ABSTRACT
Purpose: Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca2+]i). Raised [Ca2+]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2+]i and ECM gene expression in normal and glaucoma LC cells. Methods: [Ca2+]i was measured with dual-wavelength Ca2+ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-ß1 [TGFß1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. Results: Basal and hypotonic cell membrane stretch-induced [Ca2+]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2+]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2-induced elevation in profibrotic ECM genes (TGFß1, Col1A1, and periostin), both in normal and in glaucoma LC cells. Conclusions: Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.
Subject(s)
Calcium Signaling/physiology , Glaucoma/metabolism , NFATC Transcription Factors/metabolism , Optic Disk/drug effects , Optic Disk/metabolism , Aniline Compounds/metabolism , Blotting, Western , Calcineurin Inhibitors/pharmacology , Calcium/metabolism , Cells, Cultured , Collagen Type I/genetics , Collagen Type I, alpha 1 Chain , Cyclosporine/pharmacology , Extracellular Matrix/metabolism , Fura-2/analogs & derivatives , Fura-2/metabolism , Gene Expression Profiling , Humans , Hydrogen Peroxide/pharmacology , Microscopy, Confocal , Oxidants/pharmacology , Real-Time Polymerase Chain Reaction , Transforming Growth Factor beta1/genetics , Xanthenes/metabolismABSTRACT
It has been recently reported that the essential antioxidant element selenium has protective effects on cytosolic Ca(2+) levels in cell lines. However, the effects of selenium on like transient receptor potential melastatin 2 (TRPM2) in response to oxidative stress (H(2) O(2) ) are not well understood. We investigated the effects of selenium on H(2) O(2) -induced TRPM2 channel currents in the Chinese hamster ovary (CHO) cell line using patch-clamp and fura-2 fluorescence imaging techniques.
Subject(s)
Antioxidants/pharmacology , Oxidative Stress/drug effects , Selenium/pharmacology , TRPM Cation Channels/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Fluorescent Dyes/metabolism , Fura-2/metabolism , Hydrogen Peroxide/pharmacology , Patch-Clamp Techniques , TransfectionABSTRACT
In spite of the essentiality of manganese (Mn) as a trace element necessary for a variety of physiological processes, Mn in excess accumulates in the brain and has been associated with dysfunction and degeneration of the basal ganglia. Despite the high sensitivity, limited chemical interference, and multi-elemental advantages of traditional methods for measuring Mn levels, they lack the feasibility to assess Mn transport dynamics in a high-throughput manner. Our lab has previously reported decreased net Mn accumulation in a mutant striatal cell line model of Huntington's disease (STHdh(Q111/Q111)) relative to wild-type following Mn exposure. To evaluate Mn transport dynamics in these striatal cell lines, we have developed a high-throughput fluorescence-quenching extraction assay (Cellular Fura-2 Manganese Extraction Assay - CFMEA). CFMEA utilizes changes in fura-2 fluorescence upon excitation at 360 nm (Ca(2+) isosbestic point) and emission at 535 nm, as an indirect measurement of total cellular Mn content. Here, we report the establishment, development, and application of CFMEA. Specifically, we evaluate critical extraction and assay conditions (e.g. extraction buffer, temperature, and fura-2 concentration) required for efficient extraction and quantitative detection of cellular Mn from cultured cells. Mn concentrations can be derived from quenching of fura-2 fluorescence with standard curves based on saturation one-site specific binding kinetics. Importantly, we show that extracted calcium and magnesium concentrations below 10 µM have negligible influence on measurements of Mn by fura-2. CFMEA is able to accurately measure extracted Mn levels from cultured striatal cells over a range of at least 0.1-10 µM. We have used two independent Mn supplementation approaches to validate the quantitative accuracy of CFMEA over a 0-200 µM cellular Mn-exposure range. Finally, we have utilized CFMEA to experimentally confirm a deficit in net Mn accumulation in the mutant HD striatal cell line versus wild-type cells. To conclude, we have developed and applied a novel assay to assess Mn transport dynamics in cultured striatal cell lines. CFMEA provides a rapid means of evaluating Mn transport kinetics in cellular toxicity and disease models.
