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Kotaro Oka

Publications and source records attributed to Kotaro Oka.

At least 19 recordsLinked to original sources
PubMed2006

Serotonin induces the increase in intracellular Ca2+ that enhances neurite outgrowth in PC12 cells via activation of 5-HT3 receptors and voltage-gated calcium channels.

As a neurotransmitter and neuromodulator, serotonin (5-HT) influences neuronal outgrowth in the nervous systems of several species. In PC12 cells, 5-HT is known to have neuritogenic effects, although the signal transduction pathway responsible for these effects is not understood. In this study, we hypothesized that a 5-HT-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) could be involved in mediating the effects of 5-HT. Application of 5-HT to PC12 cells enhanced nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner, and the sensitivity of this neuritogenic effect was increased in differentiated PC12 cells. In accordance, an increase in [Ca(2+)](i) was observed following application of 5-HT in differentiated PC12 cells. This increase was amplified by further NGF treatment. 5-HT-induced increases in [Ca(2+)](i) were inhibited by MDL 72222, a selective 5-HT(3) receptor antagonist, and nifedipine, an L-type calcium channel blocker, but not by ketanserin, a 5-HT(2) receptor antagonist, or thapsigargin, a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase. These pharmacological tests indicated that 5-HT-induced increases in [Ca(2+)](i) are mediated by activation of voltage-gated calcium channels via 5-HT(3) receptors and that 5-HT-induced increases in [Ca(2+)](i) are likely to be independent of activation of 5-HT(2) receptors in PC12 cells. Furthermore, the neuritogenic effect of 5-HT was suppressed by MDL 72222, nifedipine, calmodulin (CaM) inhibitor, and calcineurin inhibitors. Taken together, our results indicate that 5-HT-induced increases in [Ca(2+)](i), which are mediated via 5-HT(3) receptors and L-type calcium channels in PC12 cells, and subsequent activation of CaM and calcineurin enhance NGF-induced neurite outgrowth.

Animals↗
PubMed2006

Change detection and difference detection of tone duration discrimination.

An event-related potential called mismatch negativity is known to exhibit physiological evidence of sensory memory. Mismatch negativity is believed to represent complicated neuronal mechanisms in a variety of animals and in humans. We employed the auditory oddball paradigm varying sound durations and observed two types of duration mismatch negativity in anesthetized guinea pigs. One was a duration mismatch negativity whose increase in peak amplitude occurred immediately after onset of the stimulus difference in a decrement oddball paradigm. The other exhibited a peak amplitude increase closer to the offset of the longer stimulus in an increment oddball paradigm. These results demonstrated a mechanism to percept the difference of duration change and revealed the importance of the end of a stimulus for this perception.

Acoustic Stimulation↗
PubMed2005

Single molecular multianalyte (Ca2+, Mg2+) fluorescent probe and applications to bioimaging.

Intracellular signal transduction relies on spatial and temporal signal transmitter dynamics. To clarify the correlations of these transmitter molecules, multicolor-imaging has been widely used. However, in the case of applying multiple indicators in a cell, spectral overlap of the indicators prevents accurate quantitative analysis. Moreover, the invasive (toxic) effect, the localization, the metabolism, as well as photobleaching of these indicators complicate the situation. Here, we show that single-molecular multifluorescent probes can overcome these problems. While intracellular calcium plays a critical role as a signal transmitter and magnesium acts as a cofactor in many situations, the correlations between the two cations are now the main issue. We designed and synthesized a Ca2+-Mg2+ responsive multifluorescent probe, KCM-1. KCM-1 shows a spectral blue shift upon complexation to Ca2+ and a red shift to the presence of Mg2+. With data analyzed at different excitation wavelengths, the concentrations of Ca2+ and Mg2+ are simultaneously quantified. Furthermore, by using the AM-ester method, intracellular Ca2+ and Mg2+ concentrations are simultaneously imaged. Such a type of intracellular multiple analyte imaging by a single-molecular multifluorescent probe is successfully demonstrated for the first time.

Calcium↗
PubMed2005

Influence of mRNA and protein synthesis inhibitors on the long-term memory acquisition of classically conditioned earthworms.

