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ABSTRACT: The sensitivity of post-tetanic potentiation (PTP) to high frequency stimulation (HFS) steeply decays during the first two postnatal weeks. We investigated the underlying mechanisms for the developmental change of PTP induced by HFS (100 Hz, 2 sec) at postnatal days 4-6 and 9-11 at the rat calyx of Held synapse. Low concentration tetraphenylphosphonium (2 μM), an inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, suppressed the amount of post-tetanic residual calcium and PTP to a larger extent at the immature calyx synapse, indicating a developmental reduction of mitochondrial contribution to PTP. The higher amount of mitochondrial Ca(2+) uptake during HFS and consequent post-tetanic residual Ca(2+) at the immature calyx of Held was associated with higher peak of HFS-induced Ca(2+) transients, most likely because the mitochondrial Ca(2+) uptake during HFS was supra-linearily dependent on the presynaptic [Ca(2+)] level. Probing into the contribution of Na(+)/Ca(2+) exchangers to calcium clearance, we found a specific upregulation of the K(+)-dependent Na(+)/Ca(2+) exchanger (NCKX) activity in the maturer calyx of Held. We conclude that the upregulation of NCKX limits the Ca(2+) buildup and inhibits mitochondrial Ca(2+) uptake during HFS, which in turn results in the reduction of post-tetanic residual Ca(2+) and PTP at the mature calyx of Held.
Journal of Neurophysiology 01/2013; · 3.32 Impact Factor
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ABSTRACT: We have previously reported that the surface expression of K(+)-dependent Na(+)/Ca(2+) exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the μ subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (YxxΦ) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs ((365)YGKL and (371)YDTM) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the (365)YGKL motif is essential for the interaction with the μ subunit of AP-2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.
Frontiers in Cellular Neuroscience 01/2013; 7:14. · 4.17 Impact Factor
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ABSTRACT: Neural network activity regulates the development of hippocampal newborn granule cells (GCs). Excitatory GABAergic input is known to be a key player in this regulation. Although calcium signaling is thought to be a downstream mediator of GABA, GABA-induced calcium signaling in newborn GCs is not well understood. We investigated Ca(2+) signaling and its regulatory role in axon and dendrite outgrowth in newborn GCs identified in the organotypic slice culture of early postnatal rat hippocampus. Here, we report that hippocampal network activity can induce calcium transients (CaTs) in newborn GCs during the first post-mitotic week via GABAergic inputs. The GABA-induced CaTs were mediated mainly by L-type Ca(2+) channels. Furthermore, we found that inhibiting any step in the signaling pathway, network activity → GABA → L-type Ca(2+) channels, selectively suppressed the axonal outgrowth and pruning of newborn GCs, but not dendritic outgrowth. The GABA(A) receptor blocker bicuculline significantly suppressed axonal outgrowth, despite increasing network activity, thus indicating an essential role of GABAergic inputs. Therefore, we conclude that network activity-dependent GABAergic inputs open L-type Ca(2+) channels and promote axonal outgrowth in newborn GC during the first post-mitotic week.
European Journal of Neuroscience 07/2012; 36(6):2743-52. · 3.63 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) in the early stages is characterized by memory impairment, which may be attributable to synaptic dysfunction. Oxidative stress, mitochondrial dysfunction, and Ca²⁺ dysregulation are key factors in the pathogenesis of AD, but the causal relationship between these factors and synaptic dysfunction is not clearly understood. We found that in the hippocampus of an AD mouse model (Tg2576), mitochondrial Ca²⁺ handling in dentate granule cells was impaired as early as the second postnatal month, and this Ca²⁺ dysregulation caused an impairment of post-tetanic potentiation in mossy fiber-CA3 synapses. The alteration of cellular Ca²⁺ clearance in Tg2576 mice is region-specific within hippocampus because in another region, CA1 pyramidal neuron, no significant difference in Ca²⁺ clearance was detected between wild-type and Tg2576 mice at this early stage. Impairment of mitochondrial Ca²⁺ uptake was associated with increased mitochondrial reactive oxygen species and depolarization of mitochondrial membrane potential. Mitochondrial dysfunctions in dentate granule cells and impairment of post-tetanic potentiation in mossy fiber-CA3 synapses were fully restored when brain slices obtained from Tg2576 were pretreated with antioxidant, suggesting that mitochondrial oxidative stress initiates other dysfunctions. Reversibility of early dysfunctions by antioxidants at the preclinical stage of AD highlights the importance of early diagnosis and antioxidant therapy to delay or prevent the disease processes.
