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1.
Mol Med Rep ; 17(3): 3744-3750, 2018 03.
Article in English | MEDLINE | ID: mdl-29257338

ABSTRACT

Chronic constriction injury (CCI) of the sciatic nerve may induce dorsal root ganglion (DRG) neuronal hyperexcitability and behaviorally expressed hyperalgesia. CCI is a model of neuropathic pain. To investigate the association between the expression of protease activated receptor 2 (PAR2), TMEM16A and neuropathic pain, the expression of PAR2 and TMEM16A proteins in the DRG neurons of rats following CCI of the sciatic nerve was investigated. Following the creation of the CCI model, the thermal withdrawal latency (TWL) was examined by a hot plate test. An immunofluorescence assay and western blot assay were performed to determine the expression of PAR2 and TMEM16A proteins in the ipsilateral L4­6 DRG neurons. The concentration of inositol 1,4,5­triphosphate (IP3) in the L4­6 DRG was determined by ELISA. In the CCI­D7 (7 days after CCI) and CCI­D14 (14 days after CCI) treatment groups, the TWL of rats was significantly shorter than that in the sham operated group (P<0.01; n=12). The expression of PAR2 and TMEM16A proteins in the CCI­D7 and CCI­D14 groups were significantly upregulated compared with the sham operated group (P<0.05; n=12). Additionally, it was revealed that PAR2 and TMEM16A were co­expressed in DRG neurons. It was also observed that IP3 significantly increased in the CCI­D7 and CCI­D14 groups compared with the sham operation group (P<0.05; n=6) as PAR2 and TMEM16A also increased. These findings suggest that the upregulation of PAR2 and TMEM16A in DRG neurons, the co­expression of the two proteins and increasing IP3 are critical to the development of neuropathic pain.


Subject(s)
Anoctamin-1/metabolism , Neuralgia/pathology , Receptor, PAR-2/metabolism , Animals , Enzyme-Linked Immunosorbent Assay , Ganglia, Spinal/metabolism , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/analysis , Male , Microscopy, Fluorescence , Neuralgia/metabolism , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Up-Regulation
2.
J Cell Sci ; 130(21): 3728-3739, 2017 Nov 01.
Article in English | MEDLINE | ID: mdl-28893841

ABSTRACT

The building blocks of intracellular Ca2+ signals evoked by inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ puffs, transient focal increases in Ca2+ concentration that reflect the opening of small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs evoked by photolysis of caged IP3 or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. Ca2+ puffs evoked by carbachol initiated at an estimated 65±7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged IP3 evoked Ca2+ puffs at a similar number of sites (100±35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential stimulation with carbachol or photolysed caged IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites and the sites become more likely to fire as the IP3 concentration increases; there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.


Subject(s)
Calcium Signaling , Calcium/metabolism , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Action Potentials/drug effects , Action Potentials/physiology , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate Receptors/genetics , Microscopy, Fluorescence , Photolysis , Receptors, Muscarinic/genetics , Receptors, Muscarinic/metabolism
3.
Cold Spring Harb Protoc ; 2015(3): 289-92, 2015 Mar 02.
Article in English | MEDLINE | ID: mdl-25734061

ABSTRACT

Caged IP3 is an inactive form of the second messenger IP3, consisting of the biologically active molecule linked to a cage group through a photolabile bond. This bond is cleaved by exposure to brief "flashes" of ultraviolet (UV) light, thereby releasing the active IP3 molecule. The protection of caged IP3 against metabolic transformation in combination with a defined time point of fast photoliberation of IP3 provides an efficient way to temporally and spatially control the cytosolic release of IP3 and subsequent increase of cytoplasmic Ca(2+). These properties make it an ideal method for kinetic studies and also a well-suited procedure to initiate intercellular Ca(2+) waves from a point source of IP3. This protocol describes cell loading with membrane impermeable caged IP3 and the UV flash illumination procedure.


Subject(s)
Inositol 1,4,5-Trisphosphate/analogs & derivatives , Photolysis , Ultraviolet Rays , Animals , Calcium/metabolism , Cell Line , Cytosol/chemistry , Humans , Inositol 1,4,5-Trisphosphate/radiation effects
4.
Cold Spring Harb Protoc ; 2015(3): 239-49, 2015 Mar 02.
Article in English | MEDLINE | ID: mdl-25734071

