NEUROPEPTIDE MODULATION OF TASTE SIGNALS
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 5R21DC010073-02 from National Institute On Deafness And Other Communication Disorders
Abstract: Many neuroactive peptides, acting at hypothalamic nuclei, are critically important central regulators of food intake via the gut-(blood)-brain axis. Oxytocin is a potent anorexigenic peptide that plays a key role at hypothalamic and hindbrain centers that regulate appetite, satiety and ingestion. Taste strongly influences food selection. Yet, the role of taste has not been integrated into models of appetite and satiety. The premise of this application is that oxytocin, in addition to its central effects, also modulates the peripheral taste signal. Oxytocin knockout mice show altered taste sensitivity and preference, specifically for sweeteners. Our preliminary data show that a membrane receptor for Oxytocin (OxtR) is expressed in a discrete subset of cells within the taste bud, and responds to physiological concentrations of Oxytocin. Based on published reports and our preliminary data, we hypothesize that oxytocin acts on glial-like cells within taste buds, and secondarily alters the sweet-selective sensitivity and output of taste buds. We will test this hypothesis through the following Specific Aims 1 Which taste cells express oxytocin receptor? Using single cell gene expression profiling and immunocytochemistry, we will test this because cell type has implications for function. We will conduct these analyses in wild-type and transgenic mice, PLC22-GFP, GAD-GFP and OxtR-YFP. 2 Is OxtR functional in taste buds and does it influence taste-evoked responses? We will extend our preliminary Ca2+ imaging experiments on taste cells from OxtR-YFP reporter mice. Using laser scanning confocal Ca2+ imaging on a lingual slice preparation, we will evoke responses to taste compounds, and ask whether oxytocin modulates sweet (or other) taste evoked responses. We will also examine if taste-evoked secretion of the afferent transmitter, ATP, is modulated by exposure to oxytocin. These experiments will be carried out on taste tissue from OxtR-YFP and PLC22-GFP. All proposed methodologies to achieve these aims, although highly technical, are routinely performed in our laboratories, and are in our publications. This assures the feasibility of the project. We intend to use this Exploratory Research grant (R21) to launch a new area of investigation that may have significant translational impact. Understanding how peptides (especially those implicated in central pathways for satiety) influence the peripheral taste signal may suggest new avenues to address eating disorders. Several peptides in the brain and gut regulate food intake and are intensely researched. Their malfunction results in overeating or anorexic behaviors, and/or changes in body weight. Ironically, the most intuitive driver of feeding, taste, has not been investigated as a contributor to appetite regulation. We present evidence and propose to study further, how the taste system may be modulated by at least two of the same peptides that control satiety in the brain. This understanding would suggest new pharmacological possibilities to address eating disorders
Keywords: 1, 2-Benzisothiazol-3(2H)-one, 1, 1-dioxide; Address; Altered Taste; Appetite; Appetite Regulation; Area; Behavior; Behavioral; Blood; Body Tissues; Body Weight; Brain; Carbohydrates; Cell Communication and Signaling; Cell Nucleus; Cell Signaling; Cells; Characteristics; Data; Desire for food; Eating; Eating Disorders; Electromagnetic, Laser; Encephalon; Encephalons; Exhibits; Exposure to; Food Intake; Food Selections; Gene Expression Monitoring; Gene Expression Pattern Analysis; Gene Expression Profiling; Glia; Glial Cells; Gustation; Hind Brain; Human; Human, General; Hyperphagia; Hypothalamic structure; Hypothalamus; Image; Ingestion; Intracellular Communication and Signaling; Investigation; Knock-in; Knock-in Mouse; Knockout Mice; Kolliker`s reticulum; Label; Laboratories; Lasers; Mammals, Mice; Mammals, Rodents; Man (Taxonomy); Man, Modern; Membrane; Method LOINC Axis 6; Methodology; Methods; Mice; Mice, Knock-out; Mice, Knockout; Modeling; Mouse Strains; Murine; Mus; Nerve Cells; Nerve Fibers; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; Neuropeptides; Non-neuronal cell; Nucleus; Null Mouse; OXT; Ocytocin; Output; Overeating; Oxytocin; Oxytocin Receptor; Pathway interactions; Peptides; Peripheral; Physiologic; Physiological; Play; Preparation; Profilings, Gene Expression; Publications; Publishing; R01 Mechanism; R01 Program; RPG; Radiation, Laser; Receptor Protein; Recombinant Oxytocin; Reporter; Reporting; Research; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Reticuloendothelial System, Blood; Rhombencephalon; Rodent; Rodentia; Rodentias; Role; Saccharin; Satiation; Satiations; Scanning; Scientific Publication; Sensory; Signal Transduction; Signal Transduction Systems; Signaling; Slice; Source; Stimulus; Sweeteners; Sweetening Agents; System; System, LOINC Axis 4; Taste; Taste Buds; Taste Perception; Testing; Tissues; Transcript Expression Analyses; Transcript Expression Analysis; Transgenic Mice; Type I Cell; Type I Epithelial Receptor Cell; Type III Cell; Type III Epithelial Receptor Cell; anorexigenic peptide; base; biological signal transduction; cell type; experiment; experimental research; experimental study; falls; feeding; hindbrain; hypothalamic; imaging; immunocytochemistry; interest; membrane structure; nerve cement; neuronal; pathway; polyphagia; preference; presynaptic; public health relevance; receptor; research study; response; satiety; sensor; social role; spatial relationship; sweet taste; sweet taste perception
Relevance: RELEVANCE Several peptides in the brain and gut regulate food intake and are intensely researched. Their malfunction results in overeating or anorexic behaviors, and/or changes in body weight. Ironically, the most intuitive driver of feeding, taste, has not been investigated as a contributor to appetite regulation. We present evidence and propose to study further, how the taste system may be modulated by at least two of the same peptides that control satiety in the brain. This understanding would suggest new pharmacological possibilities to address eating disorders