We investigated the process of memory consolidation following classical conditioning of earthworms. Earthworms were conditioned in paired trials by a weak vibration as a conditioned stimulus (CS), and by light as an unconditioned stimulus (US). The occurrence of a shrinking response upon exposure to the CS increased steadily with the number of paired training trials. When the training procedure was changed by increasing the intertrial interval (ITI), it was found that only those worms trained with a 68 s ITI exhibited long-term memory retention for at least 24 h. The influence of mRNA synthesis inhibition by actinomycin-D or of protein synthesis by anisomycin on memory consolidation was also examined. Induction of the long-term memory was blocked when either of these two compounds was injected into the body cavity of the worm within 25 min of conditioning with the 68 s ITI. These results demonstrate that the long-term memory is dependent upon protein synthesis in response to the upregulation of new transcription messengers.

Animals↗
PubMed2005

[Recent Topics for magnesium mobilization in neurons].

Although magnesium ion (Mg(2+)) is believed as an important factor for maintenance of normal neuron function, our understanding of its intracellular mobilization and physiological function is still not enough. We need to know the following mobilization mechanisms of Mg(2+) in neurons:Mg(2+) influx via ion channels, Mg(2+) release from intracellular pools, and Mg(2+) transporters located on cell membrane. In this short review, I show the recent findings of intracellular Mg(2+) release and also Na/Mg antiport on cell membrane in PC12 cells that have been revealed by fluorescent Mg imaging techniques.

Animals↗
PubMed2004

Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D Mg2+ imaging.

The role of intracellular magnesium ions is of high interest in the fields of pharmacology and cellular biology. To accomplish the dynamic and three-dimensional imaging of intracellular Mg2+, there is a strong desire for the development of optimized Mg2+ fluorescent probes. In this paper we describe the design, synthesis, and cellular application of the three novel Mg2+ fluorescent probes KMG-101, -103, and -104. The compounds of this series feature a charged beta-diketone as a binding site specific for Mg2+ and a fluorescein residue as the fluorophore that can be excited with an Ar+ laser such as is widely used in confocal scanning microscopy. This molecular design leads to an intensive off-on-type fluorescent response toward Mg2+ ions. The two fluorescent probes KMG-103 and -104 showed suitable dissociation constants (Kd,Mg2+ = 2 mM) and nearly a 10-fold fluorescence enhancement over the intracellular magnesium ion concentration range (0.1-6 mM), allowing high-contrast, sensitive, and selective Mg2+ measurements. For intracellular applications, the membrane-permeable probe KMG-104AM was synthesized and successfully incorporated into PC12 cells. Upon application of the mitochondria uncoupler FCCP to the probe-incorporated cells, the resulting increase in the free magnesium ion concentration could be followed over time. By using a confocal microscope, the intracellular 3D magnesium ion concentration distributions were satisfactorily observed.

Animals↗
PubMed2004

Inhibition of RANKL-induced osteoclastogenesis by (-)-DHMEQ, a novel NF-kappaB inhibitor, through downregulation of NFATc1.

