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Calcium Response (calcium + response)
Selected AbstractsEvidence that caspase-1 is a negative regulator of AMPA receptor-mediated long-term potentiation at hippocampal synapsesJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Chengbiao Lu Abstract Best known for their pivotal role in a form of programmed cell death called apoptosis, caspases may also function in more subtle physiological processes. Caspases are present in synapses and dendrites of neurons where they can be activated in response to glutamate receptor stimulation and calcium influx. Here we tested the hypothesis that caspase-1 plays a role in modulating long-term potentiation (LTP) at hippocampal synapses. We provide evidence that caspase-1 plays a role in regulating ,-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated calcium influx and synaptic plasticity in the hippocampus. LTP of excitatory postsynaptic potentials at CA1 synapses was significantly enhanced when hippocampal slices were treated with either a pan-caspase inhibitor or a selective inhibitor of caspase-1, but not by an inhibitor of caspase-6. Inhibition of caspase-1 significantly enhanced the AMPA current-mediated component of LTP without affecting the N -methyl- d -aspartate current-mediated component. Calcium responses to AMPA were enhanced in hippocampal neurons treated with a caspase-1 inhibitor suggesting that caspase-1 normally functions to reduce AMPA receptor-mediated calcium influx. These findings suggest that, by selectively reducing AMPA currents and calcium influx, caspase-1 functions as a negative regulator of LTP at hippocampal synapses. [source] Measurement of the force and torque produced in the calcium response of reactivated rat sperm flagellaCYTOSKELETON, Issue 1 2001Mark J. Moritz Abstract Rat sperm that are demembranated with Triton X-100 and reactivated with Mg-ATP show a strong mechanical response to the presence of free calcium ion. At pCa < 4, the midpiece region of the flagellum develops a strong and sustained curvature that gives the cell the overall appearance of a fishhook [Lindemann and Goltz, 1988: Cell Motil. Cytoskeleton 10:420,431]. In the present study, the force and torque that maintain the calcium-induced hook have been examined quantitatively. In addition, full-length and shortened flagella were manipulated to evaluate the plasticity of the hooks and determined the critical length necessary for maintaining the curvature. The hooks were found to be highly resilient, returning to their original configuration (>95%) after being straightened and released. The results from manipulating the shortened flagella suggest that the force holding the hook in the curved configuration is generated in the basal 60 ,m of the flagellum. The force required to straighten the calcium-induced hooks was measured with force-calibrated glass microprobes, and the bending torque was calculated from the measured force. The force and torque required to straighten the flagellum were found to be proportional to the change in curvature of the hooked region of the flagellum, suggesting an elastic-like behavior. The average torque to open the hooks to a straight position was 2.6 (±1.4) × 10 -7 dyne × cm (2.6 × 10 -14 N × m) and the apparent stiffness was 4.3 (±1.3) × 10 -10 dyne × cm2 (4.3 × 10 -19 N × m2). The stiffness of the hook was determined to be approximately one quarter the rigor stiffness of a rat sperm flagellum measured under comparable conditions. Cell Motil. Cytoskeleton 49:33,40, 2001. © 2001 Wiley-Liss, Inc. [source] Calcium and magnesium competitively influence the growth of a PMR1 deficient Saccharomyces cerevisiae strainFEMS MICROBIOLOGY LETTERS, Issue 2 2005Réka Szigeti Abstract PMR1, the Ca2+/Mn2+ ATPase of the secretory pathway in Saccharomyces cerevisiae was the first member of the secretory pathway Ca2+ ATPases (SPCA) to be characterized. In the past few years, pmr1, yeast have received more attention due to the recognition that the human homologue of this protein, hSPCA1 is defective in chronic benign pemphigus or Hailey,Hailey disease (HHD). Recent publications have described pmr1, S. cerevisiae as a useful model organism for studying the molecular pathology of HHD. Some observations indicated that the high Ca2+ sensitive phenotype of PMR1 defective yeast strains may be the most relevant in this respect. Here we show that the total cellular calcium response of a pmr1, S. cerevisiae upon extracellular Ca2+ challenge is decreased compared to the wild type strain similarly as observed in keratinocytes. Additionally, the novel magnesium sensitivity of PMR1 defective yeast is revealed, which appears to be a result of competition for uptake between Ca2+ and Mg2+ at the plasma membrane level. Our findings indicate that extracellular Ca2+ and Mg2+ competitively influence the intracellular Ca2+ homeostasis of S. cerevisiae. These observations may further our understanding of HHD. [source] Na,K-ATPase ,2 inhibition alters calcium responses in optic nerve astrocytesGLIA, Issue 3 2004April K. Hartford Abstract Experiments were conducted to test the effect of 1 ,M ouabain, an Na,K-ATPase inhibitor, on capacitative calcium entry (CCE) and calcium responses elicited by ATP in rat optic nerve astrocytes. In the rat, 1 ,M ouabain is sufficient to inhibit the ,2 Na,K-ATPase, but not the ,1. Immortalized astrocytes derived from Na,K-ATPase ,2 homozygous knockout (KO) mice and wild-type (WT) littermates were also used. Cytosolic calcium and sodium concentrations were measured using Fura-2 and SBFI, respectively. The magnitude of the increase in cytosolic calcium concentration during CCE was significantly greater in rat astrocytes exposed to 1 ,M ouabain. To measure calcium release from stores, cells were exposed to ATP in the absence of extracellular calcium. In astrocytes exposed to 1 ,M ouabain, a significantly greater calcium response to ATP was observed. 1 ,M ouabain was shown to inhibit ATP hydrolysis in membrane material containing Na,K-ATPase ,2 and ,1 isoforms (rat muscle) but not in membranes containing only Na,K-ATPase ,1 (rat kidney). In intact astrocytes, 1 ,M ouabain did not alter the cell-wide cytosolic sodium concentration. In mouse Na,K-ATPase ,2 KO astrocytes, the calcium increase during CCE was significantly higher than in WT cells, as was the magnitude of the calcium response to ATP. In KO astrocytes, but not WT, the cytosolic calcium increase during CCE was insensitive to 1 ,M ouabain. Taken together, the results suggest that selective inhibition of the Na,K-ATPase ,2 isoform has the potential to change calcium signaling and CCE. © 2003 Wiley-Liss, Inc. [source] The platelet as a peripheral marker in psychiatric illnessHUMAN PSYCHOPHARMACOLOGY: CLINICAL AND EXPERIMENTAL, Issue 3 2001Helein Plein Abstract The identification of peripheral markers of psychiatric illness is important if an improvement in the diagnosis and treatment of various diseases with overlapping symptomatology is desired. There are many disorders that not only have overlapping symptomatology, but also have similar biological disturbances. The functional capability of the neurons involved in the disease processes may be at the crux of the underlying pathology. The platelet intracellular calcium response to neurotransmitter stimulation has previously been used as a peripheral marker of psychiatric illness. This review discusses evidence in support of the extended use of the platelet as a peripheral marker. The use of the platelet intracellular calcium response to neurotransmitter stimulation as a state or trait marker in major depression, the specificity and selectivity of this response, and the possible use of the platelet as a peripheral marker in psychotic disorders such as schizophrenia, mania and psychotic depression are shown. Finally, a proposed mechanism for the association between certain psychiatric disorders and cardiovascular disease is discussed. Copyright © 2001 John Wiley & Sons, Ltd. [source] Role of annexin A6 isoforms in catecholamine secretion by PC12 cells: Distinct influence on calcium responseJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2010Paulina Podszywalow-Bartnicka Abstract Noradrenaline and adrenaline are secreted by adrenal medulla chromaffin cells via exocytosis. Exocytosis of catecholamines occurs after cell stimulation with various endogenous activators such as nicotine or after depolarization of the plasma membrane and is regulated by calcium ions. Cytosolic [Ca2+] increases in response to cell excitation and triggers a signal-initiated secretion. Annexins are known to participate in the regulation of membrane dynamics and are also considered to be involved in vesicular trafficking. Some experimental evidence suggests that annexins may participate in Ca2+ -regulated catecholamine secretion. In this report the effect of annexin A6 (AnxA6) isoforms 1 and 2 on catecholamine secretion has been described. Overexpression of AnxA6 isoforms and AnxA6 knock-down in PC12 cells were accompanied by almost complete inhibition or a 20% enhancement of dopamine secretion, respectively. AnxA6-1 and AnxA6-2 overexpression reduced ,[Ca2+]c upon depolarization by 32% and 58%, respectively, while AnxA6 knock-down increased ,[Ca2+]c by 44%. The mechanism of AnxA6 action on Ca2+ signalling is not well understood. Experimental evidence suggests that two AnxA6 isoforms interact with different targets engaged in regulation of calcium homeostasis in PC12 cells. J. Cell. Biochem. 