ABSTRACT

Many cellular functions are driven by variations in the intracellular Ca(2+) concentration ([Ca(2+)]i), which may appear as a single-event transient [Ca(2+)]i elevation, repetitive [Ca(2+)]i increases known as Ca(2+) oscillations, or [Ca(2+)]i increases propagating in the cytoplasm as Ca(2+) waves. Additionally, [Ca(2+)]i changes can be communicated between cells as intercellular Ca(2+) waves (ICWs). ICWs are mediated by two possible mechanisms acting in parallel: one involving gap junctions that form channels directly linking the cytoplasm of adjacent cells and one involving a paracrine messenger, in most cases ATP, that is released into the extracellular space, leading to [Ca(2+)]i changes in neighboring cells. The intracellular messenger inositol 1,4,5-trisphosphate (IP3) that triggers Ca(2+) release from Ca(2+) stores is crucial in these two ICW propagation scenarios, and is also a potent trigger to initiate ICWs. Loading inactive, "caged" IP3 into cells followed by photolytic "uncaging" with UV light, thereby liberating IP3, is a well-established method to trigger [Ca(2+)]i changes in single cells that is also effective in initiating ICWs. We here describe a method to load cells with caged IP3 by local electroporation of monolayer cell cultures and to apply flash photolysis to increase intracellular IP3 and induce [Ca(2+)]i changes, or initiate ICWs. Moreover, the electroporation method allows loading of membrane-impermeable agents that interfere with IP3 and Ca(2+) signaling.


Subject(s)
Calcium Signaling , Electroporation/methods , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Photolysis/radiation effects , Adenosine Triphosphate/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate/radiation effects
5.
Chembiochem ; 15(18): 2774-82, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25399672

ABSTRACT

Inositol 1,4,5-trisphosphate (IP3 ) is a universal signalling molecule that releases calcium from stores within cells by activating the IP3 receptor. Although chemical tools that modulate the IP3 receptor exist, none is ideal due to trade offs between potency, selectivity and cell permeability, and their chemical properties make them challenging starting points for optimisation. Therefore, to find new leads, we used virtual screening to scaffold hop from IP3 by using the program ROCS to perform a 3D ligand-based screen of the ZINC database of purchasable compounds. We then used the program FRED to dock the top-ranking hits into the IP3 binding pocket of the receptor. We tested the 12 highest-scoring hits in a calcium-release bioassay and identified SI-9 as a partial agonist. SI-9 competed with [(3) H]IP3 binding, and reduced histamine-induced calcium signalling in HeLa cells. SI-9 has a novel 2D scaffold that represents a tractable lead for designing improved IP3 receptor modulators.


Subject(s)
Inositol 1,4,5-Trisphosphate Receptors/agonists , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Calcium/metabolism , Drug Design , HeLa Cells , Humans , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Ligands , Molecular Docking Simulation
6.
Br J Pharmacol ; 171(3): 646-62, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24117140

ABSTRACT

BACKGROUND AND PURPOSE: While selective, bitter tasting, TAS2R agonists can relax agonist-contracted airway smooth muscle (ASM), their mechanism of action is unclear. However, ASM contraction is regulated by Ca²âº signalling and Ca²âº sensitivity. We have therefore investigated how the TAS2R10 agonists chloroquine, quinine and denotonium regulate contractile agonist-induced Ca²âº signalling and sensitivity. EXPERIMENTAL APPROACH: Airways in mouse lung slices were contracted with either methacholine (MCh) or 5HT and bronchodilation assessed using phase-contrast microscopy. Ca²âº signalling was measured with 2-photon fluorescence microscopy of ASM cells loaded with Oregon Green, a Ca²âº-sensitive indicator (with or without caged-IP3). Effects on Ca²âº sensitivity were assessed on lung slices treated with caffeine and ryanodine to permeabilize ASM cells to Ca²âº . KEY RESULTS: The TAS2R10 agonists dilated airways constricted by either MCh or 5HT, accompanied by inhibition of agonist-induced Ca²âº oscillations. However, in non-contracted airways, TAS2R10 agonists, at concentrations that maximally dilated constricted airways, did not evoke Ca²âº signals in ASM cells. Ca²âº increases mediated by the photolysis of caged-IP3 were also attenuated by chloroquine, quinine and denotonium. In Ca²âº-permeabilized ASM cells, the TAS2R10 agonists dilated MCh- and 5HT-constricted airways. CONCLUSIONS AND IMPLICATIONS: TAS2R10 agonists reversed bronchoconstriction by inhibiting agonist-induced Ca²âº oscillations while simultaneously reducing the Ca²âº sensitivity of ASM cells. Reduction of Ca²âº oscillations may be due to inhibition of Ca²âº release through IP3 receptors. Further characterization of bronchodilatory TAS2R agonists may lead to the development of novel therapies for the treatment of bronchoconstrictive conditions.


