Receptors for the detection of L-amino acids and IMP by mouse taste sensory cells

232 Pages·2016·3.15 MB·English

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Graduate College Dissertations and Theses Dissertations and Theses
2016
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Shreoshi Pal Choudhuri
University of Vermont
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RECEPTORS FOR THE DETECTION OF L-AMINO ACIDS AND IMP BY MOUSE
TASTE SENSORY CELLS

A Dissertation Presented

by
Shreoshi Pal Choudhuri
to
The Faculty of the Graduate College
of
The University of Vermont

In Partial Fulfillment of the Requirements
For the Degree of Doctorate of Philosophy
Specializing in Biology

January, 2016

Defense Date: November 2, 2015
Dissertation Examination Committee:

Eugene R. Delay, Ph.D., Advisor
Gary M. Mawe, Ph.D., Chairperson
Rona J. Delay, Ph.D.
George C. Wellman, PhD.
Cynthia J. Forehand, Ph.D., Dean of the Graduate College
ABSTRACT
The sense of taste is one of the most important factors in regulating ingestive decisions. This is
central to a number of disease conditions including but not limited to obesity, diabetes,
anorexia, hypertension, coronary artery diseases and malnutrition. The detection of the
molecules eliciting taste qualities in food is mediated by the coordinated actions of distinct
types of taste sensory cells (TSCs) housed in taste buds within specialized papillae throughout
the oral cavity. Taste receptors in the taste sensory cells that detect food molecules are the key
players in selecting dietary nutrients. One such example is L-amino acids, a critical part of one’s
diet.
L-glutamate is the prototypical umami compound and is known to increase palatability of food.
A unique characteristic of umami taste is the response potentiation of glutamate by 5’
ribonucleotide monophosphates, such as inosine 5’ monophosphate (IMP), which is also capable
of eliciting an umami taste. Candidate receptors for umami taste include a heterodimer
T1r1+T1r3, brain variants of mGluR1 and mGluR4, and the truncated variants of mGluR1 and
mGluR4. Studies using heterogeneous expression of T1r1+T1r3 suggest it is an umami and a
broadly tuned L-amino acid receptor. While much attention is devoted to understanding
glutamate transduction, the detection mechanisms for other L-amino acids by TSCs are less well
understood. Here calcium imaging of isolated TSCs and taste cell clusters from the circumvallate
and foliate papillae of C57BL/6J and T1r3 knockout mice was performed to determine if other
receptors are involved in the detection of L-amino acids and IMP. Ratiometric imaging with
Fura-2 was used to study calcium responses to IMP and four L-amino acids (monopotassium L-
glutamate, L-serine, L-arginine, and L-glutamine) with and without IMP. The results of these
experiments showed that the response patterns elicited by L-amino acids varied significantly
across TSCs. Only a small subset of cells responded to all stimuli. Interestingly, L-amino acids
other than glutamate elicited synergistic responses in a subset of TSCs. Additionally IMP alone
elicited a response in a large number of TSCs. Our data indicate that synergistic and non-
synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a
receptor complex.
Next the roles of mGluR1 and mGluR4 in the detection of the IMP and L-amino acids were
investigated. Selective agonists for mGluR1, (RS)-3, 5-dihydroxyphenylglycine (DHPG; a group I
mGluR agonist), and mGluR4, L-(+)-2-amino-4-phosphonobutyric acid (L-AP4; a group III mGluR4
agonist) elicited responses in TSCs. In addition, TSCs responsive to these agonists were also
responsive to L-amino acids and IMP. More importantly, selective antagonists against different
mGluRs such as (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; a group I mGluR antagonist), and
(RS)-α-methylserine-O-phosphate (MSOP; a group III mGluR antagonist) significantly suppressed
L-amino acid- and IMP-mediated responses in TSCs of T1r3 knockout mice. Collectively, these
data provide evidence for the involvement of taste and the brain variants of mGluR1 and
mGluR4 in L-amino acid and IMP taste responses in mice, and support the hypothesis that
multiple receptors contribute to the IMP and L-amino acid tastes.
CITATIONS

Material from this dissertation has been published in the following form:
Pal Choudhuri, S., Delay, R. J., & Delay, E. R.. (2015). L-Amino Acids Elicit Diverse
Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors. PLoS One,
10(6), e0130088. doi: 10.1371/journal.pone.0130088
Material from this dissertation has been accepted for publication in Neuroscience on
12/4/2015 in the following form:
Pal Choudhuri, S., Delay, R. J., & Delay, E. R.. (2015). Metabotropic Glutamate
Receptors Are Involved In The Detection Of IMP And L-Amino Acids By Mouse Taste
Sensory Cells. Neuroscience.

