Mitochondrial Dynamics of Bcl-2 Family Proteins during 17-ß-Estradiol-Induced Apoptosis Correlate with the Malignancy of Endometrial Cancer Cells.

Excessive fat intake leads to an increase in cholesterol. Overexposure to estrogen derived from cholesterol is known to contribute to the malignancy of endometrial adenocarcinomas. However, it is not well understood the relationship between the exposure to estrogen and the malignancy of endometrial adenocarcinomas. We investigated how estrogen affected the malignancy of endometrial cancer cells, specifically HEC1 cells (a moderately differentiated adenocarcinoma) and HEC50B cells (a poorly differentiated adenocarcinoma). Cell viability was decreased by exogenous 17-ß-estradiol (E2) in a concentration-dependent manner. E2 disturbed the mitochondrial membrane potentials by changing the localization of the B-cell lymphoma 2 (Bcl-2) family protein; however, there were significant differences in the localization of Bcl-2 family proteins between HEC1 and HEC50B cells. In HEC1 cells, E2 increased the expression of B-cell lymphoma-extra large (Bcl-XL) and the Bcl-2-associated X protein (Bax) and decreased Bcl-2 and Bcl-2-associated death promoter (Bad) expression on the outer mitochondrial membrane. Conversely, E2 increased the expression of Bad and Bax, and it decreased Bcl-2 and Bcl-XL expressions on the outer mitochondrial membrane in HEC50B cells. The disturbance of the mitochondrial membrane potential led to the release of cytochrome c from the mitochondria to the cytosolic space followed by activating caspase-9. After that, caspase-3 was activated and induced apoptosis. These results suggested that the localization of the Bcl-2 family protein observed under E2-induced apoptosis is related to the malignancy of endometrial cancer cells. We hope that the dynamics of Bcl-2 family proteins such as Bcl-XL and Bad will be used to diagnose malignant endometrial adenocarcinomas.

The endocrine disruptor bisphenol A promotes nuclear ERRγ translocation, facilitating cell proliferation of Grade I endometrial cancer cells via EGF-dependent and EGF-independent pathways.

Endocrine disruptors have become a global social and public health problem since the late 1980s. Bisphenol A (BPA) has a steroid-like skeleton similar to estrogen and progesterone, and is an endocrine disruptor that disturbs the physiological hormone balance. The potential involvement of BPA in malignancy of endometrial cancer cells caused by overexposure of steroid hormones remains incompletely understood. The present study aimed at understanding the regulatory mechanism underlying BPA-induced cell proliferation in hormone-sensitive endometrial cancer cells. BPA selectively and significantly induced cell proliferation of Grade I endometrial cancer cells such as HEC265 and Ishikawa cells. In HEC265 and Ishikawa cells, BPA induced nuclear translocation of estrogen-related receptor γ (ERRγ) in a time-dependent manner and increased expression of BPA/ERRγ-target genes. In Ishikawa cells, BPA promoted the influx of Ca2+ followed by epidermal growth factor (EGF) secretion to the extracellular space. Furthermore, EGF secreted from Ishikawa had an autocrine effect, leading to activation of the EGFR/ERK pathway. Contrastingly, in HEC265 cells, BPA increased the expression of BPA/ERRγ-target genes but did not affect Ca2+ mobilization EGF secretion. In conclusion, BPA induced cell proliferation via the BPA/ERRγ/EGF/EGFR/ERK signaling pathway in Ishikawa cells and contrastingly, in HEC265 cells, induced cell proliferation through the BPA/ERRγ signaling pathway.

Estrogen induces cell proliferation by promoting ABCG2-mediated efflux in endometrial cancer cells.

Recently, it has reported that overeating of lipid-food has led to increase the amount of estrogen in vivo and the incidence of endometrial carcinomas. It is well-known that ATP-binding cassette transporter sub-family G2 (ABCG2) is highly expressed in cancer stem cells (CSCs). CSCs possess the ability for differentiation, tumorigenesis, stem cell self-renewal, and the efflux of anti-cancer drug and these abilities affect malignancy of cancer cells. However, little is known about the relationship between the expression of ABCG2 and malignancy of cancer cells. The present study aimed at understanding the regulatory mechanism underlying 17-ß-estradiol (E2)-induced cell proliferation under the control of ABCG2. E2 increased cell viability with a peak at 1 µM and facilitated ABCG2 mRNA expression followed by the increase of ABCG2 expression level at plasma membrane. E2-induced cell proliferation was inhibited by reserpine, an inhibitor of ABCG2, and the ABCG2 siRNA treatment. Thus, these results imply that ABCG2 plays an important role in the promotion of E2-induced cell proliferation in Ishikawa cells.

