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.

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 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.

Activity of the renal Na+-K+-2Cl− cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl− transport

2006 ◽  
Vol 290 (5) ◽  
pp. F1094-F1102 ◽  
Author(s):  
Anahí Paredes ◽  
Consuelo Plata ◽  
Manuel Rivera ◽  
Erika Moreno ◽  
Norma Vázquez ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Fluorescent Protein ◽  
Functional Expression ◽  
Site Directed Mutagenesis ◽  
Intrinsic Activity ◽  
Xenopus Laevis Oocytes ◽  
Type I ◽  
Wild Type ◽  
Glycosylation Sites ◽  
Green Fluorescent

The renal-specific Na+-K+-2Cl− cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na+-Cl− cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by 86Rb+ influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced ∼20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl− affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

Lack of the Kir4.1 Channel Subunit Abolishes K+ Buffering Properties of Astrocytes in the Ventral Respiratory Group: Impact on Extracellular K+ Regulation

2006 ◽  
Vol 95 (3) ◽  
pp. 1843-1852 ◽  
Author(s):  
Clemens Neusch ◽  
Nestoras Papadopoulos ◽  
Michael Müller ◽  
Iris Maletzki ◽  
Stefan M. Winter ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Respiratory Rhythm ◽  
Brain Slices ◽  
Physiological Role ◽  
Functional Expression ◽  
Network Activity ◽  
Early Postnatal Development ◽  
Ventral Respiratory Group ◽  
Respiratory Network ◽  
Green Fluorescent

Ongoing rhythmic neuronal activity in the ventral respiratory group (VRG) of the brain stem results in periodic changes of extracellular K+. To estimate the involvement of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in extracellular K+ clearance, we examined its functional expression in astrocytes of the respiratory network. Kir4.1 was expressed in astroglial cells of the VRG, predominantly in fine astrocytic processes surrounding capillaries and in close proximity to VRG neurons. Kir4.1 expression was up-regulated during early postnatal development. The physiological role of astrocytic Kir4.1 was studied using mice with a null mutation in the Kir4.1 channel gene that were interbred with transgenic mice expressing the enhanced green fluorescent protein in their astrocytes. The membrane potential was depolarized in astrocytes of Kir4.1−/− mice, and Ba2+-sensitive inward K+ currents were diminished. Brain slices from Kir4.1−/− mice, containing the pre-Bötzinger complex, which generates a respiratory rhythm, did not show any obvious differences in rhythmic bursting activity compared with wild-type controls, indicating that the lack of Kir4.1 channels alone does not impair respiratory network activity. Extracellular K+ measurements revealed that Kir4.1 channels contribute to extracellular K+ regulation. Kir4.1 channels reduce baseline K+ levels, and they compensate for the K+ undershoot. Our data indicate that Kir4.1 channels 1) are expressed in perineuronal processes of astrocytes, 2) constitute the major part of the astrocytic Kir conductance, and 3) contribute to regulation of extracellular K+ in the respiratory network.

scholarly journals Functional properties of human ferroportin, a cellular iron exporter reactive also with cobalt and zinc

2014 ◽  
Vol 306 (5) ◽  
pp. C450-C459 ◽  
Author(s):  
Colin J. Mitchell ◽  
Ali Shawki ◽  
Tomas Ganz ◽  
Elizabeta Nemeth ◽  
Bryan Mackenzie
Keyword(s):  
Iron Homeostasis ◽  
Fluorescent Protein ◽  
Serum Zinc ◽  
Functional Expression ◽  
Extracellular Ph ◽  
Zinc Homeostasis ◽  
Affinity Constant ◽  
Protein Fusion ◽  
Zinc Levels ◽  
Green Fluorescent

Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Iron export from enterocytes or macrophages to blood plasma is thought to be mediated by ferroportin under the control of hepcidin. Although ferroportin was identified over a decade ago, little is understood about how it works. We expressed in Xenopus oocytes a human ferroportin-enhanced green fluorescent protein fusion protein and observed using confocal microscopy its exclusive plasma-membrane localization. As a first step in its characterization, we established an assay to detect functional expression of ferroportin by microinjecting oocytes with 55Fe and measuring efflux. Ferroportin expression increased the first-order rate constants describing 55Fe efflux up to 300-fold over control. Ferroportin-mediated 55Fe efflux was saturable, temperature-dependent (activation energy, Ea ≈ 17 kcal/mol), maximal at extracellular pH ≈ 7.5, and inactivated at extracellular pH < 6.0. We estimated that ferroportin reacts with iron at its intracellular aspect with apparent affinity constant < 10−7 M. Ferroportin expression also stimulated efflux of 65Zn and 57Co but not of 64Cu, 109Cd, or 54Mn. Hepcidin treatment of oocytes inhibited efflux of 55Fe, 65Zn, and 57Co. Whereas hepcidin administration in mice resulted in a marked hypoferremia within 4 h, we observed no effect on serum zinc levels in those same animals. We conclude that ferroportin is an iron-preferring cellular metal-efflux transporter with a narrow substrate profile that includes cobalt and zinc. Whereas hepcidin strongly regulated serum iron levels in the mouse, we found no evidence that ferroportin plays an important role in zinc homeostasis.

scholarly journals A novel GABA-mediated corticotropin-releasing hormone secretory mechanism in the median eminence

2016 ◽  
Vol 2 (8) ◽  
pp. e1501723 ◽  
Author(s):  
Keisuke Kakizawa ◽  
Miho Watanabe ◽  
Hiroki Mutoh ◽  
Yuta Okawa ◽  
Miho Yamashita ◽  
...  
Keyword(s):  
Median Eminence ◽  
Fluorescent Protein ◽  
Corticotropin Releasing Hormone ◽  
Releasing Hormone ◽  
Axon Terminals ◽  
Close Proximity ◽  
Excitatory Action ◽  
Gaba Content ◽  
Neuron Terminals ◽  
Green Fluorescent

Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)–containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67+/GFP), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na+-K+-2Cl− cotransporter (NKCC1), but not the K+-Cl− cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl− concentrations ([Cl−]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca2+) levels in the CRH neuron terminals but decreased the Ca2+ levels in their somata. In addition, the increases in Ca2+ concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME.

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.

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