Subject(s)
Corpus Striatum/metabolism , Fura-2/metabolism , High-Throughput Screening Assays/methods , Huntington Disease/metabolism , Manganese/deficiency , Manganese/metabolism , Animals , Cell Line , Corpus Striatum/pathology , Huntington Disease/genetics , Mice , Mice, Mutant Strains , Mutation/geneticsABSTRACT
In numerous cells, Ca2+ undershoot is commonly observed after withdrawing stimulus that release Ca2+ from intracellular stores. In airway smooth muscle (ASM), the fast intracellular Ca2+ concentration ([Ca2+]i) drop during undershoot is produced by sarcoplasmic reticulum (SR) reloading, but the mechanisms involved in the long lasting basal [Ca2+]i recovery are unknown. We investigated the post-caffeine Ca2+ undershoot recovery in ASM isolated cells from bovine trachea. [Ca2+]i determination was done by a ratiometric method by incubating cells with Fura-2/AM. After inducing a transient response, caffeine withdrawn generated a Ca2+ undershoot. SR-Ca2+ content during maximum undershoot drop was approximately 40% of SR caffeine-releasable Ca2+ (SR-Ca2+ load). Undershoot recovery rate increased in presence of cyclopiazonic acid (CPA, a SR-Ca2+ ATPase inhibitor), but SR-Ca2+ load was reduced. Genistein (a tyrosine kinase inhibitor) slowed down the Ca2+ undershoot drop and the SR-Ca2+ load but did not affect the undershoot recovery rate. Ni2+ (a capacitative Ca2+ inhibitor), but neither SKF-96365 (a passive Ca2+ entry inhibitor) nor econazole (a capacitative Ca2+ inhibitor in non-excitable cells), inhibited Ca2+ undershoot recovery and SR-Ca2+ load. Our data suggest that capacitative Ca2+ entry is involved in bovine ASM Ca2+ undershoot recovery, and that changes in Ca2+ undershoot have an impact on SR-Ca2+ loading which might affect in turn ASM excitability.
Subject(s)
Calcium/metabolism , Muscle, Smooth/metabolism , Trachea/cytology , Animals , Caffeine/metabolism , Caffeine/pharmacology , Calcium/antagonists & inhibitors , Calcium Channel Blockers/pharmacology , Calcium-Transporting ATPases/antagonists & inhibitors , Cations/pharmacology , Cattle , Cells, Cultured , Econazole/pharmacology , Fluorescent Dyes/metabolism , Fura-2/metabolism , Genistein/pharmacology , Imidazoles/pharmacology , Indoles/pharmacology , Ionomycin/pharmacology , Ionophores/pharmacology , Muscle, Smooth/drug effects , Nickel/pharmacology , Phytoestrogens/pharmacology , Sarcoplasmic Reticulum/metabolism , Time FactorsABSTRACT
Diallyl sulfide (DAS), one of the major organosulfur compounds (OSCs) of garlic, is recognized as a group of potential chemoproventive compounds. In this study, we examines the early signaling effects of DAS on renal cells loaded with Ca(2+)-sensitive dye fura-2. It was found that DAS caused an immediate and sustained rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50)=2.32 mM). DAS also induced a [Ca(2+)](i) elevation when extracellular Ca(2+) was removed, but the magnitude was reduced by 45%. Depletion of intracellular Ca(2+) stores with CCCP, a mitochondrial uncoupler, did not affect DAS's effect. In Ca(2+)-free medium, the DAS-induced [Ca(2+)](i) rise was abolished by depleting stored Ca(2+) with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). DAS-caused [Ca(2+)](i) rise in Ca(2+)-containing medium was not affected by modulation of protein kinase C activity. The DAS-induced Ca(2+) influx was blocked by nicardipine. U73122, an inhibitor of phospholipase C, abolished ATP (but not DAS)-induced [Ca(2+)](i) rise. Additionally, pretreatment with DAS for 24 h decreased cell viability in a concentration-dependent manner. Furthermore, DAS-induced cell death involved apoptotic events. These findings suggest that diallyl sulfide induced a significant rise in [Ca(2+)](i) in MDCK renal tubular cells by stimulating both extracellular Ca(2+) influx and thapsigargin-sensitive intracellular Ca(2+) release via as yet unidentified mechanisms.