Subject(s)
Bronchodilator Agents/pharmacology , Calcium Signaling/drug effects , Chloroquine/pharmacology , Lung/drug effects , Muscle, Smooth/drug effects , Quinine/pharmacology , Receptors, G-Protein-Coupled/agonists , Airway Resistance/drug effects , Animals , Caffeine/antagonists & inhibitors , Caffeine/pharmacology , Calcium Channel Blockers/chemistry , Calcium Channel Blockers/pharmacology , Drug Resistance/drug effects , Female , In Vitro Techniques , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate Receptors/agonists , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Lung/cytology , Lung/metabolism , Mice , Mice, Inbred BALB C , Muscle Relaxation/drug effects , Muscle, Smooth/cytology , Muscle, Smooth/metabolism , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacology , Quaternary Ammonium Compounds/pharmacology , Receptors, G-Protein-Coupled/metabolism , Ryanodine/antagonists & inhibitors , Ryanodine/pharmacology , Taste
7.
Cell Calcium ; 53(2): 94-101, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23131569

ABSTRACT

Ca(2+) and cAMP signalling pathways interact in a complex manner at multiple sites. This crosstalk fine-tunes the spatiotemporal patterns of Ca(2+) and cAMP signals. In salivary glands of the blowfly Calliphora vicina fluid secretion is stimulated by serotonin (5-hydroxytryptamine, 5-HT) via activation of two different 5-HT receptors coupled to the InsP(3)/Ca(2+) (Cv5-HT(2α)) or the cAMP pathway (Cv5-HT(7)), respectively. We have shown recently in permeabilized gland cells that cAMP sensitizes InsP(3)-induced Ca(2+) release to InsP(3). Here we study the effects of the cAMP signalling pathway on 5-HT-induced oscillations in transepithelial potential (TEP) and in intracellular [Ca(2+)]. We show: (1) Blocking the activation of the cAMP pathway by cinanserin suppresses the generation of TEP and Ca(2+) oscillations, (2) application of 8-CPT-cAMP in the presence of cinanserin restores 5-HT-induced TEP and Ca(2+) oscillations, (3) 8-CPT-cAMP sensitizes the InsP(3)/Ca(2+) signalling pathway to 5-HT and the Cv5-HT(2α) receptor agonist 5-MeOT, (4) 8-CPT-cAMP induces Ca(2+) oscillations in cells loaded with subthreshold concentrations of InsP(3), (5) inhibition of protein kinase A by H-89 abolishes 5-HT-induced TEP and Ca(2+) spiking and mimics the effect of cinanserin. These results suggest that activation of the cyclic AMP pathway promotes the generation of 5-HT-induced Ca(2+) oscillations in blowfly salivary glands.


Subject(s)
Calcium/metabolism , Diptera/physiology , Salivary Glands/drug effects , Serotonin/pharmacology , Animals , Cinanserin/pharmacology , Cyclic AMP/analogs & derivatives , Cyclic AMP/metabolism , Cyclic AMP/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Epithelium/drug effects , Epithelium/physiology , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/metabolism , Isoquinolines/pharmacology , Membrane Potentials/drug effects , Receptor, Serotonin, 5-HT2A/metabolism , Salivary Glands/metabolism , Signal Transduction/drug effects , Sulfonamides/pharmacology , Thionucleotides/pharmacology
8.
Nat Protoc ; 6(3): 327-37, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21372813

ABSTRACT

This protocol describes a method for efficient chemical synthesis of an analog of inositol-1,4,5-trisphosphate (IP(3)) hexakis acetoxymethyl ester having an ortho-nitroveratryl photochemical caging group on the 6-hydroxyl position. The six esters render the probe membrane permeant, such that it can be loaded into intact living cells in vitro or in vivo. Inside cells, the caged IP(3) is inert until activated by two-photon excitation at 720 nm. The photoliberated signaling molecule can mobilize release of Ca(2+) from intracellular stores on the endoplasmic reticulum. When co-loaded with the fluorescent Ca(2+) indicator rhod-2, one laser can be used for stimulating and monitoring intracellular Ca(2+) signaling with single-cell resolution. This protocol has chemistry and biology sections; the former describes the organic synthesis of the caged IP(3), which requires 12 d, and the latter an application to a day-long study of astrocyte-regulated neuronal function in living brain slices acutely isolated from rats. As Ca(2+) is the single most important intracellular second messenger and the IP(3)-Ca(2+) signaling cascade is used by many cells to produce increases in Ca(2+) concentration, this method should be widely applicable for the study of a variety of physiological processes in intact biological systems.


