Home About us Contact
|
Home About us Contact | ||
|
|
||
Thapsigargin
Kinds of Thapsigargin Selected AbstractsDirect Measurement of Hormone-Induced Acidification in Intact BoneJOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2000Glenn S. Belinsky Abstract Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6 -2,-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively. [source] Role of calcium and ROS in cell death induced by polyunsaturated fatty acids in murine thymocytesJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2010Aparna Prasad We investigated the mechanisms whereby omega-3 and -6 polyunsaturated fatty acids (PUFAs) cause cell death of mouse thymocytes using flow cytometry, focusing on the respective roles of intracellular calcium concentration, [Ca2+]i and reactive oxygen species (ROS). We applied the C-22, 20, and 18 carbon omega-3 (DHA, EPA, ALA) and omega-6 (DTA, ARA, and LNA) fatty acids to isolated thymocytes and monitored cell death using the DNA-binding dye, propidium iodide. When applied at 20,µM concentration, omega-3 fatty acids killed thymocytes over a period of 1,h with a potency of DHA,>,EPA,>,ALA. The omega-6 PUFAs were more potent. The C18 omega-6 fatty acid, LNA, was the most potent, followed by DHA and ARA. Cell death was always accompanied by an increase in the levels of [Ca2+]i and ROS. Both increases were in proportion to the potency of the PUFAs in inducing cell death. Removing extracellular calcium did not prevent the elevation in [Ca2+]i nor cell death. However, the intracellular calcium chelator, BAPTA, almost totally reduced both the elevation in [Ca2+]i and cell death, while vitamin E reduced the elevation in ROS and cell death. BAPTA also prevented the elevation in ROS, but vitamin E did not prevent the elevation in [Ca2+]i. Thapsigargin, which depletes endoplasmic reticulum calcium, blocked the elevation in [Ca2+]i, but CCCP, a mitochondrial calcium uptake inhibitor, did not. These results suggest that the six PUFAs we studied kill thymocytes by causing release of calcium from endoplasmic reticulum, which causes release of ROS from mitochondria which leads to cell death. J. Cell. Physiol. 225: 829,836, 2010. © 2010 Wiley-Liss, Inc. [source] A Gonadotropin-Releasing Hormone Insensitive, Thapsigargin-Sensitive Ca2+ Store Reduces Basal Gonadotropin Exocytosis and Gene Expression: Comparison with Agonist-Sensitive Ca2+ StoresJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2003J. D. Johnson Abstract We examined whether distinct Ca2+ stores differentially control basal and gonadotropin (GTH-II)-releasing hormone (GnRH)-evoked GTH-II release, long-term GTH-II secretion and contents, and GTH-II- , mRNA expression in goldfish. Thapsigargin (Tg)-sensitive Ca2+ stores mediated neither caffeine-evoked GTH-II release, nor salmon (s)GnRH- and chicken (c)GnRH-II-stimulated secretion; the latter responses were previously shown to involve ryanodine (Ry)-sensitive Ca2+ stores. Surprisingly, Tg decreased basal GTH-II release. This response was attenuated by prior exposure to sGnRH and caffeine, but was insensitive to the phosphatase inhibitor okadaic acid, the inhibitor of constitutive release brefeldin A and cGnRH-II. GTH-II- , mRNA expression was decreased at 24 h by 2 µm Tg, and by inhibiting (10 µm Ry) and stimulating (1 nm Ry) Ry receptors. Transient increases in GTH-II- , mRNA were observed at 2 h and 12 h following 10 µm and 1 nm Ry treatment, respectively. Effects of Tg, Ry and GnRH on long-term GTH-II secretion, contents and apparent production differed from one another, and these changes were not well correlated with changes in GTH-II- , mRNA expression. Our data show that GTH-II secretion, storage and transcription can be independently controlled by distinct Ca2+ stores. [source] Thapsigargin resistance in human prostate cancer cellsCANCER, Issue 3 2006John P. O'Neill BS Abstract BACKGROUND. Thapsigargin (TG) is a potent inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCAs). TG-based prodrugs are being developed for the treatment of prostate cancer (PC). To develop optimal TG-based therapeutics it is important to understand the mechanisms of resistance to TG that may potentially occur in cancer cells. METHODS. DU145/TG and PC3/TG cells were derived from human PC DU145 and PC3 cells, respectively, by incremental exposure to TG. Growth assays, Western blot analyses, cDNA microarrays, semiquantitative and real-time polymerase chain reaction (PCR), Northern blot analyses, and immunohistochemistry were used to study these cells. RESULTS. DU145/TG cells are 1100-fold and PC3/TG cells are 1350-fold resistant to TG. Although expression of both SERCA and p-glycoprotein can mediate TG resistance in hamster cells, neither is modulated in DU145/TG cells. In contrast, in PC3/TG cells, SERCA, and not p-glycoprotein, is significantly overexpressed but cannot by itself account for the 1350-fold resistance to TG in these cells. Several genes not previously identified to be altered by TG selection are modulated in DU145/TG and PC3/TG cells. Furthermore, the spectrum of genes modulated in DU145/TG cells are distinct from that in PC3/TG cells, even though both cells are of prostate origin and share the same TG-resistant phenotype. CONCLUSIONS. PC