatically stimulated by the application of CaMK II (300 pM). In conclusion, Ca²⁺/CaMK II-mediated phosphorylation process is involved in maintaining the K⁺ channel activity in OKP cells. [Jpn J Physiol 54 Suppl:S115 (2004)]

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Fatty acids (FA) with at least 12 carbon atoms increase intracellular Ca²⁺ ([Ca²⁺]_i) to stimulate cholecystokinin (CCK) release from enteroendocrine cells (EEC). Using the murine EEC line STC-1, we investigated whether candidate intracellular pathways transduce the FA signal, or whether FA themselves act within the cell to release Ca²⁺ directly from the intracellular store. In STC-1 cells loaded with fura-2, short (3 minutes) exposure of C12 fatty acid, but not C8 or C10, induced a dose-dependent increase in [Ca²⁺]_i, in the presence or absence of extracellular Ca²⁺. Various signaling inhibitors, including IP₃ receptor antagonists, all failed to block FA-induced Ca²⁺ responses. In permeabilized STC-1 cells loaded with the lower affinity Ca²⁺ dye magfura-2, and permeabilized by Streptolysin O, again C12 but not C8 or C10, induced release of stored Ca²⁺ dose dependently. However, 30 minutes exposure to C12 induced a sustained elevation of [Ca²⁺]_i in the presence of extracellular Ca²⁺, but only a transient response in the absence of extracellular Ca²⁺. These results suggest that at least two FA sensing mechanisms operate in enteroendocrine cells: intracellularly, FA (C12) transiently induces Ca²⁺ release from intracellular Ca²⁺ stores. However, they also induce sustained Ca²⁺ entry from the extracellular medium to maintain an elevated [Ca²⁺]_i. [Jpn J Physiol 54 Suppl:S116 (2004)]

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Short-chain fatty acids (SCFAs) are produced by bacterial fermentation in the lumen of colon and some previous studies have suggested that SCFAs enhance the motility of this organ. In the present study, effects of SCFAs (acetate, propionate, and butyrate) on the contractions of circular muscle were examined using strip preparations of rat distal colon. In non-stimulus stage, two different motility patterns were observed; one is contractions with high amplitude and low frequency (0.81 ± 0.03 g, 0.30 ± 0.01 / min) and another is contractions with low amplitude and high frequency (0.12 ± 0.01 g, 12.21 ± 0.15 / min). In the present experiment, effects of SCFAs on the former motility pattern were examined. Propionate dose-dependently increased frequency of the contraction while amplitude was slightly decreased. Mixture of SCFAs at the same ratio of physiological condition (acetate : propionate : butyrate = 15 : 7 : 4) also had the same effects. Acetate or butyrate did not increase the frequency. On the other hand, propionate or butyrate but not acetate dose-dependently induced phasic contraction immediately after the addition and the maximal responses were observed at 1 mM. The propionate(1 mM)-induced phasic contraction was inhibited by preincubation of acetate, atropine, or tetrodotoxin. These results suggest that among SCFAs, propionate most effectively enhances the motility of rat colon and a part of the responses of propionate seems to be mediated via cholinergic nerves. [Jpn J Physiol 54 Suppl:S116 (2004)]

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Ghrelin, a novel growth-hormone releasing peptide, was originally isolated from rat and human stomach. In the present study, the effect of centrally injected ghrelin on pancreatic exocrine secretion was examined in conscious rats. Moreover, an electrophysiologic study was conducted in anesthetized rats to examine whether the excitation of vagal efferent nerve could be induced by ghrelin. To determine the pancreatic exocrine secretion, rats were prepared with cannulae draining bile and pancreatic juice separately. The experiments were conducted in awake rats on day 4 or 5 after the operation. In order to exclude the involvement of gastric acid, a proton pump inhibitor omeprazole (5 mmol/kg) was administered into the duodenum 1 h before ghrelin injection. Intracerebroventricular administration of ghrelin (12, 60, and 300 pmol/10 ml) significantly increased pancreatic fluid and protein output in a dose-dependent manner. Pretreatment with the ganglion blocker hexamethonium and with atropine completely abolished the stimulatory effect of central ghrelin. In contrast, intravenous injection of ghrelin (300 pmol/10 ml) had no effect. Centrally administered ghrelin stimulated the vagal efferent nerve in anesthetized rats. In conclusion, centrally administered ghrelin stimulates pancreatic exocrine secretion through the vagal efferent nerve and the stimulatory action is independent of gastric acid secretion. [Jpn J Physiol 54 Suppl:S116 (2004)]

