We set out to study if changes of Ca²⁺-activated K⁺ (K_Ca) channels and associated signaling pathways can alter vascular reactivity during cardiac hypertrophy induced by isoprenaline. In vascular smooth muscles cells isolated from coronary artery, the whole-cell current density, single channel conductance, and open probability of K_Ca channels were significantly reduced during hypertrophy. Furthermore, K_Ca channels exhibited decreased sensitivity to [Ca²⁺]_i but pronounced block by external TEA. Consistent with the electrophysiological data, biochemical data indicated no changes in K_Ca channel expression. Furthermore, the degree of resting tension contractility increased with high [K⁺]_out and decreased with external TEA during hypertrophy. In cerebral artery, vasomotion during cardiac hypertrophy was qualitatively similar to that observed in controls but vasoconstrictor response on Angiotensin II (Ang II) was attenuated. The electrophysiological properties of K_Ca channels were significantly changed by Ang II during hypertrophy. The protein expression of Ang II receptors and associated signaling molecules also changed significantly during hypertrophy. Our findings suggest novel mechanisms for reduced coronary reserve and impaired cerebrovascular contractility during cardiac hypertrophy by modulating K_Ca channel properties and altering the gene expression of related proteins. [Jpn J Physiol 55 Suppl:S52 (2005)]

View full abstract

Mammalian circadian rhythms are generated by a feedback loop in which positive (BMAL1:CLOCK) and negative (CRYs) components activate transcription for different periods. To investigate the molecular mechanism of the circadian system, we attempted to establish a screening method to identify clock-controlling factors. The reporter construct ( Bmal1-prom-Luc) was transfected into NIH3T3. In transient expression experiments using the reporter construct, the regulatory factors that affect Bmal1 transcription were screened. Using a real-time monitoring system for Bmal1 promoter activity, we examined whether the factors that regulate Bmal1 transcription affected the circadian changes in the reporter activity of the Bmal1 promoter. The real-time monitoring system showed that the transient co-expression of RORα with the Bmal1 reporter construct in NIH3T3 cells abolished the circadian changes of the reporter activity and RNAi against RORα attenuated the circadian expression of the reporter activity. These results indicate that RORα affects not only the level of Bmal1 transcription but also its circadian expression. [Jpn J Physiol 55 Suppl:S53 (2005)]

View full abstract

The circadian rhythm is an adaptation system to a light-dark 24 hr cycle in nearly all organisms. Its center in mammals is located in the suprachismatic nucleus (SCN) of the hypothalamus, however, competitive studies for the last several years revealed that molecular clocks are located not only in SCN but also in various peripheral tissues, even in cultured cells. Moreover, molecular mechanism of circadian oscillation is based on transcriptional regulation composing of negative feedback loops of clock gene regulation. Our recent systematic analyses of canonical clock genes expression identified three clock elements (E-box, RORE, and DBPE). Using in vitro real-time oscillation monitoring system, we disclosed regulatory mechanisms of mammalian clock genes including Period, Bmal1. [Jpn J Physiol 55 Suppl:S53 (2005)]

View full abstract

To analyze the dynamics or persistence of clock gene expression, noninvasive real-time monitoring system using firefly luciferase is employed. Although this system enables longitudinal and quantitative monitoring of the gene expression in living cells, more than one gene expression cannot be monitored. To precisely analyze the molecular mechanism of circadian clock, it is critical to monitor multiple gene expressions and/or interactions with their transcription factors simultaneously. We have recently developed a novel in vitro reporter assay system, in which two gene expressions can be monitored simultaneously by splitting the emissions from green- and red-emitting luciferases. In this study, we have applied this system to real-time monitoring of clock gene expressions. To verify the system, promoter regions of mPer2 and mBmal1 were connected to the red and green luciferases, respectively, and the constructs were transiently co-transfected into rat1 fibroblasts. The bioluminescence emission from the cells was continuously measured with a luminometer equipped with an optical filter (ATTO Co.). The phases of Per2 and Bmal1 oscillations were antiphase, which is consistent with mRNA expression patterns. The identical result was also obtained when the luciferases were swapped. These results clearly indicate the system can accurately monitor respective gene expression profiles in the same cell populations even when these were measured simultaneously. We are now applying this system for analyzing interaction between clock or clock-controlled-gene products and response elements in clock gene promoters [Jpn J Physiol 55 Suppl:S53 (2005)]

View full abstract

The master circadian clock of mammals resides in the suprachiasmatic nucleus of the hypothalamus (SCN). However, most peripheral organs have also their own clocks generating circadian rhythms in clock and clock-controlled gene expressions. In order to examine the mechanisms by which the master clock regulates the peripheral clocks, we constructed a transgenic mouse line expressing firefly luciferase under the control of clock gene Bmal1 promoter and examined oscillatory mechanisms of the central and peripheral clocks by continuously monitoring bioluminescence of cultured SCN and liver. The oscillation mechanisms are also examined in the peripheral clock model of Rat-1 fibroblasts using the same Bmal1 reporter construct. Rat-1 fibroblasts showed a type 0 phase response curve not only to a brief exposure of dexamethasone but also to vehicle treatment, suggesting that desynchronized cellular oscillators are resynchronized to each other by a single perturbation. Synchronization of a number of peripheral clocks by this method seems to be advantageous for the orchestration of circadian rhythms in physiology and behavior. However, a single peripheral clock responds to multiple mediators with different sensitivities, which may cause internal desynchronization among organs and cells. [Jpn J Physiol 55 Suppl:S54 (2005)]

View full abstract

Advances in transcriptional profiling methods have allowed for the simultaneous measurement of the transcriptome of mammals. We have applied these tools to the study of the mammalian circadian clock, which governs rhythmic physiology and behavior, such as locomotor activity, to anticipated daily changes in the environment. Transcriptional profiling of the suprachiasmatic nucleus of the hypothalamus, the site of the master circadian oscillator, as well as peripheral clocks resident in the liver, aorta, and kidney, revealed a subset of genes that cycle in most tissues. Having observed that several of these ‘pan-cycling’ genes were known clock components, we used a cell-based functional assay to identify those that may regulate Bmal1 transcription within the SCN. This work revealed that Rora is required for normal Bmal1 expression, as well as for normal consolidation of daily locomotor activity. Finally, we show that Rora is itself regulated by the core clock in the SCN. These results suggest that opposing activities of the orphan nuclear receptors Rora and Rev-erb-alpha, which represses Bmal1 expression, are important in the regulation of the circadian clock. [Jpn J Physiol 55 Suppl:S54 (2005)]

View full abstract

The RBCC/TRIM family proteins are characterized by a conserved tripartitemotif, Ring finger, B-box and coiled-coil domains. We have identifiedmitsugumin53 (MG53) as a novel RBCC family member that is expressed inskeletal and cardiac muscle cells. Immunostaining and biochemical analysisindicated that MG53 is predominantly localized on the cell-surface membranein the striated muscles. Knockout mice lacking MG53 were viable and showedapparently normal health. However, (1) the mutant mice frequently showedarrhythmia including A-V block in electrocardiogram experiments. (2)cardiac myocytes from the mutant mice exhibited weak hypertrophy and showedirregular action potential, especially action potential duration. (3) Themutant myocytes showed reduction of delayed rectifier K⁺ currents, but retained normal transient outward K⁺ currents and voltage-gated Ca²⁺ currents. Moreover, we found that Kv2.1 currents were enhanced by co-expression of MG53 in cultured cells and were almost abolished by mutant MG53 lacking the ring finger domain. On the other hand, outward currents derived from Kv1.2 and Kv3.1 did not change by coexpression of MG53. These results suggest that MG53 takes part in functioning of Kv2.1 channels in striated myocytes and is essential for regulating the excitability of cardiac myocytes. [Jpn J Physiol 55 Suppl:S54 (2005)]