Subject(s)
Allyl Compounds/pharmacology , Antioxidants/pharmacology , Calcium Signaling/drug effects , Calcium/metabolism , Garlic/chemistry , Kidney Tubules/drug effects , Sulfides/pharmacology , Animals , Apoptosis/drug effects , Calcium Channel Blockers/pharmacology , Calcium Signaling/physiology , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Dogs , Dose-Response Relationship, Drug , Estrenes/pharmacology , Fluorescent Dyes/metabolism , Fura-2/metabolism , Kidney Tubules/metabolism , Kidney Tubules/pathology , Nicardipine/pharmacology , Plant Extracts/chemistry , Plant Extracts/pharmacology , Pyrrolidinones/pharmacology , Thapsigargin/pharmacology , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism , Uncoupling Agents/pharmacologyABSTRACT
Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.
Subject(s)
Multidrug Resistance-Associated Proteins/metabolism , Animals , Crangonidae/metabolism , Fura-2/analogs & derivatives , Fura-2/metabolism , Intestinal Mucosa/metabolism , Isopoda/metabolism , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Nephropidae/metabolism , Propionates/pharmacology , Quinolines/pharmacologyABSTRACT
With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.
Subject(s)
Brain/metabolism , C-Peptide/metabolism , Animals , Brain/anatomy & histology , C-Peptide/pharmacology , Calcium/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Electrophysiology/methods , Fura-2/metabolism , Hypothalamus/cytology , Iodine Isotopes/metabolism , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neurons/drug effects , Neurons/physiology , Protein Binding/drug effects , Protein Binding/physiology , Rats , Rats, Sprague-Dawley , Thiobarbiturates/metabolismABSTRACT
The lipid diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to verify the existence of DAG-sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca(2+) ([Ca(2+)]i) in nearly 35% of the KCl-responsive cells. These Ca(2+) responses disappeared in a Ca(2+)-free medium supplemented with EGTA. Mn(2+) quench experiments showed that OAG activated Ca(2+)-conducting channels that were also permeant to Ba(2+). The OAG-induced Ca(2+) responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[beta-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd(3+). Replacing Na(+) ions with N-methyl-D-glucamine diminished the amplitude of the OAG-induced Ca(2+) responses showing that the Ca(2+) entry was mediated via Na(+)-dependent and Na(+)-independent mechanisms. Experiments carried out with the fluorescent Na(+) indicator CoroNa Green showed that OAG elevated [Na(+)]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca(2+)]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca(2+) responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C-type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca(2+)]i. Whole-cell patch-clamp recordings showed that hyperforin activated non-selective cation channels. They were blocked by SKF-96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin- and OAG-sensitive Ca(2+)-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical neurons.
Subject(s)
Calcium Channels/physiology , Calcium/metabolism , Cerebral Cortex/cytology , Diglycerides/pharmacology , Neurons/drug effects , Second Messenger Systems/drug effects , TRPC Cation Channels/physiology , Aniline Compounds/metabolism , Animals , Bridged Bicyclo Compounds/pharmacology , Calcium Channel Blockers/pharmacology , Cells, Cultured , Cyclohexanones/pharmacology , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Fura-2/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Mice, Inbred C57BL , Patch-Clamp Techniques , Phloroglucinol/analogs & derivatives , Phloroglucinol/pharmacology , Potassium Chloride/pharmacology , Sodium/metabolism , Terpenes/pharmacology , Xanthenes/metabolismABSTRACT
Calcium-activated chloride channels (CaCCs) are widely expressed in mammalian tissues, including intestinal epithelia, where they facilitate fluid secretion. Potent, selective CaCC inhibitors have not been available. We established a high-throughput screen for identification of inhibitors of a human intestinal CaCC based on inhibition of ATP/carbachol-stimulated iodide influx in HT-29 cells after lentiviral infection with the yellow fluorescent halide-sensing protein YFP-H148Q/I152L. Screening of 50,000 diverse, drug-like compounds yielded six classes of putative CaCC inhibitors, two of which, 3-acyl-2-aminothiophenes and 5-aryl-2-aminothiazoles, inhibited by >95% iodide influx in HT-29 cells in response to multiple calcium-elevating agonists, including thapsigargin, without inhibition of calcium elevation, calcium-calmodulin kinase II activation, or cystic fibrosis transmembrane conductance regulator chloride channels. These compounds also inhibited calcium-dependent chloride secretion in T84 human intestinal epithelial cells. Patch-clamp analysis indicated inhibition of CaCC gating, which, together with the calcium-calmodulin data, suggests that the inhibitors target the CaCC directly. Structure-activity relationships were established from analysis of more than 1800 analogs, with IC(50) values of the best analogs down to approximately 1 muM. Small-molecule CaCC inhibitors may be useful in pharmacological dissection of CaCC functions and in reducing intestinal fluid losses in CaCC-mediated secretory diarrheas.