Project start date: 2009-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-06-181
5R21DC010073-02 (2010): $189338
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Nirupa Chaudhari
MOLECULAR PHYSIOLOGY OF GLUTAMATE IN TASTE
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5P01DC003013-04 from National Institute On Deafness And Other Communication Disorders IRG: CDRC
Abstract: Unlike vision and olfaction, taste has not been examined extensively with the modern tools of molecular biology. This Program Project brings a synthesis of molecular biology, cell biology and physiology into the field of taste by analyzing glutamate receptors in taste buds. Glutamate is an important taste stimulus (e.g. monosodium glutamate, MSG). Responses to glutamate have been recorded in sensory fibers and from the brain. Psychophysical and hedonic aspects of glutamate have been researched at great length. Yet, the initial events in glutamate taste, namely the interaction of glutamate with membrane-bound receptors, remains relatively unstudied. Consequently, critical information about glutamate receptors in taste buds--their molecular structure, their localization in taste cells, their function and modulation--is missing. The experiments outlined in this Program Project will provide these important data and will identify the first specific receptor for a taste stimulus. In the long term, this will pave the way for a comprehensive definition of the entire peripheral sequence of events in taste reception--from ligand binding to signal generation in the sensory nerves. The findings will also increase our understanding and awareness of the controversial food additive, MSG. The unifying premise underlying our Program Project is that receptors that transduce the taste of glutamate in taste cells are similar to glutamate receptors in the brain. Brain glutamate receptors form 2 large extended families of ionotropic and metabotropic receptors. We propose to take advantage of recent information about the molecular biology of these receptors to investigate glutamate receptors in taste buds. We will conduct PCR (polymerase chain reaction) with degenerate primers based on brain receptors to search for novel glutamate receptors in taste buds. Selected PCR products from lingual tissue will serve as probes to isolate full-length cDNAs encoding taste-specific glutamate receptors (Chaudhari). We will use in situ hybridization to localize mRNAs to specific taste cells and immunocytochemistry to localize receptor proteins (Roper). We will conduct functional studies of glutamate receptors endogenous to taste cells using microelectrode recording techniques. We will also express cloned glutamate receptors in oocytes and in heterologous mammalian cells and use patch clamp techniques to measure currents elicited by glutamate under different experimental conditions (Kinnamon). Lastly, we will use conditioned taste aversion to investigate the significance for taste of pharmacologically distinct receptors (Roper). By integrating these three approaches in a small, focused Program Project, we will take full advantage of the expertise and enthusiasm of 3 independent, productive, and highly interactive principal investigators. The collective insights gained on the molecular physiology of glutamate in taste will be much greater than could be achieved from the individual efforts of these researchers.
Keywords: glutamate receptor, taste bud
Project start date: 1995-09-01
Project end date: 2000-02-29
5P01DC003013-04 (1998): $486359
5P01DC003013-03 (1997): $467654
Voltage-gated Calcium Channels In Taste Buds
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5R21DC005500-02 from National Institute On Deafness And Other Communication Disorders IRG: ZDC1
Abstract: The peripheral end organs for gustation are taste buds, which transduce information on the quality and concentration of chemical taste stimuli into a coded pattern of activity in postsynaptic afferent nerve fibers. In most neurons, transmitter release at presynaptic terminals is dependent upon voltage-gated calcium channels (VGCCs). Some taste stimuli are known to cause depolarization of taste cell membranes followed by Ca++ entry. Other stimuli apparently do not lead to membrane voltage changes. The goal of this new research program is to begin to address these critical last steps of information processing in taste receptor cells. Specifically, we propose to analyze voltage-gated calcium channels in taste cells. These channels are critical for the function of most neuronal synapses but have not been examined systematically in mammalian taste cells. The Specific Aims for the proposed research are 1) To determine the molecular identities of voltage gated calcium channels present in taste buds. This aim will be carried out using reverse transcriptase-polymerase chain reaction (RT-PCR) on a mixed population of mouse taste buds isolated from circumvallate, foliate and fungiform papillae and the palate. Primer pairs used will be specific for each of the 10 known calcium channel alpha1 subunits (which form the channel pore and determine major functional properties) and for accessory beta, gamma and alpha2-delta subunits, all of which alter important functional properties, including sites for modulation by second messengers. 2) To determine whether the calcium channel types identified in Aim 1 correlate with taste specificities. We will search for co-localization of alpha1 subunits with key proteins involved in taste transduction. 3) To image voltage-gated calcium channel activity in taste receptor cells and determine if channel function is subject to modulation by second messengers relevant in taste transduction. Through these aims, we hope to gain a novel perspective on voltage-gated calcium channels, which play critical roles in all neuronal systems, but have been minimally studied in taste cells to date.