View full abstract

An abnormal regulation of intracellular Ca²⁺ by sarcoplasmic reticulum (SR) has been shown to be involved in the mechanism underlying contractile and relaxation dysfunction in heart failure. PKA-mediated hyperphosphorylation of ryanodine receptors (RyR) in SR was found to cause dissociation of FKBP12.6 from RyR in heart failure. This results in an abnormal Ca²⁺ leak through RyR, leading to increased cytosolic diastolic Ca²⁺ concentration, prolongation in the Ca²⁺ transient, and delayed/slowed diastolic decline in cytosolic Ca²⁺ concentration. We previously demonstrated that both beta-blocker and a cardioprotective drug JTV519 restores the defective FKBP12.6-mediated regulation of RyR channel gating, having an improvement of cardiac contractile and relaxation functions during the development of pacing-induced heart failure. Here, using domain-probe technique we further assessed the fundamental mechanism of abnormal Ca²⁺ regulatory process within RyR in heart failure. A synthetic peptide corresponding to the Gly2460-Pro2495 domain of the RyR2, designated DPc10, induced 1) unzipping mode of N-terminal and central domain interaction, 2) Ca²⁺ leak by itself, 3) facilitated FKBP12.6 dissociation, 4) prolongation of Ca²⁺ transient in myocyte. Thus, defective domain interaction within RyR (N-terminal and central) critically regulates the gating property of RyR2 in the pathogenic mechanism of heart failure, and therefore it may be a new therapeutic target against heart failure. [Jpn J Physiol 55 Suppl:S55 (2005)]

View full abstract

The Na⁺/Ca²⁺ exchanger (NCX) is a plasma membrane transporter that is expressed in many mammalian cell types, such as muscle, nerve, and epithelium. This exchanger is bidirectional, being controlled by membrane potential and transmembrane ion gradients, and is important in maintaining Ca²⁺ homeostasis. However, the role of NCX in cardiovascular diseases is not clear. We identified the role of NCX1 in salt-sensitive hypertension using SEA0400, a specific inhibitor for Ca²⁺ entry via NCX1, and genetically engineered mice. We found that SEA0400 lowered arterial blood pressure in salt-dependent hypertensive rat models, but not in normotensive rats or other types of hypertensive rats. Infusion of SEA0400 into the femoral artery in salt-dependent hypertensive rats increased arterial blood flow, indicating peripheral vasodilation. SEA0400 reversed ouabain-induced cytosolic Ca²⁺ elevation and vasoconstriction in arteries. Furthermore, heterozygous NCX1-deficient mice had low salt-sensitivity, whereas transgenic mice that specifically express NCX1.3 in smooth muscle were hypersensitive to salt. SEA0400 lowered the blood pressure in salt-dependent hypertensive mice expressing NCX1.3, but not in SEA0400-insensitive NCX1.3 mutants. These findings indicate that salt-sensitive hypertension is triggered by Ca²⁺ entry via NCX1 in arterial smooth muscle. In addition, we found that transgenic mice overexpressing cardiac NCX1.1 developed cardiac hypertrophy, and transgenic mice overexpressing cardiac NCX1.1 mutant, devoid of Na⁺-dependent inactivation, produced dilated cardiomyopathy. Thus, up-regulated Na⁺/Ca²⁺ exchange may be involved in various cardiovascular diseases. [Jpn J Physiol 55 Suppl:S55 (2005)]

View full abstract

In cardiac excitation-contraction (E-C) coupling, the cross-signaling between L-type Ca²⁺ channels and ryanodine receptors (RyRs) is the initial regulatory step of Ca²⁺ signaling. Small Ca²⁺ influx through L-type Ca²⁺ channels is amplified via Ca²⁺-induced Ca²⁺ release (CICR) from nearby RyRs, where the Ca²⁺-dependent inactivation of cardiac L-type Ca²⁺ channels plays a critical role in the fine-tuning of CICR and thus the SR Ca²⁺ content. The bimodal regulation of subsarcolemmal Ca²⁺ concentration via Na⁺-Ca²⁺ exchanger is also involved in the positive and negative control of CICR. The impairment of the fine-tuning mechanism of CICR evokes the compensatory mechanisms and triggers the pathological Ca²⁺ signaling, which lead to hypertrophy and electrophysiological remodeling in cardiac myocytes. The molecular mechanism underlying physiological regulation and deterioration of the fine-tuning of CICR and Ca²⁺ signaling will be further discussed. [Jpn J Physiol 55 Suppl:S55 (2005)]

View full abstract

A growing body of evidence, including studies from genetically engineered mouse models and human patients, has shown that Ca²⁺ cycling and Ca²⁺-dependent signaling pathways via sarcoplasmic reticulum (SR) play an important role in normal cardiac function as well as heart diseases. Among SR proteins, cardiac SR Ca²⁺ ATPase (SERCA2a) plays a pivotal role in regulating the rate of Ca²⁺ re-uptake during relaxation in the heart. The activity of SERCA2a is mainly regulated by its endogenous inhibitor, phospholamban (PLN). We have demonstrated that the regulation of SERCA2a activity via PLN may cause human cardiomyopathy and the inhibition of interaction between SERCA2a and PLN may have potential therapeutic value for heart diseases such as cardiomyopathy and heart failure. In addition to PLN, newly identified SR proteins such as sarcolipin and sarcalumenin also regulate Ca²⁺ re-uptake during relaxation through the interaction with SERCA2a. Sarcolipin is restrictedly expressed in the atria and its expression is down-regulated in the loaded atria. Sarcalumenin is a Ca²⁺ binding glycoprotein located in the lumen of the SR and thought to regulate Ca²⁺ transport and storage in the SR. Both heart-specific sarcolipin transgenic mice and sarcalumenin knockout mice exhibit relaxation-dominant cardiac dysfunction. The physiological role of sarcolipin and sarcalumenin will be further discussed. [Jpn J Physiol 55 Suppl:S55 (2005)]

View full abstract

Genetic transmission is suggested to be involved in the etiology of schizophrenia. Some genes and chromosomal domains have been associated with risks for the illness, although no single gene or genes have been defined as causal for this complicated illness. Individual human has one or more different sequential variations (alleles) that are determined in part by past mutations and their permanent incorporation in the genome. One frequent and non-conservative polymorphism for BDNF is a single nucleotide polymorphism (SNP) found in its pro-domain whose biological functional role remains to be fully understood. In the rodent brain, BDNF facilitate long-term potentiation in hippocampus and cortex, by enhancing synaptic transmission and vesicle docking. Therefore, we have looked for subtle cognitive and physiological differences between persons with different BDNF alleles and the underlying molecular mechanisms using several measures. Here we present our recent studies, demonstrating that different polymorphisms in the BDNF gene alter the distribution of its product, activity-dependent secretion manner, biological activity to decide neuronal survival or death, electrophysiological property to elicit synaptic transmission, and cognitive performance on an array of human brain function tests. This integrative approach is of the most importance as it suggests that a polymorphism of neuronal genes modulates cognitive performance and clinical expression of brain dysfunction. [Jpn J Physiol 55 Suppl:S56 (2005)]

View full abstract

Electroconvulsive shock (ECS) has long been used for treatment of psychiatric diseases including manic-depressive illness. Its clinical benefits have been repeatedly documented in medically resistant cases or particularly serious cases that would otherwise end up with suicide. How ECS works is not known. We have developed a rat model for studying mechanisms by which ECS is made effective. Electrophysiological recordings and calcium photometry were done in visual cortex slices obtained from ECS-subjected rats. Two remarkable findings have come out in our recent experiments. First, resting membrane potential was unusually hyperpolarized, and spike firing in response to depolarizing currents were decreased. Second, L-type voltage-dependent calcium channels were upregulated, with action potential-induced calcium influx enhanced, thereby facilitating calcium spike generation. The ECS-induced hyperpolarization is reminiscent of the effects of the minor tranquilizer benzodiazepin, which facilitates GABA-A receptors and favors hyperpolarization. The combined occurrence of calcium spike facilitation and spike firing attenuation may have a serious impact on synaptic efficiency in the framework of theoretical models of Hebbian plasticity and spike timing-dependent plasticity. According to these models, the two effects combined together are suggested to bias synaptic plasticity in favor of synaptic downregulation or downscaling. Such downscaling of synaptic efficiency might contribute to disruption of abnormal neural states. [Jpn J Physiol 55 Suppl:S56 (2005)]