Journal of Neuroscience 04/2012; 32(17):5953-63. · 7.11 Impact Factor
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ABSTRACT: We have previously shown that K(+)-dependent Na(+)/Ca(2+) exchanger (NCKX) is a major calcium clearance mechanism at the large axon terminals of central neurons, whereas their somata display little NCKX activity. We investigated mechanisms underlying the axonal polarization of NCKX2 in rat hippocampal neurons. We identified NCKX2 as the first neuron-specific cargo molecule of kinesin family member 21A (KIF21A). The intracellular loop of NCKX2 specifically interacted with the WD-40 repeats, a putative cargo-binding domain, of KIF21A. Dominant-negative mutant or depletion of KIF21A inhibited the transport of NCKX2-GFP to axon fibers. Knockdown of KIF21A caused calcium dysregulation at axonal boutons but not at somatodendritic regions. Despite the axonal polarization of the NCKX activity, both somatodendritic and axonal regions were immunoreactive to NCKX2. The surface expression of NCKX2 revealed by live-cell immunocytochemistry, however, displayed highly polarized distribution to the axon. Inhibition of endocytosis increased the somatodendritic surface NCKX2 and thus abolished the axonal polarization of surface NCKX2. These results indicate that KIF21A-mediated axonal transport and selective somatodendritic endocytosis underlie the axonal polarized surface expression of NCKX2.
Journal of Neuroscience 03/2012; 32(12):4102-17. · 7.11 Impact Factor
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ABSTRACT: Glutamatergic synaptic terminals harbor reluctant synaptic vesicles (SVs) that contribute little to synchronous release during action potentials but are release competent when stimulated by sucrose or by direct intracellular application of calcium. It has been noted that the proximity of a release-competent SV to the calcium source is one of the primary factors that differentiate reluctant SVs from fast-releasing ones at the calyx of Held synapse. It has not been known whether reluctant SVs can be converted into fast-releasing ones. Here we show that reluctant SVs are recruited rapidly in an actin-dependent manner to become fast-releasing SVs once the pool of fast-releasing SVs is depleted by a short depolarization. Recovery of the pool of fast-releasing SVs was accompanied by a parallel reduction in the number of reluctant SVs. Quantitative analysis of the time course of depletion of fast-releasing SVs during high-frequency stimulation revealed that in the early phase of stimulation reluctant SVs are converted rapidly into fast-releasing ones, thereby counteracting short-term depression. During the late phase, however, after reluctant vesicles have been used up, another process of calmodulin-dependent recruitment of fast-releasing SVs is activated. These results document that reluctant SVs have a role in short-term plasticity and support the hypothesis of positional priming, which posits that reluctant vesicles are converted into fast-releasing ones via relocation closer to Ca(2+)-channels.
Proceedings of the National Academy of Sciences 03/2012; 109(13):E765-74. · 9.68 Impact Factor
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Xi-Biao He,
Sang-Hoon Yi,
Yong-Hee Rhee,
Hyemin Kim,
Yong-Mahn Han, Suk-Ho Lee,
Hyunsu Lee,
Chang-Hwan Park,
Yong-Sung Lee,
Eric Richardson,
Byung-Woo Kim,
Sang-Hun Lee
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ABSTRACT: Understanding midbrain dopamine (DA) neuron differentiation is of importance, because of physiological and clinical implications of this neuronal subtype. We show that prolonged membrane depolarization induced by KCl treatment promotes DA neuron differentiation from neural precursor cells (NPCs) derived from embryonic ventral midbrain (VM). Interestingly, the depolarization-induced increase of DA neuron yields was not abolished by L-type calcium channel blockers, along with no depolarization-mediated change of intracellular calcium level in the VM-derived NPCs (VM-NPCs), suggesting that the depolarization effect is due to a calcium-independent mechanism. Experiments with labeled DA neuron progenitors indicate that membrane depolarization acts at the differentiation fate determination stage and promotes the expression of DA phenotype genes (tyrosine hydroxylase [TH] and DA transporter [DAT]). Recruitment of Nurr1, a transcription factor crucial for midbrain DA neuron development, to the promoter of TH gene was enhanced by depolarization, along with increases of histone 3 acetylation (H3Ac) and trimethylation of histone3 on lysine 4 (H3K4m3), and decreases of H3K9m3 and H3K27m3 in the consensus Nurr1 binding regions of TH promoter. Depolarization stimuli on differentiating VM-NPCs also induced dissociation of methyl CpG binding protein 2 and related repressor complex molecules (repressor element-1 silencing transcription factor corepressor and histone deacetylase 1) from the CpG sites of TH and DAT promoters. Based on these findings, we suggest that membrane depolarization promotes DA neuron differentiation by opening chromatin structures surrounding DA phenotype genes and inhibiting the binding of corepressors, thus allowing transcriptional activators such as Nurr1 to access DA neuron differentiation gene promoter regions.