cells can adapt to SERCA inhibition by TG. However, they demonstrate cell type-specific plasticity with respect to gene expression upon TG selection. Further, previously not described mechanisms of resistance appear to be recruited in the TG-resistant PC cells, which provide a novel model to study mechanisms of resistance and adaptation in PC on TG-mediated dysregulation of Ca2+ homeostasis. Cancer 2006. © 2006 American Cancer Society. [source] Econazole-induced Ca2+ fluxes and apoptosis in human oral cancer cellsDRUG DEVELOPMENT RESEARCH, Issue 4 2010Daih-Huang Kuo Abstract The effect of econazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability was explored in human oral cancer cells (OC2), using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations of >1,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The econazole-induced Ca2+ influx was sensitive to blockade of aristolochic acid (phospholipase A2 inhibitor) and GF109203X (PKC inhibitor). In Ca2+ -free medium, after treatment with 1,µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 30,µM econazole failed to induce a [Ca2+]i rise. Inhibition of phospholipase C with 2,µM U73122 substantially suppressed econazole-induced [Ca2+]i rise. At concentrations of 5,70,µM econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50,µM econazole was enhanced by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,,N,-tetraacetic acid (BAPTA). The ERK MAPK inhibitor, PD98059 (10,µM), also enhanced 20,µM econazole-induced cell death. Propidium iodide staining data suggest that econazole induced apoptosis between concentrations of 10,70,µM. Collectively, in OC2 cells, econazole induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2/PKC-regulated Ca2+ channels. Furthermore, econazole caused cell death appeared to be regulated by ERK MAPK. Drug Dev Res 71: 240,248, 2010. © 2010 Wiley-Liss, Inc. [source] Effect of capsaicin on Ca2+ fluxes in Madin-Darby canine renal tubular cellsDRUG DEVELOPMENT RESEARCH, Issue 2 2010Jeng-Hsien Yeh Abstract The effect of capsaicin, a transient receptor potential vanniloid-1 (TRPV1) receptor agonist, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+ -sensitive fluorescent dye. Capsaicin at concentrations between 10,100,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 80% by removing extracellular Ca2+. Capsacin induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non-selective Ca2+ entry blocker La3+, but not by store-operated Ca2+ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca2+ -free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished capsaicin-induced Ca2+ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin-induced [Ca2+]i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca2+ entry and Ca2+ release. Collectively, in MDCK cells, capsaicin induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-regulated, La3+ -sensitive Ca2+ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source] Nonylphenol-induced cytosolic Ca2+ elevation and death in renal tubular cellsDRUG DEVELOPMENT RESEARCH, Issue 5 2009Jeng-Yu Tsai Abstract Nonylphenol is an environmental endocrine disrupter. The effect of nonylphenol on intracellular free Ca2+ levels ([Ca2+]i) and viability in Madin-Darby canine kidney (MDCK) cells was explored. Nonylphenol increased [Ca2+]i in a concentration-dependent manner (EC50,0.8,,M). Nonylphenol-induced Mn2+ entry demonstrated Ca2+ influx and removal of extracellular Ca2+ partly decreased the [Ca2+]i rise. The [Ca2+]i rise was inhibited by the protein kinase C activator, phorbol 13-myristate acetate (PMA) but not by L-type Ca2+ channel blockers. In Ca2+ -free medium, nonylphenol-induced [Ca2+]i rise was partly inhibited by pretreatment with 1,,M thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Conversely, nonylphenol pretreatment abolished thapsigargin-induced Ca2+ release. Nonylphenol-induced Ca2+ release was unaltered by inhibition of phospholipase C. At concentrations of 5,100,,M, nonylphenol killed cells in a concentration-dependent manner. The cytotoxic effect of 100,,M nonylphenol was not affected by preventing [Ca2+]i rises with BAPTA/AM. Collectively, this study shows that nonylphenol induced [Ca2+]i increase in MDCK cells via evoking Ca2+ entry through protein kinase C-regulated Ca2+ channels, and releasing Ca2+ from endoplasmic reticulum and other stores in a phospholipase C-independent manner. Nonylphenol also killed cells in a Ca2+ -independent fashion. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source] Effects of Antrodia camphorata on viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation in MG63 human osteosarcoma cellsDRUG DEVELOPMENT RESEARCH, Issue 2 2007Yih-Chau Lu Abstract The present study explored the effect of Antrodia camphorata (AC) on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation, and Ca2+ regulation in MG63 human osteosarcoma cells. AC (25,50,µg/ml) did not affect cell viability, but at 100,200,µg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25,200,µg/ml did not alter basal [Ca2+]i, but at 25,µg/ml decreased [Ca2+]i increases induced by ATP, bradykinin, histamine, and thapsigargin. ATP, bradykinin, and histamine increased cell viability while thapsigargin decreased it. AC (25,µg/ml) pretreatment failed to alter bradykinin- and thapsigargin-induced effects on viability, but potentiated ATP- and histamine-induced increases in viability. Immunoblotting showed that MG63 cells did not have background phospho-JNK and phospho-p38 mitogen-activated protein kinases (MAPKs); and AC did not induce the phosphorylation of these two MAPKs. Conversely, the cells had significant background phospho-ERK MAPK that was inhibited by 200,µg/ml AC. The ERK-specific inhibitor PD98059 also induced cell death. Collectively, in MG63 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis probably via inhibition of ERK MAPK phosphorylation. Drug Dev Res 68:71,78, 2007. © 2007 Wiley-Liss, Inc. [source] Further studies on the interaction of loperamide with capacitative calcium entry in Leukemic HL-60 cells,DRUG DEVELOPMENT RESEARCH, Issue 11 2006John W. Daly Abstract Loperamide at 3,10,µM has augmentative effects on calcium levels elevated by capacitative calcium entry (CCE) in leukemic HL-60 cells after release of intracellular calcium by ATP or thapsigargin (Harper et al. [1997] Proc Natl Acad Sci USA 94:14912,14917). The effect of loperamide on calcium levels was absent at a pH value of 6.8, a pH at which CCE is not active in HL-60 cells. Further investigations of HL-60 cells in recent years revealed a great reduction in the magnitude of the loperamide response. However, when preceded by a CCE blocker, namely N-methylnitrendipine (MRS 1844) or N-propargylnitrendipine (MRS 1845), loperamide caused a significant reversal of the blockade. Six structural analogs of loperamide were synthesized, but only two showed loperamide-like activity. Drug Dev. Res. 67:842,851, 2006. Published 2007 Wiley-Liss, Inc. [source] Two distinct P2Y receptors are involved in purine- and pyrimidine-evoked Ca2+ elevation in mammalian brain astrocytic culturesDRUG DEVELOPMENT RESEARCH, Issue 1-2 2001Chiara Bolego Abstract ATP and 2-methyl-thio-ATP (2-Me-SATP) increase cytosolic calcium concentrations ([Ca2+]i) in rat striatal astrocytes (Centemeri et al. [1997] Br J Pharmacol 121:1700,1706). The aim of the present study was to: (1) characterize pyrimidine-induced [Ca2+]i increases in the same experimental system, and (2) try to identify the multiple P2Y receptor subtypes mediating Ca2+ mobilization. UDP and UTP triggered a concentration-dependent [Ca2+]i elevation (EC50s = 0.58 ,M ± 0.4 and 31 ,M ± 6, respectively). Pyrimidine-evoked [Ca2+]i elevation was solely due to mobilization from intracellular stores, because: (1) removing calcium from extracellular medium or (2) blocking its influx with Ni2+ did not modify UTP responses; (3) the store-depleting agent thapsigargin completely abolished UTP-evoked [Ca2+]i increments. Guanosine-5,-O-(2-thiodiphosphate) partially inhibited the UTP response, whereas pertussis toxin (PTx) had no effect. The phospholipase C inhibitor U-73122 significantly reduced the UTP-evoked [Ca2+]i rise. Computer-assisted analysis indicated that the UTP and UDP responses are mediated by a single receptor, while ATP and 2-Me-SATP interact with two distinct receptors. The selective P2Y1 receptor antagonist MRS2179 abolished the ATP higher potency component. Sequential challenges with the same nucleotides resulted in almost complete homologous desensitization. Pre-exposure to UTP lowered the subsequent responses to either ATP or 2-Me-SATP. Maximally active concentrations of UTP and ATP were not additive. In conclusion, [Ca2+]i elevation in astrocytes by purines and pyrimidines is mediated by two distinct P2Y receptors, likely the P2Y1 and P2Y6 subtypes. Drug Dev. Res. 52:122,132, 2001. © 2001 Wiley-Liss, Inc. [source] Synchrony of spontaneous calcium activity in mouse neocortex before synaptogenesisEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2007Jean-Claude Platel Abstract Spontaneous calcium activity can be detected in embryonic mouse cortical slices as fluorescence intensity variations, in the presence of a fluorescent calcium indicator. Current methods to detect and quantify these variations depend heavily on experimenters whose judgement may interfere with measurement. In the present work, we developed new software called CalSignal for automatic detection and tracking of cellular bodies and quantification of spontaneous calcium activity on time-series of confocal fluorescence images. Analysis of 28 neocortical slices revealed that 21.0% of detected cells displayed peaks of fluorescence corresponding to spontaneous activity, with a mean frequency of one peak per 4 min. This activity was blocked in the absence of extracellular calcium but was not modified after depletion of calcium stores with thapsigargin or blockade of voltage-gated calcium channels with Ni2+. Further, statistical analysis of calcium activity revealed concomitant activation of distant cells in 24 slices, and the existence of a significant network of synchrony based on such coactivations in 17 slices out of 28. These networks enclosed 84.3% of active cells, scattered throughout the neocortical wall (mean distance between cellular bodies, 111.7 µm). Finally, it was possible to identify specific cells which were synchronously active with more neighbouring cells than others. The identity of