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[Background & objects] EGG and ECG-RRI have been studied during the stress of MDT in human subjects. Furthermore, respiratory frequency was recorded simultaneously. [Materials & Methods] EGGs (τ=5sec, high cut=0.5Hz) were recorded from epigastric, supraumbilical and infraumbilical regions and analyzed spectrally with MEM(maximal entropy method). Stress was loaded with MDT(tracing a star figure reflected on a mirror with an electric pen, giving click alarms, when tracing is run off the star egde). Respiratory frequency was monitored by a nasal thermister. Stress responses of EGG(peak power ratio of certain spectral peaks during MDT to those during at rest) were calculated. Informed consent was obtained from all subjects, healthy volunteers(n=33). [Results] Heart rate and respiratory frequency increased in 30 out of 33 cases during MDT-stress and means of them at rest(59.4/min and 12.7/min) increased significantly during the stress(65.5 and 18.4). Mean stress resonses EGG increased, especially 1, 3 and 6 cpm groups of infraumbilical region but inhibitory responses to the stress were also encountered in all EGG frequencies and at all three regions. [Discussion] The MDT stress induced the excitatory effects on heart and respiratory rate but it induced the dual excitatory and inhibitory effects on the EGG, gastrointestinal electrical activities in healthy volunteers. [Jpn J Physiol 54 Suppl:S117 (2004)]

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The transient mechanical responses after quick change in load or in length were observed in the living single muscle fibers and these experiments contributed greatly to proposing the hypothesis of cross-bridge rotation in muscle contraction. Sugi and Tsuchiya (1981) also studied the transient length changes in living single fibers when the quick increase in load was applied during shortening. We developed the electromagnetic apparatus to perform quick change in load in the motility system consisted of the magnetizable beads with myosin thick filaments from molluscan smooth muscle or myosin of Chara and actin cables in the Chara internodal cells. In the present experiment, the quick change in load (about 10 ms) to the beads (diameter 4.5μm) was applied in the range of 0-85 pN. The movement of beads was recorded by video-system and analyzed with special software (Swallow series, Digimo). When the quick increase in load was applied during the movement of beads under no load, the beads showed the transient movement to the reverse direction before the steady slower movement to normal direction. When the application of load was stopped, the beads showed transient fast phase of movement. These velocity transients are quite similar to those observed previously in single fibers and suggesting that the velocity transients are the intrinsic properties induced by the interaction between actin and myosin without hexagonal lattice structure of filaments or regular sarcomere structure irrespective of myosin types. [Jpn J Physiol 54 Suppl:S117 (2004)]

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The biological effects of estrogen receptor(ER) had to be mediated by a receptor protein. The skeletal muscle is one of the important target tissues for estrogen, as evidenced by significant effects of estrogen on muscle contraction, muscle fatigue and cell proliferation. In this study, we investigated the regulation of ER expression by the immunoblot analysis. The ER in C2C12 cell was detected as 66 kDa and 54 kDa forms as well as in the ovary, uterus, skeletal muscle, kidney, lung, adipose in the intact mouse. Although the intensity of 66 kDa form was stronger than that of 54 kDa in intact mouse, 66 kDa form was weaker than 54 kDa form in C2C12. The effect of the 17-beta estradiol administration on the ER expression in C2C12 cell was investigated. The ER expression increased gradually after the 17-beta estradiol administration, then reaching the maximum at 12 h. This increase was compatible with the effect of proliferation and the alteration of intracellular Ca concentration. Therefore, these results suggest that the modulation of ER expression level by the extracellular situation, Ca concentration and the ligands, could be associated with the proliferation and the intracellular Ca concentration. The functional role of 66 kDa and 54 kDa forms in some tissues and cells will be clarified by another analysis. [Jpn J Physiol 54 Suppl:S117 (2004)]