Isolation of side population cells from endometrial cancer cells using a violet laser diode.

Cancer stem cells (CSCs) possess the ability for self-renewal, differentiation, and tumorigenesis and play a role in cancer recurrence and metastasis. CSCs are usually sorted in analysis into side population (SP) cells using ultraviolet (UV) laser (350 nm) excitation; they cannot be stained with Hoechst 33342 because of their efflux ability. However, it is difficult to avoid cell damage using a UV laser. Therefore, we attempted to isolate CSCs using a violet laser (407 nm) excitation to avoid cellular DNA damage. We sorted SP cells and main population (MP) cells from a human endometrial cancer cell line using the FACSAria system equipped with a violet laser and analyzed the biological properties of these cells. SP cells exhibited drug efflux, self-renewal, differentiation abilities, and tumorigenicity. It was found that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) expression was significantly higher in SP cells than in MP cells. Our results suggest that CSCs exist in the SP fraction sorted using the FACSAria system equipped with a violet laser, which presents a useful tool to isolate small populations of viable putative CSCs from solid tumors and can be used to identify and characterize CSCs.

Tumorigenesis in cells derived from induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells are an attractive source for potential cell-replacement therapy. However, transplantation of differentiated products harbors the risk of teratoma formation, presenting a serious health risk. Thus, we characterized Nanog-expressing (undifferentiated) cells remaining after induction of differentiation by cytological examination. To induce differentiation of iPS cells, we generated embryoid bodies (EBs) derived from iPS cells carrying a Nanog­green fluorescent protein(GFP) reporter and then injected GFP-positive and GFP negative EBs into nude mice. GFP-positive EB transplantation resulted in the formation of immature teratoma grade 3, but no tumors were induced by GFP-negative EB. GFP positive cells revealed significantly lower cytoplasmic area and higher nucleus/cytoplasm ratio than those of GFP negative cells. Our results suggest that morphological analysis might be a useful method for distinguishing between tumorigenic and nontumorigenic iPS cells.

8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid and its diastereomers improve age-related cognitive deterioration.

Racemic 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a linoleic acid derivative with cyclopropane rings instead of cis-double bonds, contains possible four diastereomers such as α,α-, α,ß-, ß,α-, and ß,ß-DCP-LA. The present study examined the effect of racemic and diastereomeric DCP-LA on age-related learning and memory disorders using accelerated-senescence-prone mice 8 (SAMP8) and accelerated-senescence-resistant mice 1 (SAMR1). In the water maze test, the acquisition and retention latencies for SAMP8 mice were significantly longer than the latency for SAMR1 mice, indicating spatial learning and memory impairment for SAMP8 mice. All the racemic (1 mg/kg, per os) and diastereomeric DCP-LA (0.25 mg/kg, per os) significantly shortened the acquisition latency for SAMP8 mice, and racemic, α,α- and α,ß-DCP-LA significantly shortened the retention latency, with the advantage greater than the acetylcholine (ACh) esterase inhibitor galanthamine. The results of the present study show that all the racemic and diastereomeric DCP-LA, has the potential to improve age-related learning and memory deterioration, the potential varying among them.

Indomethacin activates protein kinase C and potentiates α7 ACh receptor responses.