111: 168,178, 2010. © 2010 Wiley-Liss, Inc. [source] Negative regulation of endogenous protein kinase C, on the dynamic change of carbachol-induced intracellular calcium response in different melanoma cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2009Huan Wang Regulations of intracellular protein kinase C (PKC) on carbachol (CCh)-induced intracellular calcium ([Ca2+]i) responses were investigated in different stages of melanoma cells. We found that CCh (1,mM) significantly increased [Ca2+]i with 6-, 4-, 4-, and 25-folds intensities in WM793B, 451Lu, SK-MEL-5, and A2058 melanoma cells, respectively. Pretreatment of phorbol 12, 13-dibutyrate (PDBu, 2,µM), an activator of intracellular PKC, significantly suppressed CCh-induced peak reactions in WM793B, SK-MEL-5, and A2058 cells. RT-PCR data showed that mRNA levels of PKC, were 12-, 4-, 6-, and 0.9-folds higher in above four melanoma cells. Short interfering RNA (siRNA) targeting to PKC, in WM793B cells enhanced CCh-induced peak calcium reactions. Present data indicated that CCh-induced [Ca2+]i responses were dynamically changed in different stages of melanoma progression. Moreover, intracellular PKC, activated by exogenous agonist and expressed through endogenous gene transcription negatively regulated CCh-induced calcium responses. The functional analysis on the relationship between CCh-induced calcium response and endogenous PKC, expression might be helpful to predict the development of melanoma. J. Cell. Physiol. 221: 276,282, 2009. © 2009 Wiley-Liss, Inc. [source] Varicella-zoster virus isolates, but not the vaccine strain OKA, induce sensitivity to alpha-1 and beta-1 adrenergic stimulation of sensory neurones in cultureJOURNAL OF MEDICAL VIROLOGY, Issue S1 2003Michaela Schmidt Abstract The reactivation of varicella-zoster virus (VZV) from its persistent state in sensory neurones causes shingles and induces severe, long-lasting pain and hyperalgesia that often lead to postherpetic neuralgia. To investigate the VZV-induced neuropathic changes, we established conditions for the active infection of sensory neurones from rat dorsal root ganglia in vitro. After 2 days of culture, up to 50% of the cells expressed viral antigens of the immediate-early and late replication phase. The intracellular calcium ion concentration was monitored in individual cells by microfluorimetry. Whereas the calcium response to capsaicin was preserved, the VZV-infected neurones gained an unusual sensitivity to noradrenaline stimulation in contrast to non-infected cells. The adrenergic agonists phenylephrine and isoproterenol had a similar efficacy demonstrating that both ,1 - and ,1 -adrenoreceptors were involved. The sensitivity to adrenergic stimulation was observed after infection with different wildtype isolates, but not with the attenuated vaccine strain OKA. The lack of noradrenaline sensitivity of vaccine-infected neurones demands a structural comparison of wildtype and vaccine viruses with and without phenotype. A partial sequence evaluation (26 kb) of the European OKA vaccine strain surprisingly revealed a series of nucleotide exchanges in comparison to presumably identical OKA strains from other sources, although VZV is generally considered genetically stable. In summary, we report that the infection with wildtype VZV isolates, but not with the vaccine strain, induces noradrenaline sensitivity in sensory neurones, which correlates with clinical and experimental observations of adrenergic effects involved in VZV-induced neuralgia. J. Med. Virol. 70:S82,S89, 2003. © 2003 Wiley-Liss, Inc. [source] Impaired long-term depression in P2X3 deficient mice is not associated with a spatial learning deficitJOURNAL OF NEUROCHEMISTRY, Issue 5 2006Yue Wang Abstract The hippocampus is a brain region critical for learning and memory processes believed to result from long-lasting changes in the function and structure of synapses. Recent findings suggest that ATP functions as a neurotransmitter or neuromodulator in the mammalian brain, where it activates several different types of ionotropic and G protein-coupled ATP receptors that transduce calcium signals. However, the roles of specific ATP receptors in synaptic plasticity have not been established. Here we show that mice lacking the P2X3 ATP receptor (P2X3KO mice) exhibit abnormalities in hippocampal synaptic plasticity that can be restored by pharmacological modification of calcium-sensitive kinase and phosphatase activities. Calcium imaging studies revealed an attenuated calcium response to ATP in hippocampal neurons from P2X3KO mice. Basal synaptic transmission, paired-pulse facilitation and long-term potentiation are normal at synapses in hippocampal slices from