Stem Cells 09/2011; 29(11):1861-73. · 7.78 Impact Factor
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ABSTRACT: Oxidative stress remodels Ca(2+) signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca(2+) signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca(2+) current (I(Ca,L)) in rat cardiomyocytes. A 5-min exposure of 1mM H(2)O(2) induced an increase in I(Ca,L), and this increase was sustained for ~1h. The CaMKII inhibitor KN-93 fully reversed H(2)O(2)-induced LTF of I(Ca,L), indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca(2+) release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H(2)O(2) via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.
Free radical biology & medicine 08/2011; 51(9):1708-16. · 5.42 Impact Factor
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Yong-Hee Rhee,
Ji-Yun Ko,
Mi-Yoon Chang,
Sang-Hoon Yi,
Dohoon Kim,
Chun-Hyung Kim,
Jae-Won Shim,
A-Young Jo,
Byung-Woo Kim,
Hyunsu Lee, Suk-Ho Lee,
Wonhee Suh,
Chang-Hwan Park,
Hyun-Chul Koh,
Yong-Sung Lee,
Robert Lanza,
Kwang-Soo Kim,
Sang-Hun Lee
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ABSTRACT: Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.
The Journal of clinical investigation 06/2011; 121(6):2326-35. · 15.39 Impact Factor
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ABSTRACT: Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5) exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca(2+). In this study, we characterized the cellular mechanisms underlying Ca(2+) mobilization induced by (RS)-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist) in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca(2+) from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR), while the PLC/IP(3) signaling pathway was not involved in Ca(2+) mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4), led to transient Ca(2+) mobilization by mGluR5 and Ca(2+) influx through L-type Ca(2+) channels. We found no evidence that mGluR5-mediated Ca(2+) release and Ca(2+) influx through L-type Ca(2+) channels interact to generate supralinear Ca(2+) transients. Our study provides novel insights into the mechanisms of intracellular Ca(2+) mobilization by mGluR5 in the somata of hippocampal neurons.
PLoS ONE 01/2011; 6(10):e26625. · 4.09 Impact Factor
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ABSTRACT: Post-tetanic potentiation (PTP) at the calyx of Held synapse is caused by increases not only in release probability (P(r)) but also in the readily releasable pool size estimated from a cumulative plot of excitatory post-synaptic current amplitudes (RRP(cum)), which contribute to the augmentation phase and the late phase of PTP, respectively. The vesicle pool dynamics underlying the latter has not been investigated, because PTP is abolished by presynaptic whole-cell patch clamp. We found that supplement of recombinant calmodulin to the presynaptic pipette solution rescued the increase in the RRP(cum) after high-frequency stimulation (100 Hz for 4-s duration, HFS), but not the increase in P(r). Release-competent synaptic vesicles (SVs) are heterogeneous in their releasing kinetics. To investigate post-tetanic changes of fast and slowly releasing SV pool (FRP and SRP) sizes, we estimated quantal release rates before and 40 s after HFS using the deconvolution method. After HFS, the FRP size increased by 19.1% and the SRP size decreased by 25.4%, whereas the sum of FRP and SRP sizes did not increase. Similar changes in the RRP were induced by a single long depolarizing pulse (100 ms). The post-tetanic complementary changes of FRP and SRP sizes were abolished by inhibitors of myosin II or myosin light chain kinase. The post-tetanic increase in the FRP size coupled to a decrease in the SRP size provides the first line of evidence for the idea that a slowly releasing SV can be converted to a fast releasing one.
The Journal of General Physiology 09/2010; 136(3):259-72. · 3.84 Impact Factor
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Tae-Hee Lee,
Sun-Hwa Song,
Koung Li Kim,
Ji-Yeun Yi,
Ga-Hee Shin,
Ji Yeon Kim,
Jihoon Kim,
Yong-Mahn Han,
Sang Hun Lee, Suk-Ho Lee,
Sung Han Shim,
Wonhee Suh
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ABSTRACT: Generation of induced pluripotent stem (iPS) cells has been intensively studied by a variety of reprogramming methods, but the molecular and functional properties of the cells differentiated from iPS cells have not been well characterized.