UNLABELLED: (-)-DHMEQ, a newly designed NF-kappaB inhibitor, inhibited RANKL-induced osteoclast differentiation in mouse BMMs through downregulation of the induction of NFATc1, an essential transcription factor of osteoclastogenesis. INTRODUCTION: Bone destruction is often observed in advanced case of rheumatoid arthritis and neoplastic diseases, including multiple myeloma. Effective and nontoxic chemotherapeutic agents are expected for the suppression of these bone destructions. RANKL induces activation of NF-kappaB and osteoclastogenesis in bone marrow-derived monocyte/macrophage precursor cells (BMMs). Targeted disruption or pharmacological suppression of NF-kappaB result in impaired osteoclastogenesis, but how NF-kappaB is involved in the regulation of osteoclastogenesis is not known. MATERIALS AND METHODS: The effect of (-)-dehydroxymethylepoxyquinomicin [(-)-DHMEQ] on osteoclast differentiation was studied using a culture system of mouse BMMs stimulated with RANKL and macrophage colony-stimulating factor. The mechanism of the inhibition was studied by biochemical analysis such as immunoblotting and retroviral transfer experiments. RESULTS: (-)-DHMEQ strongly inhibited RANKL-induced NF-kappaB activation in BMMs and inhibited RANKL-induced formation of TRACP(+) multinucleated cells. Interestingly, (-)-DHMEQ specifically inhibited the RANKL-induced expression of NFATc1 but not the expressions of TRAF6 or c-fos. Inhibition of osteoclast differentiation by (-)-DHMEQ was rescued by overexpression of NFATc1, suggesting that the inhibition is not caused by a toxic effect. Moreover, pit formation assays showed that (-)-DHMEQ also inhibited the bone-resorbing activity of mature osteoclasts. CONCLUSION: The inhibition of NF-kappaB suppresses osteoclastogenesis by downregulation of NFATc1, suggesting that NFATc1 expression is regulated by NF-kappaB in RANKL-induced osteoclastogenesis. Our results also indicate the possibility of (-)-DHMEQ becoming a new therapeutic strategy against bone erosion.

Animals↗
PubMed2004

Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells.

To determine the nature of intracellular Mg2+ stores and Mg2+ release mechanisms in differentiated PC12 cells, Mg2+ and Ca2+ mobilizations were measured simultaneously in living cells with KMG-104, a fluorescent Mg2+ indicator, and fura-2, respectively. Treatment with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), increased both the intracellular Mg2+ concentration ([Mg2+]i) and the [Ca2+]i in these cells. Possible candidates as intracellular Mg2+ stores under these conditions include intracellular divalent cation binding sites, endoplasmic reticulum (ER), Mg-ATP and mitochondria. Given that no change in [Mg2+]i was induced by caffeine application, intracellular IP3 or Ca2+ liberated by photolysis, it appears that no Mg2+ release mechanism thus exists that is mediated via the action of Ca2+ on membrane-bound receptors in the ER or via the offloading of Mg2+ from binding sites as a result of the increased [Ca2+]i. FCCP treatment for 2 min did not alter the intracellular ATP content, indicating that Mg2+ was not released from Mg-ATP, at least in the first 2 min following exposure to FCCP. FCCP-induced [Mg2+]i increase was observed at mitochondria localized area, and vice versa. These results suggest that the mitochondria serve as the intracellular Mg2+ store in PC12 cell. Simultaneous measurements of [Ca2+]i and mitochondrial membrane potential, and also of [Ca2+]i and [Mg2+]i, revealed that the initial rise in [Mg2+]i followed that of mitochondrial depolarization for several seconds. These findings show that the source of Mg2+ in the FCCP-induced [Mg2+]i increase in PC12 cells is mitochondria, and that mitochondrial depolarization triggers the Mg2+ release.

Adenosine Triphosphate↗
PubMed2004

Directional sensitivity of dendritic calcium responses to wind stimuli in the cricket giant interneuron.

We examined directional sensitivities in the dendritic activity of the identified giant interneurons (GIs) in the cricket, using in vivo Ca(2+) imaging during different directional air-current stimuli. Air current stimulus evoked action potential burst and quick Ca(2+) increase in GI. The stimulus direction of the maximal Ca(2+) responses corresponded to that of the maximal voltage response. However, the shapes of the directional tuning curves based on the Ca(2+) responses for each dendritic branch were different from the overall tuning curve based on spike counts for the cell. Moreover, different dendritic branches displayed distinct directional sensitivity profiles to the air-current stimuli. We propose that postsynaptic activities will influence the local Ca(2+) signals in the distal dendrites, and produce the difference in directional sensitivity of the dendritic Ca(2+) response.

Action Potentials↗
PubMed2004

Modulation of motor patterns by sensory feedback during earthworm locomotion.