Keywords: biological signal transduction, calcium channel, neural information processing, taste, taste bud, voltage gated channel, neural transmission, palate, protein localization, protein structure function, second messenger, confocal scanning microscopy, immunocytochemistry, in situ hybridization, laboratory mouse, polymerase chain reaction, tissue /cell preparation, western blotting
Project start date: 2002-04-01
Project end date: 2005-03-31
5R21DC005500-02 (2003): $75750
MOLECULAR BIOLOGY OF GLUTAMATE RECEPTORS IN TASTE BUDS
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5P01DC003013-070001 from National Institute On Deafness And Other Communication Disorders
Abstract: The monosodium salt of L-glutamate (MSG) is a distinctive taste stimulus found naturally in protein-rich and other foods. A metabotropic receptor for glutamate, mGluR4, is expressed in rat taste buds and appears to function as a taste receptor for MSG. Ligands acting at mGluR4 mimic the taste of MSG. Recently, we have cloned a novel taste-specific form of mGluR4, that has a severely truncated glutamate-binding site. The receptor, taste-mGluR4, is activated only by high concentrations of glutamate, as expected for a taste receptor. We postulate that the altered binding site accounts for some of the unique features of MSG taste. Functional and molecular evidence also exists for ionotropic glutamate receptors (iGluRs) in taste cells. The goal of Project #1 is to use molecular techniques to address whether taste-mGluR4 alone is the taste receptor for MSG for whether activation of iGluRs also contributes to transduction. We will address this question by comparing the functional properties of cloned receptors from taste cells against the functions of taste cells themselves, to identify the key players in MSG taste transduction. Specifically, we will 1. Test the hypothesis that taste-mGluR4 is a taste receptor for MSG. We will express taste-mGluR4 in CHO cells and measure its response to glutamate, GluR agonists and nucleotides that enhance MSG taste. We will also use Western blots and promoter analyses to test whether taste buds are able to express this novel GluR. 2. Determine if cAMP and/or cGMP are 2nd messengers in glutamate taste transduction as predicted in taste mGluR4 is a taste receptor for MSG. 3. Conduct detailed molecular and functional analyses of a novel iGluR- like sequence we have cloned from taste tissue. We will examine whether this receptor underlies an unusual glutamate-gated ionic conductance found in taste cells, and whether this receptor plays a role in MSG taste. We will also search for additional GluRs in taste tissue in order to achieve a comprehensive of the taste transduction of MSG. The data from these molecular and cellular approaches will be closely coordinated with results from electrophysiological and behavioral analyses of the same questions, conducted from Projects #2 and #3.
Keywords: glutamate, glutamate receptor, molecular biology, receptor expression, taste, taste bud, biological signal transduction, cyclic AMP, cyclic GMP, membrane potential, receptor binding, CHO cell, laboratory rat, polymerase chain reaction, transfection, western blotting
MOLECULAR BASIS OF TASTE CELL SIGNALING
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 5R01DC006308-05 from National Institute On Deafness And Other Communication Disorders
Abstract: Recent studies have identified G protein coupled receptors (GPCRs) that respond to umami, bitter and sweet taste stimuli. Downstream signaling pathways for these GPCRs are beginning to be understood through powerful combinations of biochemical and genetic analyses. With the advances, have come significant discrepancies between physiological/behavioral analyses and molecular studies, especially for umami taste. The mechanisms underlying sour taste are far less understood, and many candidate transducer channels remain as candidates. How taste cells process taste signals and transmit information to sensory afferent fibers is virtually unknown. A critical discrepancy exists between physiological evidence that taste cells respond to multiple taste qualities, and molecular evidence that taste cells appear to express GPCRs for only one quality. The present application addresses these key open questions using newly developed methods to examine the gene expression profile of functionally defined taste cells. We hypothesize that signals from receptor cells converge onto a separate class of output cells within taste buds; only output cells form synapses with sensory afferent fibers. Critical tests of this hypothesis may resolve the current controversy on the breadth of tuning of taste cells. For umami and acid tastes, we will carry out functional imaging on isolated taste cells, using criteria derived from detailed studies in the slice preparation. Such functionally defined taste cells will then be subjected to single-cell RT-PCR and/or differential library screening to identify molecules associated with the functional phenotype. To test our hypothesis on output cells, We will employ mice in which functional cell lineages for cells (a) that express PLCb2 or (b) that synthesize biogenic amines are transgenically labeled with Green Fluorescent Protein (GFP) or b-galactosidase. Functional in situ imaging of taste cells from these mice will allow us to test whether there is a separate category of taste bud output cells, akin to ganglion cells in the retina. Differential library screening will then allow us to begin defining the functional relationship between receptor and output cells