To address this issue, we generated iPS cells from human aortic vascular smooth muscle cells (HASMCs) using lentiviral transduction of defined transcription factors and differentiated these iPS cells back into smooth muscle cells (SMCs).
Established iPS cells were shown to possess properties equivalent to human embryonic stem cells, in terms of the cell surface markers, global mRNA and microRNA expression patterns, epigenetic status of OCT4, REX1, and NANOG promoters, and in vitro/in vivo pluripotency. The cells were differentiated into SMCs to enable a direct, comparative analysis with HASMCs, from which the iPS cells originated. We observed that iPS cell-derived SMCs were very similar to parental HASMCs in gene expression patterns, epigenetic modifications of pluripotency-related genes, and in vitro functional properties. However, the iPS cells still expressed a significant amount of lentiviral transgenes (OCT4 and LIN28) because of partial gene silencing.
Our study reports, for the first time, the generation of iPS cells from HASMCs and their differentiation into SMCs. Moreover, a parallel comparative analysis of human iPS cell-derived SMCs and parental HASMCs revealed that iPS-derived cells possessed representative molecular and in vitro functional characteristics of parental HASMCs, suggesting that iPS cells hold great promise as an autologous cell source for patient-specific cell therapy.
Circulation Research 12/2009; 106(1):120-8. · 9.49 Impact Factor
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ABSTRACT: The Ca(2+)-dependent facilitation (CDF) of L-type Ca(2+) channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca(2+)/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca(2+) currents (I(Ca,L)) by H(2)O(2) and whether Ca(2+) is required in this process. Using patch clamp, I(Ca)(,L) was measured in rat ventricular myocytes. H(2)O(2) induced an increase in I(Ca,L) amplitude and slowed inactivation of I(Ca)(,L). This oxidation-dependent facilitation (ODF) of I(Ca)(,L) was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca(2+) with Ba(2+) or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca(2+) stores using caffeine and thapsigargin. Alkaline phosphatase, beta-iminoadenosine 5'-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca(2+) channels is mediated by oxidation-dependent CaMKII activation, in which local Ca(2+) increases induced by SR Ca(2+) release is required.
Journal of Molecular and Cellular Cardiology 10/2009; 48(4):773-80. · 5.17 Impact Factor
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ABSTRACT: AMP-activated protein kinase (AMPK) and the ATP-sensitive K(+) (K(ATP)) channel are metabolic sensors that become activated during metabolic stress. AMPK is an important regulator of metabolism, whereas the K(ATP) channel is a regulator of cellular excitability. Cross talk between these systems is poorly understood.
Rat pancreatic beta-cells or INS-1 cells were pretreated for 2 h at various concentrations of glucose. Maximum K(ATP) conductance (G(max)) was monitored by whole-cell measurements after intracellular ATP washout using ATP-free internal solutions. K(ATP) channel activity (NPo) was monitored by inside-out patch recordings in the presence of diazoxide. Distributions of K(ATP) channel proteins (Kir6.2 and SUR1) were examined using immunofluorescence imaging and surface biotinylation studies. Insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay.
G(max) and NPo in cells pretreated with glucose-free or 3 mmol/l glucose solutions were significantly higher than in cells pretreated in 11.1 mmol/l glucose solutions. Immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of Kir6.2 without affecting the total cellular amount. Increases in G(max) and the surface level of Kir6.2 were inhibited by compound C, an AMPK inhibitor, and siAMPK transfection. The effects of glucose deprivation on K(ATP) channels were mimicked by an AMPK activator. Glucose deprivation reduced insulin secretion, but this response was attenuated by compound C.
K(ATP) channel trafficking is regulated by energy status via AMPK, and this mechanism may play a key role in inhibiting insulin secretion under low energy status.
Diabetes 09/2009; 58(12):2813-9. · 8.29 Impact Factor
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ABSTRACT: Calcium is a key regulator for expression of genes relevant to survival and maturation of newborn neurons. Mammalian hippocampal dentate gyrus generates new granule cells (GCs) throughout adult life. We identified young and mature GCs in hippocampi of young adult mice according to their electrical properties, and investigated contributions of Na/Ca exchanger (NCX), sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), plasma membrane Ca(2+)-ATPase (PMCA) and mitochondria to Ca(2+) clearance in somata of GCs. Somatic Ca(2+) clearance was increased by about 50% as GCs matured. NCX activity increased proportionally during maturation with its relative contribution kept about 40% both in young and mature GCs. On the other hand, the developmental increases in activities of mitochondria and SERCA resulted in higher contributions to Ca(2+) clearance in mature GCs than in young GCs. Especially mitochondrial function was most highly enhanced during maturation. PMCA activity, however, did not increase during maturation. Low Ca(2+) clearance in immature GCs might facilitate higher Ca(2+) accumulation during network activity, which in turn help survival of young GCs.