P2X3KO. However, long-term depression is severely impaired at CA1, CA3 and dentate gyrus synapses. Long-term depression can be partially rescued in slices treated with a protein phosphatase 1,2 A activator or by postsynaptic inhibition of calcium/calmodulin-dependent protein kinase II. Despite the deficit in hippocampal long-term depression, P2X3KO mice performed normally in water maze tests of spatial learning, suggesting that long-term depression is not critical for this type of hippocampus-dependent learning and memory. [source] von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2003J. J. Briedé Summary., Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet-rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin-free planar fibrin layers and washed platelets to examine the relationship between platelet,fibrin interaction and exposure of coagulation-stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s,1 requires fibrin-bound VWF. The cytosolic calcium concentration ([Ca2+]i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 ± 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 ± 14%) along with a transient elevation in [Ca2+]i from 0.05 µmol L,1 up to 1.1 ± 0.2 µmol L,1. Platelet adhesion to fibrin at a shear rate of 50 s,1 is mediated by thrombin but not by fibrin-bound VWF. The [Ca2+]i of these thrombin-activated platelets was elevated (0.2 ± 0.1 µmol L,1), but only a minority of the platelets (11 ± 8%) exposed PS. The essential role of VWF in this thrombin-induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 ± 23%) exposed PS and had peak values of [Ca2+]i of about 0.6 µmol L,1. Taken together, these results demonstrate that thrombin-induced exposure of PS and high calcium response on fibrin-adherent platelets depends on shear- or botrocetin-induced VWF,platelet interaction. [source] Both ADP and Thrombin Regulate Arteriolar Thrombus Stabilization and Embolization, but Are Not Involved in Initial Hemostasis as Induced by MicropunctureMICROCIRCULATION, Issue 3 2007Miriam A. Van Gestel ABSTRACT Objective: Thrombosis and embolization are main causes of morbidity and mortality. Up to now, the relative importance of mediators involved is only partly known. It was the aim of this study to investigate the involvement of ADP and thrombin in subsequent phases of arteriolar hemostasis and thromboembolism in vivo. Methods: Rabbit mesenteric arterioles were punctured, which induced bleeding, hemostasis, and subsequent thromboembolism. This reaction as well as the activation state of platelets involved ([Ca2 +]i), was monitored in real time by intravital (fluorescence) microscopy. Results: Neither inhibition of thrombin formation or thrombin activity nor blockade of platelet ADP receptors P2Y1 and P2Y12 influenced the initial hemostatic reaction: in all experiments initial bleeding was stopped by a primary thrombus within 2,3 s. On the other hand, both thrombin inhibition and P2Y1 blockade increased rebleeding frequency, which indicates reduced thrombus stability in the long term. Finally, inhibition of either thrombin or ADP (via both receptors) reduced aggregate formation during the embolization phase by at least 90%. While most participating platelets exhibited a transient increase in [Ca2 +]i during embolization, an increased percentage of platelets showed no calcium response at all during P2Y1 blockade, which was accompanied by reduced platelet,platelet interaction strength. Conclusions: Whereas thrombin and ADP are not involved in the initial hemostatic reaction, both substances appear to be essential to prevent rebleedings in the long term. During subsequent embolization, ADP (via both receptors) and small amounts of thrombin are involved in platelet activation. [source] Functional Expression, Targeting and Ca2+ Signaling of a Mouse Melanopsin-eYFP Fusion Protein in a Retinal Pigment Epithelium Cell Line,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Maikel E. Giesbers Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11- cis retinal or all- trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin. [source] Identification and characterisation of GPR100 as a novel human G-protein-coupled bradykinin receptorBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2003Katrin Boels G-protein-coupled receptor 100 (GPR100) was discovered by searching the human genome database for novel G-protein-coupled peptide receptors. Full-length GPR100 was amplified from a cDNA library of the neuroendocrine cell line BON, which is derived from a human pancreas carcinoid. The open-reading frame, present on a single exon, coded for a protein of 374 amino acids with highest sequence identity (43%) to the human orphan somatostatin- and angiotensin-like peptide receptor. The