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The accumulation of glutamate in the placental syncytiotrophoblast as a driver of membrane transport

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UNIVERSITY OF SOUTHAMPTON
Faculty of Medicine
Human Development and Health
The accumulation of glutamate in the placental
syncytiotrophoblast as a driver of membrane
transport
by
Emma Michelle Lofthouse
Thesis for the degree of Doctor of Philosophy
February 2014
UNIVERSITY OF SOUTHAMPTON
ABSTRACT
FACULTY OF MEDICINE
Thesis for the degree of Doctor of Philosophy
THE ACCUMULATION OF GLUTAMATE IN THE
PLACENTAL SYNCYTIOTROPHOBLAST AS A DRIVER
OF MEMBRANE TRANSPORT
Emma Michelle Lofthouse
Impaired fetal growth is associated with increased prevalence of chronic diseases in
later life. One of the major causes of impaired fetal growth is suboptimal placental
function. The placenta has multiple roles including the transport of nutrients and waste
products. Identifying the mechanisms underlying placental function is essential in
gaining a better understanding of the processes that determine fetal growth and
development.
The metabolically important amino acid glutamate is not transferred across the placenta
and there is net uptake from both the maternal and fetal circulations. There is a high
concentration of glutamate within the placenta which is used in metabolism. Recent
observations indicate that while there is net glutamate uptake on both sides of the
placenta there is also an unidentified glutamate efflux route. This thesis investigates the
mechanisms of glutamate efflux from the syncytiotrophoblast and what role this efflux
may have in determining placental function.
Efflux of glutamate was hypothesised to be mediated by exchange transporters and
channels. These hypotheses were initially tested using the isolated perfused human
placenta. On the microvillous membrane (MVM) of the syncytiotrophoblast, glutamate
efflux was observed in response to boli of N-acetylcysteine, suggesting efflux via the
exchanger xCT and following a urea bolus, suggesting cellular swelling-induced efflux
by the Maxi chloride channel. xCT expression had not previously been demonstrated in
human placenta and may be an important source of cysteine for placental glutathione
synthesis. Interestingly, pre-treatment of the placenta and of BeWo choriocarcinoma
cells with the xCT substrate N-acetylcysteine was shown to inhibit activity of the Maxi
chloride channel. Additionally, glutamate release was stimulated by hydrogen peroxide,
potentially suggesting a role for reactive oxygen species in the regulation of the Maxi
chloride channel.
On the basal membrane (BM) of the syncytiotrophoblast, glutamate efflux was observed
in response to glutamate boli but not in response to boli of other known exchanger
substrates. I hypothesised that this exchange may be mediated by the organic anion
transporter OAT4 and/or OATP2B1, neither of which had previously been shown to
transport glutamate. OAT4 and OATP2B1 were expressed in Xenopus laevis oocytes
and it was demonstrated that both transporters could mediate glutamate efflux. I suggest
that glutamate efflux on the BM is mediated in exchange for uptake of fetal organic
anions including waste products, xenobiotics and steroid synthesis precursor molecules.
These findings suggest that the placenta establishes a transmembrane gradient of
glutamate that is linked to biologically important transport processes, which are critical
to normal placental development. The glutamate gradient is likely to be driving
antioxidant and hormone synthesis as well as protecting the fetus from waste product
and xenobiotic exposure. I propose that glutamate gradients play previously
unrecognised roles in determining placental function and these require further study.
It is unclear whether changes in the transmembrane glutamate gradient will alter fetal
growth and development. If the gradient is altered, placental transporters such as xCT
may be affected, resulting in a decreased uptake of antioxidant precursors. This in turn
may lead to high levels of oxidative stress, as observed in pre-eclampsia. Future work
should aim to determine whether the glutamate gradient and glutamate transporters are
altered in pathological placentas, with the aim to design interventions to improve
placental function.
iii
Contents
ABSTRACT ................................................................................................................. i
Contents iii
List of tables ............................................................................................................... ix
List of figures .............................................................................................................. x
DECLARATION OF AUTHORSHIP ................................................................... xvi
Acknowledgements ................................................................................................. xvii
Definitions and Abbreviations .............................................................................. xviii
Chapter 1: General Introduction............................................................................... 1
1.1 Introduction ......................................................................................................... 2
1.2 Development of the human placenta ................................................................... 3
1.2.1 Implantation and invasion of the placenta into the uterine wall .................... 4
1.2.2 First trimester placental development ............................................................ 4
1.2.3 Placental blood flow ....................................................................................... 6
1.2.4 Second trimester placental development ........................................................ 6
1.2.5 Third trimester placental development ........................................................... 7
1.2.6 Placental structure at term .............................................................................. 8
1.2.7 The term placental barrier ............................................................................ 11
1.3 Endocrine roles of the placenta ......................................................................... 12
1.3.1 Developmental origins of health and disease ............................................... 15
1.4 Placental influences on fetal development ........................................................ 16
1.5 Mechanisms of placental transport .................................................................... 18
1.5.1 Active transport ............................................................................................ 18
1.5.1.1 Primary active transporters in human placenta ........................................ 19
1.5.1.2 Secondary active transport ....................................................................... 20
1.5.1.3 Exchangers ............................................................................................... 20
1.5.2 Facilitated transport ...................................................................................... 21