Cell calcium 05/2009; 45(5):465-73. · 4.29 Impact Factor
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ABSTRACT: At the immature calyx of Held, the fast decay phase of a Ca(2+) transient induced by tetanic stimulation (TS) was followed by a period of elevated [Ca(2+)](i) for tens of seconds, referred to as posttetanic residual calcium (Ca(res)). We investigated the source of Ca(res) and its contribution to posttetanic potentiation (PTP). After TS (100 Hz for 4 s), posttetanic Ca(res) at the calyx of Held was largely abolished by tetraphenylphosphonium (TPP(+)) or Ru360, which inhibit mitochondrial Na(+)-dependent Ca(2+) efflux and Ca(2+) uniporter, respectively. Whereas the control PTP lasted longer than Ca(res), inhibition of Ca(res) by TPP(+) resulted in preferential suppression of the early phase of PTP, the decay time course of which well matched with that of Ca(res). TS induced significant increases in release probability (P(r)) and the size of the readily releasable pool (RRP), which were estimated from plots of cumulative EPSC amplitudes. TPP(+) or Ru360 suppressed the posttetanic increase in P(r), whereas it had little effect on the increase in RRP size. Moreover, the posttetanic increase in P(r), but not in RRP size, showed a linear correlation with the amount of Ca(res). In contrast, myosin light chain kinase (MLCK) inhibitors and blebbistatin reduced the posttetanic increase in RRP size with no effect on the increase in P(r). Application of TPP(+) in the presence of MLCK inhibitor peptide caused further suppression of PTP. These findings suggest that Ca(res) released from mitochondria and activation of MLCK are primarily responsible for the increase in P(r) and that in the RRP size, respectively.
Journal of Neuroscience 09/2008; 28(32):7945-53. · 7.11 Impact Factor
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Jiho Jang,
Seung Yup Ku,
Jung Eun Kim,
Kyunghee Choi,
Yoon Young Kim,
Hee Sun Kim,
Sun Kyung Oh,
Eun Ju Lee,
Hyun-Jai Cho,
Young Hwan Song,
Sang Hun Lee, Suk Ho Lee,
Chang Suk Suh,
Seok Hyun Kim,
Shin Yong Moon,
Young Min Choi
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ABSTRACT: The roles of Notch signaling in cardiac differentiation from murine embryonic stem cells have been well documented. We investigated whether Notch signaling plays a similar role in human embryonic stem cells (hESCs). Although, as previously reported, blocking Notch signaling via the addition of gamma-secretase inhibitor (GSI) alone failed to affect hESC differentiation, we found that GSI plus reduced-volume culture medium (GSI/RVCM) accelerated mesodermal differentiation. GSI/RVCM conditions simultaneously suppressed commitment toward neuroectodermal lineages. Furthermore, sustained inhibition of Notch signaling further enhanced differentiation into cardiac mesoderm. Spontaneous beating activity was typically observed from 12 days after initiation of GSI treatment in RVCM. Moreover, hESC-derived cardiomyocytes expressed connexin 43 and possessed spontaneous calcium oscillations and cardiomyocyte beats coupled to neonatal rat cardiomyocytes when cocultured. These findings strongly suggest a distinct role for Notch signaling in the induction and specification of hESC-derived cardiac mesoderm in vitro. Disclosure of potential conflicts of interest is found at the end of this article.