Keywords: Acids; Address; Behavioral; Biochemical Genetics; Biogenic Amines; Categories; Cell Communication and Signaling; Cell Function; Cell Lineage; Cell Process; Cell Signaling; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Complex; Fiber; Functional Imaging; G Protein-Complex Receptor; G-Protein-Coupled Receptors; GFP; Galactosidase; Gene Expression Profile; Genetic analyses; Genetic, Biochemical; Green Fluorescent Proteins; Gustation; Image; In Situ; Intracellular Communication and Signaling; Investigators; Ion Channel; Ionic Channels; Knockout Mice; Label; Laboratories; Libraries; Mammalia; Mammals; Mammals, General; Mammals, Mice; Membrane Channels; Methods; Mice; Mice, Knock-out; Mice, Knockout; Molecular; Murine; Mus; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurons; Null Mouse; Output; Phenotype; Physiologic; Physiologic Imaging; Physiological; Population; Preparation; Process; Programs (PT); Programs [Publication Type]; Proteins; RT-PCR; RTPCR; Receptor Cell; Receptor Protein; Recovery; Reporting; Research Personnel; Researchers; Retina; Reverse Transcriptase Polymerase Chain Reaction; Screening procedure; Sensory; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Slice; Stimulus; Subcellular Process; Synapses; Synaptic; Taste; Taste Buds; Taste Perception; Testing; Transducers; base; biological signal transduction; cDNA Library; computerized data processing; data processing; gangliocyte; ganglion cell; gene expression signature; gene product; genetic analysis; imaging; neuronal; novel; programs; receptor; reverse transcriptase PCR; screening; screenings; sensor; signal processing; sweet taste; sweet taste perception; transcriptome
Project start date: 2005-04-01
Project end date: 2011-02-28
Budget start date: 1-MAR-2009
Budget end date: 28-FEB-2011
5R01DC006308-05 (2009): $311920
5R01DC006308-03 (2007): $316029
Sponsored Links Excellgen http://Excellgen.com
5R01DC006308-02 (2006): $325467
1R01DC006308-01A2 (2005): $333300
Nirupa Chaudhari
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 2R01DC006308-06A1 from National Institute On Deafness And Other Communication Disorders
Abstract: Recent findings indicate that taste buds are much more complex than previously held. Mature taste cells display diverse properties and may communicate among themselves. There are suggestions that chemo-sensory detection by taste receptors is followed by a shaping of the sensory signal within the taste bud prior to transmission to the brain, much as occurs in other sensory system. Yet, the significance of cellular heterogeneity and intercellular signals within the taste bud remain to be explained. We will ask the following questions focused on the most prevalent cells in taste buds, the glial-like Type I cells 1. Do Type I taste cells regulate the ionic environment in taste buds? Spatially buffering K+ is a key function performed by astrocytes in the CNS. Our data suggest that ROMK and certain other apical channels may form such a K+ clearance pathway in taste buds. We will use confocal Ca imaging of taste buds in lingual slices, afferent nerve recordings and behavioral assays to test the effects of K+ accumulation using physiological, pharmacological, and genetic manipulations of ROMK and other regulators of K+ efflux. 2. Do Type I taste cells use GABA as a gliotransmitter to modulate taste signals? Our pilot data suggest that Type I cells are key players in GABAergic circuits in taste buds. We will complete production of a unique transgenic mouse strain in which yellow fluorescent protein (YFP) is expressed only in Type I cells of taste buds. We will then use these and our other transgenic mice to identify the cell-type selective pattern of receptors and confocal Ca2+ imaging to analyze GABA-mediated responses in these cells types, especially with respect to the impact on taste-evoked responses. 3. Do Type I cells arise from local epithelium and what is their lifespan? Cells in taste buds have an estimated average lifespan of 10-14 days. Earlier studies did not confidently resolve taste cell types. We will employ a new technology for birth-dating cells to assess if the different taste cell types have different lifespans and lineages. Through this series of testable hypotheses and powerful new technologies focused on Type I taste bud cells, we will begin to address the larger, and decades-old question why do chemosensory taste cells form communities (i.e. taste buds)? What signals are processed within taste buds and what role in taste reception does ongoing communication among the cells serve? Taste buds detect nutritive and potentially poisonous materials through the coordinated action of distinct types of cells housed in taste buds throughout the oral cavity. We will investigate how cells located in taste buds, but resembling glia, may influence the sensitivity of taste buds. Because the proteins responsible for regulation are in an accessible location, understanding these mechanisms introduces the possibility of pharmacological manipulation of aversive and appetitive taste sensations
Keywords: (beta 1-(2-Deoxyribopyranosyl))thymidine; 4-Aminobutanoic Acid; 4-Aminobutyric Acid; Ablation; Action Potentials; Address; Aminalon; Aminalone; Apical; Astrocytes; Astrocytus; Astroglia; Behavior; Behavioral Assay; Brain; Buccal Cavity; Buffers; Butanoic acid, 4-amino-; Cavitas Oris; Cell Communication and Signaling; Cell Signaling; Cell membrane; Cells; Classification; Communication; Communities; Complex; Cytoplasmic Membrane; Data; Date of birth; Deoxyuridine; Detection; Encephalon; Encephalons; Environment; Enzymes; Epithelium; Esthesia; Expression Profiling; Expression Signature; Funding; GABA; Genetic; Glia; Glial Cells; Goals; Gustation; Head and Neck, Buccal Cavity; Heterogeneity; Housing; Image; Individual; Intracellular Communication and Signaling; Ion Channels, Potassium; K channel; KCNJ1 gene; Kidney; Kolliker`s reticulum; Label; Length; Length of Life; Location; Longevity; Mediating; Membrane Transport Proteins; Membrane Transporters; Molecular Fingerprinting; Molecular Profiling; Mouse Strains; Mouth; Nerve Cells; Nerve Unit; Nervous System, Brain; Neural Cell; Neural Stem Cell; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; Non-neuronal cell; Oral cavity; Output; Pathway interactions; Pattern; Physiologic; Physiological; Plasma Membrane; Potassium