Role of sensory feedback to motor pattern activity concerning locomotion in the earthworm, Eisenia fetida, was investigated. We have previously reported that bath application of octopamine induces fictive locomotion in the earthworm. In this study, we have examined the role of sensory feedback during fictive locomotion by analyzing electrical activities from the cut end and intact first lateral nerves of the ventral nerve cord (VNC). From the cut end recordings, motor activity associated with fictive locomotion was measured. A mixture of sensory and motor activities was measured from the intact first lateral nerve using en passant recordings, and sensory activity was separated by subtraction of the cut end recording (mainly motor activities) from the intact first lateral nerve recording. We estimated the effect of sensory feedback from the earthworm body wall by comparing recordings that made when the preparation was in-contact with a substrate or suspended above it. Motor pattern activities and the coefficient of variation for inter-spike-interval of motor outputs were increased under suspended conditions during circular muscle contraction. These results indicate that sensory feedback modulates the pattern of motor activity in the earthworm during locomotion.

Action Potentials↗
PubMed2004

Investigation of intracellular magnesium mobilization pathways I PC12 cells B simultaneous Mg-Ca fluorescent imaging.

OBJECTIVE AND METHODS: PC12 cells were loaded with a novel Mg indicator KMG-104 and Ca indicator fura-2, and intracellular Mg was studied in the endoplasmic reticulums (ERs), mitochondria, and Mg-ATP. Under coexistence of the two indicators, fluorescent signals of Mg and Ca can be measured separately. Mg release from the ER was investigated by photolysis of caged compounds. RESULTS: Transient [Ca]i increase by uncaging of caged Ca or caged IP3 or bath-application of caffeine (10 mM) induced no [Mg]i increase. These results suggest that there is no mechanism for Mg release from the ER through ryanodine receptors or IP3 receptors. In order to investigate the possibility of Mg release from Mg-ATP by energy consumption, we depleted ATP by oligomycin, an inhibitor of mitochondrial ATP synthase. Treating with oligomycin (4 microM) for several minutes showed no change of [Mg]i and [Ca]i. CONCLUSIONS: This result shows that Mg-ATP is not a Mg store. Since, when cells were treated by an uncoupler FCCP (3 microM), [Mg]i and [Ca]i increased, we concluded that mitochondria participate in maintenance of intracellular Mg stores.

Calcium↗
PubMed2004

Differences in pO2 peaks of a murine fibrosarcoma between carbon-ion and X-ray irradiation.

We measured and compared the oxygen partial pressure (pO(2)) profiles in experimental tumors after irradiation with carbon ions and with X-rays. The NFSa fibrosarcomas grown in the hind legs of C3H male mice received isoeffect single doses of carbon ions or X-rays. Coaxial oxygen microelectrodes of high spatial resolution were inserted into the tumor with 20 microm steps by a computerized micromanipulator. The number of pO(2) peaks that reached 15 mmHg were at least 0.45 per 3,000 microm in unirradiated tumors and significantly increased to 1.55 per 3,000 microm as early as day 1 of carbon-ion irradiation (p < 0.001). The tumors that received X-ray irradiation also significantly increased pO(2) peaks, but as late as day 3. The time course of pO(2) peak appearance in the present study coincides with a previous report where reoxygenation was measured by paired growth delay assay. The pO(2) peaks appeared selectively in peripheral regions of X-ray irradiated tumors, but they appeared rather homogeneously in the tumor after carbon-ion irradiation. It is concluded that carbon-ion irradiation reoxygenated the NFSa fibrosarcomas earlier in time and deeper in space than the X-ray irradiation did.

Animals↗
PubMed2004

In vivo nitric oxide measurements using a microcoaxial electrode.

Nitric oxide (NO) is a vital molecule contributing to numerous physiological phenomena in various biological systems. To investigate the physiological role of NO, a range of NO-specific electrodes allowing direct and continuous NO measurement have been developed for in vitro and in vivo NO detection. A microcoaxial electrode has also been developed for the measurement of real-time NO levels. Because the working and reference electrodes in this device are situated close together, the microcoaxial electrode is considered to be ideal for the measurement of local NO concentrations with high spatial resolution. The microcoaxial electrode has been successfully applied to the real-time measurement of NO in endothelial cells, thereby demonstrating its effectiveness as an NO-specific electrode. In this chapter, we describe our experimental protocol for performing real-time NO measurements that was developed during physiological experiments using the microcoaxial electrode.