Subject(s)
Chloride Channels/antagonists & inhibitors , Drug Evaluation, Preclinical/methods , Adenosine Triphosphate/antagonists & inhibitors , Adenosine Triphosphate/pharmacology , Calcium/analysis , Carbachol/antagonists & inhibitors , Carbachol/pharmacology , Chloride Channels/chemical synthesis , Chloride Channels/chemistry , Chloride Channels/genetics , Chlorides/metabolism , Dose-Response Relationship, Drug , Fluorescent Dyes/metabolism , Fura-2/metabolism , Genetic Vectors , HT29 Cells , Humans , Inhibitory Concentration 50 , Intestinal Mucosa/metabolism , Intestines/drug effects , Intestines/pathology , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Kinetics , Lentivirus/genetics , Models, Biological , Molecular Structure , Patch-Clamp Techniques , Reproducibility of Results , Structure-Activity RelationshipABSTRACT
We describe a thalamocortical slice preparation in which connectivity between the mouse lateral geniculate nucleus (LGN) and primary visual cortex (V1) is preserved. Through DiI injections in fixed brains we traced and created a three-dimensional model of the mouse visual pathways. From this computer model we designed a slice preparation that contains a projection from LGN to V1. We prepared brain slices with these predicted coordinates and demonstrated anatomical LGN-V1 connectivity in these slices after LGN tracer injections. We also revealed functional LGN-V1 connectivity by stimulating LGN electrically and detecting responses in layer 4 of V1 using calcium imaging, field potential recordings and whole-cell recordings. We also identified layer-4 neurons that receive direct thalamocortical input. Finally, we compared cortical activity after LGN stimulation with spontaneous cortical activity and found significant overlap of the spatiotemporal dynamics generated by both types of events.
Subject(s)
Cerebral Cortex/physiology , Microtomy/methods , Thalamus/physiology , Visual Cortex/physiology , Visual Pathways/physiology , Animals , Calcium/analysis , Calcium/metabolism , Cerebral Cortex/anatomy & histology , Electric Stimulation , Electrophysiology , Fura-2/analogs & derivatives , Fura-2/metabolism , Geniculate Bodies/anatomy & histology , Geniculate Bodies/physiology , Imaging, Three-Dimensional/methods , Methylamines/chemistry , Mice , Microscopy, Fluorescence , Models, Neurological , Neurons/cytology , Neurons/physiology , Staining and Labeling/methods , Thalamus/anatomy & histology , Tissue Fixation , Visual Cortex/anatomy & histology , Visual Pathways/anatomy & histologyABSTRACT
GABA(A) receptor activation during brain development is a critical source of excitation. This is due to the positive equilibrium potential for chloride relative to resting membrane potential, resulting in membrane depolarization sufficient to open voltage sensitive calcium channels. The gonadal steroid estradiol has pronounced trophic effects on the developing hippocampus, promoting cell survival and synaptogenesis. In the current study, we investigated the effect of estradiol on GABA(A) receptor-mediated calcium transients in cultured neonatal hippocampal neurons, from Sprague-Dawley rats, using the calcium sensitive dye, Fura-2-AM. Treatment of hippocampal neurons with physiological levels of estradiol significantly increased the peak amplitude of calcium transients, increased the number of cells responding to the GABA(A) agonist muscimol with membrane depolarization, and delayed the rate of clearance of free intracellular calcium. These effects were significantly attenuated by pretreatment with the oestrogen receptor antagonist ICI-182,780. This suggests that estradiol, via its action on the oestrogen receptor, prolongs the developmental duration of depolarizing GABA. Estradiol likely maintains GABA-mediated excitation by promoting increased protein levels of the active/phosphorylated form of the chloride cotransporter Na+K+2CL- and L-type voltage sensitive calcium channels containing the alpha1C subunit. We propose that a component of the trophic effects of estradiol on hippocampal development results from enhanced calcium influx subsequent to GABA(A) receptor activation.