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We examined the role of nitric oxide (NO) in the development of muscle damage. A standardized eccentric stretch injury was created to the tibialis anterior (TA) muscle of 40 male Wistar rats (220 to 250 g), using a customized isokinetic test device and electrical stimulation. We evaluated the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME; 85 mg/kg/day) on skeletal muscle damage and repair. The TA muscle was isolated immediately, 1, 3 and 7 days after muscle stretch injury. The animals were divided into two groups: (1) structural muscle damage, without L-NAME infusion (SMD/-L-NAME) (n=5 at each day); (2) structural muscle damage, with L-NAME infusion (SMD/+L-NAME) (n=5 at each day). NO content was greater in the SDM/-L-NAME group at all time points. Additionally, significant differences in NO content were observed on day 0 (p<0.05), and day 3 (p<0.05), SMD/+L-NAME vs. SMD/-L-NAME. The first day following injury, muscle protein degradation, as characterized by m-calpain mRNA levels and calpain activity, was increased in both groups. However, Myo-D and Myogenin mRNA levels were both increased at 3 days in the SMD/-L-NAME group. Our results demonstrate that NO influenced both muscle degradation and regeneration following structural damage in rat skeletal muscle. [Jpn J Physiol 54 Suppl:S118 (2004)]

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Acetaldehyde alters Ca²⁺-release channel gating and muscle contraction in a dose-dependent mannerToshiharu Oba and Yoshitaka Maeno¹, Depts.Reg.Cell Physiol., and ¹Forensic Med. Sci., Nagoya City Univ. Grad. Sch. Med. Sci., Nagoya 467-8601, JapanWe studied whether acetaldehyde, which would be produced by alcohol consumption, impacts ryanodine receptor (RyR) activity and muscle force. Exposure to 50-200 μM acetaldehyde enhanced channel activity of frog RyR and rabbit RyR1 incorporated into lipid bilayers. An increase in acetaldehyde to 1 mM modified channel activity in a time-dependent manner, with a brief activation and then inhibition. Application of 200 μM acetaldehyde to frog fibers increased twitch tension. The maximum rate of rise of tetanus tension was accelerated to 1.5 and 1.74 times the control rate on exposure of fibers to 50 and 200 μM acetaldehyde, respectively. Fluorescence monitoring using Fluo-3 demonstrated that 200-400 μM acetaldehyde induced Ca²⁺ release from the sarcoplasmic reticulum (SR) in frog muscles. Acetaldehyde at 1 mM inhibited twitch tension by about 12% with an increased relaxation time after a small and transient twitch potentiation. These results suggest that moderate concentrations of acetaldehyde can elicit Ca²⁺ release from the SR by increasing the open probability of the RyR channel, resulting in increased tension. However, the effects of acetaldehyde at clinical doses (1-30 μM) are unlikely to mediate alcohol-induced acute muscle dysfunction. [Jpn J Physiol 54 Suppl:S118 (2004)]

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We found a very interesting contraction in flexor carpi radialis muscle (FCR) of forelimb in the frog, Rana japonica, during the breeding season. This contraction has a characteristic 'long-lasting relaxation phase after contraction'. In the present experiment, we investigated the mechanical properties of FCR by measuring the change in force and stiffness of the muscle contraction at 4°C. The total time from the end of stimulation to the end of relaxation was 1797±732 s (n=13) and the relaxation was composed of three phases, which were separated clearly by two changing points on the force curve. The time of first changing point was 4.0±1.0 s (n=13) and the second one was 77±23 s (n=13) after the end of stimulation. When a quick release was applied to each phase, the force recovered slowly but not completely. The magnitude of stiffness decreased quickly in the first relaxation phase, but in the second and third phases they decreased slowly, and the third phase was the slowest. Another specific property of FCR was that it contracted spontaneously, resulting in the appreciable increase in stiffness without stimulation. Present results indicate that cross-bridge cycles would turn slowly in the relaxation and that three different types of relaxations would exist at least in the muscle fibers of FCR, and these might be related to the mechanism of the change in the intracellular Ca concentration. [Jpn J Physiol 54 Suppl:S118 (2004)]