View full abstract

ADHD is one of the most prevalent childhood psychiatric disorders. Although ADHD is not caused by a single cause, many studies suggest that ADHD can be explained as prefrontal dysfunctions. Since the prefrontal cortex participates in working memory, attentional control, inhibition of inappropriate behavior, and regulation of emotion and motivation, the fact that many children exhibiting ADHD show disinhibition of behavioral responses and difficulty in sustained attention suggests strong contribution of the prefrontal cortex to ADHD. Studies also suggest that ADHD is related to deficits of dopamine-related functions, because methylphenidate, which is a dopamine transporter inhibitor, is the most effective drug for the treatment of ADHD and because genetic studies suggest the relations between ADHD and dopamine functions. The prefrontal cortex is the cortical area where the most strong dopamine innervation is observed. In addition, local injection of dopamine-related drugs to the prefrontal cortex produces modulation of task-related activity and behavioral deficits in cognitive task performances. Thus, the dysfunction of dopamine-related modulation in the prefrontal cortex could be possible candidates of biological causes of ADHD. To prove this notion, it is now necessary to make a primate model of ADHD by artificially disturbing dopamine functions in the prefrontal cortex clonically and examine its behavior and neural activities. [Jpn J Physiol 55 Suppl:S56 (2005)]

View full abstract

Dopamine dysfunction is generally recognized as a trigger that produces loss of motor control and various cognitive dysfunctions. In particular, the importance of dopamine D1 receptors in regulating working memory has been emphasized ever since experimental studies found that working memory performance demonstrates an inverted U-shape property according to dopamine D1 receptor activation in the prefrontal cortex (PFC). In this context, the D1 receptor activation level in the prefrontal cortex should be regulated at the optimum to perform a working memory task. Because the regulation of the optimal D1 receptor activation in the PFC depends on the dopamine release in the PFC, controlling the dopamine release and the activity of the mesofrontal dopaminergic neurons must be necessary to acquire accurate working memory activity of the cortical neurons. Based on the neuroanatomical findings, we have recently shown that this closed-loop system can regulate working memory activity in the prefrontal cortex by stabilized optimum dopamine release. We here report our recent study which is designed to investigate computationally 1) qualitative and quantitative aspects of the dynamics of the fronto-mesofrontal system; and 2) functional difference in regulation of frontal dopamine release between the fronto-mesofrontal system and the tonic disinhibitory circuit. [Jpn J Physiol 55 Suppl:S57 (2005)]

View full abstract

Voltage-dependent Ca²⁺ channels (VDCCs) mediate Ca²⁺ inflow into the presynaptic terminals in responses to arrival of action potential. In the cerebral cortex and hippocampus, both N- and P/Q-type VDCCs are involved in the fast transmitter release from the presynaptic terminal. How these VDCC subtypes are distributed among individual presynaptic terminals was investigated for mossy fibre (MF) synapses in mouse hippocampal slices. The individual large MF terminals were loaded with dextran conjugates of a calcium-sensitive indicator and identified under confocal microscopy. The strontium influx of individual large MF terminals was fluorometrically investigated for neurotoxin sensitivities, using strontium as a calcium substitute. The relative contributions of N- and R-type VDCCs were found to be variable among large MF terminals whereas that of P/Q-type was rather uniform. Even two adjacent large MF presynaptic terminals differed in the relative contributions of N- and R-type VDCCs. These results provide direct evidence that large MF synapses are non-uniform in the composition of presynaptic VDCC subtypes. Since large MF synapses are the most efficient known excitatory inputs in the hippocampus, the heterogeneous expressions of presynaptic VDCC subtypes provide the hippocampal network with space-time variability. [Jpn J Physiol 55 Suppl:S57 (2005)]

View full abstract

Monitoring neuronal activities from multiple neurons is essential for understanding neuronal network activities. While calcium imaging from a population of cells is an effective method to study the network dynamics of a neural structure, it has been difficult to image from densely packed structures, such as the granule cell layer of the dentate gyrus, due to overlap of the cells. We have developed a novel method to label multiple granule cells retrogradely with a Ca²⁺ indicator in rat hippocampal slices using Oregon Green 488 BAPTA-1 AM. Synchronized burst activities (0.3–1.4 Hz), which were induced by applying 50 μM 4-aminopyridine, were monitored extracellularly with a glass electrode placed at the granule cell layer in the dentate gyrus. During the burst activities, action potential-induced Ca²⁺ transients from multiple (4–12) granule cells were monitored simultaneously with a cooled CCD camera with single-cell resolution. Temporal structures of firing patterns from the multiple neurons were determined from Ca²⁺ imaging data. In each single burst event recorded from the extracellular electrode, each neuron fired synchronously within a 200 ms time window. The latency and its variance from the onset time of the single burst events to one of the Ca²⁺ transients were decreased over time (< 7.5 min). These results indicate that the temporal synchrony of the action potentials within the single burst event was enhanced as the burst activities proceeded. The progressive synchronization may be a key feature to make self-organizing network activities. [Jpn J Physiol 55 Suppl:S57 (2005)]

View full abstract

Our laboratory investigates mechanisms underlying the operation of neuronal circuits. In order to understand information processing in neuronal circuits, we need to expand studies at the single cell level to those at the network level. Optical imaging techniques provide the spatial and temporal information that is required for studies both at the single cell and at the network level. At the level of single cells, we have used the sodium-sensitive dye SBFI and the calcium sensitive fluorescent dye Oregon green BAPTA-1 to investigate intrinsic properties and mechanisms of integration of synaptic inputs in cerebellar Purkinje cells and olfactory mitral cells. At the level of neuronal circuits, we have used voltage sensitive dyes (di-4-ANEPPS), activity dependent autofluorescence signals (flavin) and a genetically-encoded calcium sensor protein to image the synaptic information flow in the cerebellar mossy fiber-granule cell-Purkinje cell pathway. The above research results will be used to exemplify issues related to the methodology and application of optical imaging of CNS networks. [Jpn J Physiol 55 Suppl:S58 (2005)]

View full abstract

We studied activity-dependent cortical plasticity in C57BL/6 mice using transcranial flavoprotein autofluorescence imaging. In the auditory cortex, we tested the effect of acoustic environment on the cortical activities. Neural responses in the auditory cortex elicited by sound stimuli (5-20 kHz) exhibited tonotopic maps. In mice exposed to environmental stimuli (10 kHz, 70 dB), the cortical responses to 10 kHz stimuli were augmented in a frequency-specific manner. In the visual cortex, effects of monocular deprivation for 4 days in the critical period were evaluated a few weeks after the deprivation. Monocular deprivation selectively diminished the autofluorescence responses to light stimuli applied to the deprived eyes in the binocular zone but not in the monocular zone of the visual cortex. In the somatosensory cortex, effects of tail amputation were investigated. Neonatal mice were anesthetized with ether, and the tail was cut at the base. Cortical activities were evaluated 2-13 weeks after the tail amputation. In control mice, an activity-dependent autofluorescence increase was observed in the hindpaw, tail base and tail tip regions, which were adjacent to each other in the posterio-medial direction. In the tail-amputated group, the tail base region shifted toward the region corresponding to the tail tip in control mice. Furthermore, the tail base and hindpaw regions were expanded toward the border between them. The present results indicate the usefulness of transcranial autofluorescence imaging for investigating activity-dependent plasticity of mouse sensory cortices. [Jpn J Physiol 55 Suppl:S58 (2005)]