Subject(s)
Estradiol/administration & dosage , Hippocampus/cytology , Hippocampus/enzymology , Neurons/drug effects , Receptors, GABA-A/physiology , Animals , Bicuculline/pharmacology , Blotting, Western/methods , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/metabolism , Cells, Cultured , Diagnostic Imaging/methods , Diltiazem/pharmacology , Dizocilpine Maleate/pharmacology , Drug Administration Schedule , Drug Interactions , Embryo, Mammalian , Excitatory Amino Acid Antagonists/pharmacology , Female , Fura-2/analogs & derivatives , Fura-2/metabolism , GABA Agonists/pharmacology , GABA Antagonists/pharmacology , Glutamic Acid/pharmacology , Muscimol/pharmacology , Neurons/metabolism , Nimodipine/pharmacology , Potassium Chloride/pharmacology , Pregnancy , Quinoxalines/pharmacology , Rats , Sodium-Potassium-Chloride Symporters/metabolism , Solute Carrier Family 12, Member 2 , Symporters/metabolism , Time Factors , K Cl- CotransportersABSTRACT
Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.
Subject(s)
Calcium Channels/physiology , Calcium/metabolism , Mushroom Bodies/cytology , Neurons/drug effects , Spider Venoms/pharmacology , Valine/analogs & derivatives , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Age Factors , Analysis of Variance , Animals , Caffeine/pharmacology , Cells, Cultured , Chlorine/pharmacology , Cobalt/pharmacology , Curare/pharmacology , Diagnostic Imaging/methods , Dose-Response Relationship, Radiation , Drosophila , Drug Combinations , Drug Interactions , Electric Stimulation/methods , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Fura-2/metabolism , GABA Antagonists/pharmacology , Green Fluorescent Proteins/metabolism , Iodine/pharmacology , Neurons/physiology , Nicotine/pharmacology , Nicotinic Antagonists/pharmacology , Patch-Clamp Techniques/methods , Phenols/pharmacology , Picrotoxin/pharmacology , Pupa , Salicylates/pharmacology , Tetrodotoxin/pharmacology , Thapsigargin/pharmacology , Time Factors , Valine/pharmacology , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Agrin has been implicated in multiple aspects of central nervous system (CNS) neuron differentiation and function including neurite formation, synaptogenesis, and synaptic transmission. However, little is known about the signaling mechanisms whereby agrin exerts its effects. We have recently identified a neuronal receptor for agrin, whose activation induces expression of c-fos, and provided evidence that agrin binding to this receptor is associated with a rise in intracellular Ca2+, a ubiquitous second messenger capable of mediating a wide range of effects. To gain further insight into agrin's role in brain, we used Ca2+ imaging to explore agrin signal transduction in cultured cortical neurons. Bath application of either z+ or z-agrin isoforms resulted in marked changes in intracellular Ca2+ concentration specifically in neurons. Propagation of the Ca2+ response was a two-step process characterized by an initial increase in intracellular Ca2+ mediated by ryanodine receptor (RyR) release from intracellular stores, supplemented by influx through voltage-gated calcium channels (VGCCs). Agrin-induced increases in intracellular Ca2+ were blocked by genistein and herbimycin, suggesting that the agrin receptor is a tyrosine kinase. Ca2+ release from intracellular stores activates both calcium/calmodulin-dependent kinase II (CaMKII) and mitogen activated protein kinase (MAPK). Activation of CaMKII is required for propagation of the Ca2+ wave itself, whereas both MAPK and CaMKII play a role in mediating long latency responses such as induction of c-fos. These results suggest that an agrin-dependent tyrosine kinase could play a critical role in modulating levels of intracellular Ca2+ and activity of MAPK and CaMKII in CNS neurons.