Channel; Presynaptic Receptors; Production; Property; Property, LOINC Axis 2; Proteins; Pump; ROMK; Receptor Protein; Regulation; Role; Self-Help Groups; Sensation; Sensory; Series; Shapes; Signal Transduction; Signal Transduction Systems; Signaling; Slice; Stimulus; Suggestion; Support Groups; Synapses; Synaptic; System; System, LOINC Axis 4; Systematics; Taste; Taste Bud Cell; Taste Buds; Taste Perception; Testing; Time; Transgenic Mice; Transmission; Type I Cell; Type I Epithelial Receptor Cell; Type II Cell; Type II Epithelial Receptor Cell; Type III Cell; Type III Epithelial Receptor Cell; Uridine, 2`-deoxy-; Urinary System, Kidney; Work; ing; afferent nerve; base; biological signal transduction; cell type; computerized data processing; data processing; gamma-Aminobutyric Acid; gene product; genetic manipulation; imaging; interest; life span; lifespan; molecuar profile; molecular signature; nerve cement; nerve stem cell; neural progenitor cells; neuronal; neuronal progenitor; neuronal progenitor cells; new technology; paracrine; pathway; plasmalemma; presynaptic; receptor; renal; renal outer medullary potassium channel; response; self help organization; sensor; sensory nerve; sensory system; signal processing; social role; transmission process
Relevance: RELEVANCE Taste buds detect nutritive and potentially poisonous materials through the coordinated action of distinct types of cells housed in taste buds throughout the oral cavity. We will investigate how cells located in taste buds, but resembling glia, may influence the sensitivity of taste buds. Because the proteins responsible for regulation are in an accessible location, understanding these mechanisms introduces the possibility of pharmacological manipulation of aversive and appetitive taste sensations
Project start date: 2003-07-01
Project end date: 2015-08-31
Budget start date: 30-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-10-067
2R01DC006308-06A1 (2010): $325125
MECHANISMS OF SWEET TRANSDUCTION IN MAMMALIAN TASTE BUDS
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 5R01DC006021-07 from National Institute On Deafness And Other Communication Disorders
Abstract: Owing to a worldwide epidemic of obesity, there is enormous interest in understanding physiological mechanisms that regulate body weight. Sweet taste sensitivity is likely to play a significant role in food selection, calorie balance and the onset and progression of disorders such as type II diabetes and obesity. In recent years, taste research has focused on the identity of sweet taste receptors, T1R2+T1R3, and their binding sites for sugars and other natural and synthetic sweeteners. Yet, the downstream transduction events within taste cells following sweet receptor activation are incompletely understood. And, mechanisms modulating the primary sensory signal to produce adaptation are unexplored. In this competing renewal, we will extend studies begun during the previous funding period on mechanisms of sweet transduction. These provide a foundation for understanding the interplay of signaling pathways for sweet taste. In particular, we will focus on the role of cAMP in both transduction and adaptation for sweet stimuli. We will achieve this through the use of a novel transgenic mouse, that we developed, that expresses an inducible fluorescent reporter for cAMP in selected populations of cells. Functional studies on taste buds will include real-time imaging for cAMP in individual taste cells, patch-clamp recordings, and Ca2+ imaging (taste buds that are either isolated from the tongue, or retained in a semi-intact preparation). These will reveal cellular functions in individual taste cells as they respond to sucrose and synthetic sweeteners. We will answer the following questions in two specific aims 1. Is cAMP modulated in sweet-sensitive taste cells? Our transgenic, inducible cAMP reporter will allow us gain spatial and temporal resolution of cAMP modulation in mammalian taste cells. 2. How is the cAMP signal produced and what is its downstream consequence for sweet sensing? We will test whether the cAMP opposes, complements, or refines the well-characterized Ca2+ signal, and whether cAMP plays a role in sweet taste adaptation. These studies will provide important new information about the relative roles of cAMP and phospho- inositide signaling in sweet taste transduction and adaptation, and should provide a foundation for future studies on the role of sweet taste in obesity
Keywords: 3`5`-cyclic ester of AMP; 3, 5 cyclic AMP synthetase; ATP pyrophosphate-lyase (cyclizing); Adenosine Cyclic 3`, 5`-Monophosphate; Adenosine Cyclic Monophosphate; Adenosine, cyclic 3`, 5`-(hydrogen phosphate); Adenyl Cyclase; Adenylate Cyclase; Adenylyl Cyclase; Altered Taste; Animals; Attenuated; Binding; Binding (Molecular Function); Binding Sites; Biochemical; Body Tissues; Body Weight; Cell Communication and Signaling; Cell Function; Cell Process; Cell Signaling; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Combining Site; Complement; Complement Proteins; Cyclic AMP; Data; Diabetes Mellitus, Adult-Onset; Diabetes Mellitus, Ketosis-Resistant; Diabetes Mellitus, Non-Insulin-Dependent; Diabetes Mellitus, Noninsulin Dependent; Diabetes Mellitus, Slow-Onset; Diabetes Mellitus, Stable; Diabetes Mellitus, Type 2; Diabetes Mellitus, Type II; Disease; Disorder; Enzymes; Epidemic; Epithelial; Equilibrium; Event; Food Selections; Foundations; Functional Imaging; Funding; Future; G Protein-Complex Receptor; G-Protein-Coupled Receptors; G-Proteins; GTP-Binding Proteins; GTP-Regulatory Proteins; Genetic; Gnat3 protein, rat; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Gustation; Heterogeneity; Image; Immunofluorescence; Immunofluorescence Immunologic; Immunologic, Immunofluorescence; Individual; Intracellular Communication and Signaling; Intracellular Second Messengers; Investigators; Knockout Mice; Lecithinase C; MODY; Maturity-Onset Diabetes