View full abstract

Electrically-excitable cells of which firing rate is increased in response to glucose are called glucose-responsive (GR) cells. They are present in several tissues such as endocrine pancreas, gut and brain. In pancreatic β-cells, ATP-sensitive K⁺ (K_ATP) channels are essential for the regulation of cellular excitability by glucose and glucose-induced insulin secretion. In gut, K_ATP channels are detected in GR cholinergic neurons and are suggested to be involved in the glucose-induced membrane depolarization of these peripheral neurons. In central nervous system, GR neurons exist at the highest density in the ventromedial hypothalamus (VMH), the region important in sensing ambient glucose levels. K_ATP channels in the VMH are identical to that of pancreatic β-cells, which is composed of Kir6.2 and SUR1. Analysis of the mice lacking Kir6.2 (Kir6.2^−/− mice) have clarified that the channels are essential for regulating the firing rate of GR neurons in response to glucose. Thus, GR neurons of the VMH and pancreatic β-cell not only possess the same K_ATP channels but also are equipped with similar glucose-sensing apparatus. Kir6.2^−/− mice exhibit an impaired glucagon secretion in response to hypoglycemia and attenuated feeding response to neuronal glycopenia induced by 2-deoxyglucose. K_ATP channels in the skeletal muscles are also involved in the glucose uptake. Thus, the K_ATP channels, as central and peripheral glucose sensors, are critical in the maintenance of glucose homeostasis. [Jpn J Physiol 55 Suppl:S58 (2005)]

View full abstract

The midbrain nucleus substantia nigra pars reticulata (SNr) consists mainly of GABAergic projection neurons. These SNr GABAergic neurons exhibit the highest spontaneous activity in the brain, suggesting their very high demand for energy substrates such as oxygen and glucose. SNr neurons are extremely sensitive to hypoxia, ceasing firing upon opening of their ATP-sensitive potassium (K_ATP) channels, well before the hypoxia-induced general self-defense reactions in other neuron types. Unexpectedly, approximately two thirds of SNr GABAergic neurons showed increased spontaneous activity as the extracellular glucose concentration was lowered from 10 to 4-6 mM in acute slice, indicating that SNr contains glucose-sensitive neurons. In addition, a small proportion of SNr neurons exhibited multiple, abrupt increses in firing rate with periods ranging in minutes. At 1-3 mM glucose, the firing rate ceased after a rapid increase. No significant change in firing was observed by increasing the glucose from 10 to 20 mM. These responses were independent of the K_ATP channels, and persisted under blockade of GABA_A, NMDA, and non-NMDA receptors, suggesting a postsynaptic mechanism. Thus, the altered activity of the SNr, a major output neucleus of the basal ganglia, itself might signal the metabolic state of the brain to its target nuclei. [Jpn J Physiol 55 Suppl:S59 (2005)]

View full abstract

Intracerebroventricular (icv) injection of glucose inhibited food intake, while that of 2-deoxy-glucose and a glucokinase inhibitor enhanced it in rats. Lowering glucose concentrations from 5-10 mM to 1-3 mM increased cytosolic Ca²⁺ concentration ([Ca²⁺]i) in 20% of isolated neurons from the hypothalamic arcuate nucleus (ARC), exhibiting the property of glucose-sensitive (GS) neurons. More than 90%of these GS neurons were neuropeptide Y (NPY)-containing neurons. Orexin neurons in the lateral hypothalamus (LH) are also GS neurons, and orexin innervates and activates NPY neurons in ARC. Thus, lowering glucose both directly and indirectly via LH orexin neurons stimulates ARC NPY neurons. Ghrelin also increased [Ca²⁺]i in GS and NPY neurons in ARC. [Ca²⁺]i increases in ARC GS and NPY neurons induced by lowering glucose, orexin and ghrelin were all inhibited by insulin and leptin at physiological concentrations in brain. Fasting-induced increase in NPY mRNA level in ARC was reduced by repeated icv injection of insulin, showing a long-term inhibitory effect. Thus, GS-NPY neurons in ARC appear to receive both orexigenic and anorexigenic factors including glucose and hormones, integrate their information, and produce appropriate outputs to downstream neural circuits and effector organs, thereby playing a central role in regulation of feeding. [Jpn J Physiol 55 Suppl:S59 (2005)]

View full abstract

The glucose (G) concentration in CSF is 2-3 mM and increases twice during food intake. When G was injected into the hippocampus to turn to 7 mM, spatial learning and memory were facilitated. To make clear this, neurophysiological measurement using hippocampal slice preparations were carried out. The G concentration in perfusate was changed from 3.5 to 7 mM for 15 min and returned to 3.5 mM. The CA1 synaptic potentials produced by Schaffer collateral stimuli were augmented, started from 3-4 min after the change to 7mM G. This augmentation continued for 40 min even after returning to 3.5 mM G. This was like a long-term potentiation (LTP) without a tetanic stimulation. LTP produced by tetanic stimulation (100 Hz, 1 s) just after returning to 3.5 mM G was significantly facilitated, while only short-term potentiation was produced in 3.5 mM G. Presynaptic transmitter release measured by paired pulse facilitation method and postsynaptic response to NMDA applied at the apical dendrites were also facilitated by 7 mM G. The membrane potential and input resistance were a little changed by 7 mM G. The phosphorylation of presynaptic synapsin I-3 and of postsynaptic CaMkII of CA1 neurons were facilitated by 7 mM G. These evidences indicate that food intake is necessary not only for keeping body homeostasis but also reinforcing the higher brain plasticity. [Jpn J Physiol 55 Suppl:S59 (2005)]

View full abstract

Ethics Committee Symposium Recently various restrictions have been added in the environment surrounding biomedical scientists. For example, the Law Concerning the Humane Care and Control of Animals will be revised in May, 2005. To facilitate productive biomedical researches to protect human health, we should appeal to social community for appropriate support. For these support, we would propose a reasonable self-control system to assure public of biomedical ethics. It is also necessary to maintain stable supply of experimental animals in good condition. Prof. A. Kaneko, the president of this Society, will give a lecture on a proposal issued on July 14, 2004, by the Science Council of Japan, entitled as the promotion of support and understanding by the social community for animal experiments. The second speaker is Prof. T. Isa, who gave a lecture on the current status of the Japanese Monkey Bio-resource Project. [Jpn J Physiol 55 Suppl:S60 (2005)]

View full abstract

Needless to say, animal experiments are indispensable in medical and animal sciences, medical practice and pharmaceutical industries. On the other hand, there are many criticisms against animal experiments from the view point of animal welfare and ethics. There are many countries in Europe where animal experiments are strongly retarded by highly severe regulations. Our country is not an exception where animal supply is getting to be limited, and the anti-vivisections movement is getting more active. Under these circumstances, it is important to promote support and understanding by the social community for animal experiments and to tell them that animal experiments are conducted scientifically and ethically. The Science Council of Japan (SCJ) has proposed, on July 14, 2004, to establish nation-wide guidelines for animal experiments and a third-party evaluating organization composed of experts. SCJ is calling active participation of scientific organizations committed in animal experiments to establish these self-regulatory systems. In this symposium presentation, the detailed process and circumstances discussed in SCJ will be reported. [Jpn J Physiol 55 Suppl:S60 (2005)]

View full abstract

The “Japanese Monkey Bioresource Project” has been launched since 2002 as a part of the National Bioresource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT). The project is aimed at establishing a stable breeding and supply system of the Japanese monkeys for experimental use. The project is run by the committee set in the National Institute for Physiological Sciences in Okazaki, and actual breeding is proceeded in Kyoto University Primate Research Center in Inuyama and a breeding facility of a private company. Although the project has met a counteractive action by an antivivisectionist group, we have made efforts to establish good public relations by organizing open symposiums and other publishing activities. So far, the project is expanding on schedule. I will talk on the current state of the project in this symposium. [Jpn J Physiol 55 Suppl:S60 (2005)]