analysis of chromosomal localisation mapped the GPR100 gene to chromosome 1q21.2,q21.3. The stable expression of GPR100 in Chinese hamster ovary cells together with aequorin as calcium sensor and the promiscuous G-protein subunit ,16 as signal transducer revealed bradykinin and kallidin as effectors to elicit a calcium response. Dose,response curves yielded EC50 values for both ligands in the low nanomolar range, while the respective analogues without arginine at the carboxy-terminus were inactive. Calcium mobilisation was inhibited by the phospholipase C blocker U73122, but not by pertussis toxin, suggesting the involvement of the G-protein subunit ,q and not ,i or ,o in signal transduction. In line with the main function of kinins as peripheral hormones, we found that GPR100 was expressed predominantly in tissues like pancreas, heart, skeletal muscle, salivary gland, bladder, kidney, liver, placenta, stomach, jejunum, thyroid gland, ovary, and bone marrow, but smaller amounts were also detected in the brain and in cell lines derived from tumours of various origins. British Journal of Pharmacology (2003) 140, 932,938. doi:10.1038/sj.bjp.0705521 [source] The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1)BRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2000D Smart The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 ,M) and capsaicin (1 ,M) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 ,M). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC50 values of 5.94±0.06 (n=5) and 7.13±0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pKB of 7.40±0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1. British Journal of Pharmacology (2000) 129, 227,230; doi:10.1038/sj.bjp.0703050 [source] The molecular receptive range of an olfactory receptor in vivo (Drosophila melanogaster Or22a)DEVELOPMENTAL NEUROBIOLOGY, Issue 14 2006Daniela Pelz Abstract Understanding how odors are coded within an olfactory system requires knowledge about its input. This is constituted by the molecular receptive ranges (MRR) of olfactory sensory neurons that converge in the glomeruli of the olfactory bulb (vertebrates) or the antennal lobe (AL, insects). Aiming at a comprehensive characterization of MRRs in Drosophila melanogaster we measured odor-evoked calcium responses in olfactory sensory neurons that express the olfactory receptor Or22a. We used an automated stimulus application system to screen [Ca2+] responses to 104 odors both in the antenna (sensory transduction) and in the AL (neuronal transmission). At 10,2 (vol/vol) dilution, 39 odors elicited at least a half-maximal response. For these odorants we established dose-response relationships over their entire dynamic range. We tested 15 additional chemicals that are structurally related to the most efficient odors. Ethyl hexanoate and methyl hexanoate were the best stimuli, eliciting consistent responses at dilutions as low as 10,9. Two substances led to calcium decrease, suggesting that Or22a might be constitutively active, and that these substances might act as inverse agonists, reminiscent of G-protein coupled receptors. There was no difference between the antennal and the AL MRR. Furthermore we show that Or22a has a broad yet selective MRR, and must be functionally described both as a specialist and a generalist. Both these descriptions are ecologically relevant. Given that adult Drosophila use approximately 43 ORs, a complete description of all MRRs appears now in reach. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Interneuron subtype specific activation of mGluR1/5 during epileptiform activity in hippocampusEPILEPSIA, Issue 8 2010Nathalie T Sanon Summary Purpose:, Specific inhibitory interneurons in area CA1 of the hippocampus, notably those located in stratum oriens,alveus (O/A-INs), are selectively vulnerable in patients and animal models of temporal lobe epilepsy (TLE). The excitotoxic mechanisms underlying the selective vulnerability of interneurons have not been identified but could involve group I metabotropic glutamate receptor subtypes (mGluR1/5), which have generally proconvulsive actions and activate prominent cationic currents and calcium responses specifically in O/A-INs. Methods:, In this study, we examine the role of mGluR1/5 in interneurons during epileptiform activity using whole-cell recordings from CA1 O/A-INs and selective antagonists of mGluR1, (LY367385) and mGluR5 (MPEP) in a disinhibited rat hippocampal slice model of epileptiform activity. Results:, Our data indicate more prominent epileptiform burst discharges and paroxysmal depolarizations (PDs) in O/A-INs than in interneurons located at the border of strata radiatum and lacunosum/moleculare (R/LM-INs). In addition, mGluR1 and mGluR5 