Mellitus; Measures; Mediating; Membrane; Methods; Mice, Knock-out; Mice, Knockout; Molecular Genetic; Molecular Genetics; Molecular Interaction; NIDDM; Non-Insulin Dependent Diabetes; Non-Insulin-Dependent Diabetes Mellitus; Null Mouse; Obesity; PDE; Pathway interactions; Phosphodiesterases; Phospholipase C; Phosphorylation; Physiologic; Physiologic Imaging; Physiological; Play; Population; Preparation; Production; Programs (PT); Programs [Publication Type]; Protein Phosphorylation; Reactive Site; Receptor Activation; Receptor Protein; Relative; Relative (related person); Reporter; Research; Research Personnel; Researchers; Resolution; Role; Saccharose; Second Messenger Systems; Second Messengers; Sensory; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Stimulus; Subcellular Process; Sucrose; Sweeteners; Sweetening Agents; T2D; T2DM; Taste; Taste Buds; Taste Perception; Testing; Time; Tissue Sample; Tissues; Tongue; Transgenic Mice; Transgenic Organisms; Type 2 diabetes; Type II diabetes; Work; adenosine 3`5` monophosphate; adenylcyclase; adiposity; adult onset diabetes; alpha-D-Glucopyranoside, beta-D-fructofuranosyl; balance; balance function; biological signal transduction; cAMP; cell type; corpulence; corpulency; corpulentia; disease/disorder; gustducin; gustducin alpha-3 chain protein, rat; imaging; interest; ketosis resistant diabetes; lipophosphodiesterase I; maturity onset diabetes; membrane structure; novel; obese; obese people; obese person; obese population; patch clamp; pathway; phosphatidylcholine cholinephosphohydrolase; phosphoric diester hydrolase; programs; rat Gnat3 protein; receptor; response; second messenger; social role; sugar; sweet receptor; sweet taste; sweet taste perception; tool; transgenic
Project start date: 2003-04-04
Project end date: 2012-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5R01DC006021-07 (2010): $368294
5R01DC006021-06 (2009): $361181
5R01DC006021-05 (2008): $357080
2R01DC006021-04A1 (2007): $382683
NEUROPEPTIDE MODULATION OF TASTE SIGNALS
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 1R21DC010073-01A1 from National Institute On Deafness And Other Communication Disorders
Abstract: Many neuroactive peptides, acting at hypothalamic nuclei, are critically important central regulators of food intake via the gut-(blood)-brain axis. Oxytocin is a potent anorexigenic peptide that plays a key role at hypothalamic and hindbrain centers that regulate appetite, satiety and ingestion. Taste strongly influences food selection. Yet, the role of taste has not been integrated into models of appetite and satiety. The premise of this application is that oxytocin, in addition to its central effects, also modulates the peripheral taste signal. Oxytocin knockout mice show altered taste sensitivity and preference, specifically for sweeteners. Our preliminary data show that a membrane receptor for Oxytocin (OxtR) is expressed in a discrete subset of cells within the taste bud, and responds to physiological concentrations of Oxytocin. Based on published reports and our preliminary data, we hypothesize that oxytocin acts on glial-like cells within taste buds, and secondarily alters the sweet-selective sensitivity and output of taste buds. We will test this hypothesis through the following Specific Aims 1 Which taste cells express oxytocin receptor? Using single cell gene expression profiling and immunocytochemistry, we will test this because cell type has implications for function. We will conduct these analyses in wild-type and transgenic mice, PLC22-GFP, GAD-GFP and OxtR-YFP. 2 Is OxtR functional in taste buds and does it influence taste-evoked responses? We will extend our preliminary Ca2+ imaging experiments on taste cells from OxtR-YFP reporter mice. Using laser scanning confocal Ca2+ imaging on a lingual slice preparation, we will evoke responses to taste compounds, and ask whether oxytocin modulates sweet (or other) taste evoked responses. We will also examine if taste-evoked secretion of the afferent transmitter, ATP, is modulated by exposure to oxytocin. These experiments will be carried out on taste tissue from OxtR-YFP and PLC22-GFP. All proposed methodologies to achieve these aims, although highly technical, are routinely performed in our laboratories, and are in our publications. This assures the feasibility of the project. We intend to use this Exploratory Research grant (R21) to launch a new area of investigation that may have significant translational impact. Understanding how peptides (especially those implicated in central pathways for satiety) influence the peripheral taste signal may suggest new avenues to address eating disorders. Several peptides in the brain and gut regulate food intake and are intensely researched. Their malfunction results in overeating or anorexic behaviors, and/or changes in body weight. Ironically, the most intuitive driver of feeding, taste, has not been investigated as a contributor to appetite regulation. We present evidence and propose to study further, how the taste system may be modulated by at least two of the same peptides that control satiety in the brain. This understanding would suggest new pharmacological possibilities to address eating disorders
