Taste Receptor Cell Responses to the Bitter Stimulus Denatonium Involve Ca2+ Influx Via Store-Operated Channels

2002 ◽  
Vol 87 (6) ◽  
pp. 3152-3155 ◽  
Author(s):  
Tatsuya Ogura ◽  
Robert F. Margolskee ◽  
Sue C. Kinnamon
Keyword(s):  
Prolonged Exposure ◽  
Fluorescent Protein ◽  
Bitter Taste ◽  
Taste Receptor ◽  
Taste Receptor Cell ◽  
Cell Responses ◽  
Necturus Maculosus ◽  
Taste Cells ◽  
Intracellular Ca ◽  
Green Fluorescent

Previous studies in rat and mouse have shown that brief exposure to the bitter stimulus denatonium induces an increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores, rather than Ca2+influx. We report here that prolonged exposure to denatonium induces sustained increases in [Ca2+]i that are dependent on Ca2+ influx. Similar results were obtained from taste cells of the mudpuppy, Necturus maculosus, as well as green fluorescent protein (GFP) tagged gustducin-expressing taste cells of transgenic mice. In a subset of mudpuppy taste cells, prolonged exposure to denatonium induced oscillatory Ca2+responses. Depletion of Ca2+ stores by thapsigargin also induced Ca2+ influx, suggesting that Ca2+store-operated channels (SOCs) are present in both mudpuppy taste cells and gustducin-expressing taste cells of mouse. Further, treatment with thapsigargin prevented subsequent responses to denatonium, suggesting that the SOCs were the source of the Ca2+ influx. These data suggest that SOCs may contribute to bitter taste transduction and to regulation of Ca2+ homeostasis in taste cells.

scholarly journals Nasal Solitary Chemoreceptor Cell Responses to Bitter and Trigeminal Stimulants In Vitro

2008 ◽  
Vol 99 (6) ◽  
pp. 2929-2937 ◽  
Author(s):  
Brian D. Gulbransen ◽  
Tod R. Clapp ◽  
Thomas E. Finger ◽  
Sue C. Kinnamon
Keyword(s):  
Fluorescent Protein ◽  
Bitter Taste ◽  
Taste Receptor ◽  
Receptor Ligands ◽  
Isolated Cells ◽  
Taste System ◽  
Intracellular Ca ◽  
Green Fluorescent

Nasal trigeminal chemosensitivity in mice and rats is mediated in part by epithelial solitary chemoreceptor (chemosensory) cells (SCCs), but the exact role of these cells in chemoreception is unclear. Histological evidence suggests that SCCs express elements of the bitter taste transduction pathway including T2R (bitter taste) receptors, the G protein α-gustducin, PLCβ2, and TRPM5, leading to speculation that SCCs are the receptor cells that mediate trigeminal nerve responses to bitter taste receptor ligands. To test this hypothesis, we used calcium imaging to determine whether SCCs respond to classic bitter-tasting or trigeminal stimulants. SCCs from the anterior nasal cavity were isolated from transgenic mice in which green fluorescent protein (GFP) expression was driven by either TRPM5 or gustducin. Isolated cells were exposed to a variety of test stimuli to determine which substances caused an increase in intracellular Ca2+ ([Ca2+]i). GFP-positive cells respond with increased [Ca2+]i to the bitter receptor ligand denatonium and this response is blocked by the PLC inhibitor U73122. In addition, GFP+ cells respond to the neuromodulators adenosine 5′-triphosphate and acetylcholine but only very rarely to other bitter-tasting or trigeminal stimuli. Our results demonstrate that TRPM5- and gustducin-expressing nasal SCCs respond to the T2R agonist denatonium via a PLC-coupled transduction cascade typical of T2Rs in the taste system.

scholarly journals Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic β-cells

2005 ◽  
Vol 185 (1) ◽  
pp. 57-67 ◽  
Author(s):  
L B Hays ◽  
B Wicksteed ◽  
Y Wang ◽  
J F McCuaig ◽  
L H Philipson ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Zymogen Granule ◽  
Β Cells ◽  
Rat Islets ◽  
Intracellular Ca ◽  
Specific Localization ◽  
Glucagon Like Peptide 1 ◽  
Green Fluorescent ◽  
Insulin Exocytosis