View full abstract

The premotor neurons for cardiovascular sympathetic nerves are located in the rostral ventrolateral medulla (RVLM) and are called RVLM neurons. Activities of peripheral sympathetic nerves are not always changed in a uniform way and this phenomenon is known as a ‘regional difference’. For example, the cutaneous vasoconstrictor fibers participate in thermoregulation while vasoconstrictor fibers for the visceral and muscular vessels play an important role in regulation of arterial pressure. No neural mechanism of the regional difference has been established. We tried to identified reticulospinal neurons in the rostral medulla of the anesthetized rabbits; 1) premoter neurons for cutaneou vasoconstrictors and 2) ones for the cardiac sympathetic nerve. Warm stimulation of the preoptic area and anterior hypothalamus, the thermoregulatory center, inhibited activity of the cutaneous sympathetic nerve activity. We found reticulospinal neurons in the rostral ventromedial medulla that responded to the same stimulation with similar temporal response pattern of the nerve, suggesting that these neurons were premotor neurons for cutaneous vasoconstrictors. Hypoxia inhibited the activity of the cardiac sympathetic nerve and the same stimulation concurrently inhibited some of the RVLM neurons. We thought these neurons were premotor neurons for the cardiac sympathetic nerve. These data indicated that the premotor neurons for the sympathetic nervous system were grouped in according to their function and this grouping is the fundamental mechanism of the regional difference. [Jpn J Physiol 55 Suppl:S61 (2005)]

View full abstract

It has been written in a textbook of physiology that the cardiac adaptation during low to moderate exercise is controlled by cardiac parasympathetic withdrawal, whereas the cardiovascular responses to a higher intensity of exercise are induced not only by the parasympathetic withdrawal but also by activation of the sympathetic nervous system, which is assumed to be relatively uniform among various organs irrespective of different physiological function. This classical view was challenged by measuring cardiac and renal sympathetic outflows (CSNA and RSNA) during treadmill exercise (speed, 10-60m/min) in conscious cats. CSNA augmented by 168-297% at the onset of and during the later period of exercise even though the exercise intensity was low. The initial and late increases in CSNA were dependent on the intensity of exercise. Simultaneously with the initial CSNA, RSNA augmented by 185% at the onset of exercise but the increase was independent of the exercise intensity, suggesting that central command may cause the initial responses in CSNA and RSNA in the different manner. In contrast, RSNA returned near the control level during the later period of exercise at 10-20m/min but increased by 90-280% at a higher speed. Thus CSNA was stimulated during exercise in proportion to the exercise intensity of exercise whereas RSNA was not driven until a certain exercise level was exceeded, suggesting that sympathetic outflows to the heart and the kidneys are differentially controlled during dynamic exercise, probably depending on their physiological function. [Jpn J Physiol 55 Suppl:S61 (2005)]

View full abstract

We have recently established a method to measure renal (RSNA) and lumber (LSNA) sympathetic nerve activities simultaneously and continuously in freely moving rats and examined whether patterning of sympathetic outflow occurs in regionally different manner during changes in natural behavioural activities in rats. We observed a regional diversity of the sympathetic outflow during rapid eye movement (REM) sleep. RSNA decreased in a step manner during REM sleep that was accompanied by a step increase in LSNA. This indicates that the sympathetic regulation did not consist of a simple unidirectional readjustment, and sympatho-inhibition on the kidney coexists with the sympatho-excitation on the muscle during REM sleep in rats. This is consistent with the fact that renal blood flow and vascular conductance were increased while iliac blood flow and vascular conductance decreased during REM sleep. During non-REM sleep, quiet awake, moving and grooming states, LSNA and RSNA increased in parallel manner in proportion to the increase in physical activity. Systemic arterial pressure and heart rate increased associated with the increase in physical activity. The parallel increases in LSNA and RSNA seems favorable to the concomitant increases in systemic arterial pressure and heart rate. Together the data during REM sleep and other behavioural states, it would be seen that each behavioural state may generate a different pattern of the changes in RSNA and LSNA, which may be involved in state-related changes in cardiovascular functions during natural behavioural change in rats. [Jpn J Physiol 55 Suppl:S61 (2005)]

View full abstract

Human thermoregulatory system is a feedback system that maintains core temperature (Tcore) within normal range. In this system, the central nervous system receives afferent information regarding skin (Tskin) and core temperatures, and then outputs sympathetic nerve activity (SNA) to muscular vessels (Muscle SNA) and skin (Skin SNA). Both SNAs may regulate heat loss. Muscle SNA regulates blood redistribution from central to cutaneous circulations, whereas skin SNA regulates skin blood flow and sweat. However, the transfer function in the central nervous system from Tskin and Tcore to these SNAs remains unclear. To examine the transfer functions, we largely changed environmental room temperature from 0 to 50°C while measuring muscle and skin SNA by microneurography, Tskin, and Tcore. By using 2 input 1 output models, we investigated the transfer function from both Tskin and Tcore inputs to either muscle or skin SNA output. The transfer functions controlling muscle and skin SNAs have following similar characteristics; 1) the gain from Tcore to each SNA was over 10 times larger than that from Tskin to it, 2) the transfer function has high-pass filter characteristics with approximately 20 dB/dec. However, these transfer functions have regionally different characteristics as follows; 1) the transfer gain from either Tskin or Tcore to skin SNA was 2-3 times larger than that to muscle SNA, 2) coherence between both temperature to skin SNA (0.7-0.8) was smaller than that to muscle SNA (0.8-0.9). [Jpn J Physiol 55 Suppl:S62 (2005)]

View full abstract

The brainstem respiratory network, which involves a variety of respiratory neurons and constitutes the respiratory center, generates the automatic rhythmicity of respiration and the spatio-temporally organized contraction of respiration-related muscles. In vivo electrophysiological and neuroanatomical studies using largely cats and rats, have greatly increased our knowledge about the overall respiratory system and its network mechanisms. Currently, a limited region of the ventrolateral medulla, which spans the areas called the Boetzinger complex and the pre-Boetzinger complex, is the focus of our attention. In this region, a number of excitatory and inhibitory neurons with specific firing patterns and characteristic morphological features have been identified. These neurons form the networks that involve 1) inhibitory connections between inspiratory and expiratory neurons, 2) excitatory connections between inspiratory neurons, 3) inhibitory connections between expiratory neurons, and 4) inhibitory connections between inspiratory neurons. Some of these synaptic connections seem to be essential for the generation of respiratory rhythm, and some for the formation of patterned respiratory outputs. On the other hand, at the level of individual neurons it is often impossible to unequivocally determine whether some specific neurons are essential either for rhythm generation or for pattern formation, suggesting that such classifications may not always be meaningful. As an introductory talk of this symposium, these topics will be discussed in relation to other speakers’ analyses in in vitro preparations. [Jpn J Physiol 55 Suppl:S62 (2005)]

View full abstract

In the medulla, there are at least two respiratory-related rhythm generators that are neuron networks periodically producing intrinsic burst activity under some conditions. One is an inspiratory (Insp) neuron network, which produces Insp activity, and whose main center in the ventrolateral medulla is called as the pre-Boetzinger complex. Another is a pre-inspiratory (Pre-I) neuron network, which produces activity preceding Insp burst, and is located in the more rostral ventrolateral medulla including the para-facial respiratory group (pFRG). These rhythm generators could be distinguished by the differences in the burst phase and in their distribution of the main population of those neurons in the ventral medulla. Our current hypothesis for the respiratory rhythm generation is that the pFRG-Pre-I neuron network is responsible for the primary respiratory rhythm generator (RRG) that periodically triggers Insp burst generation in the pre-Boetzinger complex-Insp neuron network (i.e., inspiratory pattern generator, IPG) and thus determines the frequency of inspiratory motor output. Although these rhythm generators interact via reciprocal connections, they also could produce independently rhythmic burst after transverse section of the medulla at the level between the pre-Boetzinger complex and pFRG. On the basis of results from our transection experiments in brainstem-spinal cord preparations from newborn rats, we will show noticeable differences in the bursting properties of these neuron networks and discuss rhythm generation in the intact network. [Jpn J Physiol 55 Suppl:S62 (2005)]