Subject(s)
Agrin/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium/metabolism , Cerebral Cortex/cytology , Fura-2/analogs & derivatives , Mitogen-Activated Protein Kinases/metabolism , Neurons/enzymology , Signal Transduction/physiology , 2-Amino-5-phosphonovalerate/pharmacology , Agrin/chemistry , Agrin/pharmacology , Animals , Animals, Newborn , Blotting, Western/methods , Calcium Channel Blockers/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Cells, Cultured , Chlorocebus aethiops , Conotoxins/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Extracellular Space/drug effects , Extracellular Space/metabolism , Fura-2/metabolism , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry/methods , Mice , Mice, Inbred ICR , Models, Neurological , Neurons/drug effects , Nifedipine/pharmacology , Peptide Fragments/pharmacology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Transfection/methods , Tubocurarine/pharmacologyABSTRACT
Orexin-A and -B (hypocretin-1 and -2) have been implicated in the stimulation of feeding. Here we show the effector neurons and signaling mechanisms for the orexigenic action of orexins in rats. Immunohistochemical methods showed that orexin axon terminals contact with neuropeptide Y (NPY)- and proopiomelanocortin (POMC)-positive neurons in the arcuate nucleus (ARC) of the rats. Microinjection of orexins into the ARC markedly increased food intake. Orexins increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the isolated neurons from the ARC, which were subsequently shown to be immunoreactive for NPY. The increases in [Ca(2+)](i) were inhibited by blockers of phospholipase C (PLC), protein kinase C (PKC) and Ca(2+) uptake into endoplasmic reticulum. The stimulation of food intake and increases in [Ca(2+)](i) in NPY neurons were greater with orexin-A than with orexin-B, indicative of involvement of the orexin-1 receptor (OX(1)R). In contrast, orexin-A and -B equipotently attenuated [Ca(2+)](i) oscillations and decreased [Ca(2+)](i) levels in POMC-containing neurons. These effects were counteracted by pertussis toxin, suggesting involvement of the orexin-2 receptor and Gi/Go subtypes of GTP-binding proteins. Orexins also decreased [Ca(2+)](i) levels in glucose-responsive neurons in the ventromedial hypothalamus (VMH), a satiety center. Leptin exerted opposite effects on these three classes of neurons. These results demonstrate that orexins directly regulate NPY, POMC and glucose-responsive neurons in the ARC and VMH, in a manner reciprocal to leptin. Orexin-A evokes Ca(2+) signaling in NPY neurons via OX(1)R-PLC-PKC and IP(3) pathways. These neural pathways and intracellular signaling mechanisms may play key roles in the orexigenic action of orexins.
Subject(s)
Calcium Signaling/drug effects , Carrier Proteins/pharmacology , Glucose/metabolism , Hypothalamus/cytology , Intracellular Signaling Peptides and Proteins , Leptin/pharmacology , Neuropeptide Y/metabolism , Neuropeptides/pharmacology , Pro-Opiomelanocortin/metabolism , Animals , Dose-Response Relationship, Drug , Drug Interactions , Eating/drug effects , Enzyme Inhibitors , Fura-2/metabolism , Immunohistochemistry , Models, Neurological , Neurons/classification , Neurons/drug effects , Neurons/metabolism , Orexins , Pertussis Toxin/pharmacology , Rats , Rats, Sprague-Dawley , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Calcium homeostasis was studied in dunce, a Drosophila mutant that is defective in learning and memory. Fura 2-AM fluorescence photometry was used to measure the intracellular calcium concentration in wild type and dunce cleavage-arrested neurons under resting conditions and in response to neurotransmitters. After acetylcholine application, the peak [Ca2+]i was greater in dunce (693 +/- 125 nM) than in wild type neurons (464 +/- 154 nM), but half decay time was shorter in dunce (66 +/- 15 s) than in wild type neurons (104 +/- 40 s). In contrast, the application of glutamate, NMDA, dopamine, and serotonin had no effect on [Ca2+]i. These results indicate that calcium influx through acetylcholine receptors is increased in dunce, compared to wild type neurons. The results also suggest that calcium extrusion to the outside and/or calcium buffering are enhanced in dunce, compared to wild type neurons. This disturbance in the homeostasis of cytosolic calcium concentration in dunce may be implicated in defective associative learning in Drosophila, and may play a role in acute and chronic neurodegenerative disorders in the mammalian brain.