BACKGROUND/AIMS: We have earlier found that indomethacin activates CaMKII, as a novel action distinct from COX inhibition. To explore further indomethacin actions, the present study focused upon PKC and examined the effect of indomethacin on α7 ACh receptor responses and hippocampal synaptic transmission through PKC. METHODS: We recorded currents through α7 ACh receptors expressed in Xenopus oocytes, quantified PKC activity in the in situ and cell-free PKC assay, and monitored field excitatory postsynaptic potentials (fEPSPs) and miniature excitatory postsynaptic currents (mEPSCs) from the CA1 region of rat hippocampal slices. RESULTS: Indomethacin potentiated α7 ACh receptor currents in a bell-shaped concentration (100 nM-1 mM)-dependent manner, and the potentiating effect was inhibited by the PKC inhibitor GF109203X. Indomethacin activated PKC in a concentration (1-100 µM)-dependent manner for cultured rat hippocampal neurons. Additionally, indomethacin (100 µM) significantly activated PKC-ε under the cell-free conditions. Indomethacin (100 µM) induced a transient huge increase in the fEPSP slope followed by persistent increase, and the former effect was attenuated by the α7 ACh receptor antagonist α-bungarotoxin or GF109203X. Indomethacin (100 µM) also increased the rate of nicotine-evoked mEPSCs, and the effect was prevented by α-bungarotoxin or GF109203X. CONCLUSION: The results of the present study show that indomethacin activates PKC, possibly PKC-e in the brain, thereby potentiating α7 ACh receptor responses to stimulate presynaptic glutamate release, which in part contributes to facilitation of hippocampal transmission. This extends our knowledge about diverse indomethacin actions.

PDGF-D/PDGF-ßß receptor-regulated chemotaxis of malignant mesothelioma cells.

BACKGROUND/AIMS: Our earlier study suggested that platelet-derived growth factor (PDGF)- ßß receptor regulates chemotaxis of human malignant mesothelioma cells such as MSTO-211H, NCIH-2052, NCIH-2452, and NCIH-28 cells, but not non-malignant Met5A cells. The present study was designed to gain further insight into the PDGF-ßß receptor signals underlying the chemotaxis. METHODS: PDGF-D secreted from cells, activation of Akt and ERK, and cell migration were monitored for cells with and without knocking-down PDGF-ßß receptor. RESULTS: FBS significantly stimulated PDGF-D secretion from malignant mesothelioma cells, but not Met5A cells. PDGF-D activated Akt and ERK in both the non-malignant and malignant cells. PDGF-D significantly facilitated migration of malignant mesothelioma cells, but not Met5A cells, with the extent varying among the cell types. The facilitatory action of PDGF-D was clearly prevented by knocking-down PDGF-ßß receptor or inhibitors of PI3 kinase, PDK1, Akt, Rac1, ROCK, and MEK. CONCLUSION: The results of the present study indicate that PDGF-D promotes malignant mesothelioma cell chemotaxis through PDGF-ßß receptor signaling pathways along a PI3 kinase/PDK1/Akt/Rac1/ROCK axis and relevant to ERK activation.

Resveratrol induces apoptosis MH7A human rheumatoid arthritis synovial cells in a sirtuin 1-dependent manner.

Resveratrol, a phytoalexin, reduced the viability of MH7A cells, a human rheumatoid arthritis synovial cell line. In the apoptosis assay, resveratrol increased TUNEL-positive cells and stimulated H2A.X phosphorylation. Resveratrol disrupted mitochondrial membrane potentials in MH7A cells and stimulated cytochrome c release from the mitochondria to the cytosol. Resveratrol activated caspase-3 and caspase-9 but not caspase-8 in MH7A cells. Resveratrol upregulated the expression of the NAD-dependent deacetylase sirtuin 1 mRNA and downregulated the expression of the Bcl-X(L) mRNA, and resveratrol-induced MH7A cell death, mitochondrial damage, and caspase-3/-9 activation were prevented by sirtinol, an inhibitor of sirtuin 1. The results of the present study show that resveratrol induces MH7A cell apoptosis by activating caspase-9 and the effector caspase-3 along mitochondrial disruption as a result of reduced Bcl-X(L) expression, allowing cytochrome c release from the mitochondria into the cytosol, in a sirtuin 1-dependent manner. This suggests that resveratrol could suppress hyperplasia of synovial cells, a critical factor of rheumatoid arthritis.

Indomethacin enhances learning and memory potential by interacting with CaMKII.