View full abstract

Spontaneous respiratory rhythm is produced in in vitro medullary slice preparations in the neonatal rodents. It is hypothesized that the site of respiratory rhythm generation resides in the pre-Boetzinger complex (pre-BOT) localized in the rostral ventrolateral medulla. To analyze the changes of respiratory bursts of the bilateral pre-BOT after severing the mutual connections, we employed reduced medullary slice preparations (mid- or para-sagittal transection sparing the raphe nuclei). Respiratory bursts originated in the bilateral pre-BOT were recorded extracellulary or through the hypoglossal (XII) nerves before and after mid- or para-sagittal transection. After mid- or para-sagittal transection, desynchronized respiratory bursts in both half slices persisted with slightly decreased amplitudes and frequencies. This confirmed that the pre-BOT can generate respiratory bursts without mutual connections. In reduced slices, the respiratory bursts were facilitated when 5-HT was applied. This suggests that 5-HT neurons in the raphe nuclei located at the midline region exert facilitatory influences on pre-BOT neurons. In a following series of experiments, by using brainstem-spinal cord preparations, we recorded rhythmic bursts from the C1-C2 and C4 ventral roots synchronized with respiratory activity of the XII nerve. After transection just above the C1 segment, small and slow rhythmic bursts still remained in C1-C2 and C4 ventral roots. The fact that bursting rhythm remained in C1-C2 slices suggests that the spinal respiratory rhythm generator is localized in the upper cervical segments. [Jpn J Physiol 55 Suppl:S63 (2005)]

View full abstract

To clarify the role and identify the substrate of central chemoreceptors, we conducted in vivo and in vitro experiments using rodents. Central chemoreceptors are mainly located in the superficial rostral ventral medulla overlapping with the para-facial respiratory group (pFRG) region. Two types of cells are supposed to be important on the basis of c-fos immunohistochemistry; surface small (type I) cells are primary receptor cells, and sensed information is transmitted to the deep respiratory rhythm generator via subsurface relay (type II) neurons. Because type I cells are cholineacetyltransferase-positive, acetylcholine (ACh) is likely a neurotransmitter at the synapse from type I to II cells. To investigate the role of nicotinic ACh receptors (nAChRs) in chemoreception, we applied nicotine to the medulla and observed increases of respiratory output, similarly to the responses to CO₂. Application of nAChRs-antagonists then indicated that the nAChRs-subtype α₄β₂ is important. Analyses of ventilation in each subtype of muscarinic ACh receptors (mAChRs) gene knockout mice showed that M1/M3-compound subtypes were also important. We succeeded to augment chemoresponsiveness by increasing endogenous ACh action with a cholinesterase inhibitor donepezil, which is promising as a therapeutic medicine for blunted chemoresponsiveness. We conclude that both nAChRs and mAChRs are importantly involved in the chemical regulation and maintenance of respiratory rhythm as sources of excitatory neural drive. [Jpn J Physiol 55 Suppl:S63 (2005)]

View full abstract

The central chemoreceptor activation evokes the abdominal expiratory activity in the in vitro brainstem-spinal cord-rib preparation from neonatal rats (Iizuka, J Physiol, 551.2: 617-633, 2003). This expiratory activity is limited to the first part of the expiratory phase in most of preparations. In the anesthetized, vagotomized, paralyzed and artificially ventilated adult rat, the abdominal activity showed a plateau pattern during the expiratory phase or a gradually augmenting pattern with abrupt termination upon the onset of the inspiratory activity under hypercapnia. Similar results were obtained in the anesthetized neonatal rat. Although the expiratory motor pattern in detail was different between the in vitro and in vivo conditions, thus, the fundamental alternating pattern between inspiratory and expiratory motor activity was well preserved in the in vitro preparation. Since the abdominal muscle showed weak or no expiratory activity during the anoxia-induced gasping in the in vivo neonatal rat preparation, the respiratory activity in the in vitro preparation is not equal to the gasping. Furthermore, our recent study suggested that the neuronal mechanisms needed to generate the rostrocaudal gradient in the inspiratory and expiratory motor output to various rib-cage muscles should be intact in the in vitro preparation (Iizuka, Neurosci Res, 50: 263-269, 2004). The advantages and limitations of the in vitro preparation for the study of the respiratory motor pattern generation will be discussed. I will also refer the possible factors for such distortion of the expiratory motor pattern under the in vitro conditions. [Jpn J Physiol 55 Suppl:S63 (2005)]

View full abstract

It is well-known that skeletal muscle has a greater plasticity. It is speculated that proliferation and differentiation of satellite cells play a major role in the hypertrophy and de novo formation of muscle fibers. These properties of satellite cells may be also associated with growth/development, aging, and/or damage-regeneration cycle of skeletal muscles. However, the precise mechanism responsible for the role or plasticity of satellite cells associated with growth/development, aging, or damage-regeneration cycle is not fully elucidated. Therefore, we will discuss about the molecular mechanism for the proliferation and differentiation of muscle satellite cells, roles of satellite cells in the growth/development and in age-related atrophy of skeletal muscles. Further, the discussion will be extended to the application of satellite cells or bone marrow derived cells for regeneration of muscles. [Jpn J Physiol 55 Suppl:S64 (2005)]

View full abstract

Sarcopenia, the decline in muscle mass and functional capacity associated with aging, is an important component of frailty in the elderly. One potential contributor to sarcopenia is a gradual loss of muscle fibers during senescence. Although skeletal muscle has the capacity to regenerate itself, this process is not activated by the gradual age-related loss of muscle fibers. Satellite cells are a population of mononuclear myogenic cells, which are located between the sarcolemma and the basal lamina of the skeletal muscle fiber. Satellite cells in adult skeletal muscle are mitotically quiescent; however, in response to stimuli such as injury, these cells become activated, proliferate, and fuse to existing muscle fibers or fuse together to form new myofibers for the regeneration of damaged skeletal muscle. The proliferation potential of satellite cells decreases with aging, which is of clinical concern as this could contribute to sarcopenia. However, the cellular mechanisms responsible for the age-associated decrease in satellite cell proliferation are still unclear. Thus, the hypothesis of this study was that proteins involved in cell cycle arrest and the related transcription factor would be elevated in nuclei of primary cultured satellite cells from old skeletal muscle. Further, the roles of satellite cells and haemopoietic stem cells for the regeneration and repair of damaged skeletal muscle in old rats were determined in vivo. [Jpn J Physiol 55 Suppl:S64 (2005)]

View full abstract

There are some animals which live in the high-altitude and cold environments. To learn how the wild animals adapt to and survive in such extreme environments is an important and useful approach not only for the progress of science but also for health promotion and prosperity of the human. The pika, Ochotona, living in cold zone or in high mountains prefers the low temperatures and is considered to be remained species of the glacial periods. The pika's high body temperature, high metabolic rate and high fur insulation are advantageous for survival in the cold. The pika's unique behavioral strategies to adapt to cold are living in burrows under the ground or in the shelter among rocks and hoarding grasses for winter foods. The pika do not hibernate, though many mammals living nearby hibernate. On the other hand, the pika is heat intolerant due to poor heat loss ability such as poor panting, small ear pinnae and the absence of thermal salivation. The pikas reared in the laboratory showed neither diurnal nor nocturnal body temperature rhythm. Our fieldwork using bio-telemetry devices in their natural habitat in Qinghai, China demonstrated that the pikas are essentially diurnal. Taking the ecologists' field observation together into consideration, it is suggested that the pikas may vary their activity rhythm from diurnal in the relatively cool environment to a crepuscular (dawn and dusk) pattern in the relatively hot environment to avoid the heat during midday. The pika is also of interesting because of its communication with calls and its territorial social organization. [Jpn J Physiol 55 Suppl:S65 (2005)]