Subject(s)
Calcium Signaling , Cell Membrane Permeability , Cell Membrane/metabolism , Inositol 1,4,5-Trisphosphate , Neurons , Animals , Astrocytes/drug effects , Astrocytes/physiology , Calcium/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Fluorescent Dyes , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/chemical synthesis , Neurons/drug effects , Neurons/physiology , Photolysis , Rats , Rats, Sprague-Dawley , Time Factors
9.
Chem Commun (Camb) ; 47(1): 242-4, 2011 Jan 07.
Article in English | MEDLINE | ID: mdl-20938532

ABSTRACT

The syntheses of four D-myo-inositol 1,4,5-trisphosphate (InsP(3)) derivatives, incorporating phosphate bioisosteres at the 5-position, are reported. The methyl phosphate ester and sulfate derivatives retain InsP(3) receptor (InsP(3)R) agonist activity; the compounds that possess a methylphosphonate or a carboxymethyl moiety are InsP(3)R antagonists.


Subject(s)
Inositol 1,4,5-Trisphosphate Receptors/antagonists & inhibitors , Inositol 1,4,5-Trisphosphate/pharmacology , Carbohydrate Conformation , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/chemistry , Structure-Activity Relationship
10.
ACS Chem Biol ; 6(3): 223-8, 2011 Mar 18.
Article in English | MEDLINE | ID: mdl-21158426

ABSTRACT

Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer's disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable K(m) and V(max) with the endogenous PIP(2). This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high-throughput screening of PLC inhibitors.


Subject(s)
Aminoquinolines/chemistry , Enzyme Assays/methods , Fluorescent Dyes/chemistry , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Molecular Probes/chemistry , Type C Phospholipases/analysis , Type C Phospholipases/metabolism , Aminoquinolines/metabolism , Biocatalysis , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , Diglycerides/biosynthesis , Diglycerides/chemistry , Fluorescent Dyes/metabolism , Humans , Inositol 1,4,5-Trisphosphate/biosynthesis , Inositol 1,4,5-Trisphosphate/chemistry , Inositol 1,4,5-Trisphosphate/metabolism , Isoenzymes/analysis , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/metabolism , Kinetics , Molecular Probes/metabolism , Molecular Structure , Molecular Weight , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphatidylinositol 4,5-Diphosphate/metabolism , Stereoisomerism , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/chemistry
11.
ACS Chem Neurosci ; 1(8): 575-85, 2010 Aug 18.
Article in English | MEDLINE | ID: mdl-22778846

ABSTRACT

We have developed a caged IP(3) analogue for two-photon photolysis in living animals. This probe is a cell permeable version and was coloaded with a fluorescent Ca(2+) dye into astrocytes in layer 1 of the somatosensory cortex of anesthetized mice. Two-photon irradiation of single cells at 720 nm produced rapid and robust increases in intracellular Ca(2+) concentrations monitored using two-photon microscopy at 950 nm. The photoevoked intracellular Ca(2+) waves were similar in magnitude to intrinsic signals in wild type mice. These waves did not propagate to other cells beyond the targeted astrocyte. In contrast, we observed intercellular astrocytic Ca(2+) waves in two mouse models of familial Alzheimer's disease. These data suggest that Alzheimer's might perturb gliotransmission but not IP(3) signaling per se in mouse models of the disease.


Subject(s)
Astrocytes/drug effects , Calcium Signaling/drug effects , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Microscopy, Fluorescence, Multiphoton/methods , Alzheimer Disease/genetics , Animals , Astrocytes/physiology , Astrocytes/radiation effects , Calcium Signaling/radiation effects , Disease Models, Animal , Esterases/metabolism , Fluorescent Dyes/analysis , Green Fluorescent Proteins/analysis , Humans , Inositol 1,4,5-Trisphosphate/adverse effects , Inositol 1,4,5-Trisphosphate/pharmacology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Photolysis , Photons , Pia Mater/ultrastructure , Presenilin-1/genetics , Presenilin-2/genetics , Transgenes
12.
Neuron ; 62(6): 826-38, 2009 Jun 25.
Article in English | MEDLINE | ID: mdl-19555651

ABSTRACT

Bursts of spikes triggered by sensory stimuli in midbrain dopamine neurons evoke phasic release of dopamine in target brain areas, driving reward-based reinforcement learning and goal-directed behavior. NMDA-type glutamate receptors (NMDARs) play a critical role in the generation of these bursts. Here we report LTP of NMDAR-mediated excitatory transmission onto dopamine neurons in the substantia nigra. Induction of LTP requires burst-evoked Ca2+ signals amplified by preceding metabotropic neurotransmitter inputs in addition to the activation of NMDARs themselves. PKA activity gates LTP induction by regulating the magnitude of Ca2+ signal amplification. This form of plasticity is associative, input specific, reversible, and depends on the relative timing of synaptic input and postsynaptic bursting in a manner analogous to the timing rule for cue-reward learning paradigms in behaving animals. NMDAR plasticity might thus represent a potential neural substrate for conditioned dopamine neuron burst responses to environmental stimuli acquired during reward-based learning.