1.5.3 Passive diffusion .......................................................................................... 22
1.6 Placental transport ............................................................................................. 23
1.7 Regulation of membrane transporters in the placenta ....................................... 25
1.8 Placental glutamate ............................................................................................ 27
1.8.1 Roles of placental glutamate ........................................................................ 29
1.9 Aim .................................................................................................................... 33
iv
1.9.1 Hypothesis .................................................................................................... 33
Chapter 2: General Methods ................................................................................... 35
2.1 Methods ............................................................................................................. 36
2.2 Placental perfusion methodology ...................................................................... 36
2.2.1 Equipment and reagents for placental perfusions ........................................ 36
2.2.2 Working solutions for placental perfusions ................................................. 37
2.2.3 Isolated perfused human placental cotyledon .............................................. 38
2.3 Sampling during placental perfusions ............................................................... 39
2.4 Liquid scintillation counting ............................................................................. 40
2.5 Creatinine infinity assay .................................................................................... 42
2.6 RNA extraction, purification and quantification ............................................... 43
2.7 Reverse transcription ......................................................................................... 44
2.8 Reverse transcriptase polymerase chain reaction .............................................. 44
2.9 Gel electrophoresis ............................................................................................ 45
2.10 Purification of PCR products .......................................................................... 45
2.11 Basal and microvillous membrane preparation ............................................... 46
2.12 Determining membrane preparation protein concentration ............................ 46
2.13 Western Blotting ............................................................................................. 48
2.13.1 Equipment and reagents for Western blotting ........................................... 48
2.13.2 Working reagents for Western blotting ..................................................... 49
2.13.3 Western blotting protocol .......................................................................... 49
2.13.4 Western blotting quantification ................................................................. 50
2.14 Xenopus laevis oocyte preparation and injection ............................................ 51
2.14.1 Bacterial transformation ............................................................................ 51
2.14.2 Maxi-prep of plasmid DNA ....................................................................... 52
2.14.3 Complementary RNA (cRNA) synthesis and purification ........................ 53
2.14.4 Quantification of cRNA ............................................................................. 53
2.14.5 Xenopus laevis oocyte microinjection ....................................................... 54
2.14.6 Efflux studies ............................................................................................. 55
2.15 BeWo cell culture ............................................................................................ 56
2.15.1 Equipment and reagents for cell culture .................................................... 56
2.15.2 Working solutions for cell culture ............................................................. 57
2.15.3 Retrieving BeWo cells from liquid nitrogen ............................................. 57
2.15.4 Splitting BeWo cells .................................................................................. 58
2.15.5 Transfection ............................................................................................... 58
2.16 Data analysis ................................................................................................... 59
2.16.1 Calculating area under the curve for perfusion data .................................. 59
v
2.16.2 Transfer, uptake and retention of
14
C-glutamate and
3
H-proline in
perfusions ................................................................................................................... 60
2.16.3 Xenopus oocyte data .................................................................................. 61
Chapter 3: The role of xCT
in mediating placental glutamate efflux .................. 63
3.1 Introduction ....................................................................................................... 64
3.1.1 Aims ............................................................................................................. 66
3.2 Methods ............................................................................................................. 67
3.2.1 rtPCR to determine gene expression in placenta .......................................... 67
3.2.2 Perfusions to investigate glutamate efflux from the placenta ...................... 67
3.2.3 Creatinine Assays ......................................................................................... 69
3.2.4 Western blotting to localise xCT to MVM and BM ..................................... 69
3.2.5 DH5α transformation with xCT-GFP ........................................................... 70
3.2.6 xCT-GFP restriction digest .......................................................................... 70
3.2.7 xCT-4F2hc cRNA synthesis ......................................................................... 70
3.2.8 Injection of xCT-4F2hc cRNA into Xenopus laevis oocytes ....................... 71
3.2.9 Oocyte uptake experiments to investigate xCT activity ............................... 71
3.2.10 Oocyte efflux experiments ......................................................................... 71
3.2.11 xCT knockdown studies ............................................................................ 72
3.2.12 Statistical analysis ...................................................................................... 72
3.2.12.1 Placental perfusions and area under the curve ....................................... 72
3.2.12.2 Maternal arterial inflow and uptake from maternal circulation ............. 73
3.2.12.3 xCT mediated glutamate efflux in Xenopus oocytes ............................. 73
3.3 Results ............................................................................................................... 75