these nodal cells remains to be investigated to fully comprehend the role of spontaneous calcium activity, before synaptogenesis, in shaping cortical neurogenesis. [source] Muscarinic control of graded persistent activity in lateral amygdala neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Alexei V. Egorov Abstract The cholinergic system is crucially involved in several cognitive processes including attention, learning and memory. Muscarinic actions have profound effects on the intrinsic firing pattern of neurons. In principal neurons of the entorhinal cortex (EC), muscarinic receptors activate an intrinsic cation current that causes multiple self-sustained spiking activity, which represents a potential mechanism for transiently sustaining information about novel items. The amygdala appears to be important for experience-dependent learning by emotional arousal, and cholinergic muscarinic influences are essential for the amygdala-mediated modulation of memory. Here we show that principal neurons from the lateral nucleus of the amygdala (LA) can generate intrinsic graded persistent activity that is similar to EC layer V cells. This firing behavior is linked to muscarinic activation of a calcium-sensitive non-specific cation current and can be mimicked by stimulation of cholinergic afferents that originate from the nucleus basalis of Meynert (n. M). Moreover, we demonstrate that the projections from the n. M. are essential and sufficient for the control and modulation of graded firing activity in LA neurons. We found that activation of these cholinergic afferents (i) is required to maintain and to increase firing rates in a graded manner, and (ii) is sufficient for the graded increases of stable discharge rates even without an associated up-regulation of Ca2+. The induction of persistent activity was blocked by flufenamic acid or 2-APB and remained intact after Ca2+ -store depletion with thapsigargin. The internal ability of LA neurons to generate graded persistent activity could be essential for amygdala-mediated memory operations. [source] Multiple P2 Receptors Contribute to a Transient Increase in Intracellular Ca2+ Concentration in Atp-Stimulated Rat Brown AdipocytesEXPERIMENTAL PHYSIOLOGY, Issue 6 2002Mariko Omatsu-Kanbe Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca2+ concentration ([Ca2+]i), which arises primarily from a release of Ca2+ from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca2+]i elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2,,4,-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 ,M) almost completely depressed the thapsigargin (100 nM)-evoked [Ca2+]i elevation mediated through store-operated Ca2+ entry. The inhibitory effect of ATP was antagonized by PPADS with IC50 of 0.7 ,M. In the presence of PPADS at concentrations of more than 5 ,M, the ATP-induced [Ca2+]i elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca2+]i elevation was reduced in a concentration-dependent manner by PPADS with an IC50 of 200 ,M. In contrast, the ATP-induced [Ca2+]i elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca2+]i responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca2+ stores, which is probably followed by the activation of store-operated Ca2+ entry. Extracellular ATP, however, would inhibit this Ca2+ entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca2+]i elevation in response to extracellular ATP. [source] Calcium and polyamine regulated calcium-sensing receptors in cardiac tissuesFEBS JOURNAL, Issue 12 2003Rui Wang Activation of a calcium-sensing receptor (Ca-SR) leads to increased intracellular calcium concentration and altered cellular activities. The expression of Ca-SR has been identified in both nonexcitable and excitable cells, including neurons and smooth muscle cells. Whether Ca-SR was expressed and functioning in cardiac myocytes remained unclear. In the present study, the transcripts of Ca-SR were identified in rat heart tissues using RT-PCR that was further confirmed by sequence analysis. Ca-SR proteins were detected in rat ventricular and atrial tissues as well as in isolated cardiac myocytes. Anti-(Ca-SR) Ig did not detect any specific bands after preadsorption with standard Ca-SR antigens. An immunohistochemistry study revealed the presence of Ca-SR in rat cardiac as well as other tissues. An increase in extracellular calcium or gadolinium induced a concentration-dependent sustained increase in [Ca2+]i in isolated ventricular myocytes from adult rats. Spermine (1,10 mm) also increased [Ca2+]i. Pre-treatment of cardiac myocytes with thapsigargin or U73122 abolished the extracellular calcium, gadolinium or spermine-induced increase in [Ca2+]i. The blockade of Na+/Ca2+ exchanger or voltage-dependent calcium channels did not alter the extracellular calcium-induced increase in [Ca2+]i. Finally, extracellular calcium, gadolinium and spermine all increased intracellular inositol 1,4,5-triphosphate (IP3) levels. Our results demonstrated that Ca-SR was expressed in cardiac tissue and cardiomyocytes and its function was regulated by extracellular calcium and spermine. [source] Ca2+ - and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: From microtubules to the plasma membraneGLIA, Issue 12 2009J. W. Ou Abstract Large-conductance, voltage- and Ca2+ -activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming ,-subunits (MaxiK,) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiK, is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiK, was visualized by three independent labeling approaches: (1) MaxiK,-specific antibodies, (2) expressed EGFP-labeled MaxiK,, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiK, association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiK, labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+ -release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiK, surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a ,3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK,. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK, stores to the plasma membrane of developing murine astrocytes. © 2009 Wiley-Liss, Inc. [source] Chlorotoxin-sensitive Ca2+ -activated Cl, channel in type R2 reactive astrocytes from adult rat brainGLIA, Issue 4 2003Stanislava Dalton Abstract Astrocytes express four types of Cl, or anion channels, but Ca2+ -activated Cl, (ClCa) channels have not been described. We studied Cl, channels in a morphologically distinct subpopulation (, 5% of cells) of small (10,12 ,m, 11.8 ± 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, ,57.1 ± 4.0 mV, polarized to ,72.7 ± 4.5 mV with BAPTA-AM, an intracellular Ca2+ chelator, and depolarized to ,30.7 ± 6.1 mV with thapsigargin, which mobilizes Ca2+ from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl, reversal potential, which was blocked by Cl, channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn2+, by I, (10 mM), and by chlorotoxin (EC50 = 47 nM). With conventional whole-cell clamp, NPPB- and Zn2+ -sensitive currents became larger with increasing [Ca2+]i (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca2+ sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive ClCa channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive ClCa channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte. GLIA 42:325,339, 2003. © 2003 Wiley-Liss, Inc. [source] Effect of tauroursodeoxycholic acid on endoplasmic reticulum stress,induced caspase-12 activationHEPATOLOGY, Issue 3 2002Qing Xie Activation of death receptors and mitochondrial damage are well-described common apoptotic pathways. Recently, a novel pathway via endoplasmic reticulum (ER) stress has been reported. We assessed the role of tauroursodeoxycholic acid (TUDCA) in inhibition of caspase-12 activation and its effect on calcium homeostasis in an ER stress-induced model of apoptosis. The human liver-derived cell line, Huh7, was treated with thapsigargin (TG) to induce ER stress. Typical morphologic changes of ER stress preceded development of apoptotic changes, including DNA fragmentation and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP), as well as activation of caspase-3 and -7. Elevation of intracellular calcium levels without loss of mitochondrial membrane potential (MMP) was shown using Fluo-3/Fura-red labeling and flow cytometry, and confirmed by induction of Bip/GRP78, a calcium-dependent chaperon of ER lumen. These changes were accompanied by procaspase-12 processing. TUDCA abolished TG-induced markers of ER stress; reduced calcium efflux, induction of Bip/GRP78, and caspase-12 activation; and subsequently inhibited activation of effector caspases and apoptosis. In conclusion, we propose that mitochondria play a secondary role in ER-mediated apoptosis and that TUDCA prevents apoptosis by blocking a calcium-mediated apoptotic pathway as well as caspase-12 activation. This novel mechanism of TUDCA action suggests new intervention methods for ER stress-induced liver disease. [source] Dual Mechanism of Intercellular Communication in HOBIT Osteoblastic Cells: A Role for Gap-Junctional HemichannelsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2001Milena Romanello Abstract Intercellular communication allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks, which favors the intercellular exchange of nutrients and second messengers. Intercellular Ca2+ signaling was investigated in human osteoblast-like initial transfectant (HOBIT) cells, a human osteoblastic cell line in which cells retain most of the osteoblastic differentiation markers. HOBIT cells express connexin43 (Cx43) clustered at the cell-to-cell boundary and display functional intercellular coupling as assessed by the intercellular transfer of Lucifer yellow. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading and dye transfer were inhibited by 18,-glycyrrhetinic acid (18-GA), showing the involvement of gap junctions in signal propagation. Pretreatment of cells with suramin or with apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signaling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake, and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate (InsP3) from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18-GA. A role for Cx hemichannels in adenosine triphosphate (ATP) release and paracrine stimulation is suggested. [source] Basic Fibroblast Growth Factor Stimulates Vascular Endothelial Growth Factor Release in Osteoblasts: Divergent Regulation by p42/p44 Mitogen-Activated Protein Kinase and p38 Mitogen-Activated Protein KinaseJOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Haruhiko Tokuda Abstract We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O -aminophinoxy)-ethane- N,N,N,N -tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase. [source] Nerve growth factor blocks thapsigargin-induced apoptosis at the level of the mitochondrion viaregulation of BimJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6a 2008E. Szegezdi Abstract This study examined how the neurotrophin, nerve growth factor (NGF), protects PC12 cells against endoplasmic reticulum (ER) stress-induced apoptosis. ER stress was induced using thapsigargin (TG) that inhibits the sarcoplasmic/ER Ca2+ -ATPase pump (SERCA) and depletes ER Ca2+ stores. NGF pre-treatment inhibited translocation of Bax to the mitochondria, loss of mitochondrial transmembrane potential, cytochrome c release, activation of caspases (,3, ,7 and ,9) and apoptosis induction by TG. Notably, TG also caused a marked induction of Bimel mRNA and protein, and knockdown of Bim with siRNA protected cells against TG-induced apoptosis. NGF delayed the induction and increased the phosphorylation of Bimel. NGF-mediated protection was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bimel protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF had no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. [source] Overexpression of Par-4 enhances thapsigargin-induced apoptosis via down-regulation of XIAP and inactivation of Akt in human renal cancer cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Tae-Jin Lee Abstract The prostate-apoptosis-response-gene-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor-mediated cell death pathways. We found that overexpressing Par-4 by stable transfection sensitizes Caki cells to induction of apoptosis by TRAIL and drugs that induce endoplasmic reticulum (ER) stress [thapsigargin (TG), tunicamycin (TU) and etoposide]. Ectopic expression of Par-4 is associated with decreased levels of XIAP protein in TG-treated cells, caused in part by XIAP protein instability and caspase activation. Levels of phospho-Akt are decreased in Caki/Par-4 cells to a significantly greater extent than in Caki/Vector cells by treatment with TG, and this is in turn associated with decreased levels of phospho-PDK1, the kinase upstream of Akt. In conclusion, we provide evidence that ectopic expression of Par-4 sensitizes Caki cells to TG and that XIAP protein instability and inactivation of Akt are important in cellular pathways affected by Par-4. J. Cell. Biochem. 103: 358,368, 2008. © 2007 Wiley-Liss, Inc. [source] CREB-dependent cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression is mediated by protein kinase C and calciumJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2006Hung Pham Abstract Cellular production of prostaglandins (PGs) is controlled by the concerted actions of cyclooxygenases (COX) and terminal PG synthases on arachidonic acid in response to agonist stimulation. Recently, we showed in an ileal epithelial cell line (IEC-18), angiotensin II-induced COX-2-dependent PGI2 production through p38MAPK, and calcium mobilization (J. Biol. Chem. 280: 1582,1593, 2005). Agonist binding to the AT1 receptor results in activation of PKC activity and Ca2+ signaling but it is unclear how each pathway contributes to PG production. IEC-18 cells were stimulated with either phorbol-12,13-dibutyrate (PDB), thapsigargin (TG), or in combination. The PG production and COX-2 and PG synthase expression were measured. Surprisingly, PDB and TG produced PGE2 but not PGI2. This corresponded to induction of COX-2 and mPGES-1 mRNA and protein. PGIS mRNA and protein levels did not change. Activation of PKC by PDB resulted in the activation of ERK1/2, JNK, and CREB whereas activation of Ca2+ signaling by TG resulted in the delayed activation of ERK1/2. The combined effect of PKC and Ca2+ signaling were prolonged COX-2 and mPGES-1 mRNA and protein expression. Inhibition of PKC activity, MEK activity, or Ca2+ signaling blocked agonist induction of COX-2 and mPGES-1. Expression of a dominant negative CREB (S133A) blocked PDB/TG-dependent induction of both COX-2 and mPGES-1 promoters. Decreased CREB expression by siRNA blocked PDB/TG-dependent expression of COX-2 and mPGES-1 mRNA. These findings demonstrate a coordinated induction of COX-2 and mPGES-1 by PDB/TG that proceeds through PKC/ERK and Ca2+ signaling cascades, resulting in increased PGE2 production. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Effect of selenium-supplement on the calcium signaling in human endothelial cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2005Yi Zheng Intracellular Ca2+ signaling controls many cellular functions. Understanding its regulation by selenoproteins is essential for understanding the role of selenoproteins in regulating cell functions. The activity of thioredoxin reductase (TrxR), thioredoxin (Trx) content, and the activity of glutathione peroxidase (GPx) in the human endothelial cells cultured in selenium-supplemented medium (refer as Se+ cells) was found 70%, 40%, and 20% higher, respectively than those in the cells cultured in normal medium (refer as Se0 cells). The intracellular Ca2+ signaling initiated by inositol 1,4,5-trisphosphate (IP3), histamine, thapsigargin (TG), carbonyl cyanide p -(tri-fluoromethoxy) phenyl-hydrazone (FCCP), and cyclosporin A (CsA) was investigated in both Se+ and Se0 cells. It was interestingly found that the higher activity of selenoproteins reduced the sensitivity of IP3 receptor to the IP3 -triggered Ca2+ release from intracellular stores, but enhanced activation of the receptor-coupled phospholipase C in histamine-stimulated Se+ cells by showing much more generation of IP3 and higher elevation of cytosolic Ca2+. The higher selenoprotein activity also reduced susceptibility of the uniporter to the mitochondrial uncoupler, susceptibility of the permeability transition pore (PTP) to its inhibitor, and the vulnerability of endoplasmic reticulum (ER) Ca2+ -ATPase to its inhibitor in selenium-supplementing cells. The results suggest that cell calcium signaling is subjected to thiol-redox regulation by selenoproteins. © 2005 Wiley-Liss, Inc. [source] Early nuclear exclusion of the transcription factor max is associated with retinal ganglion cell death independent of caspase activityJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Hilda Petrs-Silva We examined the behavior of the transcription factor Max during retrograde neuronal degeneration of retinal ganglion cells. Using immunohistochemistry, we found a progressive redistribution of full-length Max from the nucleus to the cytoplasm and dendrites of the ganglion cells following axon damage. Then, the axotomized cells lose all their content of Max, while undergoing nuclear pyknosis and apoptotic cell death. After treatment of retinal explants with either anisomycin or thapsigargin, the rate of nuclear exclusion of Max accompanied the rate of cell death as modulated by either drug. Treatment with a pan-caspase inhibitor abolished both TUNEL staining and immunoreactivity for activated caspase-3, but did not affect the subcellular redistribution of Max immunoreactivity after axotomy. The data show that nuclear exclusion of the transcription factor Max is an early event, which precedes and is independent of the activation of caspases, during apoptotic cell death in the central nervous system. J. Cell. Physiol. 198: 179,187, 2004© 2003 Wiley-Liss, Inc. [source] Characterization of Ca2+ signaling pathways in mouse adrenal medullary chromaffin cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2010Pei-Chun Wu J. Neurochem. (2010) 112, 1210,1222. Abstract In the present study, we characterized the Ca2+ responses and secretions induced by various secretagogues in mouse chromaffin cells. Activation of the acetylcholine receptor (AChR) by carbachol induced a transient intracellular Ca2+ concentration ([Ca2+]i) increase followed by two phases of [Ca2+]i decay and a burst of exocytic events. The contribution of the subtypes of AChRs to carbachol-induced responses was examined. Based on the results obtained by stimulating the cells with the nicotinic receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, high K+ and the effects of thapsigargin, it appears that activation of nAChRs induces an extracellular Ca2+ influx, which in turn activate Ca2+ -induced Ca2+ release via the ryanodine receptors. Muscarine, a muscarinic receptor (mAChRs) agonist, was found to induce [Ca2+]i oscillation and sustained catecholamine release, possibly by activation of both the receptor- and store-operated Ca2+ entry pathways. The RT-PCR results showed that mouse chromaffin cells are equipped with messages for multiple subtypes of AChRs, ryanodine receptors and all known components of the receptor- and store-operated Ca2+ entry. Furthermore, results obtained by directly monitoring endoplasmic reticulum (ER) and mitochondrial Ca2+ concentration and by disabling mitochondrial Ca2+ uptake suggest that the ER acts as a Ca2+ source, while the mitochondria acts as a Ca2+ sink. Our results show that both nAChRs and mAChRs contribute to the initial carbachol-induced [Ca2+]i increase which is further enhanced by the Ca2+ released from the ER mediated by Ca2+ -induced Ca2+ release and mAChR activation. This information on the Ca2+ signaling pathways should lay a good foundation for future studies using mouse chromaffin cells as a model system. [source] ,-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2009Nishani T. Hettiarachchi Abstract Parkinson's disease (PD) is characterized in part by the presence of ,-synuclein (,-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the ,-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type ,-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of ,-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the ,-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+]i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of ,-synuclein. However, only WT ,-syn transfected cells displayed significantly impaired viability. Our findings suggest that ,-syn regulates Ca2+ entry pathways and, consequently, that abnormal ,-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis. [source] Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathwaysJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Mami Noda Abstract Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane- N,N,N,,N,-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function. [source] A2A