Several proteins play a role in the mechanism of insulin exocytosis. However, these ‘exocytotic proteins’ have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet β-cells as experimental tools. Syncollin is not normally expressed in β-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of β-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5μM). Consistent with specific localization of syncollin to β-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in β-cells has highlighted the importance of intralumenal β-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a ‘fluorescent tag’ to track β-granule transport and exocytosis in real time.

Ca2+ mobilization through dorsal root ganglion Ca2+-sensing receptor stably expressed in HEK293 cells

2007 ◽  
Vol 292 (5) ◽  
pp. C1895-C1905 ◽  
Author(s):  
Emmanuel M. Awumey ◽  
Allyn C. Howlett ◽  
James W. Putney ◽  
Debra I. Diz ◽  
Richard D. Bukoski
Keyword(s):  
Dorsal Root Ganglion ◽  
Fusion Protein ◽  
Dorsal Root ◽  
Fluorescent Protein ◽  
Hek293 Cells ◽  
Pcr Analysis ◽  
Hek 293 ◽  
Intracellular Ca ◽  
Egfp Fusion Protein ◽  
Green Fluorescent

The rat dorsal root ganglion (DRG) Ca2+-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca2+ concentration ([Ca2+]i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca2+] ([Ca2+]e) from 0.5 to 1 mM resulted in increases in [Ca2+]i levels, which were blocked by 30 μM 2-aminoethyldiphenyl borate. [Ca2+]e-response studies indicate a Ca2+ sensitivity with an EC50 of 1.75 ± 0.10 mM. NPS R-467 and Gd3+ activated the CaR. When [Ca2+]e was successively raised from 0.25 to 4 mM, peak [Ca2+]i, attained with 0.5 mM, was reduced by ∼50%. Similar reductions were observed with repeated applications of 10 mM Ca2+, 1 and 10 μM NPS R-467, or 50 and 100 μM Gd3+, indicating desensitization of the response. Furthermore, Ca2+ mobilization increased phosphorylated protein kinase C (PKC)α levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca2+ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Cae2+. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca2+]i transients by 49.9 ± 5.2% (at 1 mM Ca2+) and 40.5 ± 6.5% (at 2 mM Ca2+), compared with controls. The findings suggest involvement of PKC in the pathway for Ca2+ mobilization following CaR activation.

scholarly journals Dynamin Regulates Focal Exocytosis in Phagocytosing Macrophages

2003 ◽  
Vol 14 (5) ◽  
pp. 2016-2028 ◽  
Author(s):  
Anke Di ◽  
Deborah J. Nelson ◽  
Vytautas Bindokas ◽  
Mary E. Brown ◽  
Frances Libunao ◽  
...  
Keyword(s):  
Cell Surface ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Glutathione S Transferase ◽  
Particle Uptake ◽  
Cytoplasmic Vesicles ◽  
Intracellular Ca ◽  
Particle Ingestion ◽  
Green Fluorescent

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion (“focal” exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5′-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase•amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein•dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.

Mouse mandibular retromolar taste buds associated with a mucus salivary gland

2021 ◽  
Author(s):  
Quan T Nguyen ◽  
Grace E Beck Coburn ◽  
Amber Valentino ◽  
Bekir Karabucak ◽  
Marco Tizzano
Keyword(s):  
Salivary Gland ◽  
Fluorescent Protein ◽  
Minor Salivary Gland ◽  
Immunohistochemical Analysis ◽  
Taste Buds ◽  
Taste Bud ◽  
Neuronal Marker ◽  
Taste Cells ◽  
Green Fluorescent ◽  
A Minor