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It has been reported that hardness palpated from the body surface is related to muscle tonus. The aim of this study, an experimental model of muscle hardness was investigated under tetanic contractions of the gastrocnemius (GS) muscle using electrical stimulation of the sciatic nerve (50Hz, 100μs, 2min) in rats (n=10). The muscle hardness was measured by a hardness meter. The M-wave was recorded from a pair of wire electrode inserted into the muscle. Muscle tension was measured by a push-pull gauge after cutting the tendon at the distal end. The M-wave and the tension of GS muscle were monitored to clarify a component of the muscle hardness. The maximum amplitude of the M-wave was observed immediately after the onset of stimulation, whereas the hardness and tension reached their maximum at 5 sec after the stimulation. The amplitude of M-wave markedly decreased compared to the hardness and tension during the stimulation. The hardness was significantly and positively correlated to tension (R=0.82, P<0.01) and the amplitude of the M-wave (R=0.63, P<0.01). After pancuronium (2mg/ml : 0.4ml, i.v.) injection, the increases in the hardness and the tension were completely suppressed. These results suggest that the muscle hardness in tetanic contractions induced by electrical nerve stimulation might mostly reflect the changes in the muscle tension. [Jpn J Physiol 54 Suppl:S118 (2004)]

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Calcineurin plays important signaling pathways in gene regulation and biological responses to external stimuli in many organisms and in many types of cell. Recently, we found that heat stress induced the increment of calcineurin expression and hypertrophy in skeletal muscle in vivo. However, the precise role of a calcineurin-dependent signaling pathway in skeletal muscle hypertrophy is still unclear. Therefore, this study was carried out to elucidate a role of a calcineurin-dependent signaling pathway in heat stress-induced muscle hypertrophy. Wistar strain male rats were divided into three groups; control (CC), heat stress (HC) and heat stress with the inhibition of calcineurin by using cyclosporine A (HI), a potent inhibitor for calcineurin. The heat-stressed groups were exposed to environmental heat stress (41°C for 60 min). Injection of cyclosporine A was administrated for animals in HI group once a day for 14 days. Soleus and extensor digitorum longus muscles were dissected from both hindlimbs 1, 7 and 14 days after heat exposure. Effects of inhibitions of calcineurin on heat-stresses muscle hypertrophy were evaluated. A partial depression of heat stress-associated muscle hypertrophy was observed in rats with inhibition of calcineurin expression. [Jpn J Physiol 54 Suppl:S119 (2004)]

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It is considered that muscle satellite cells are myogenic precursor cells and are responsible for the postnatal growth and regeneration of skeletal muscles. Recently, we have discovered that heat stress induces skeletal muscle hypertrophy in vitro and in vivo. Our results suggest that heat stress activates Akt signaling pathway and facilitates the proliferation and differentiation of satellite cells. However, it is still unclear how the heat stress influences the morphological properties (satellite cell activation) of skeletal muscle. Therefore, the current study was performed to investigate the responses of skeletal muscles to heat stress by using histochemical techniques. Wistar strain male rats were divided into two groups; control and heat stress. Heat stress group was exposed to environmental heat stress (41°C for 60 min). Soleus muscle was dissected from both hindlimbs 1 and 7 days after heat exposure. Administration of 10% Evans blue dye (100 μl/100 g BW, intravenously) were performed to some of the rats in both groups 24 hr prior to the dissection of muscles. A few muscle fibers in which accumulated Evans blue dye were noted in heat stress, but not in control, group. The data suggested that activation of satellite cells was induced following heat exposure. [Jpn J Physiol 54 Suppl:S119 (2004)]

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