The present study examined the effect of indomethacin (IM), a cyclooxygenase inhibitor, on learning and memory functions. IM activated Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) in cultured rat hippocampal neurons. IM (100 µM) significantly increased the rate of spontaneous AMPA receptor-mediated miniature excitatory postsynaptic currents elicited from CA1 pyramidal neurons of rat hippocampal slices, without affecting the amplitude, and enhanced extracellular high K(+) (20 mM)-induced glutamate release from rat hippocampal slices, indicating that IM stimulates presynaptic glutamate release. Those IM effects were clearly inhibited by the CaMKII inhibitor KN-93. IM persistently facilitated synaptic transmission monitored from the CA1 region of rat hippocampal slices in a concentration (1-100 µM)-dependent manner that was also abolished by KN-93. In the water maze test, IM (1 mg/kg, i.p.) enhanced spatial learning and memory ability for normal rats, and ameliorated scopolamine-induced spatial learning and memory impairment or age-related spatial learning and memory deterioration for senescence-accelerated mouse-prone 8 mice. In the test to learn 15 numbers consisting of three patterns of five digit number for healthy human subjects, oral intake with IM (25 mg/kg) significantly raised the scores of correct number arrangements that subjects memorized 5 min and 3 days after the test. The results of the present study indicate that IM could enhance learning and memory potential by facilitating hippocampal synaptic transmission as a result from stimulating presynaptic glutamate release under the control of CaMKII.

Adenosine promotes GATA-2-regulated p53 gene transcription to induce HepG2 cell apoptosis.

BACKGROUND/AIMS: In our earlier study, adenosine induced apoptosis in HepG2 human hepatoma cells by tuning of apoptosis-mediator gene transcription. The present study aimed at understanding the regulatory mechanism underlying the apoptosis-mediator gene transcription under the control of adenosine. METHODS: For HepG2 cells with and without knocking-down p53 or GATA-2, cell viability, mitochondrial membrane potentials, caspase activity, and transcriptional activity were monitored, and Western blotting, RT-PCR, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were carried out. RESULTS: Extracellular adenosine upregulated expression of the p53 mRNA and protein in HepG2 human hepatoma cells. Adenosine induced apoptosis, disrupted mitochondrial membrane potentials, and activated caspase-3, -8 and -9 in HepG2 cells, and those effects were inhibited by silencing the p53-targetd gene. In the assay of transcriptional activity using full-length p53 gene promoter and 5' deletion mutants combined with the luciferase reporter vector, adenosine enhanced transcriptional activity for full-length p53 gene promoter, that was prevented by deleting from -240 to -146 bp on the promoter. In the EMSA using a (32)P-labeled DNA probe to detect binding to the putative GATA-2 biding site on the p53 gene promoter, adenosine produced (32)P-positive signals in nuclear extracts from HepG2 cells. In the Western blot analysis, adenosine increased presence of GATA-2 in nuclear extracts. In the ChIP assay, adenosine increased PCR products for the p53 gene promoter in chromosomal extracts from HepG2 cells, immunoprecipitated using an anti-GATA-2 antibody. Adenosine-induced upregulation of the p53 mRNA expression was suppressed by knocking-down GATA-2. CONCLUSION: The results of the present study show that p53 is a transcriptional target of GATA-2 and that adenosine upregulates GATA-2-regulated p53 expression, thereby activating caspase-3, -8, and -9 to induce HepG2 cell apoptosis.

AMID mediates adenosine-induced caspase-independent HuH-7 cell apoptosis.

BACKGROUND/AIMS: The mechanism underlying extracellular adenosine-induced caspase-independent apoptosis in HuH-7 human hepatoma cells is not fully understood. The present study investigated the role for apoptosis-inducing factor (AIF)-homologous mitochondrion-associated inducer of death (AMID) in the pathway. METHODS: To see the implication of AMID in adenosine-induced HuH-7 cell apoptosis, real-time reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescent cytochemistry, time-laps GFP monitoring, cell cycle analysis, flow cytometry, Western blotting, cell viability assay, and TUNEL staining were carried out. RESULTS: Adenosine upregulated AMID expression in HuH-7 cells, and translocated AMID from the cytosol into the nucleus. Adenosine induced HuH-7 cell apoptosis, and the effect was further enhanced by overexpressing AMID. Adenosine-induced HuH-7 cell apoptosis, alternatively, was inhibited by knocking-down AMID. CONCLUSION: The results of the present study provide evidence for AMID as a critical factor for adenosine-induced caspase-independent HuH-7 cell apoptosis.

Adenosine activates AMPK to phosphorylate Bcl-XL responsible for mitochondrial damage and DIABLO release in HuH-7 cells.