Animals↗
PubMed2004

[Simultaneous Fluorescent Imaging of Mg2+ and Ca2+].

Simultaneous fluorescent imaging technique with several indicators is a powerful bio-imaging tool for a comprehensive survey about molecular functions in living cells. There are several interactions between Mg(2+) and Ca(2+) such as a counter transport or a competition at binding sites of divalent cation. To measure Mg-Ca images simultaneously, indicators with high selectivity to each divalent cation and multi-color fluorescent imaging system are essential. Here we introduce a setup for Mg-Ca fluorescent imaging we construct, and some reports about Mg-Ca imaging.

Animals↗
PubMed2003

Dual responses of tissue partial pressure of oxygen after functional stimulation in rat somatosensory cortex.

To compare the spatial heterogeneity of brain tissue partial pressure of oxygen (pO(2)) among local brain regions, we focused on functional and anatomical variations in rat somatosensory cortex. Tissue pO(2) was measured by using an oxygen microelectrode with high spatio-temporal resolution, and investigated in three somatosensory areas including hindlimb (HL), forelimb (FL), and trunk region (Tr). Their anatomical structures were determined with histological techniques (Nissl stain). In addition to the measurement of baseline tissue pO(2), we examined temporal shifts in tissue pO(2) distribution elicited by functional stimulation using the brushing stimulation to the hindlimb, forelimb, and trunk regions of the body. We observed that average tissue pO(2) in the Tr (14+/-10 Torr) was significantly lower than those in the HL (25+/-13 Torr) and FL (24+/-13 Torr). Such regional differences in tissue pO(2) were closely related to the cytoarchitectonic variations among these three areas. In addition, the functional stimulation enlarged the regional differences in the pO(2) depending on each somatosensory area; the pO(2) in the HL increased by 3.6+/-2.9% after the stimulation to hindlimb, whereas that in the Tr decreased by -2.9+/-2.5% after the stimulation to trunk region. Such dual responses of tissue pO(2) (i.e. increase or decrease) after the functional stimulation to the corresponding body regions may provide a criterion to clinically predict regions susceptible to tissue hypoxia, because considerable decrease in tissue pO(2) occurred in the Tr showing the lowest baseline pO(2).

Animals↗
PubMed2003

Na+/Mg2+ transporter acts as a Mg2+ buffering mechanism in PC12 cells.

Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.

Adrenergic Uptake Inhibitors↗
PubMed2003

Kinetic simulation of signal transduction system in hippocampal long-term potentiation with dynamic modeling of protein phosphatase 2A.

We modeled and analyzed a signal transduction system of long-term potentiation (LTP) in hippocampal post-synapse. Bhalla and Iyengar [Science 283(1999) 381] have developed a hippocampal LTP model. In the conventional model, the concentration of protein phosphatase 2A (PP2A) was fixed. However, it was reported that dynamic inactivation of PP2A was essential for LTP [J. Neurochem. 74 (2000) 807]. We introduced a dynamic modeling of PP2A; inactivation (phosphorylation) of PP2A by calcium/calmodulin-dependent protein kinase II (CaMKII) in the presence of calcium/calmodulin, self-activation (autodephosphorylation) of PP2A, and inactivation (dephosphorylation) of CaMKII by PP2A. This model includes complex feedback loops; both CaMKII and PP2A are autoactivated, while they inactivate each other. Moreover, we proposed an analysis strategy for model validation by applying the results of sensitivity analysis. In our system, calcineurin (CaN) played an essential role, rather than the activation of protein kinase C (PKC) as documented in the conventional model. From results of the analysis of our model, we found the following robustness as characteristics of bistability in our model: (1). PP2A reactions against calcium ion (Ca(2+)) perturbation; (2). PP2A inactivation against PP2A increase; (3). protein phosphatase 1 (PP1) activation against PF2A increase; and (4). PP2A reactions against PP2A initial concentration. These properties facilitated LTP induction in our system. We showed that another mechanism could introduce bistable behavior by adding dynamic reactions of PP2A.

Animals↗