Stem Cells 09/2008; 26(11):2782-90. · 7.78 Impact Factor
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ABSTRACT: Previous studies indicate that boutons from the same axon exhibit distinct Ca2+ dynamics depending on the postsynaptic targets. Mossy fibers of hippocampal granule cells innervate synaptic targets via morphologically distinct boutons. We investigated mitochondrial involvement in the generation of post-tetanic residual Ca2+ (Ca(res)) at large and small en passant mossy fiber boutons (MFBs). Mitochondria limited the [Ca2+]i build-up during high-frequency stimulation (HFS) at large MFBs, but not at small MFBs. The amount of Ca(res), quantified as a time integral of residual [Ca2+]i, was significantly larger at large MFBs than at small MFBs, and that at large MFBs was substantially attenuated by inhibitors of mitochondrial Ca2+ uniporter and mitochondrial Na+/Ca2+ exchanger (mitoNCX). In contrast, blockers of mitoNCX had no effect on the Ca(res) at small MFBs. Post-tetanic Ca(res) has been proposed as a mechanism for post-tetanic potentiation (PTP). We examined mitochondrial involvement in PTP at mossy fiber synapses on hilar mossy cells (MF-->MC synapse) and on hilar interneurons (MF-->HI synapse), which are presumably innervated via large and small MFBs, respectively. Consistent with the differential contribution of mitochondria to Ca(res) at large and small MFBs, mitoNCX blockers significantly reduced the PTP at the MF-->MC synapse, but not at the MF-->HI synapse. In contrast, protein kinase C (PKC) inhibitors significantly reduced the PTP at MF-->HI synapse, but not at the MF-->MC synapse. These results indicate that mitochondria- and PKC-dependent PTP are expressed at distinct hilar mossy fiber synapses depending on postsynaptic targets.
Journal of Neuroscience 12/2007; 27(50):13603-13. · 7.11 Impact Factor
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ABSTRACT: We showed in our previous study that in hippocampal CA1 neurons the stimulation of muscarinic receptors inhibited the GIRK current (I(GIRK)) via a PLC/PKC pathway, whereas group I metabotropic glutamate receptors (mGluR) inhibited I(GIRK) via a PLA(2)/arachidonic acid pathway. In this study, we present evidence that receptor-mediated signalling pathways activated by the two G(q)-coupled receptors (G(q)PCRs) converge on the inhibition of GIRK channel-PIP(2) interaction. I(GIRK) was activated in acutely isolated hippocampal CA1 neurons by repetitive application of baclofen, a GABA(B) receptor agonist, with a 2-3 min interval. When both CCh and DHPG were pretreated before the second I(GIRK) activation, the magnitude of the second I(GIRK) was 52.2 +/- 2.5% of the first I(GIRK), which was not significantly different from the magnitude of inhibition by CCh or DHPG alone. This result shows that the effects of muscarinic receptor and group I mGluR stimulation on I(GIRK) are not additive but occlusive, suggesting that each pathway may converge to a common mechanism that finally regulates I(GIRK). To test the involvement of PIP(2) in this mechanism, the effect of CCh and DHPG on I(GIRK) was tested in cells loaded with exogenous PIP(2). The inhibition of I(GIRK) by CCh or DHPG was almost completely abolished in PIP(2)-loaded cells. We confirmed that the inhibition of I(GIRK) by direct application of phorbol ester or arachidonic acid was also completely reversed in PIP(2)-loaded cells. These results indicate that the decrease in PIP(2)-channel interactions is the final common mechanism responsible for G(q)PCR-induced inhibitions of I(GIRK) mediated by PKC and arachidonic acid.
The Journal of Physiology 09/2007; 582(Pt 3):1037-46. · 4.72 Impact Factor
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ABSTRACT: Supraoptic magnocellular neurons (SMNs) undergo dramatic changes in morphological and electrical properties during postnatal development. We investigated the developmental change in Ca2+ homeostasis in SMNs. The decay rate of Ca2+ transients markedly increased during the third postnatal week (PW3) to an adult level. This increase in the Ca2+ decay rate was paralleled by hypertrophy of the SMN somata. Activity of Na+/Ca2+ exchanger (Na/CaX) and sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) was quantified as a decrement in the Ca2+ decay rate caused by extracellular [Na+] reduction and that by thapsigargin, respectively. SERCA activity was negligible during PW2, and markedly increased during PW3. SERCA activity and soma size remained stable thereafter. Na/CaX activity was a major Ca2+-clearance mechanism (CCM) during PW2, increased further during PW3, but was negligible in mature SMNs (PW10). In parallel with the decrease in Na/CaX activity, endogenous Ca2+ buffering capacity declined, resulting that the apparent Ca2+ decay rate remained relatively constant between PW4 and PW10. Replacement of intracellular K+ with Li+ had no effect on Na/CaX activity, suggesting that NCX rather than NCKX comprises Na/CaX. These findings indicate a developmental shift in the balance of CCMs from Ca2+ extrusion via NCX toward Ca2+ sequestration into endoplasmic reticulum via SERCA.
Cell Calcium 06/2007; 41(5):441-50. · 3.77 Impact Factor