3.3.1 Detection of SLC7A11 / SLC7A13 mRNA expression ............................... 75
3.3.2 Determining xCT functionality in the perfused placenta ............................. 76
3.3.2.1 Transfer of
14
C-glutamate and
3
H-proline ................................................ 76
3.3.2.2 Effect of xCT boli on
14
C-glutamate and
3
H-proline efflux ..................... 80
3.3.3 xCT protein expression ................................................................................ 85
3.3.4 xCT-GFP 4F2hc microinjection into oocytes .............................................. 87
3.3.4.1 Glutamate efflux from xCT-GFP 4F2hc expressing oocytes ................... 89
3.4 Discussion ......................................................................................................... 96
3.4.1 Glutamate exchange on the MVM ............................................................... 96
3.4.2 Transport of proline and creatinine in the perfused placenta ....................... 98
3.4.3 Evidence that N-acetylcysteine is an xCT substrate .................................. 100
3.4.4 The role of xCT in the human placenta ...................................................... 103
3.4.5 Glutamate efflux by exchange on the BM ................................................. 106
3.4.6 A role for N-acetylcysteine in pre-eclampsia? ........................................... 106
vi
3.4.7 Future work ................................................................................................ 108
3.4.8 Conclusion .................................................................................................. 110
Chapter 4: Volume regulated glutamate release from the human placenta ..... 111
4.1 Introduction ..................................................................................................... 112
4.1.1 Aims ........................................................................................................... 115
4.2 Methods ........................................................................................................... 116
4.2.1 Placental perfusions ................................................................................... 116
4.2.2 Maxi chloride channel activity in BeWo cells ........................................... 117
4.2.2.1 Maxi chloride channel activity in response to urea and NAC ................ 117
4.2.2.2 Maxi chloride channel activity in response to H
2
O
2
and NAC .............. 118
4.2.3 xCT knockdown ......................................................................................... 121
4.2.4 Statistical analysis ...................................................................................... 121
4.3 Results ............................................................................................................. 123
4.3.1 Maxi chloride channel glutamate efflux in placental perfusions ............... 123
4.3.2 Urea stimulates Maxi chloride channel activity in BeWo cells ................. 130
4.3.2.1 Hydrogen peroxide alters Maxi Chloride channel activity .................... 133
4.4 Discussion ....................................................................................................... 138
4.4.1 Osmotically induced glutamate release from the MVM ............................ 138
4.4.2 N-acetylcysteine inhibits Maxi chloride channel like activity ................... 139
4.4.3 What is the physiological role of the placental Maxi chloride channel? ... 143
4.4.4 The γ-glutamylcysteine pathway ................................................................ 146
4.4.5 Future work ................................................................................................ 147
4.4.6 Conclusion .................................................................................................. 149
Chapter 5: The glutamate gradient and placental organic anion transport ..... 151
5.1 Introduction ..................................................................................................... 152
5.1.1 Aims ........................................................................................................... 156
5.2 Methods ........................................................................................................... 157
5.2.1 Placental perfusions ................................................................................... 157
5.2.2 Creatinine assays ........................................................................................ 158
5.2.3 DH5α transformation with OAT4 and OATP2B1 ..................................... 158
5.2.4 Transfection of OAT4 into BeWo cells ..................................................... 159
5.2.5 OAT4 and OATP2B1 restriction digest ..................................................... 159
5.2.6 OAT4 and OATP2B1 fragment purification by PCR ................................ 159
5.2.7 OAT4 and OATP2B1 cRNA synthesis ...................................................... 160
5.2.8 Injection of OAT4 cRNA into Xenopus laevis oocytes ............................. 160
5.2.9 OAT4 /OATP2B1 Xenopus oocyte
14
C-glutamte efflux studies ................ 162
vii
5.2.10 OAT and OATP PCR .............................................................................. 162
5.2.11 Western Blotting ...................................................................................... 165
5.2.12 Statistical analysis .................................................................................... 165
5.2.12.1 Placental perfusions ............................................................................. 165
5.2.12.2 Maternal arterial inflow and uptake from maternal circulation ........... 166
5.2.12.3 OAT4 and OATP2B1 oocyte efflux studies ........................................ 166
5.3 Results ............................................................................................................. 167
5.3.1 OAT/OATP substrates did not initiate exchange for glutamate in placental
perfusions ................................................................................................................. 167
5.3.2 Trans-stimulation of glutamate efflux via OAT4 oocytes ......................... 170
5.3.3 Trans-stimulation of glutamate efflux via OATP2B1 ................................ 176
5.3.4 OAT mRNA expression in the placenta ..................................................... 181
5.3.5 OATP mRNA expression in the human placenta ....................................... 184
5.3.6 Protein localisation of OAT4 to the basal membrane ................................ 186
5.4 Discussion ....................................................................................................... 187
5.4.1 Perfusions investigating OAT/OATP activity ............................................ 187
5.4.2 Glutamate is an OAT4 and OATP2B1 substrate ....................................... 188
5.4.3 OAT4 and OATP2B1 kinetics ................................................................... 189
5.4.4 Roles of OAT4 and OATP2B1 in the placenta .......................................... 190
5.4.5 Do other OATs and OATPs transport glutamate? ..................................... 193