BACKGROUND/AIMS: Accumulating evidence has pointed to AMP-activated protein kinase (AMPK) as an inducer of apoptosis in a variety of cancer cells. The present study aimed at understanding AMPK signals for adenosine-induced HuH-7 cell apoptosis. METHODS: Cell viability, AMPK activity, mitochondrial membrane potentials, phosphorylation of Bcl-X(L), in situ DIABLO mobilizations, and caspase-3 activity were monitored in HuH-7 cells. Plasmid DNAs for DIABLO-GFP, mutant Bcl-X(L), dominant negative mutant AMPKα2 and the siRNAs to silence the AMPKα1 or AMPKα2 targeted gene were constructed and transfected. RESULTS: Adenosine or the AMPK activator AICAR induced apoptosis in HuH-7 cells, and no synergistic effect was obtained with co-treatment. Adenosine activated AMPK, to phosphorylate Bcl-X(L). Adenosine or AICAR disrupted mitochondrial membrane potentials, and the effect was inhibited by knocking-down AMPKα1 and/or AMPKα2, expressing dominant negative mutant AMPKα2 or mutant Bcl-X(L) lacking Ser/Thr phosphorylation sites. AICAR stimulated DIABLO release from the mitochondria, and the release was suppressed by expressing the mutant Bcl-X(L). AICAR activated caspase-3, which was also inhibited by expressing the mutant Bcl-X(L). CONCLUSION: Adenosine activates AMPK, to disrupt mitochondrial membrane potentials through Bcl-X(L) phosphorylation, allowing DIABLO release from the mitochondria, as a factor for caspase-3 activation to induce HuH-7 cell apoptosis.

DL- and PO-phosphatidylcholines as a promising learning and memory enhancer.

In the water maze test, oral administration with 1,2-dilynoleoyl-sn-glycero-3-phosphocholine (DLPhtCho)(5 mg/kg) alone or DLPhtCho (5 mg/kg) plus 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPhtCho)(5 mg/kg) significantly shortened the prolonged acquisition latency for rats intraperitoneally injected with scopolamine, with more efficient effect than (POPhtCho)(5 mg/kg) alone, arachidonic acid (AA)(5 mg/kg) alone, docosahexaenoic acid (DHA)(5 mg/kg) alone, or 1-palmitoyl-2-linoleil-sn-glycero-3-phosphoserine (PLPhtSer)(5 mg/kg) alone. POPhtCho (5 mg/kg) alone or DLPhtCho (5 mg/kg) plus POPhtCho (5 mg/kg) also significantly shortened the prolonged retention latency for rats intraperitoneally injected with scopolamine, but otherwise no significant effect was obtained with DLPhtCho (5 mg/kg) alone, AA (5 mg/kg) alone, DHA (5 mg/kg) alone, or PLPhtSer (5 mg/kg) alone. Oral co-administration with DLPhtCho (5 mg/kg) and POPhtCho (5 mg/kg) significantly shortened the acquisition latency for rats untreated with scopolamine as compared with the latency for administration with polyethylene glycol (PEG), DLPhtCho alone at doses of 5 and 10 mg/kg, or POPhtCho alone at doses of 5 and 10 mg/kg, while no efficient effect on the retention latency was obtained. To assess the effect of DLPhtCho and POPhtCho on cognitive functions for humans, Mini Mental State Examination (MMSE) test was performed in subjects with cognitive disorders (the average MMSE score, 15). Oral co-intake with DLPhtCho (50 mg) and POPhtCho (45 mg) once after breakfast everyday raised the score to over 20, corresponding to normal cognitive functions, throughout 5 months after intake, and the increase in the score was significantly greater than that for oral intake with DLPhtCho (100 mg/day) alone or POPhtCho (90 mg/kg) alone. Taken together, the results of the present study show that co-intake with DLPhtCho and POPhtCho could enhance learning and memory ability and improve cognitive disorders for both the animals and humans with a promising efficacy.

1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine ameliorates age-related spatial memory deterioration by preventing neuronal cell death.