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DEDICATION

This is dedicated to
my mother Ushasi Pal Choudhuri and my father Subrata Pal Choudhuri

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ACKNOWLEDGEMENTS
I would like to thank all the people who made possible to develop this project.
First of all, I am very grateful to my advisor, Dr. Eugene R. Delay (more popularly
known as Dr. D), who had been indispensable during my development as a scientist. I
would like to thank Dr. D and my co-advisor Dr. Rona J. Delay for their patience,
assistance, supervision, guidance, and encouragement to develop and finish this project.
They taught me that patience, persistence and diligence is the key. I am thankful to my
committee members, Dr. Gary M. Mawe and Dr. George C. Wellman, who have patiently
guided me through a challenging project over the past several years. I also want to thank
all the professors in the Biology, especially Dr. Judith Van Houten and Dr. Bryan Ballif
who were always available to help with their advice whenever I needed.
I also want to thank our lab technician Diane Morgan for her help with lab work
and for the fun times we spent together. I have a number of friends and colleagues I have
made that supported me and made this journey enjoyable. Thank you Dr. Nabanita
Mukherjee, Dr. Jonathan Vick, Dr. Suraj Cherian, and Dave Harris for we are, or have
been, fortunate to have one another as friends and learning partners. I also want to thank
Nora Awadallah, Brenden Chandler, and Kyle Joseph, for their friendship and support
and for all the fun chats that we had.
I am also very thankful to my mother, Ushasi Pal Choudhuri, for always being
the most supportive person, for always giving me encouragement to follow the dreams,
and for teaching me to never give up. And thanks Dad (Subrata Pal Choudhuri) for
always being there to support, and for the courage you infused in me. I also want to thank
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my sweet brother Sayanta Pal Chowdhury for believing in me and always supporting me
(No questions asked).
I also want to thank my husband Paresh Panchal for giving me the push when I
needed them most. Thank you for listening to my endless complaints about experimental
failures and giving me the strength to keep going.
Last but not the least, I want to thank my extended Vermont family, all my
friends, for giving me the family away from home.