5.5 Future work ..................................................................................................... 195
5.6 Conclusion ....................................................................................................... 197
Chapter 6: Glutamine efflux from human placental syncytiotrophoblast ........ 199
6.1 Introduction ..................................................................................................... 200
6.1.1 Aims ........................................................................................................... 204
6.2 Methods ........................................................................................................... 205
6.2.1 RNA extraction and cDNA synthesis ......................................................... 205
6.2.2 PCR ............................................................................................................ 205
6.2.3 DNA sequencing ........................................................................................ 205
6.2.4 Basal and microvillous membrane preparations ........................................ 207
6.2.5 Western blotting ......................................................................................... 207
6.2.6 Statistical analysis ...................................................................................... 208
6.3 Results ............................................................................................................. 209
6.3.1 SNAT3/SNAT5 mRNA expression in human placenta ............................. 209
6.3.2 SNAT3 and SNAT5 protein expression in human placenta ...................... 214
6.4 Discussion ....................................................................................................... 217
6.4.1 The role of SNAT3/SNAT5 in placental glutamine efflux ........................ 217
viii
6.4.2 Factors which may determine placental glutamine uptake / efflux ............ 219
6.4.3 Placental expression of other SLC38 family transporters .......................... 222
6.4.4 Future work ................................................................................................ 222
6.5 Conclusion ....................................................................................................... 223
Chapter 7: General Discussion .............................................................................. 225
7.1 Overview ......................................................................................................... 226
7.2 Why is the transmembrane glutamate gradient important? ............................. 226
7.2.1 Is the glutamate gradient important for fetal development?......................... 228
7.2.1.1 Role of intracellular glutamate in placental metabolism ........................... 229
7.2.2 Is the glutamate gradient involved in pregnancy pathologies? .................... 232
7.3 Does the glutamate gradient have roles beyond the placenta? ........................ 234
7.4 Future directions .............................................................................................. 236
7.5 Implications for the future ............................................................................... 239
Bibliography ........................................................................................................... 241
Appendix 1: List of abstracts ................................................................................ 265
Appendix 2: Publications ....................................................................................... 267
ix
List of tables
Table 1.1 Key hormones produced by the placenta and their functions in pregnancy.14
Table 1.2 - Transporters and channels known to mediate glutamate efflux ................... 29
Table 2.1 Standards used for BCA protein assay ........................................................ 47
Table 3.1 - Summary of all substrates injected into the fetal and maternal circulations
throughout the placental perfusion experiments including substrates for
ASCT2 and xCT ........................................................................................ 84
Table 4.1 - Properties of the VRAC and Maxi Chloride channel . .............................. 114
Table 5.1 - OAT forward and reverse primers used for PCR ....................................... 164
Table 5.2 - Results of OAT PCR runs with a variety of temperature gradients ........... 183
Table 5.3 - Results of OATP PCR runs with a variety of temperature gradients ........ 184
Table 6.1 - Substrate specificity & human protein localisation of the SLC38 family.. 202
Table 6.2 - SLC38 forward and reverse primers used for PCR.................................... 206
Table 6.3 Presence or absence of novel SLC38 genes in placenta ............................ 213
x
List of figures
Figure 1.1 - Early placental development during the first trimester. ............................... 5
Figure 1.2 Differentiation of the villi in first trimester and third trimester. .................. 8
Figure 1.3 - Placental structure and circulation at term ................................................... 9
Figure 1.4 - Structure of the placental villi at term ....................................................... 10
Figure 1.5 Structure of the syncytiotrophoblast at term showing the exchange barriers
involved in nutrient transfer ....................................................................... 12
Figure 1.6 - Mechanisms of nutrient transfer across the syncytiotrophoblast ............... 22
Figure 1.7 Amino acid transporter localisation on the MVM and BM of the placental
syncytiotrophoblast . .................................................................................. 24
Figure 1.8 - Localisation of the SLC and ABC transporters on the BM and MVM of the
syncytiotrophoblast .................................................................................... 25
Figure 1.9 The involvement of glutathione (GSH) with reactive oxygen species ...... 31
Figure 1.10 - The metabolic fate of glutamate within the human placenta .................... 32
Figure 1.11 Potential glutamate efflux mechanisms from the MVM and BM of the
placental syncytiotrophoblast .................................................................... 33
Figure 2.1 - Placental perfusion methodology ............................................................... 39
Figure 2.2 - Optimal sampling times for placental perfusion experiments .................... 40
Figure 2.3 Linear relationship between
14
C-glutamate counts (cpm) and serially
diluted
14
C-glutamate stocks ..................................................................... 41
Figure 2.4 - Validation of creatinine assay using a standard curve ............................... 43
Figure 2.5 Example BSA standard curve used in BCA protein assay ........................ 47
Figure 2.6 Structure of pCMV6 vectors....………………………………….…...…..52
Figure 2.7 Example cRNA visualisation on an agarose gel ........................................ 54
xi
Figure 2.8 - Xenopus laevis microinjection setup with stage V/VI oocytes showing the