BACKGROUND: Accumulating evidence has pointed that a variety of lipids could exert their beneficial actions against dementia including Alzheimer disease and age-related cognitive decline via diverse signaling pathways. Endoplasmic reticulum (ER) stress-induced neuronal apoptosis, on the other hand, is a critical factor for pathogenesis of neurodegenerative diseases such as Alzheimer disease and Parkinson disease, senile dementia, and ischemic neuronal damage. The present study examined the effects of 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPhtEtn), a phospholipid, on ER stress-induced neuronal death and age-related cognitive disorders. METHODS: PC-12 cell viability was assayed before and after treatment with amyloid-ß(1-40) peptide or thapsigargin in the presence and absence of DLPhtEtn. A series of behavioral tests were performed for senescence-accelerated mouse-prone 8 (SAMP8) mice after 7-month oral administration with polyethylene glycol (PEG) or DLPhtEtn and then, the number of hippocampal neurons was counted. RESULTS: Amyloid-ß(1-40) peptide or thapsigargin is capable of causing ER stress-induced apoptosis. DLPhtEtn (30 µM) significantly inhibited PC-12 cell death induced by amyloid-ß(1-40) peptide or thapsigargin. In the water maze test, oral administration with DLPhtEtn (1 mg/kg) for 7 months (three times a week) significantly shortened the prolonged retention latency for SAMP8 mice. In contrast, DLPhtEtn had no effect on the acquisition and retention latencies in both the open field test and the passive avoidance test for SAMP8 mice. Oral administration with DLPhtEtn (1 mg/kg) for 7 months prevented a decrease in the number of hippocampal neurons for SAMP8 mice. CONCLUSION: The results of the present study show that DLPhtEtn ameliorates age-related spatial memory decline without affecting motor activities or fear memory, possibly by protecting hippocampal neuronal death. DLPhtEtn, thus, might exert its beneficial action against senile dementia and neurodegenerative diseases such as Alzheimer disease.

Higher concentrations of extracellular ATP suppress proliferation of Caco-2 human colonic cancer cells via an unknown receptor involving PKC inhibition.

BACKGROUND/AIMS: Adenosine 5'-triphosphate (ATP) mediates a variety of signal transductions via ATP receptors such as P2X and P2Y receptors. The present study aimed at understanding the mechanism underlying extracellular ATP-induced suppression of Caco-2 human colonic cancer cell proliferation. METHODS: Caco-2 cells were cultured. To examine cell viability and cell cycling, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, fluorescent cytochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and flow cytometry were carried out. To see mRNA expression of ATP receptors, reverse transcription-polymerase chain reaction (RT-PCR) was performed. To examine PKC activity and mitogen-activated protein (MAP) kinase activity, in situ PKC assay and Western blotting using an anti-extracellular signal-regulated kinase 1 (ERK1)-antibody and an anti-phospho-ERK antibody were carried out. RESULTS: Extracellular ATP or the unhydrolyzed ATP analogue 5'-adenylyimido-diphosphate (AMP-PNP) reduced Caco-2 cell viability in a concentration (10 microM-10 mM)-dependent manner at 48-h treatment, and the effect was not affected by caspase inhibitors. Caco-2 cells were little reactive to propidium iodide and Hoechst 33342 or little positive to TUNEL after 48-h treatment with ATP (1 mM). In the flow cytometry, 48-h treatment with ATP (1 mM) arrested cell cycling at the S phase in Caco-2 cells. P(2) purinoceptor agonists reduced Caco-2 cell viability with the order of potency: 2-methylthio ATP>UTP>beta, gamma-methylene ATP, and the ATP effect was partially inhibited by suramin, a non-selective inhibitor of P(2) purinoceptors. The PKC inhibitor GF109203X or the MAP kinase kinase inhibitor PD98059 reduced Caco-2 cell viability to an extent similar to that achieved by ATP (1 mM), and no further reduction was obtained with co-treatment with ATP. ATP and its ATP analogues such as AMP-PNP and ATPgammaS, at higher concentrations (1-10 mM), inhibited PKC activation in Caco-2 cells in a fashion that mimics the effect of GF109203X, but PD98059 exhibited no effect on PKC activation. The inhibitory effect of ATP on PKC activation was not found with SK-N-SH cells, a human neuroblastoma cell line, but the cells expressed all the mRNAs for P2X and P2Y receptors that Caco-2 cells did. ATP (10 mM) or GF109203X inhibited activation of ERK, a MAP kinase, in Caco-2 cells. CONCLUSION: Extracellular ATP, at higher concentrations, suppresses Caco-2 cell proliferation at the S phase of cell cycling by inhibiting PKC, possibly as mediated via an unknown ATP receptor, followed by MAP kinase.

Auto-positive feedback regulation for nicotinic acetylcholine receptors by protein kinase C activation.