Subject(s)
Action Potentials/physiology , Dopamine/metabolism , Mesencephalon/cytology , Neuronal Plasticity/physiology , Neurons/physiology , Receptors, N-Methyl-D-Aspartate/physiology , 2-Amino-5-phosphonovalerate/pharmacology , Action Potentials/drug effects , Animals , Biophysics , Calcium/metabolism , Dose-Response Relationship, Drug , Electric Stimulation/methods , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , In Vitro Techniques , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/pharmacology , Intracellular Signaling Peptides and Proteins/pharmacology , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Male , Methoxyhydroxyphenylglycol/analogs & derivatives , Methoxyhydroxyphenylglycol/pharmacology , Neural Pathways/physiology , Neuronal Plasticity/drug effects , Neurons/drug effects , Peptide Fragments/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Time Factors
13.
J Neurosci ; 29(24): 7833-45, 2009 Jun 17.
Article in English | MEDLINE | ID: mdl-19535595

ABSTRACT

In most neurons postsynaptic [Ca(2+)](i) changes result from synaptic activation opening voltage gated channels, ligand gated channels, or mobilizing Ca(2+) release from intracellular stores. In addition to these changes that result directly from stimulation we found that in pyramidal cells there are spontaneous, rapid, Ca(2+) release events, predominantly, but not exclusively localized at dendritic branch points. They are clearest on the main apical dendrite but also have been detected in the finer branches and in the soma. Typically they have a spatial extent at initiation of approximately 2 microm, a rise time of <15 ms, duration <100 ms, and amplitudes of 10-70% of that generated by a backpropagating action potential at the same location. These events are not caused by background electrical or synaptic activity. However, their rate can be increased by repetitive synaptic stimulation at moderate frequencies, mainly through metabotropic glutamate receptor mobilization of IP(3). In addition, their frequency can be modulated by changes in membrane potential in the subthreshold range, predominantly by affecting Ca(2+) entry through L-type channels. They resemble the elementary events ("sparks" and "puffs") mediated by IP(3) receptors and ryanodine receptors that have been described primarily in non-neuronal preparations. These spontaneous Ca(2+) release events may be the fundamental units underlying some postsynaptic signaling cascades in mature neurons.


Subject(s)
Calcium/metabolism , Dendrites/physiology , Membrane Potentials/physiology , Pyramidal Cells/cytology , Synapses/physiology , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Animals , Animals, Newborn , Biophysical Phenomena , Caffeine/pharmacology , Calcium Channel Agonists/pharmacology , Computer Simulation , Dendrites/drug effects , Electric Stimulation , Excitatory Amino Acid Agents/pharmacology , GABA Antagonists/pharmacology , Hippocampus/cytology , In Vitro Techniques , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/pharmacology , Membrane Potentials/drug effects , Models, Neurological , Organic Chemicals/metabolism , Patch-Clamp Techniques , Phosphodiesterase Inhibitors/pharmacology , Picrotoxin/pharmacology , Rats , Rats, Sprague-Dawley , Sodium Channel Blockers/pharmacology , Synapses/drug effects , Tetrodotoxin/pharmacology
14.
Am J Physiol Gastrointest Liver Physiol ; 296(6): G1248-57, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19359429