animal pole and the vegetal pole ............................................................... 55
Figure 2.9 Schematic diagram illustrating how AUC was calculated from raw data . 60
Figure 3.1 Glutamate efflux may be mediated by amino acid transporters including
ASCT2, AGT-1 or xCT ............................................................................. 66
Figure 3.2 Experimental outline for xCT perfusions .................................................. 69
Figure 3.3 - PCR products from SLC7A11 and SLC7A13 primers .............................. 75
Figure 3.4 Uptake of
3
H-proline,
14
C-glutamate and creatinine from the maternal
circulation in xCT perfusions. ................................................................... 77
Figure 3.5
14
C-glutamate,
3
H-proline and creatinine measured in the fetal circulation
as a percentage of maternal arterial inflow, averaged over all experiments78
Figure 3.6 Retention of
14
C-glutamate and
3
H-proline in the placenta ....................... 79
Figure 3.7 - Example experiments showing the appearance of
14
C-glutamate and
3
H-
proline from the perfused placenta ............................................................ 81
Figure 3.8 - NAC stimulates exchange for
14
C-glutamate in the maternal circulation .. 82
Figure 3.9 - xCT and ASCT2 substrates did not initiate exchange for
3
H-proline ........ 83
Figure 3.10 - xCT protein localisation in placental MVM and BM samples of the
syncytiotrophoblast . .................................................................................. 86
Figure 3.11 - Example Xenopus laevis injection with and without xCT-GFP ............... 87
Figure 3.12 -
14
C-glutamate uptake into xCT-4F2hc injected oocytes in the presence
and absence of 10 mmol/l glutamate ......................................................... 88
Figure 3.13 Example raw
14
C-glutamate efflux data from one oocyte efflux
experiment ................................................................................................ 90
Figure 3.14 -
14
C-glutamate efflux from oocytes injected with xCT-4F2hc cRNA ....... 91
Figure 3.15 Uptake of
14
C-glutamate in xCT-4F2hc, 4F2hc, non-injected and water
injected oocytes .......................................................................................... 92
xii
Figure 3.16 - Efflux of
14
C-glutamate from oocytes over 5 min in the presence of 10
mmol/l unlabelled glutamate in xCT-4F2hc, non-injected, 4F2hc injected
and water injected oocytes. ........................................................................ 93
Figure 3.17 Raw data from a time course of
14
C-glutamate efflux from xCT-4F2hc
injected oocytes in response to ND91 and 10 mmol/l NAC between 2 and
6 min .......................................................................................................... 94
Figure 3.18 Summary data of
14
C-glutamate efflux from xCT-4F2hc injected oocytes
in response to ND91 and 10 mmol/l NAC ................................................. 95
Figure 3.19 xCT is expressed on the MVM of the syncytiotrophoblast and mediates
glutamate exchange for NAC .................................................................... 96
Figure 3.20 - The possible conversion of NAC into cystine in the Xenopus oocyte. .. 102
Figure 3.21 - Molecular structures of xCT substrates and NAC.................................. 103
Figure 3.22 - The role of glutamate transporters in the provision of substrates for
glutathione synthesis ................................................................................ 105
Figure 4.1 - Does the maxi chloride channel mediate glutamate efflux into the maternal
and fetal circulations? .............................................................................. 115
Figure 4.2 Experimental outline for Maxi chloride placental perfusions ................. 116
Figure 4.3 - Experimental outline for Maxi chloride cell swelling cell culture
experiments .............................................................................................. 118
Figure 4.4 - Experimental outline for Maxi chloride urea and hydrogen peroxide cell
culture experiments .................................................................................. 119
Figure 4.5 - Experimental outline for Maxi chloride hydrogen peroxide cell culture
experiments .............................................................................................. 120
Figure 4.6 - Representative experiment showing hypo-osmotically induced
14
C-
glutamate efflux. ...................................................................................... 125
Figure 4.7 The effect of NAC pre-treatment on
14
C-glutamate efflux into the maternal
circulation in response to a maternal urea bolus. ..................................... 126
xiii
Figure 4.8 - The effect of NAC pre-treatment on
3
H-proline efflux into the maternal
circulation in response to a maternal urea bolus. ..................................... 127
Figure 4.9 - Transfer of
14
C-glutamate and
3
H-proline into the fetal circulation. ........ 128
Figure 4.10 - The relationship between maternal AUC in response to a maternal urea
bolus and cotyledon weight. .................................................................... 129
Figure 4.11 -
14
C-glutamate release from BeWo cells in response to urea / NAC . ..... 131
Figure 4.12 -
14
C-glutamate remaining in BeWo cells at the end of the experiment,
adjusted for protein. ................................................................................. 132
Figure 4.13 -
14
C-glutamate release from BeWo cells in response to urea and hydrogen
peroxide / NAC preincubation ................................................................. 134
Figure 4.14 -
14
C-glutamate remaining in BeWo cells at the end of the hydrogen
peroxide preincubation experiments. ....................................................... 135
Figure 4.15 -
14
C-glutamate release from BeWo cells in response to hydrogen peroxide
alone ......................................................................................................... 136
Figure 4.16 -
14
C-glutamate remaining in BeWo cells preincubated with different
concentrations of hydrogen peroxide ....................................................... 137
Figure 4.17 - MVM expression of xCT and Maxi chloride channel. ........................... 143
Figure 4.18 - Substrates of Maxi chloride channel may act as autocrine factors. ........ 145
Figure 5.1 - Potential mechanisms for glutamate efflux from the BM into the fetal
circulation ................................................................................................ 153
Figure 5.2 - Phylogenetic tree representation of the human OAT (SLC22) and OATP
(SLCO) family members ......................................................................... 154
Figure 5.3 The glutamate gradient may drive OAT4 and OATP2B1 activity. ......... 156
Figure 5.4 Typical experimental outline for OAT perfusions. .................................. 158
Figure 5.5 OAT4 and OATP2B1 restriction digest................................................... 161
Figure 5.6 - OAT/OATP substrates did not initiate exchange for