Keywords: 1, 2-Benzisothiazol-3(2H)-one, 1, 1-dioxide; Address; Altered Taste; Appetite; Appetite Regulation; Area; Behavior; Behavioral; Blood; Body Tissues; Body Weight; Brain; Carbohydrates; Cell Communication and Signaling; Cell Nucleus; Cell Signaling; Cells; Characteristics; Data; Desire for food; Eating; Eating Disorders; Electromagnetic, Laser; Encephalon; Encephalons; Exhibits; Exposure to; Food Intake; Food Selections; Gene Expression Monitoring; Gene Expression Pattern Analysis; Gene Expression Profiling; Glia; Glial Cells; Gustation; Hind Brain; Human; Human, General; Hyperphagia; Hypothalamic structure; Hypothalamus; Image; Ingestion; Intracellular Communication and Signaling; Investigation; Knock-in; Knock-in Mouse; Knockout Mice; Kolliker`s reticulum; Label; Laboratories; Lasers; Mammals, Mice; Mammals, Rodents; Man (Taxonomy); Man, Modern; Membrane; Method LOINC Axis 6; Methodology; Methods; Mice; Mice, Knock-out; Mice, Knockout; Modeling; Mouse Strains; Murine; Mus; Nerve Cells; Nerve Fibers; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; Neuropeptides; Non-neuronal cell; Nucleus; Null Mouse; OXT; Ocytocin; Output; Overeating; Oxytocin; Oxytocin Receptor; Pathway interactions; Peptides; Peripheral; Physiologic; Physiological; Play; Preparation; Profilings, Gene Expression; Publications; Publishing; R01 Mechanism; R01 Program; RPG; Radiation, Laser; Receptor Protein; Recombinant Oxytocin; Reporter; Reporting; Research; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Reticuloendothelial System, Blood; Rhombencephalon; Rodent; Rodentia; Rodentias; Role; Saccharin; Satiation; Satiations; Scanning; Scientific Publication; Sensory; Signal Transduction; Signal Transduction Systems; Signaling; Slice; Source; Stimulus; Sweeteners; Sweetening Agents; System; System, LOINC Axis 4; Taste; Taste Buds; Taste Perception; Testing; Tissues; Transcript Expression Analyses; Transcript Expression Analysis; Transgenic Mice; Type I Cell; Type I Epithelial Receptor Cell; Type III Cell; Type III Epithelial Receptor Cell; anorexigenic peptide; base; biological signal transduction; cell type; experiment; experimental research; experimental study; falls; feeding; hindbrain; hypothalamic; imaging; immunocytochemistry; interest; membrane structure; nerve cement; neuronal; pathway; polyphagia; preference; presynaptic; public health relevance; receptor; research study; response; satiety; sensor; social role; spatial relationship; sweet taste; sweet taste perception
Relevance: RELEVANCE Several peptides in the brain and gut regulate food intake and are intensely researched. Their malfunction results in overeating or anorexic behaviors, and/or changes in body weight. Ironically, the most intuitive driver of feeding, taste, has not been investigated as a contributor to appetite regulation. We present evidence and propose to study further, how the taste system may be modulated by at least two of the same peptides that control satiety in the brain. This understanding would suggest new pharmacological possibilities to address eating disorders
Project start date: 2009-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
PFA/PA: PA-06-181
1R21DC010073-01A1 (2009): $229500
MOLECULAR PHYSIOLOGY OF GLUTAMATE IN TASTE
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5P01DC003013-07 from National Institute On Deafness And Other Communication Disorders IRG: ZDC1
Abstract: L-Glutamate, in the form of its monosodium salt (MSG), is a distinctive and potent taste stimulus found naturally in many foods. The goal of the first funding period for this Program Project was to determine if glutamate receptors (GluRs) resembling those at synapses in the brain are found in taste receptor cells and might transduce MSG taste. This goal was approached using molecular biological (Project #1, cell biological metabotropic and one or more ionotropic GluRs are expressed in rat taste bud cells. These receptors are similar to CNS GluRs but have important sequence and functional differences that may imply a role in taste transduction. Lastly, we obtained critical molecular and functional evidence for the role of a novel taste-specific metabotropic glutamate receptor (taste-mGluR4) in transducing the taste of MSG. In the next period, we propose more stringent tests to discriminate whether taste-mGluR4 is necessary and sufficient for MSG taste transduction, or whether additional GluRs might also be involved. This question will be addressed through molecular and functional analysis of taste bud cells and heterologous cells transfected with GluRs cloned from taste buds. We will obtain detailed pharmacological profiles of cloned receptors (Project #1) and compare these data with the responses to pharmacological agents used as taste stimulate in isolated tissues (Project #3) and intact tongues (Project #2). Functional studies will include biochemical measurements of second messengers (Project #1), patch- clamp and calcium imaging of taste receptor cells (Project #2), electrophysiological recordings of gustatory afferent fiber activity (Project #2), and animal behavioral tests (Project #2). This converted multi-disciplinary effort from molecular and cellular levels to animals behavior will serve to elucidate the transduction of MSG s a taste stimulus. The long term of this Program Project is to provide a comprehensive analysis of taste transduction for an important gustatory stimulus.
Keywords: glutamate, glutamate receptor, taste, taste bud
Project start date: 1995-09-01
Project end date: 2004-02-28
5P01DC003013-07 (2002): $679131
5P01DC003013-06 (2001): $659148
Sponsored Links Excellgen http://Excellgen.com
2P01DC003013-05 (2000): $717610
PHYSIOLOGY OF CALCIUM CHANNEL GENES
Nirupa Chaudhari, Professor
Psychologycolorado State University-fort Collins
fort Collins, Co 80523
Grant 3R01GM042652-03S1 from National Institute Of General Medical Sciences IRG: PHY