Subject(s)
Calcium/metabolism , Fura-2/analogs & derivatives , Neurons/metabolism , Receptors, Cholinergic/metabolism , Acetylcholine/pharmacology , Analysis of Variance , Animals , Cadmium Chloride/pharmacology , Cells, Cultured , Curare/pharmacology , Dopamine/pharmacology , Drosophila , Drug Interactions , Excitatory Amino Acid Agonists/pharmacology , Extracellular Space/drug effects , Extracellular Space/metabolism , Fura-2/metabolism , Glutamic Acid/pharmacology , Mutation , N-Methylaspartate/pharmacology , Neurons/drug effects , Nicotinic Antagonists/pharmacology , Potassium Chloride/pharmacology , Receptors, Cholinergic/drug effects , Serotonin/pharmacology , Time Factors , Vasodilator Agents/pharmacologyABSTRACT
The effects of compounds with the stilbene pharmacophore [diethylstilbestrol (DES), DES derivatives, tetrahydrochrysene (THC), and THC derivatives] were examined for their ability to inhibit thrombin-induced Ca(2+) influx in human platelets. DES derivatives (DES dimethyl ether, DES dipropionate, dienestrol, and hexestrol) had lower inhibitory activity than DES. Esterification of DES with the bulky monobenzyl group eliminated inhibitory activity. Unsubstituted THC diol had the lowest inhibitory activity in the series of the THC derivatives bearing substituents in the 5,11 positions. These derivatives, either diethyl or dipropyl, cis or trans, were potent inhibitors of thrombin-induced [Ca(2+)](i) elevation (near 100% inhibition at 10 microM). Therefore, stilbene pharmacophore having bulk out of the plane of the double bond (from the twisting of the two aromatic rings or from addition of all substituents) seems to be requirement for the inhibitory activity. Free hydroxyl groups are also required for inhibitory activity, most likely for hydrogen bonding, since trans-diethyl tetrahydrochrysene dimethyl ether was inactive. Compounds bearing ethyl substituents (DES and THC derivatives) inhibited thrombin-induced release of calcium from the endoplasmic reticulum. These compounds also inhibited thapsigargin-induced Ca(2+) influx. This result implies that these compounds also block store-operated Ca(2+) influx directly, as well as internal Ca(2+) release. Compounds without ethyl substituents (trans-resveratrol, genistein, daidzein, and THC diol) only inhibited calcium influx into platelets.
Subject(s)
Blood Platelets/drug effects , Calcium Channel Blockers/pharmacology , Chrysenes/pharmacology , Diethylstilbestrol/pharmacology , Adult , Barium/metabolism , Blood Platelets/metabolism , Calcium/metabolism , Calcium Channels/drug effects , Calcium Channels/metabolism , Calcium Signaling/drug effects , Chrysenes/chemistry , Diethylstilbestrol/chemistry , Female , Fura-2/metabolism , Humans , Hydrogen Bonding , Ion Transport/drug effects , Ionophores/chemistry , Ionophores/pharmacology , Isoflavones/chemistry , Isoflavones/pharmacology , Male , Middle Aged , Molecular Structure , Phytoestrogens , Plant Preparations/chemistry , Plant Preparations/pharmacology , Platelet Activation/drug effects , Strontium/metabolism , Structure-Activity Relationship , Substrate Specificity , Thapsigargin/pharmacology , Thrombin/pharmacologyABSTRACT
Alternative medicines such as herbal products are increasingly being used for preventive and therapeutic purposes. Ginseng is the best known and most popular herbal medicine used worldwide. In spite of some beneficial effects of ginseng on the CNS, little scientific evidence shows at the cellular level. In the present study, we have examined the direct modulation of ginseng on the activation of glutamate, especially NMDA, receptors in cultured hippocampal neurons. Using fura-2-based digital imaging techniques, we found ginseng total saponins inhibited NMDA-induced but less effectively glutamate-induced increase in [Ca2+]i. Ginseng total saponins also modulated Ca2+ transients evoked by depolarization with 50mM KCl along with its own effects on [Ca2+]i. Furthermore, we demonstrated that ginsenoside Rg3 is an active component for ginseng actions on NMDA receptors. The data obtained suggest that the inhibition of NMDA receptors by ginseng, in particular by ginsenoside Rg3, could be one of the mechanisms for ginseng-mediated neuroprotective actions.