14
C-glutamate ........ 168
xiv
Figure 5.7 - OAT/OATP substrates did not initiate exchange for
3
H-proline ............. 169
Figure 5.8 - Estrone sulphate and BSP trans-stimulate efflux of
14
C-glutamate. ........ 172
Figure 5.9 -
14
C-glutamate uptake in OAT4, non-injected and water injected oocytes.173
Figure 5.10 - The raw efflux of
14
C-glutamate in response to 10 mmol/l unlabelled
glutamate in OAT4, non-injected and water injected oocytes. ................ 174
Figure 5.11 -
14
C-glutamate efflux over time from OAT4 injected oocytes in response
to 10 mmol/l glutamate and ND91 buffer ............................................... 175
Figure 5.12 - BSP, estrone sulphate and pravastatin trans-stimulate efflux of
14
C-
glutamate from OATP2B1 injected oocytes. ........................................... 177
Figure 5.13 -
14
C-glutamate uptake in in OATP2B1 and non-injected oocytes. .......... 178
Figure 5.14 - The efflux of
14
C-glutamate in response to 10 mmol/l unlabelled
glutamate in OATP2B1, non-injected and water injected oocytes. ......... 179
Figure 5.15 -
14
C-glutamate efflux over time from OATP2B1 injected oocytes in
response to 20 mmol/l BSP and ND91 buffer . ....................................... 180
Figure 5.16 - OAT PCR products from cytotrophoblast tissue and placental tissue .. 182
Figure 5.17 OAT PCR products from placental and positive control tissue ............. 183
Figure 5.18 - OATP PCR products from placental cytotrophoblast and positive control
cDNA samples. ........................................................................................ 185
Figure 5.19 OAT4 is expressed on the BM of the syncytiotrophoblast. ................... 186
Figure 5.20 Proposed mechanism by which the transmembrane glutamate gradient
drives the uptake of organic anions. ........................................................ 193
Figure 6.1 - An unknown transporter mediates glutamate efflux ................................ 203
Figure 6.2 - SLC38A3, SLC38A5 and SLC38A6 PCR products ................................ 210
Figure 6.3 - SLC38A7 to SLC38A11 PCR products. .................................................. 211
xv
Figure 6.4 - SLC38 PCR products from placental mRNA, No template control, BeWo
and BeWo control samples ...................................................................... 212
Figure 6.5 SNAT5 protein expression. ..................................................................... 214
Figure 6.6 - SNAT3 protein expression ....................................................................... 215
Figure 6.7 SNAT3 and SNAT5 protein expression in MVM and BM samples,
normalised to β-actin ............................................................................... 216
Figure 6.8 - Proposed mechanism of glutamate efflux via SNAT3 and SNAT5 ......... 221
Figure 7.1 Roles of the transmembrane glutamate gradient...................................... 227
Figure 7.2 - The transmembrane glutamate gradient results in the formation of
secondary glutamine gradients which may drive the uptake of other amino
acids into the placenta .............................................................................. 230
Figure 7.3 - Overall findings showing the potential functions of the transmembrane
glutamate gradient .................................................................................... 231
Figure 7.4 - Potential relationship between xCT and the Maxi chloride channel, based
on oxidative stress. ................................................................................... 234
xvi
DECLARATION OF AUTHORSHIP
I, Emma Michelle Lofthouse,
declare that the thesis entitled
The accumulation of glutamate in the placental syncytiotrophoblast as a driver of
membrane transport’
and the work presented in the thesis are both my own, and have been generated by me as the
result of my own original research. I confirm that:
this work was done wholly or mainly while in candidature for a research degree at
this University;
where any part of this thesis has previously been submitted for a degree or any other
qualification at this University or any other institution, this has been clearly stated;
where I have consulted the published work of others, this is always clearly attributed;
where I have quoted from the work of others, the source is always given. With the
exception of such quotations, this thesis is entirely my own work;
I have acknowledged all main sources of help;
where the thesis is based on work done by myself jointly with others, I have made
clear exactly what was done by others and what I have contributed myself;
or parts of this work have been published as:
Day PE, Cleal JK, Lofthouse EM, Goss V, Koster G, Postle A, Jackson JM, Hanson
MA, Jackson AA, & Lewis RM (2013). Partitioning of glutamine synthesised by the
isolated perfused human placenta between the maternal and fetal circulations. Placenta
34, 1223-1231.
Signed:……………………………………………………………………..
Date:…………………………………………………………………………
xvii
Acknowledgements
There are numerous people that I would like to thank for contributing to my PhD.
First and foremost I would like to express my thanks and gratitude to my supervisor, Dr
Rohan Lewis, for his incredible level of supervision, help and guidance. Thank you for
your encouragement and constructive criticism!
My secondary supervisors, Dr Kirsten Poore and Professor Mark Hanson, for their
additional support, guidance and advice.
The Placenta group: particularly Dr. Jane Cleal for her advice and encouragement and
Dr. Priscilla Day for her teaching of the placental perfusion technique at the very
beginning of my PhD. I would also like to thank Dr. Ita O’Kelly for her oocyte expertise
and the use of her laboratory equipment during my final year.
Miss Claire Simner: with whom my PhD experience would have been much more
stressful and less amusing without. I would like to thank her for being a great friend, for
putting up with the tears, tantrums and antics and for being my ‘Sprinkles’ partner.
Miss Liz Halstead: who has been of great support to me over the last year. I would like
to thank her for all of her encouragement and support and for always being on the end
of the phone whenever necessary!
Dr. James Atherton: my lab and desk partner, who was taken from this world far too
early and whose support and great friendship I greatly miss. This thesis was written in
his memory.
The midwives and patients at the Princess Anne Maternity Hospital: I would like to
thank them for allowing me to trawl the corridors of labour ward at all times of the day
in the search for placentas!
My family and friends: for all of their support and love over the years up until this point.
Sprinkles Gelato: for making incredibly good ice cream and crêpes.
Most importantly I would like to thank my husband, Nige, who has put up with constant
placenta and syncytiotrophoblast talk for the last three years. I would like to thank him
for all that he does for me and for his unwavering supply of love, encouragement and
emotional support.
Lastly, I would like to thank the Gerald Kerkut Trust for their funding over the course
of my PhD.