significantly contributed to epileptiform responses in O/A-INs but not in R/LM-INs. Epileptiform burst discharges in O/A-INs were partly dependent on mGluR5. PDs and associated postsynaptic currents were dependent on both mGluR1, and mGluR5. These receptors contributed differently to postsynaptic currents underlying PDs, with mGluR5 contributing to the fast and slow components and mGluR1, to the slow component. Discussion:, These findings support interneuron subtype-specific activation and differential contributions of mGluR1, and mGluR5 to epileptiform activity in O/A-INs, which could be important for their selective vulnerability in TLE. [source] Calcium/calcineurin signaling in primary cortical astrocyte cultures: Rcan1-4 and cyclooxygenase-2 as NFAT target genesGLIA, Issue 7 2008Andrea Canellada Abstract The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway mediates important cell responses to calcium, but its activity and function in astrocytes have remained unclear. We show that primary cortical astrocyte cultures express the regulatory and catalytic subunits of the phosphatase calcineurin as well as the calcium-regulated NFAT family members (NFATc1, c2, c3, and c4). NFATs are activated by calcium-mobilizing agents in astrocytes, and this activation is blocked by the calcineurin inhibitor cyclosporine A. Microarray screening identified cyclooxygenase-2 (Cox-2), which is implicated in brain injury, and Rcan 1-4, an endogenous calcineurin inhibitor, as genes up-regulated by calcineurin-dependent calcium signals in astrocytes. Mobilization of intracellular calcium with ionophore potently augments the promoter activity and mRNA and protein expression of Rcan 1-4 and Cox-2 induced by combined treatment with phorbol esters. Moreover, Rcan 1-4 expression is efficiently induced by calcium mobilization alone. For both the genes, the calcium signal component is dependent on calcineurin and is replicated by exogenous expression of a constitutively active NFAT, strongly suggesting that the calcium-induced gene activation is mediated by NFATs. Finally, we report that calcineurin-dependent expression of Cox-2 and Rcan 1-4 is induced by physiological calcium mobilizing agents, such as thrombin, agonists of purinergic and glutamate receptors, and L-type voltage-gated calcium channels. These findings provide insights into calcium-initiated gene transcription in astrocytes, and have implications for the regulation of calcium responses in astrocytes. © 2008 Wiley-Liss, Inc. [source] Na,K-ATPase ,2 inhibition alters calcium responses in optic nerve astrocytesGLIA, Issue 3 2004April K. Hartford Abstract Experiments were conducted to test the effect of 1 ,M ouabain, an Na,K-ATPase inhibitor, on capacitative calcium entry (CCE) and calcium responses elicited by ATP in rat optic nerve astrocytes. In the rat, 1 ,M ouabain is sufficient to inhibit the ,2 Na,K-ATPase, but not the ,1. Immortalized astrocytes derived from Na,K-ATPase ,2 homozygous knockout (KO) mice and wild-type (WT) littermates were also used. Cytosolic calcium and sodium concentrations were measured using Fura-2 and SBFI, respectively. The magnitude of the increase in cytosolic calcium concentration during CCE was significantly greater in rat astrocytes exposed to 1 ,M ouabain. To measure calcium release from stores, cells were exposed to ATP in the absence of extracellular calcium. In astrocytes exposed to 1 ,M ouabain, a significantly greater calcium response to ATP was observed. 1 ,M ouabain was shown to inhibit ATP hydrolysis in membrane material containing Na,K-ATPase ,2 and ,1 isoforms (rat muscle) but not in membranes containing only Na,K-ATPase ,1 (rat kidney). In intact astrocytes, 1 ,M ouabain did not alter the cell-wide cytosolic sodium concentration. In mouse Na,K-ATPase ,2 KO astrocytes, the calcium increase during CCE was significantly higher than in WT cells, as was the magnitude of the calcium response to ATP. In KO astrocytes, but not WT, the cytosolic calcium increase during CCE was insensitive to 1 ,M ouabain. Taken together, the results suggest that selective inhibition of the Na,K-ATPase ,2 isoform has the potential to change calcium signaling and CCE. © 2003 Wiley-Liss, Inc. [source] Negative regulation of endogenous protein kinase C, on the dynamic change of carbachol-induced intracellular calcium response in different melanoma cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2009Huan Wang Regulations of intracellular protein kinase C (PKC) on carbachol (CCh)-induced intracellular calcium ([Ca2+]i) responses were investigated in different stages of melanoma cells. We found that CCh (1,mM) significantly increased [Ca2+]i with 6-, 4-, 4-, and 25-folds intensities in WM793B, 451Lu, SK-MEL-5, and A2058 