Subject(s)
Central Nervous System Agents/pharmacology , Neurons/drug effects , Panax/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism , Saponins/pharmacology , Animals , Animals, Newborn , Calcium Signaling/physiology , Cells, Cultured , Excitatory Amino Acid Agonists/metabolism , Fluorescent Dyes/metabolism , Fura-2/metabolism , Ginsenosides , Glutamic Acid/metabolism , Herbal Medicine , Hippocampus/cytology , Hippocampus/metabolism , N-Methylaspartate/metabolism , Neurons/cytology , Neurons/metabolism , Patch-Clamp Techniques , Plants, Medicinal , Rats , Rats, Sprague-DawleyABSTRACT
The effect of gossypol, a compound found in cottonseed, on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. Gossypol (0.2-5microM) increased [Ca2+](i) in a concentration-dependent manner with an EC(50) value of 1.5microM. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca2+ markedly reduced the [Ca2+](i) signals by 80+/-2%. Preincubation with 0.1mM La3+ or 10microM nimodipine abolished the Ca2+ influx. Gossypol (5microM)-induced release of intracellular Ca2+ was reduced by 75% by pretreatment with 1microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. After pretreatment with 5microM gossypol in Ca2+-free medium for several min, addition of 3mM Ca2+ induced a [Ca2+](i) increase of a magnitude nine-fold greater than control. Gossypol (5microM)-induced Ca2+ release was not affected by inhibiting phospholipase C with 2microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca2+](i) increases in Chang liver cells by releasing Ca2+ from intracellular pools in a phospholipase C-dissociated fashion and by causing La3+- and nimodipine-sensitive Ca2+ influx.
Subject(s)
Calcium/metabolism , Cottonseed Oil , Cytosol/drug effects , Gossypol/toxicity , Hepatocytes/drug effects , Calcium Channel Blockers/pharmacology , Calcium Signaling/drug effects , Cytosol/metabolism , Dose-Response Relationship, Drug , Drug Combinations , Estrenes/pharmacology , Fluorescent Dyes/metabolism , Fura-2/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Nimodipine/pharmacology , Pyrrolidinones/pharmacology , Thapsigargin/pharmacology , Type C Phospholipases/antagonists & inhibitorsABSTRACT
The effects of five lignans (epi-aschantin, epi-magnolin, epi-yangambin, deoxypodophyllotoxin, yatein) isolated from Hernandia nymphaeifolia (Presl.) Kubitzki (Hernandiaceae) on intracellular Ca2+ levels ([Ca2+]i) in human neutrophils were investigated by using fura-2 as a fluorescent probe. In both Ca2+-containing and Ca2+-free media, the lignans (50-100 microM) did not alter basal [Ca2+]i but inhibited the [Ca2+]i increase induced by platelet activating factor (PAF, 10 microM), leukotriene B4 (LTB4, 0.2 microM), and thapsigargin (1 microM) to different extents. In Ca2+-free medium, after depleting stores of Ca2+ with PAF, LTB4 or thapsigargin, addition of 3 mM Ca2+ induced Ca2+ influx. Each of the lignans (50-100 microM) caused 39-89% inhibition of PAF-induced Ca2+ influx; whereas only epi-aschantin was able to inhibit LTB4- and thapsigargin-induced Ca2+ influx by 54-79%. Together, the results suggest that in human neutrophils, these lignans did not alter basal [Ca2+]i but inhibited Ca2+ movement induced by Ca2+ mobilizing agents.
Subject(s)
Calcium Signaling/drug effects , Drugs, Chinese Herbal/pharmacology , Lignans/pharmacology , Magnoliopsida/chemistry , Neutrophils/drug effects , Calcium/metabolism , Calcium/pharmacology , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/isolation & purification , Fluorescent Dyes/metabolism , Fura-2/metabolism , Humans , Leukotriene B4/pharmacology , Neutrophils/metabolism , Platelet Activating Factor/pharmacology , Thapsigargin/pharmacologyABSTRACT
Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+ )waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.