xviii
Definitions and Abbreviations
Abbreviation
Definition
4F2hc 4F2 cell-surface antigen heavy chain
AGT-1 Aspartate glutamate transporter 1
Ala Alanine (A)
ANOVA Analysis of variance
Arg Arginine
ASCT2 Alanine, serine, cysteine transporter 2
Asn Asparagine (N)
Asp Aspartate (D)
ATP Adenine triphosphate
AUC Area under the curve
BCA Bicinchoninic acid assay
BM Basal membrane
bp Base pair
BSA Bovine serum albumin
BSP Bromosulphothalein
cAMP Cyclic adenosine monophosphate
cDNA Complementary deoxyribonucleic acid
cpm Counts per minute
cRNA Complementary ribonucleic acid
Cys Cysteine
DAPI 4',6-diamidino-2-phenylindole
DHEAS Dehydroepiandrosterone sulphate
DIDS 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid
DMEM Dulbecco's modified eagle medium
xix
DNA Deoxyribonucleic acid
DOHaD Developmental origins of health and disease
DTT Dithiothreitol
E Efficiency
e- Electron
EAAT Excitatory amino acid transporter
EBB Earle's bicarbonate buffer
ECL Enhanced chemiluminescent
EDTA Ethylenediaminetetraacetic acid
ES Estrone sulphate
FBS Fetal bovine serum
FGR Fetal growth restriction
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GFP Green fluorescent protein
Gln Glutamine (Q)
Glu Glutamate (E)
Gly Glycine (G)
GPX Glutathione peroxidase
GR Reduced glutathione
GSH Glutathione
GSSG Glutathione disulfide
H
2
O
2
Hydrogen peroxide
hCG Human chorionic gonadotropin
His Histidine (H)
Hr Hour
IGF I Insulin like growth factor II
IGF II Insulin like growth factor II
xx
IGFBP3 Insulin growth factor binding protein 3
IGFIR Insulin like growth factor I receptor
IRS1 Insulin receptor substrate 1
Ile Isoleucine (I)
kDa Kilodalton
Km Substrate concentration that gives half maximal velocity of a reaction
LAT3 L-type amino acid transporter 3
LAT4 L-type amino acid transporter 4
LCMS Liquid chromatograph-mass spectrometry
Leu Leucine (L)
LGA Large for gestational age
Lys Lysine (K)
MAPK Mitogen activated protein kinase
Met Methionine (M)
min Minute
mRNA Messenger ribonucleic acid
MRP Multidrug resistance protein
mTOR Mammalian target of rapamycin
mV Millivolt
MVM Microvillous membrane
NADPH Nicotinamide adenine dinucleotide phosphate
NEC No enzyme control
NSAID Non-steroidal anti-inflammatory drug
NTC No template control
OAT Organic anion transporter
OATP Organic anion transporter polypeptide
OCT Organic cation transporter
xxi
ONOO- Peroxynitrite
OR2 Oocyte Ringer's 2 buffer
ORF Open reading frame
P5C Pyrroline-5-carboxylate
PAGE Polyacrylamide gel electrophoresis
PAH Para-aminohippuric acid
PBS Phosphate buffered saline
Phe Phenylalanine (F)
PI3K Phosphatidylinositide 3-kinase
POX Proline oxidase
Pro Proline (P)
pS Picosiemen
PVDF Polyvinylidene fluoride
qPCR Quantitative PCR
RIPA Radioimmunoprecipitation assay buffer
RNA Ribonucleic acid
ROS Reactive oxygen species
rtPCR Real time PCR
RVD Regulatory volume decrease
s Second
SDS Sodium dodecyl sulphate
SEM Standard error of the mean
Ser Serine (S)
siRNA Small interfering RNA
SLC Solute carrier
SNAT Sodium dependent neutral amino acid transporter
TAE Tris-acetate-ethylenediaminetetraacetic acid
xxii
TAT1 T-type amino acid transporter
Thr Threonine (T)
Trp Tryptophan (W)
Tyr Tyrosine (Y)
URAT1 Urate transporter 1
Val Valine (V)
Vmax Maximum rate of reaction
VRAC Volume regulated anion channel
xCT Cystine transporter
Chapter 1: General Introduction
Chapter One: Introduction
2
1.1 Introduction
The placenta is the interface between the maternal and fetal circulations. Its specialised
structure and functions enable the maintenance of pregnancy and the support of fetal
growth and development. The placenta is the barrier between mother and fetus and
selectively mediates nutrient transfer to the fetus. The placenta also protects the fetus
from toxins / infections and removes waste products from the fetal circulation. The
environment in which the fetus develops can affect its susceptibility to chronic diseases
later in life (Gluckman & Hanson, 2004; Lewis et al., 2013a). Changes in placental
function may influence fetal development and may therefore have long term
implications for disease risk (Powe et al., 2011). Understanding the mechanisms behind
normal placental function and nutrient transfer is, therefore, crucial if fetal outcome
from pathological pregnancies is to be improved.
Suboptimal placental transfer of any nutrient may result in fetal development being
compromised. Nutrients from the maternal circulation must be transported across the
maternal facing microvillous membrane (MVM) and the fetal facing basal membrane
(BM) of the placental syncytiotrophoblast in order to enter the fetal circulation (Lewis
et al., 2013a). Small lipophilic molecules such as oxygen can cross the placenta by
simple diffusion but larger hydrophilic molecules including amino acids require
membrane transporters to enter the fetal circulation (Alberts et al., 2002; Lodish et al.,
2000). The amino acid glutamate is unusual in the sense that there is no net transfer
across the placenta. Glutamate uptake from the maternal and fetal circulations is
mediated by secondary active transporters (excitatory amino acid transporters (EAAT)).
There are also efflux routes for glutamate into the maternal and fetal circulations but the
mechanisms behind this are unknown (Cleal et al., 2011; Day, 2012).
Chapter One: Introduction
3
The purpose of glutamate efflux from the placenta is unclear. Placental uptake of
glutamate by the placenta is known to have important roles in metabolism by producing
glutamine for the developing fetus as well as intermediates for the Kreb’s cycle (Day et
al., 2013). In other tissues such as the brain, glutamate plays a role in antioxidant
synthesis and is an osmolyte (Lushchak, 2012; Musante et al., 1999). It is possible that
there are unidentified glutamate efflux transporters within the human
syncytiotrophoblast. This thesis aims to investigate the mechanisms of glutamate efflux
from the syncytiotrophoblast and determine whether the transmembrane glutamate
gradient has unidentified roles.
1.2 Development of the human placenta
The human placenta is essential for fetal growth and development. Its primary function
is to act as a barrier, protecting the fetus from substances in the maternal circulation but
selectively allowing the flow of maternal nutrients to the fetus and the transfer of waste
products back into the maternal circulation. The placenta is also a highly active
endocrine organ, producing hormones that are necessary for the continuation of
pregnancy.
Development of the human placenta begins from the trophectoderm, the outer layer of
the blastocyst. Once implantation into the uterus occurs during the early stages of the
first trimester, placental development is tightly regulated. Throughout gestation, the
placenta develops in order to meet the increasing demands of the growing fetus. Normal
placental development is crucial for placental function so abnormalities in development
may directly impair or compromise the growth of the developing fetus.