Abstract We have characterized a recently rediscovered chemosensory structure at the rear of the mandibular mucosa in the mouse oral cavity originally reported in the 1980s. This consists of unorganized taste buds, not contained within troughs, associated with the ducts of an underlying minor salivary gland. Using whole-mount preparations of transgenic mice expressing green fluorescent protein under the promoter of taste-signaling-specific genes, we determined that the structure contains taste bud clusters and salivary gland orifices at the rear of each mandible, distal to the last molar and anterior to the ascending ramus. Immunohistochemical analysis show in the retromolar taste buds expression of the taste receptors Tas2R131 and T1R3 and taste cascade molecules TrpM5, PLCβ2, and GNAT3, consistent with type II taste cells, and expression of GAD1, consistent with type III taste cells. Furthermore, the neuronal marker CGRP in retromolar mucosa tissue wrapping around TrpM5+ taste buds was observed. RT-PCR showed that retromolar taste buds express all three mouse tas1r genes, 28 of the 35 tas2r genes, and taste transduction signaling genes gnat3, plcb2, and trpm5, making the retromolar TBs similar to other lingual and palate taste buds. Finally, histochemistry demonstrated that the mandibular retromolar secretory gland is a minor salivary gland of mucous type. The mandibular retromolar taste structure may thus play a role in taste sensation and represent a potential novel pharmacological target for taste disorders.

scholarly journals Membrane properties of isolated mudpuppy taste cells.

1988 ◽  
Vol 91 (3) ◽  
pp. 351-371 ◽  
Author(s):  
S C Kinnamon ◽  
S D Roper
Keyword(s):  
Bitter Taste ◽  
Taste Receptor ◽  
Membrane Properties ◽  
Basal Cells ◽  
Patch Clamp Technique ◽  
Inward Current ◽  
Taste Cells ◽  
Voltage Dependent ◽  
Inward Currents ◽  
K Currents

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.

Acetylcholine Increases Intracellular Ca2+ Via Nicotinic Receptors in Cultured PDF-Containing Clock Neurons of Drosophila

2004 ◽  
Vol 91 (2) ◽  
pp. 912-923 ◽  
Author(s):  
Christian Wegener ◽  
Yasutaka Hamasaka ◽  
Dick R. Nässel
Keyword(s):  
Fluorescent Protein ◽  
Biological Clock ◽  
Functional Expression ◽  
Muscarinic Agonists ◽  
Larval Brain ◽  
Axon Terminals ◽  
Voltage Dependent ◽  
Dissociated Cell Culture ◽  
Intracellular Ca ◽  
Green Fluorescent

Light entrains the biological clock both in adult and larval Drosophila melanogaster. The Bolwig organ photoreceptors most likely constitute one substrate for this light entrainment in larvae. Acetylcholine (ACh) has been suggested as the neurotransmitter in these photoreceptors, but there is no evidence that ACh signaling is involved in photic input onto circadian pacemaker neurons. Here we demonstrate that the putative targets of the Bolwig photoreceptors, the PDF-containing clock neurons (LNs), in the larval brain express functional ACh receptors (AChRs). With the use of GAL4-UAS-driven expression of green fluorescent protein (GFP), we were able to identify LNs in dissociated cell culture. After loading with the Ca2+-sensitive dye fura-2, we monitored changes in intracellular Ca2+ levels ([Ca2+]i) in GFP-marked LNs while applying candidate neurotransmitters. ACh induced transient increases in [Ca2+]i at physiological concentrations. These increases were dependent on extracellular Ca2+ and Na+ and were likely caused by activation of voltage-dependent Ca2+ channels. Application of nicotinic and muscarinic agonists and antagonists showed that the AChRs on cultured LNs have a nicotinic pharmacology. Antibodies to several subunits of nicotinic AChRs (nAChRs) labeled the putative contact site of the Bolwig organ axon terminals with the dendrites of LNs, as well as dissociated LNs in culture. Our findings support a role of ACh as input factor onto the LNs and suggest that Ca2+ is used as a second messenger mediating cholinergic input within the LNs. Experiments using a more general GAL4-UAS-driven expression of GFP showed that functional expression of nAChRs is a widespread phenomenon in peptidergic neurons.

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