melanoma cells, respectively. Pretreatment of phorbol 12, 13-dibutyrate (PDBu, 2,µM), an activator of intracellular PKC, significantly suppressed CCh-induced peak reactions in WM793B, SK-MEL-5, and A2058 cells. RT-PCR data showed that mRNA levels of PKC, were 12-, 4-, 6-, and 0.9-folds higher in above four melanoma cells. Short interfering RNA (siRNA) targeting to PKC, in WM793B cells enhanced CCh-induced peak calcium reactions. Present data indicated that CCh-induced [Ca2+]i responses were dynamically changed in different stages of melanoma progression. Moreover, intracellular PKC, activated by exogenous agonist and expressed through endogenous gene transcription negatively regulated CCh-induced calcium responses. The functional analysis on the relationship between CCh-induced calcium response and endogenous PKC, expression might be helpful to predict the development of melanoma. J. Cell. Physiol. 221: 276,282, 2009. © 2009 Wiley-Liss, Inc. [source] Role of calcium in the gating of isoproterenol-induced arylalkylamine N- acetyltransferase gene expression in the mouse pineal glandJOURNAL OF PINEAL RESEARCH, Issue 1 2006Mathieu Chansard Abstract:, Melatonin and its autonomic regulation serve important physiological functions. We recently demonstrated that stimulation of beta-adrenergic receptors only increases nighttime arylalkylamine N- acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) mRNA levels in mouse pineal gland in vitro, which suggests that pineal clocks may gate Aa-Nat gene expression. In the present study, our data reveal that cAMP analog increased Aa-Nat at any time of day but only in the presence of ionomycin. Using Fura-2AM in ratiometric calcium measurements, we show that isoproterenol stimulation increased intracellular free calcium levels at night, contrary to previous reports. Further, intra- or extracellular calcium depletion suppressed the isoproterenol-induced calcium responses as well as Aa-Nat gene expression. These results suggest calcium may be a critical factor in isoproterenol-induced Aa-Nat gene expression, which may be limited in the daytime. We also found that basal intracellular calcium levels were lower during the night and responses to isoproterenol and KCl depolarization were more robust. In addition, pineals of Cryptochrome mutant mice exhibited no significant difference between day and nighttime basal calcium or isoproterenol response. Together, these results suggest that basal calcium levels in the pineal may be controlled by the endogenous pineal clock, which may influence calcium dynamics, cellular homeostasis and sensitivity to external stimulation. Although the mechanism underlying Aa-Nat gene expression has been well studied, the role of calcium as a link between the pineal clock and Aa-Nat gene expression has been underestimated in rodent pineals. [source] High glucose levels enhance platelet activation: involvement of multiple mechanismsBRITISH JOURNAL OF HAEMATOLOGY, Issue 3 2006Dzana Sudic Summary Diabetes mellitus (DM) and hyperglycaemia are associated with platelet activation. The present study was designed to investigate how high glucose levels influence platelet function. Fasting human blood was incubated with different concentrations of d -glucose (5, 15 and 30 mmol/l) and other sugars without or with in vitro stimuli. Platelet activation was monitored by whole blood flow cytometry. High glucose levels enhanced adenosine diphosphate (ADP)- and thrombin receptor-activating peptide (TRAP)-induced platelet P-selectin expression, and TRAP-induced platelet fibrinogen binding. Similar effects were seen with 30 mmol/l l -glucose, sucrose and galactose. Hyperglycaemia also increased TRAP-induced platelet-leucocyte aggregation. Protein kinase C (PKC) blockade did not counteract the enhancement of platelet P-selectin expression, but abolished the enhancement of TRAP-induced platelet fibrinogen binding by hyperglycaemia. Superoxide anion scavenging by superoxide dismutase (SOD) attenuated the hyperglycaemic enhancement of platelet P-selectin expression, but did not counteract the enhancement of TRAP-induced platelet fibrinogen binding. Hyperglycaemia did not alter platelet intracellular calcium responses to agonist stimulation. Blockade of cyclo-oxygenase (COX), phosphotidylinositol-3 (PI3) kinase, or nitric oxide synthase, or the addition of insulin did not influence the effect of hyperglycaemia. In conclusion, high glucose levels enhanced platelet reactivity to agonist stimulation through elevated osmolality. This occurred via superoxide anion production, which enhanced platelet P-selectin expression (secretion), and PKC signalling, which enhanced TRAP-induced fibrinogen binding (aggregablity). [source] | ||||||||||||||||||||||||||||