Abstract: Calcium channels regulate a wide range of cellular activities. Based on physiological and pharmacological evidence, diverse calcium channel types exist and are presumably derived from independent genes. The regulation of these calcium channel genes during normal development and their effects on other genes are poorly understood. This project utilizes naturally occurring mutations in the calcium channel genes of vertebrates to gain insights into the structure of calcium channels and the modifications in electrophysiological and cellular functions that ensue from the altered structures. Muscular dysgenesis (mdg) of mice is a mutation in the gene for the alpha 1 subunit of the skeletal muscle dihydropyridine (DHP) receptor and leads to loss of normal calcium channels and excitation-contraction coupling. However, dysgenic muscle does contain a low level of a mRNA hybridizable with DHP receptor alpha 1 cDNA and also expresses an unusual calcium current. Detailed molecular characterizations (via cDNA cloning) of this DHP receptor mRNA from dysgenic muscle will be carried out to obtain insights into the structural domains within the normal DHP receptor which give rise to its electrophysiological properties. Expression of the "dysgenic mRNA" in heterologous systems and antisense RNA inhibition of expression in dysgenic myotubes will be used to establish definitively whether this mRNA encodes a novel calcium channel. The crooked neck dwarf (cn) chicken and the cardiomyopathic (cm) hamster are known to exhibit functional alterations in their calcium currents. These mutants strains will be investigated along similar molecular genetic lines to determine if the functional alterations arise from mutations in the calcium channel genes in these mutants also. Finally, the effects of the DHP receptor on the accumulation of other mRNAs involved in muscle differentiation will be examined in normal myotubes developing under experimental paralysis in culture. These multi-disciplinary and collaborative studies will provide valuable new insights into the structure-function relationships, and the regulation, of calcium channel genes
Keywords: calcium channel, chemical structure function, cytogenetics, dihydropyridine, electrophysiology, genetic regulation antisense nucleic acid, bioenergetics, biological transport, cell differentiation, complementary DNA, gene expression, gene mutation, hormone receptor, messenger RNA, molecular genetics, muscle cell, muscle disorder chicken, hamster, myotube, paralysis, western blotting
Project start date: 1989-07-01
Project end date: 1995-05-31
3R01GM042652-03S1 (1994): $51831
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine, Po Box 016960 (r-64), Miami, Fl 33101
Grant 2R56DC006308-06 from National Institute On Deafness And Other Communication Disorders
Keywords: (beta 1-(2-Deoxyribopyranosyl))thymidine; 4-Aminobutanoic Acid; 4-Aminobutyric Acid; 5-BrdU; 5-Bromo-2`-deoxyuridine; 5-Bromodeoxyuridine; 5-Bromouracil deoxyriboside; 5-Bromouracil-2-deoxyriboside; 5-Budr; Ablation; Address; Age; Aminalon; Aminalone; Apical; BUdR; Behavior; Biology; Body Tissues; BrdU; Bromodeoxyuridine; Bromodeoxyuridine (BUDR); Bromouracil Deoxyriboside; Broxuridine; Buccal Cavity; Butanoic acid, 4-amino-; Cavitas Oris; Cell Communication and Signaling; Cell Signaling; Cell membrane; Cell/Tissue, Immunohistochemistry; Cells; Characteristics; Chemicals; Classification; Communication; Competence; Cytoplasmic Membrane; Data; Deoxyuridine; Electric Stimulation; Electrical Stimulation; Environment; Enzymes; Epithelium; Esthesia; Expression Profiling; Expression Signature; Funding; GABA; Gene Expression Monitoring; Gene Expression Pattern Analysis; Gene Expression Profiling; Genetic; Glia; Glial Cells; Gustation; Head and Neck, Buccal Cavity; Housing; IHC; IRK1 channel; Image; Immunofluorescence; Immunofluorescence Immunologic; Immunohistochemistry; Immunohistochemistry Staining Method; Immunologic, Immunofluorescence; Individual; Intracellular Communication and Signaling; Inward Rectifier K+ Channels; Inwardly Rectifying Postassium Channels; Ion Channel; Ionic Channels; K+ Channels, Inwardly Rectifying; KCNJ1 gene; Kolliker`s reticulum; Label; Length; Length of Life; Life; Location; Longevity; Mammals, Mice; Measurement; Measures; Mediating; Membrane Channels; Membrane Transport Proteins; Membrane Transporters; Methods; Mice; Minority; Molecular Fingerprinting; Molecular Profiling; Mouse Strains; Mouth; Murine; Mus; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; Non-neuronal cell; Oral cavity; Organ; Plasma Membrane; Potassium Channels, Inwardly Rectifying; Preparation; Presynaptic Receptors; Production; Profilings, Gene Expression; Property; Property, LOINC Axis 2; Proteins; Pump; ROMK; Receptor Protein; Regulation; Resolution; Role; Sensation; Sensory; Series; Signal Transduction; Signal Transduction Systems; Signaling; Slice; Stimulus; System; System, LOINC Axis 4; Systematics; Taste; Taste Buds; Taste Perception; Technology; Testing; Time; Tissues; Transcript Expression Analyses; Transcript Expression Analysis; Transgenic Mice; Type I Cell; Type I Epithelial Receptor Cell; Type II Cell; Type II Epithelial Receptor Cell; Type III Cell; Type III Epithelial Receptor Cell; Uridine, 2`-deoxy-; Uridine, 5-bromo-2`-deoxy-; Work; ing; base; behavior test; behavioral test; biological signal transduction; cell type; experiment; experimental research; experimental study; extracellular; gamma-Aminobutyric Acid; gene product; imaging; immunocytochemistry; interest; inward rectifier potassium channel; life span; lifespan; molecuar profile; molecular signature; nerve cement; neuronal; new technology; novel; plasmalemma; presynaptic; receptor; renal outer medullary potassium channel; research study; response; social role
Relevance: Taste buds detect nutritive and potentially poisonous materials through the coordinated action of distinct types of cells housed in taste buds throughout the oral cavity. We will investigate how cells located in taste buds, but resembling glia, may influence the sensitivity of taste buds. Because the proteins responsible for regulation are in an accessible location, understanding these mechanisms introduces the possibility of pharmacological manipulation of aversive and appetitive taste sensations
Project start date: 2003-07-01
Project end date: 2011-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: PA-07-070
2R56DC006308-06 (2010): $50000
MOLECULAR PHYSIOLOGY OF GLUTAMATE IN TASTE
Nirupa Chaudhari, Professor
University Of Miami School Of Medicine 1507 Levante Avenue Coral Gables, Fl 33124
Grant 5P01DC003013-02 from National Institute On Deafness And Other Communication Disorders IRG: CDRC
Project start date: 1995-09-01
Project end date: 1999-08-31
5P01DC003013-02 (1996): $449670