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TABLE OF CONTENTS
CITATIONS ....................................................................................................................... ii
DEDICATION ................................................................................................................... iii
ACKNOWLEDGEMENTS ............................................................................................... iv
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................ x
Chapter 1 : TASTE TRANSDUCTION – A COMPREHENSIVE LITERATURE
REVIEW ............................................................................................................................. 1
1.1 Taste ....................................................................................................................................... 1
1.1.1 Sense of Taste: ................................................................................................................ 1
1.1.2 Taste and Physiological Functions: ................................................................................. 5
1.1.3 Taste Confusion: ............................................................................................................. 5
1.2 Taste System .......................................................................................................................... 7
1.2.1 The Structure of the Primary Sensory Organ, Tongue: ................................................... 7
1.2.2. Taste Bud Cells: ............................................................................................................ 10
1.3 Transduction Mechanisms of Taste Molecules by the TSCs ................................................ 16
1.3.1 Salt Taste Transduction: ................................................................................................ 16
1.3.2 Sour Taste Transduction: .............................................................................................. 17
1.3.3 Sweet, Bitter, and Umami Taste Transduction: ............................................................ 18
1.3.4 Cell-Cell Communication in Taste Buds: ....................................................................... 25
1.4 Taste Signaling from Tongue to Brain .................................................................................. 26
1.4.1 Neural Coding of Taste: ................................................................................................. 28
1.5 Taste Receptors in a Myriad of Other Tissues ..................................................................... 30
1.5.1 Taste Receptors in the Gut: .......................................................................................... 31
1.5.2 T1rs and T2rs in the Airway Cells: ................................................................................. 32
1.5.3 T1R2+T1R3 Receptors in the Bladder: .......................................................................... 33
1.5.4 Taste Receptors in the Brain: ........................................................................................ 34
1.6 Coming Back to the L-Amino Acid Story .............................................................................. 39
1.6.1 Amino Acid Taste in Human: ......................................................................................... 39
1.6.2 In Search of L-amino Acid Receptor(s) – Rodent Story: ................................................ 42
1.6.3 Taste Potentiation: ........................................................................................................ 46
1.7 Research Hypothesis ............................................................................................................ 46
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1.8 References ........................................................................................................................... 54
Chapter 2 : L-AMINO ACIDS ELICIT DIVERSE RESPONSE PATTERNS IN TASTE
SENSORY CELLS: A ROLE FOR MULTIPLE RECEPTORS ..................................... 87
2.1 Abstract ................................................................................................................................ 88
2.2 Introduction ......................................................................................................................... 89
2.3 Materials and Methods ........................................................................................................ 92
2.3.1 Ethical Consideration: ................................................................................................... 92
2.3.2 Animals:......................................................................................................................... 93
2.3.3 Solutions: ...................................................................................................................... 93
2.3.4 Taste cell isolation:........................................................................................................ 94
2.3.5 Calcium (Ca2+) imaging: ................................................................................................. 95
2.3.6 Quantification of calcium responses: ........................................................................... 97
2.4 Results .................................................................................................................................. 98
2.4.1 Single TSCs from WT Mice Respond to Multiple, but Not All L-Amino Acids: .............. 98
2.4.2 In some but not all cases, L-amino acids elicit synergy when presented with IMP: ... 102
2.4.3 MIX-Responsive TSCs Respond Differently to IMP and L-Amino Acids: ..................... 105
2.4.4 Synergistic and non-synergistic responses are mediated by different receptors: ..... 107
2.4.5 Some TSCs were broadly tuned: ................................................................................. 109
2.4.6 TSCs from T1r3 KO mice responded to different L-amino acids: ................................ 112
1.3.6 Receptor(s) other than T1r1+T1r3 may be involved in synergistic responses: .......... 114
2.4.7 TSCs in Clusters: .......................................................................................................... 119
2.5 Discussion........................................................................................................................... 121
2.5.1 Multiple receptors and/or transduction mechanisms are involved in L-amino acids and
IMP taste .............................................................................................................................. 124
2.5.2 In posterior tongue, receptor(s) other than T1r1+T1r3 can generate synergistic
responses: ............................................................................................................................ 125
2.5.3 IMP alone elicits responses in TSCs: ........................................................................... 128
2.6 Conclusions ........................................................................................................................ 129
2.7 Acknowledgements ............................................................................................................ 131
2.8 References ......................................................................................................................... 132
Chapter 3 : METABOTROPIC GLUTAMATE RECEPTORS ARE INVOLVED IN
THE DETECTION OF IMP AND L-AMINO ACIDS BY MOUSE TASTE SENSORY
CELLS ............................................................................................................................ 146
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3.1 Abstract .............................................................................................................................. 147
3.2 Introduction ....................................................................................................................... 149
3.3 Experimental Procedures ................................................................................................... 153
3.3.1 Ethical Consideration: ................................................................................................. 153
3.3.2 Animals:....................................................................................................................... 153
3.3.3 Solutions and Chemicals: ............................................................................................ 154
3.3.4 Taste Cell Isolation: ..................................................................................................... 155
3.3.5 Calcium (Ca2+) imaging: ............................................................................................... 156
3.3.6 Quantification of Calcium Responses: ........................................................................ 157
3.3.7 Statistical Analysis: ...................................................................................................... 158
3.4 Results ................................................................................................................................ 158
3.4. 1 mGluR1-mediates responses to L- amino acid and IMP: ........................................... 162
3.4.2 mGluR4-mediates responses to L- amino acid and IMP: ............................................ 168
3.5 Discussion........................................................................................................................... 175
3.5.1 mGluR1 detects L-amino acids and IMP in mouse TSCs ............................................. 179
3.5.2 L-amino acids and IMP can activate mGluR4 in mouse TSCs ...................................... 181
3.5 Conclusions ........................................................................................................................ 184
3.7 Acknowledgement ............................................................................................................. 185
3.8 References ......................................................................................................................... 186
Chapter 4 : CONCLUSIONS AND FUTURE DIRECTIONS ....................................... 198
4.1 Are L-Amino Acids and IMP Detected by Multiple or a Single Receptor? ......................... 199
4.2 mGluRs Detect L-Amino Acids and IMP Taste: .................................................................. 200
4.3 Speculations about possible receptors: ............................................................................. 203
4.4 Preliminary Studies and Future Directions ........................................................................ 204
4.4.1 Preliminary Studies and Future Direction Concerning Chapter 2: .............................. 204
4.4.2 Can mGluRs Elicit Synergistic Responses? .................................................................. 207
4.4.3 Are L-Amino Acid and IMP Responses Mediated by PLCβ2 Dependent pathway? .... 208
4.4.4 Is cAMP Pathway Involved in L-Amino Acid and IMP Detection? ............................... 209
4.4.5 Does cAMP Pathway Interact with PLCβ2-Mediated Pathway? ................................. 211
4.5 Conclusions ........................................................................................................................ 212
4.6 References ......................................................................................................................... 214

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