View full abstract

When the animals are exposed to high-altitude over a long period, the pulmonary hypertension and right ventricular hypertrophy are observed. These responses are typical singes for the adaptation to high-altitude. Main factors for affecting to living body are hypoxia and cold in the high-altitude. These two factors affect to the lung and blood sides. In the lung side, the pulmonary capillaries constrict due to hypoxia. Its call hypoxic pulmonary vasoconstriction or HPV. In the blood side, RBC and Ht increase by exposure to high-altitude and it cause increasing blood viscosity. These two factors, increased HPV and blood viscosity, are very important to cause the pulmonary hypertension or right ventricular hypertrophy. The pika, blue sheep, and yak are living in the Tibetan Highlands to an altitude of 6100m above sea level and are typical animals adapted to high-altitude among mammals of the world. These mammals have a long history of habitation at high-altitude and are considered to be animals completely adapted to high altitude. The purpose of this study is to make clear the physiological characteristics of pika, blue-sheep, and yak as high-altitude adapted animals. [Jpn J Physiol 55 Suppl:S65 (2005)]

View full abstract

It is well known that sheep can successfully inhabit highlands to an altitude, but not pig. We compared the effects of endogenous nitric oxide synthase (NOS) inhibition on pulmonary vascular tone in response to several hypoxic conditions in sheep and pig to evaluate the role of NO in adaptation to acute and chronic hypoxia. Unanaesthetized male sheep and pig in three areas; Matsumoto, Japan (680 m above sea level), Xing, China (2,300 m), and Maxin, China (3,750 m), were prepared for measurements of pulmonary artery (Ppa) and vascular resistance (PVR) before and after the NOS inhibition. The non-selective NOS inhibitor, Nw-nitro-L-argine (NLA, 20mg/kg) was used. Baseline Ppa became elevated with an increase in altitude in both animals. Ppa and PVR at baseline in pigs were significantly higher than those in sheep. The increase in PVR after NLA in sheep at 3,750 m was significantly higher than those in other groups, but there were no significant differences in three grade altitudes of pig. Furthermore, we examined the effects of acute hypoxic exposure from 2,300 m to 4,500 m in both animals using a hypobaric/hyperbaric controlled chamber with and without NLA. The enhanced pulmonary vasoconstriction after NO inhibition observed during hypoxic exposure in sheep but not pig. The increased endogenous NO production may contribute to regulating the pulmonary vascular tone in the adaptation to high-altitude in sheep. We conclude that NO plays a significant role in the pulmonary vascular tone in adaptable animal to hypoxia. [Jpn J Physiol 55 Suppl:S65 (2005)]

View full abstract

Nitric oxide (NO) possesses a variety of biological actions in the kidney, including regulation of renal hemodynamics, renin secretion and tubular transport of solutes and water. However, effects of NO on the K channel activity in proximal tubule cells have not been elucidated. We recently reported that NO had a biphasic effect on activity of a 40 pS K channel in cultured human proximal tubule cells. In this study, we tried to clarify which isoforms of NO synthase (NOS) were involved in the endogenous production of NO and modulation of the K channel activity, using the cell-attached mode of the patch-clamp technique and RT-PCR. Under the control condition, a NOS substrate, L-arginine (500μM), increased channel activity, which was abolished by an nNOS/iNOS-specific inhibitor, TRIM (100μM). Consistent with this observation, mRNA expression of iNOS alone was detected by RT-PCR. However, after the cells were incubated with atrial natriuretic peptide (ANP, 300nM) for 24 h, eNOS expression was also evident. In addition to this long-term effect, ANP increased channel activity within 5 min under the control condition, which was not affected by TRIM or an inhibitor of soluble guanylate cyclase (ODQ, 10μM), suggesting that the acute stimulatory effect of ANP would be independent of the NO pathway. In conclusion, iNOS is the major source of endogenous NO in cultured human proximal tubule cells under the control condition, whereas eNOS also participates after prolonged exposure to ANP. [Jpn J Physiol 55 Suppl:S68 (2005)]

View full abstract

We reported previously that the major effector cells essential for allograft (e.g., BALB/c skin and Meth A tumor; both H-2D^d) rejection were allograft-induced macrophages (AIM; H-2D^b) and that AIM exhibited the cytotoxic activity against the allograft with MHC haplotype specificity. In the last meeting, we reported that we isolated a cDNA clone, 15-4-4 (261-bp fragment), by the expression cloning method using a monoclonal antibody, R15, specific for AIM and an H-2D^d tetramer. In the present study, we further characterized the cDNA. In order to know whether the protein encoded by 15-4-4 cDNA is functional or not, we ligated a full-length cDNA (1181-bp) with pEGFPNI vector; and the cDNA or vector alone was stably transfected into 293T cells. After selection of the transfectants by both EGFP expression and neomycin (G418) resistance, binding abilities of the expressed protein to an H-2D^d or H-2D^b tetramer were examined under a confocal microscope or by cell sorter. The analyses revealed that the 293T cells transfected with full-length 15-4-4 cDNA did react with R15 antibody and H-2D^d tetramer and that the cells transfected with vector alone did not. In contrast, H-2D^b tetramer did not react with 293T transfectants. These results suggest that the protein encoded by 15-4-4 cDNA may be a putative receptor for allogeneic MHC, H-2D^d. [Jpn J Physiol 55 Suppl:S68 (2005)]

View full abstract

Medium-chain acyl-CoA dehydrogenase (MCAD) oxidizes acyl-CoA to 2-enoyl-CoA. The acyl-CoAs with C6-C10 chain length are good substrates for MCAD. The hydrogen bonds of C(1)=O of the thioester with ribityl-2'-OH of FAD and with amide N-H of Glu376 are important to activate the thioester. We investigated the molecular mechanism for the substrate specificity of MCAD. MCAD reconstituted with 8-NH₂-FAD can bind acyl-CoA, but does not oxidize it, because the oxidizing power is weak. Therefore the complex is a good material to investigate the nature of ES complex (the complex of oxidized MCAD with acyl-CoA). The substrate chain-length dependency on the absorption maximum for 8-NH₂-FAD in ES complex is correlated well with the substrate specificity of MCAD. FT-IR study suggests the existence of multiple conformations, i.e., a strong and weak hydrogen bonding forms, in the cases of long-chain (>C12) substrate acyl-CoAs. Isovaleryl-CoA (IV-CoA), which has a branched chain, is a poor substrate for MCAD, but the band of C(1)=O stretch shifted downward by 42 cm⁻¹ in the bound form. The large shift indicates the existence of a strong hydrogen bond(s) in the active site, as in the case of octanoyl-CoA. The low activity of MCAD for IV-CoA is due to deviation from an appropriate orientation of IV-CoA in the active site, rather than the destruction of the hydrogen bond(s) by steric hindrance of isovaleryl group in the active site. [Jpn J Physiol 55 Suppl:S68 (2005)]

View full abstract

Frog parathyroid cells displayed transient inward currents in response to depolarizing pulses from a holding potential of -84 mV. We analyzed the biophysical properties of the inward currents using perforated and conventional whole-cell patch-clamp techniques. The inward currents disappeared by the replacement of external Na⁺ with NMDG⁺ and were reversibly inhibited by 3 μM TTX, indicating that the currents occur through the TTX-sensitive voltage-gated Na⁺ channels. Current density elicited by a voltage step from -84 to -24 mV was -80 pA/pF in perforated mode and -55 pA/pF in conventional mode. Internal GTPγS (0.5 mM) decreased the density to -12 pA/pF, but internal GDPβS (1 mM) did not affect the density. The voltage (V_1/2) of half-maximum activation was -46 mV in both of perforated and conventional modes. The voltage (V_1/2) of half-maximum inactivation was -80 mV in perforated mode and -86 mV in conventional mode, respectively. Internal GTPγS (0.5 mM) shifted the V_1/2 for activation to -36 mV and the V_1/2 for inactivation to -98 mV. The results suggest that the Na⁺ currents in frog parathyroid cells can be modulated by a G protein-dependent mechanism. [Jpn J Physiol 55 Suppl:S69 (2005)]