BACKGROUND/AIMS: Protein kinase C (PKC) is well-recognized to modify ligand-gated ion channels such as nicotinic ACh (nACh) receptors by phosphorylating the receptors. The aim of the present study was to obtain direct evidence for PKC activation through nACh receptors. METHODS: Two-electrode voltage-clamp was made to Xenopus oocytes expressing wild-type and mutant Torpedo nACh receptors. Western blotting using antibodies against phospho-serine and phospho-threonine was carried out in oocytes expressing Torpedo nACh receptors. In situ PKC activation was monitored in cultured rat skeletal muscle cells expressing nACh receptors. RESULTS: In the Xenopus oocyte expression systems, ACh-evoked whole-cell membrane currents through wild-type Torpedo nACh receptors were depressed by GF109203X, a PKC inhibitor, while currents through mutant receptors lacking PKC phosphorylation sites was not affected. In the Western blot analysis, ACh produced immunoreactive bands against an anti-phospho-serine or an anti-phospho-threonine antibody in oocytes expressing wild-type Torpedo nACh receptors, and those signals were attenuated by alpha-bungarotoxin, an inhibitor of nACh receptors, or GF109203X. In the in situ PKC assay using cultured rat muscle cells that expressed all the mRNAs for muscle nACh receptor subunits such as the alpha, beta, gamma, delta, and e subunit, ACh activated PKC in the presence of atropine, an inhibitor of muscarinic ACh receptors, and the activation was abolished by alpha-bungarotoxin or GF109203X. CONCLUSION: The results of the present study show that ACh activates PKC through nACh receptors and that in turn, activated PKC constantly enhances ACh receptor responses by phosphorylating the receptors. This may represent a new auto-positive feedback regulation for nACh receptors by PKC activation.

Noradrenaline stimulates ATP release from DRG neurons by targeting beta(3) adrenoceptors as a factor of neuropathic pain.

Noradrenaline (NA), released in association with sympathetic nerve sprouting into the dorsal root ganglion (DRG) after peripheral nerve injury, may enhance neuropathic pain. ATP serves as a pain mediator; however, NA-regulated ATP mobilizations in the DRG is far from understanding. In the present study, we analyzed ATP mobilizations in acutely dissociated rat DRG neurons by recording single-channel currents through P2X receptor channels as an ATP biosensor in an outside-out patch-clamp configuration and by monitoring real-time enzymatic NADPH fluorescent imaging, and examined the role for beta(3) adrenoceptors in allodynia using an in vivo rat model. We show here that NA stimulates ATP release from DRG neurons as mediated via beta(3) adrenoceptors linked to G(s) protein involving PKA activation, to cause allodynia. This represents a fresh regulatory pathway for neuropathic pain relevant to noradrenergic transmission in the DRG.

Extracellular high K+ stimulates vesicular glutamate release from astrocytes by activating voltage-dependent calcium channels.

Extracellular high K(+) (75 mM) increased intracellular Ca(2+) concentrations in cultured rat hippocampal astrocytes, and the Ca(2+) rise was abolished by deleting extracellular Ca(2+) or cadmium, a non-selective inhibitor of voltage-dependent calcium channels (VDCCs). In the reverse transcription-polymerase chain reaction analysis, cultured astrocytes expressed mRNAs for L type-VDCC subunits such as alpha1B, alpha1C, alpha1D, and alpha1E. Extracellular high K(+) (75 mM) stimulated glutamate release from astrocytes. The glutamate release was not prevented by the glutamate transporter inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), or deleting extracellular Na(+), but otherwise it was clearly inhibited by deleting extracellular Ca(2+), cadmium, vesicular transport inhibitors such as brefeldin A, bafilomycin A1, and latrunculin B, or botulinum toxin-A, an exocytosis inhibitor. Extracellular high K(+) (75 mM) bleached fluorescent signals of FM1-43, taken up into the vesicular membrane in astrocytes, that was also inhibited by deleting extracellular Ca(2+), cadmium, brefeldin A, bafilomycin A1, latrunculin B, or botulinum toxin-A, but not by PDC. Taken together, the results of the present study indicate that extracellular high K(+)-evoked depolarization activates VDCCs expressed in astrocytes, causing an increase in intracellular Ca(2+) concentrations through VDCCs, which triggers vesicular glutamate release from astrocytes, independently of reverse transport through glutamate transporters.