Adenosine Receptor Facilitation of Neuromuscular TransmissionJOURNAL OF NEUROCHEMISTRY, Issue 6 2000Influence of Stimulus Paradigm on Calcium Mobilization Abstract: The influence of stimulus pulse duration on calcium mobilization triggering facilitation of evoked [3H]acetylcholine ([3H]ACh) release by the A2A adenosine receptor agonist CGS 21680C was studied in the rat phrenic nerve-hemidiaphragm. The P-type calcium channel blocker ,-agatoxin IVA (100 nM) decreased [3H]ACh release evoked with pulses of 0.04-ms duration, whereas nifedipine (1 ,M) inhibited transmitter release with pulses of 1-ms duration. Depletion of intracellular calcium stores by thapsigargin (2 ,M) decreased [3H]ACh release evoked by pulses of 1 ms, an effect observed even in the absence of extracellular calcium. With short (0.04-ms) stimulation pulses, when P-type calcium influx triggered transmitter release, facilitation of [3H]ACh release by CGS 21680C (3 nM) was attenuated by both thapsigargin (2 ,M) and nifedipine (1 ,M). With longer stimuli (1 ms), a situation in which both thapsigargin-sensitive internal stores and L-type channels are involved in ACh release, pretreatment with either ,-agatoxin IVA (100 nM) or nifedipine (1 ,M) reduced the facilitatory effect of CGS 21680C (3 nM). The results suggest that A2A receptor activation facilitates ACh release from motor nerve endings through alternatively mobilizing the available calcium pools (thapsigargin-sensitive internal stores and/or P- or L-type channels) that are not committed to the release process in each stimulation condition. [source] Interleukin-1, Release in the Supraoptic Nucleus Area During Osmotic Stimulation Requires Neural FunctionJOURNAL OF NEUROENDOCRINOLOGY, Issue 11 2008J. Y. Summy-Long Interleukin (IL)-1, is present throughout the magnocellular neuroendocrine system and co-depletes with oxytocin and vasopressin from the neural lobe during salt-loading. To examine whether IL-1, is released from the dendrites/soma of magnocellular neurones during osmotic stimulation, microdialysis adjacent to the supraoptic nucleus (SON) in conscious rats was combined with immunocapillary electrophoresis and laser-induced fluorescence detection to quantify cytokine in 5-min dialysates collected before (0,180 min; basal), and after (180,240 min), hypertonic saline injected s.c. (1.5 m NaCl). Osmotic release of IL-1, was compared after inhibiting local voltage-gated channels for Na+ (tetrodotoxin) and Ca2+ (cadmium and nickel) or by reducing intracellular Ca2+ stores (thapsigargin). Immunohistochemistry combined with microdialysis was used to localise cytokine sources (IL-1,+) and microglia (OX-42+). Under conditions of microdialysis, the basal release of IL-1,+ in the SON area was measurable and stable (pg/ml; mean ± SEM) from 0,60 min (2.2 ± 0.06), 60,120 min (2.32 ± 0.05) and 120,180 min (2.33 ± 0.06), likely originating locally from activated microglia (OX42+; IL-1,+; ameboid, hypertrophied) and magnocellular neurones expressing IL-1,. In response to osmotic stimulation, IL-1, increased progressively in dialysates of the SON area by a mechanism dependent on intracellular Ca2+ stores sensitive to thapsigargin and, similar to dendritic secretion of oxytocin and vasopressin, required local voltage-gated Na+ and Ca2+ channels for activation by osmoregulatory pathways from the forebrain. During osmotic stimulation, neurally dependent release of IL-1, in the SON area likely upregulates osmosensitive cation currents on magnocellular neurones (observed in vitro by others), to facilitate dendritic release of neurohypophysial hormones. [source] Bradykinin and Angiotensin II-Induced [Ca2+]i Rise in Cultured Rat Pituitary Folliculo-Stellate CellsJOURNAL OF NEUROENDOCRINOLOGY, Issue 11 2001T. Sudo Abstract Folliculo-stellate cells of the anterior pituitary are thought to modulate pituitary hormone secretion through a paracrine mechanism. Angiotensin II and pituitary adenylate cyclase-activating polypeptide (PACAP) have previously been shown to increase the intracellular Ca2+ concentration ([Ca2+]i) of these cells. In the present study, we examined the effects of various peptides such as bradykinin, angiotensin II, endothelin-1, PACAP, galanin and neurotensin by Ca2+ -imaging of folliculo-stellate cells in primary culture. Bradykinin and angiotensin II increased [Ca2+]i in folliculo-stellate cells. Both responses were completely suppressed by thapsigargin and were significantly suppressed by the phospholipase C inhibitor, U-73122. Ryanodine did not significantly modify the responses. A B2 antagonist and angiotensin II receptor antagonist inhibited the response induced by bradykinin and angiotensin II, respectively. Endothelin-1 and PACAP increased [Ca2+]i in fewer than 50% of folliculo-stellate cells but galanin and neurotensin did not influence [Ca2+]i in any of the folliculo-stellate cells tested. These results indicate that bradykinin and angiotensin II increase [Ca2+]i in folliculo-stellate cells by activating phospholipase C through B2 receptor and AT1 receptor, respectively, and that endothelin-1 and PACAP also increase [Ca2+]i in some folliculo-stellate cells. [source] | |||||||||||||