ABSTRACT

The inflammasome is a cytoplasmic multiprotein complex that has recently been identified in immune cells as an important sensor of signals released by cellular injury and death. Analogous to immune cells, hepatic stellate cells (HSC) also respond to cellular injury and death. Our aim was to establish whether inflammasome components were present in HSC and could regulate HSC functionality. Monosodium urate (MSU) crystals (100 microg/ml) were used to experimentally induce inflammasome activation in LX-2 and primary mouse HSC. Twenty-four hours later primary mouse HSC were stained with alpha-smooth muscle actin and visualized by confocal microscopy, and TGF-beta and collagen1 mRNA expression was quantified. LX-2 cells were further cultured with or without MSU crystals for 24 h in a transwell chemotaxis assay with PDGF as the chemoattractant. We also examined inhibition of calcium (Ca(2+)) signaling in LX-2 cells treated with or without MSU crystals using caged inositol 1,4,5-triphosphate (IP(3)). Finally, we confirmed an important role of the inflammasome in experimental liver fibrosis by the injection of carbon tetrachloride (CCl(4)) or thioacetamide (TAA) in wild-type mice and mice lacking components of the inflammasome. Components of the inflammasome are expressed in LX-2 cells and primary HSC. MSU crystals induced upregulation of TGF-beta and collagen1 mRNA and actin reorganization in HSCs from wild-type mice but not mice lacking inflammasome components. MSU crystals inhibited the release of Ca(2+) via IP(3) in LX-2 cells and also inhibited PDGF-induced chemotaxis. Mice lacking the inflammasome-sensing and adaptor molecules, NLRP3 and apoptosis-associated speck-like protein containing CARD, had reduced CCl(4) and TAA-induced liver fibrosis. We concluded that inflammasome components are present in HSC, can regulate a variety of HSC functions, and are required for the development of liver fibrosis.


Subject(s)
Hepatic Stellate Cells/physiology , Inflammation/physiopathology , Actins/metabolism , Animals , Apoptosis Regulatory Proteins , CARD Signaling Adaptor Proteins , Calcium Signaling/drug effects , Carbon Tetrachloride/pharmacology , Carrier Proteins/genetics , Cell Line, Transformed , Chemotaxis/drug effects , Chemotaxis/genetics , Collagen Type I/genetics , Cytoskeletal Proteins/genetics , Gene Expression/drug effects , Gene Expression/genetics , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/drug effects , Humans , Inflammation/chemically induced , Inflammation/metabolism , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/pharmacology , Liver/metabolism , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/etiology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , Platelet-Derived Growth Factor/pharmacology , Thioacetamide/pharmacology , Transforming Growth Factor beta/genetics , Uric Acid/pharmacology
15.
J Neurosci ; 28(1): 133-42, 2008 Jan 02.
Article in English | MEDLINE | ID: mdl-18171931

ABSTRACT

Associative long-term depression (LTD) at cerebellar parallel fiber-Purkinje cell synapses is sensitive to the temporal order in which the parallel fiber is coactivated with the climbing fiber input, but how order sensitivity is achieved is unknown. Here we show that the cerebellar inositol-1,4,5-trisphosphate (IP3) receptor, whose activation is required for LTD induction, is sensitive in situ to the order of presentation of its coagonists, IP3 and cytoplasmic calcium. By focally photolyzing a novel caged IP3 compound in dendritic spines, we find that pairing IP3 with climbing fiber-mediated calcium entry leads to a large calcium release transient if the climbing fiber is activated up to 100 ms before or up to 500 ms after IP3 uncaging. This asymmetric timing window for coactivation follows the kinetics of calcium removal and IP3 unbinding from the receptor and is not limited by IP3 metabolism. IP3 receptor binding thus acts as an eligibility trace that can drive temporal order-dependent calcium release and LTD induction in Purkinje cells and event order-dependent sensory plasticity in the whole animal.


Subject(s)
Cerebellum/cytology , Inositol 1,4,5-Trisphosphate Receptors/physiology , Purkinje Cells/physiology , Animals , Animals, Newborn , Calbindins , Calcium/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Dendritic Spines/metabolism , Electric Stimulation/methods , In Vitro Techniques , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate/pharmacokinetics , Inositol 1,4,5-Trisphosphate/pharmacology , Long-Term Synaptic Depression/physiology , Mice , Mice, Knockout , Patch-Clamp Techniques/methods , Purkinje Cells/cytology , Purkinje Cells/drug effects , Rats , S100 Calcium Binding Protein G/genetics , Time Factors
16.
Bioorg Med Chem ; 16(6): 3419-27, 2008 Mar 15.
Article in English | MEDLINE | ID: mdl-17981044