View full abstract

Capacitation is one of important event in mammalian sperm. Although ejaculated sperm can not fertile to egg, capacitated sperm can fertile. Generally, capacitated sperm show acrosomal reaction and hyperactivation. Acrosomal reaction occurs at sperm head during capacitation. On the other hand, hyperactivation occurs at sperm flagellum after acrosomal reaction. From many studies, it is accepted that those reaction related to capacitation are regulated with protein modification, such as phosphorylation, fragmentation and etc... So, we examined protein modification associated with sperm capacitation using SELDI protein chip system® in the present study. SELDI protein chip system® is new technology to analyze proteins and consists of protein chip and time of flight mass spectrometry (TOF MS).Using ion exchange chips such as weak anion exchange chip (CM) and strong cation exchange chip (Q), we detected protein fragmentation during capacitation. In several experiments, we found many fragments of sperm proteins. Those fragments time dependently appeared during capacitation. Moreover, we detected phosphorylation during capacitation using immobilized metal affinity capture (IMAC) gallium (III) and iron (III). From the experiment, we found many phosphorylations and dephosphorylations of sperm proteins. Those proteins were time dependently phosphorylated or dephosphorylated during capacitation. [Jpn J Physiol 55 Suppl:S69 (2005)]

View full abstract

The conductance of voltage-gated proton channels (Hv channels) has a strong temperature dependence (Q₁₀ ≥2), which is distinct from other water-filled pore channels. The temperature dependence of Hv channels in rat microglia (brain phagocyte) was evaluated over a wide range of temperatures (15–45°C) under various cellular conditions. The gating time constants exhibited a monotonic response, with high values of Q₁₀ (4–6), but the temperature dependence of the conductance showed a breakpoint at the transition temperature ( T_trans ). The Q₁₀ below T_trans was about ∼2.5 (the high Q₁₀ phase) and above T_trans it was about ∼1.3 (the low Q₁₀ phase). An increase in the pH gradient across the cell membrane shifted T_trans to lower temperatures, and cell swelling lowered T_trans further. Moreover, the value of T_trans fluctuated even in a single cell from ≤ 20°C to ≥ 33°C. Thus, T_trans is not likely the phase transition temperature of the membrane lipid. We conclude that the two Q₁₀ phases are based on the distinct physical processes: The low Q₁₀ phase represents H⁺ conductance through the maximal number of the open channels and the high Q₁₀ phase is due to the recruitment of ready-to-activate channels. Under physiological conditions the high Q₁₀ phase predominates, thus, temperature serves as a critical regulatory factor for the Hv channel through the recruitment mechanism. [Jpn J Physiol 55 Suppl:S69 (2005)]

View full abstract

By using MRI, we studied the correlation between saturation transfer ratio (STR) and physical state of water in copolymer gels, and that between STR and histologic parameters of human breast carcinomas. Fourteen copolymer gels were composed of any two or three monomers among HEMA, GMA, N-VP and MMA (water contents: 18.4–83.0%). Twenty-seven patients with histologically confirmed invasive ductal carcinomas of the breast were participated. These carcinomas were classified based on their histologic features. To evaluate STRs, an off-resonance MR technique for preferential saturation of the immobile protons was used (irradiation at 4 and 19 ppm). In the copolymer gels, there was a good correlation between STR values in MRI and the hydrophilicity of various copolymer gels, especially the STR-4 (STR at 4 ppm) values were found to be more separable parameter depending on the different hydrophilicity of the samples. In the breast carcinomas, STR-4 values were correlated well with the intracellular characteristics, whereas STR-19 values were correlated with the histologic intercellular structure. Intracellular macromolecules contain a large number of OH groups. STR-4 values are increased with increasing dysplastic changes of nuclei and mitotic index, suggesting that STR-4 values might reflect the amount of bound water or the degree of hydration with the intracellular macromolecules. [Jpn J Physiol 55 Suppl:S69 (2005)]

View full abstract

In non-excitable cells, the underlying mechanism for Ca²⁺ influx is thought to be mediated by newly generated Ca²⁺ selective channels through the depletion of Ca²⁺ storage pools (store-operated Ca²⁺ influx: SOC). A unique SOC with high Ca²⁺ selectivity has been well studied in mast cells and the like, however, SOC appears to vary in cell types and play its role in cell specific manner. Here we studied SOC in fura-2 loaded mouse submandibular acinar cells with microfluorimetry. After depleting the Ca²⁺-stores with thapsigargin or a high concentration of ACh under Ca²⁺ free condition, we measured SOC signal by the readmission of Ca²⁺. The signal possessed two phases. That is, an initial large transient and the subsequent long-lasting phase. External Zn²⁺ inhibited the former but not the latter. External Ni²⁺ or excess of outside K⁺ (90mM) markedly reduced both. The results suggest that two SOCs, differentiable by the divalent cations, exist in salivary gland acinar cells. [Jpn J Physiol 55 Suppl:S70 (2005)]

View full abstract

In contrast to the Ca²⁺-dependent contraction of vascular smooth muscle (VSM) which regulates physiological vascular tone, the Rho-kinase (ROK)-mediated Ca²⁺-sensitization of VSM contraction contributes to abnormal VSM contraction such as vasospasm. We previously found that SPC is an upstream messenger for the ROK-mediated Ca²⁺-sensitization and that inhibitors of Src family tyrosine kinase (Src-TK) blocked the SPC-induced contraction and activation of ROK. In the present study, we attempted to determine the enzyme molecule in a family of Src-TK which contributes to the Ca²⁺-sensitization mediated by a SPC/ROK pathway. In order to accomplish this purpose, we performed the selective knockdown of the target molecule by siRNA, which was transfected into the human coronary artery smooth muscle cells (CASMC) with the transfection efficiency of about 100%. The siRNA-mediated knockdown of Fyn inhibited the contraction of CASMC induced by SPC, without affecting the expression of other proteins such as MAP kinase, whereas non-silencing control siRNA lacked any effect. In contrast, siRNA-mediated knockdown of MAP kinase had no effect on the expression of Fyn and the cell shape of CASMC. These results produce the first direct evidence that Fyn mediates the Ca²⁺-sensitization of VSM contraction induced by a SPC/ROK pathway. In poster presentation, the effects of transfection of Fyn constructs (wild and mutated) on the VSM contraction of CASMC will be also discussed. [Jpn J Physiol 55 Suppl:S70 (2005)]

View full abstract

We have developed the system to measure many fluorescent components in their mixtures, based on the microspectroscopic recording of excitation-emission matrix (EEM) fluorescence data and on parallel factor analysis (PARAFAC) as the blind spectral decomposition method. This EEM/PARAFAC system can easily separate >10 heavily overlapping spectral components, without any preknowledge about the individual component spectra. It has proven to be very effective in the simultaneous measurement of multiple fluorescence components (either intrinsic or extrinsic) in single cells (Shirakawa and Miyazaki, Biophys J, 2004). In the present study, we tried to apply the same algorithm to multispectral imaging data for the analysis of the fluorescent components in living cells. The EEM fluorescence images were acquired with the conventional fluorescence microscope, changing and emission wavelengths by switching bandpass filters, or with the laser-scanning confocal microscope that equipped a multichannel spectrometric sensor as the fluorescence detector. PARAFAC modeling applied to time-series data of EEM fluorescence images for single mouse eggs could blindly separate signals of, e.g., functional proteins labeled with GFP and conventional calcium indicators, as well as autofluorescence components. The results showed that this approach will be a powerful methodology for the simultaneous measurement of many intracellular molecules in physiological experiments. [Jpn J Physiol 55 Suppl:S70 (2005)]

View full abstract