ABSTRACT

Inositol phosphates, such as 1D-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], are cellular second messengers with potential roles in cancer prevention and therapy. It typically is difficult to attribute specific pharmacological activity to a single inositol phosphate because they are rapidly metabolized by phosphatases and kinases. In this study, we have designed stable analogs of myo-inositol 4,5-bisphosphate [Ins(4,5)P(2)] and Ins(1,4,5)P(3) that retain the cyclohexane scaffold, but lack hydroxyl groups that might be phosphorylated and have phosphate groups replaced with phosphatase-resistant phosphorothioates. An Ins(1,4,5)P(3) analog, 1D-2,3-dideoxy-myo-inositol 1,4,5-trisphosphorothioate, was synthesized from (-)-quebrachitol, and an Ins(4,5)P(2) analog, 1D-1,2,3-trideoxy-myo-inositol 4,5-bisphosphorothioate, was prepared from cyclohexenol. The Ins(1,4,5)P(3) analog was recognized by Ins(1,4,5)P(3) receptor with a binding constant (K(d)) of 810 nM, compared with 54 nM for the native ligand Ins(1,4,5)P(3), and was resistant to dephosphorylation by alkaline phosphatase under conditions in which Ins(1,4,5)P(3) is extensively hydrolyzed. Analogs developed in this study are potential chemical probes for understanding mechanisms of inositol phosphate actions that may be elucidated by eliciting specific and prolonged activation of the Ins(1,4,5)P(3) receptor.


Subject(s)
Alkaline Phosphatase/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/chemistry , Inositol Phosphates/chemistry , Animals , Drug Stability , Humans , Phosphates , Protein Binding
17.
J Neurosci ; 27(50): 13813-21, 2007 Dec 12.
Article in English | MEDLINE | ID: mdl-18077693

ABSTRACT

The inositol 1,4,5-trisphosphate (InsP3) receptor type I (InsP3R-I) is the principle channel for intracellular calcium (Ca2+) release in many cell types, including central neurons. It is regulated by endogenous compounds like Ca2+ and ATP, by protein partners, and by posttranslational modification. We report that the InsP3R-I is modified by O-linked glycosylation of serine or threonine residues with beta-N-acetylglucosamine (O-GlcNAc). The level of O-GlcNAcylation can be altered in vitro by the addition of the enzymes which add [OGT (O-GlcNActransferase)] or remove (O-GlcNAcase) this sugar or by loading cells with UDP-GlcNAc. We monitored the effects of this modification on InsP3R function at the single-channel level and on intracellular Ca2+ transients. Single-channel activity was monitored with InsP3R incorporated into bilayers; Ca2+ signaling was monitored using cells loaded with a Ca2+-sensitive fluorophore. We found that channel activity was decreased by the addition of O-GlcNAc and that this decrease was reversed by removal of the sugar. Similarly, cells loaded with UDP-GlcNAc had an attenuated response to uncaging of InsP3. These results show that O-GlcNAcylation is an important regulator of the InsP3R-I and suggest a mechanism for neuronal dysfunction under conditions in which O-GlcNAc is high, such as diabetes or physiological stress.


Subject(s)
Glucosamine/analogs & derivatives , Inositol 1,4,5-Trisphosphate Receptors/chemistry , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Interneurons/metabolism , Animals , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cells, Cultured , Cerebellum/chemistry , Cerebellum/metabolism , Glucosamine/chemistry , Glucosamine/metabolism , Glycosylation , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate/pharmacology , Interneurons/drug effects , Lipid Bilayers/chemistry , Mice , Microsomes/chemistry , N-Acetylglucosaminyltransferases/chemistry , N-Acetylglucosaminyltransferases/metabolism , Organ Culture Techniques , Patch-Clamp Techniques , Rats , Ultraviolet Rays , Uridine Diphosphate N-Acetylglucosamine/metabolism , Uridine Diphosphate N-Acetylglucosamine/pharmacology , beta-N-Acetylhexosaminidases/chemistry
18.
J Biol Chem ; 282(35): 25517-26, 2007 Aug 31.
Article in English | MEDLINE | ID: mdl-17540776

ABSTRACT

Biological messengers can be "caged" by adding a single photosensitive group that can be photolyzed by a light flash to achieve spatially and temporally precise biochemical control. Here we report that photolysis of a double-caged form of the second messenger inositol 1,4,5-trisphosphate (IP3) triggers focal calcium release in Purkinje cell somata, dendrites, and spines as measured by two-photon microscopy. In calbindin knock-out Purkinje cells, peak calcium increased with flash energy with higher cooperativity for double-caged IP3 than for conventional single-caged IP3, consistent with a chemical two-photon effect. Spine photolysis of double-caged IP3 led to local calcium release. Uncaging of glycerophosphoryl-myo-inositol 4,5-bisphosphate (gPIP2), a poorly metabolizable IP3 analog, led to less well localized release. Thus, IP3 breakdown is necessary for spine-specificity. IP3- and gPIP2-evoked signals declined from peak with similar, slow time courses, indicating that release lasts hundreds of milliseconds and is terminated not by IP3 degradation but by intrinsic receptor dynamics. Based on measurements of spine-dendrite coupling, IP3-evoked calcium signals are expected to be at least 2.4-fold larger in their spine of origin than in nearby spines, allowing IP3 to act as a synapse-specific second messenger. Unexpectedly, single-caged IP3 led to less release in somata and was ineffective in dendrites and spines. Calcium release using caged gPIP2 was inhibited by the addition of single-caged IP3, suggesting that single-caged IP3 is an antagonist of calcium release. Caging at multiple sites may be an effective general approach to reducing residual receptor interaction.


Subject(s)
Calcium Signaling/drug effects , Calcium/metabolism , Dendrites/metabolism , Inositol 1,4,5-Trisphosphate/pharmacology , Inositol Phosphates/pharmacology , Purkinje Cells/metabolism , Spine/metabolism , Animals , Calbindins , Calcium/antagonists & inhibitors , Calcium Signaling/physiology , Calcium-Binding Proteins/deficiency , Calcium-Binding Proteins/metabolism , Evoked Potentials/drug effects , Evoked Potentials/physiology , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/chemistry , Inositol Phosphates/chemistry , Mice , Mice, Knockout , Microscopy, Fluorescence, Multiphoton , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/metabolism , Organ Specificity/physiology , Photolysis , Purkinje Cells/cytology , Rats , S100 Calcium Binding Protein G , Spine/cytology
19.
ChemMedChem ; 2(9): 1281-9, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17589888

ABSTRACT

We describe the synthesis of four novel metabolically stabilized analogues of Ins(1,4,5)P(3) based on the known cyclopentane pentaol tris(phosphate) 2: tris(phosphorothioate) 3, tris(methylenephosphate) 4, tris(sulfonamide) 5, and tris(sulfate) 6. Of these analogues, only the tris(phosphorothioate) 3 and parent tris(phosphate) 2 bound to the type I InsP(3)R construct. In addition, both the tris(phosphorothioate) 3 and parent tris(phosphate) 2 elicited calcium release in MDA MB-435 breast cancer cells. The Ins(1,4,5)P(3) agonist activities of these two compounds can be rationalized on the basis of computational docking of the ligands to the binding domain of the type I InsP(3)R.


Subject(s)
Inositol 1,4,5-Trisphosphate/analogs & derivatives , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/pharmacology , Crystallography, X-Ray , Inositol 1,4,5-Trisphosphate/chemical synthesis , Inositol 1,4,5-Trisphosphate/pharmacology , Magnetic Resonance Spectroscopy , Organophosphorus Compounds/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
20.
Biochim Biophys Acta ; 1761(9): 1114-31, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16956790

ABSTRACT

Thromboxane (TX) A(2) is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA(2) mediated through the TPbeta isoform of the human TXA(2) receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPbeta. It was established that TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA(2) mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser(145) within intracellular domain (IC)(2) has been identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser(239) and Ser(357) within its IC(3) and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser(239) or Ser(357), through site directed mutagenesis, impaired desensitization while mutation of both Ser(239) and Ser(357) almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser(145) partially impairs. TPbeta signalling while GRK2/3 phosphorylation at both Ser(239) and Ser(357) within its IC(3) and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization. Thromboxane (TX) A(2) is a potent stimulator of platelet aggregation and smooth muscle contraction and contributes to a variety of vascular pathologies. Herein the mechanism whereby the cellular responses to TXA(2) mediated through the TPbeta isoform of the human TXA(2) receptor (TP) are dynamically regulated was investigated by examining the mechanism of its agonist-induced desensitization of intracellular signalling and second messenger generation. TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA(2) mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser(145) within intracellular domain (IC)(2) was identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser(239) and Ser(357) within its IC(3) and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser(239) or Ser(357), through site directed mutagenesis, impaired desensitization while mutation of both Ser(239) and Ser(357) almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser(145) partially impairs TPbeta signalling while GRK2/3 phosphorylation at both Ser(239) and Ser(357) within its IC(3) and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization.


Subject(s)
Receptors, Thromboxane A2, Prostaglandin H2/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Amino Acid Sequence , Arrestins/metabolism , Calcium/metabolism , Cell Line , G-Protein-Coupled Receptor Kinase 3 , Humans , Indoles/pharmacology , Inositol 1,4,5-Trisphosphate/analogs & derivatives , Inositol 1,4,5-Trisphosphate/metabolism , Maleimides/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Protein Isoforms/agonists , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Receptors, Thromboxane A2, Prostaglandin H2/agonists , Receptors, Thromboxane A2, Prostaglandin H2/antagonists & inhibitors , Second Messenger Systems , Serine/metabolism , Signal Transduction , beta-Arrestins
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