Chapter One: Introduction
4
1.2.1 Implantation and invasion of the placenta into the uterine wall
After fertilisation, the conceptus enters the uterus where it develops into a blastocyst
and implants into the uterine wall (James et al., 2012). The blastocyst comprises of the
inner cell mass and the trophectoderm. The trophectoderm layer develops into the
placenta and external membranes and the inner cell mass develops into the fetus
(Kaufmann & Castellucci, 2006) (Figure 1.1A).
When the blastocyst implants into the wall of the uterus, the trophectoderm layer
proliferates and differentiates into the mononuclear cytotrophoblast, which
differentiates into the multinucleated syncytiotrophoblast. The syncytiotrophoblast
secretes proteolytic enzymes that degrade decidual epithelia and aid the invasion
process (Figure 1.1B).
1.2.2 First trimester placental development
After 9-10 days, the lacunar stage occurs where vacuoles appear within the
syncytiotrophoblast, forming lacunae (Benirschke et al., 2012). During the first weeks
of gestation the embryo is able to receive its nourishment by simple diffusion. Once the
placenta has formed, but before maternal blood flow begins, nutrition is derived from
secretions from uterine glands (histotrophic nutrition) but as growth continues, a
powerful nutrient and exchange system is required for growth and development (Burton
et al., 2002). The lytic activity of the syncytiotrophoblast results in maternal vessels in
the uterine tissue bursting resulting in the lacunae filling with blood as well as
secretions from endometrial glands (Burton et al., 2002) (Figure 1.1C).
Cytotrophoblastic projections then extend into the lacunae, forming the basis of the
primary villi, which have a cytotrophoblast core and a covering syncytiotrophoblast
Chapter One: Introduction
5
external layer (Benirschke et al., 2012) (Figure 1.1C). The secondary villi form
approximately 2 weeks after conception when mesenchymal cells from the extra-
embryonic mesenchyme layer invade the primary villi. The mesenchyme core continues
to expand towards the end of the villous, new branches and fetal capillaries are created,
signifying the transformation of secondary villi into tertiary villi (Robin et al., 2009)
(Figure 1.1D).
Around day 16 post conception, mesenchymal villi, formed from the tertiary villi,
undergo differentiation to form different villous types (immature intermediate villi, stem
villi, mature intermediate villi and terminal villi) that have different structures and
functions (Benirschke et al., 2012).
Figure 1.1 - Early placental development during the first trimester A) Structure of the
blastocyst showing the inner cell mass, which develops into the fetus and the trophectoderm. B)
Implantation of the blastocyst into the maternal endometrium of the uterine wall. C) The
syncytiotrophoblast envelops the embryo and cytotrophoblast columns form the primary villi.
D) Development and differentiation of the different types of villus during first trimester.
Chapter One: Introduction
6
1.2.3 Placental blood flow
Maternal blood flow to the placenta does not begin until 10-12 weeks post conception at
the end of the first trimester. In addition to the invasion of the placenta into the uterine
wall, extravillous trophoblast migrates into the maternal tissue and remodels the spiral
arteries by eroding the smooth muscle surrounding them. The remodelling of spiral
arteries was thought to be important for reducing vascular resistance so that more blood
flows to the placenta. However, it is thought that spiral artery remodelling allows the
delivery of blood to be less forceful, preserving villous tree structure (Burton et al.,
2009b).
Once the cytotrophoblastic plugs are removed from the opening of the spiral arteries at
10-12 weeks post conception, blood flow to the placenta is initiated resulting in the
levels of oxygen rising rapidly and the utero-placental circuit becoming complete. The
fetal circulation enters the placenta through the umbilical arteries of the umbilical cord
and branches out across the chorionic plate before exchange occurs between the
intervillous maternal blood and the fetal blood. The maternal circulation flows through
the remodelled maternal spiral arteries into the intervillous space which is in contact
with the microvillous membrane of the syncytiotrophoblast. Blood containing fetal
waste products that has passed from the chorionic villi into the intervillous space is then
returned back to the maternal circulation via the maternal uterine veins (Wang & Zhao,
2010).
1.2.4 Second trimester placental development
In the second trimester, the mesenchymal villi continue to differentiate into immature
intermediate villi before differentiating into stem villi. The stem villi are characterised
Chapter One: Introduction
7
by their dense stroma and thick walled vessels. The stem villi then differentiate into the
mature intermediate villi which contain capillaries, arterioles and venules, have fewer
cytotrophoblast cells and a thinner layer of syncytiotrophoblast. By the end of the
second trimester, terminal villi begin to differentiate from the mature intermediate villi
and these have highly dilated capillaries and a much thinner cytotrophoblast layer that
contains syncytial knots, providing a reduced diffusion distance (Kaufmann &
Castellucci, 2006).
1.2.5 Third trimester placental development
During the third trimester, the number of mature intermediate villi and terminal villi are
higher than they were in the second trimester. The terminal villi have a discontinuous
cytotrophoblast layer, highly dilated capillaries and the thinnest syncytiotrophoblast
layer of all types of villi (Figure 1.2).
These characteristics provide a minimal diffusion distance for gases and nutrients. The
fetal capillaries and syncytiotrophoblast are separated by the basal membrane of the
syncytiotrophoblast, providing a minimal diffusion distance of 3.7 microns, maximising
surface area and decreasing diffusion distance for gases and nutrients (Wang & Zhao,
2010). At term, the terminal villi account for nearly 40% of the villous volume of the
placenta (Kaufmann & Castellucci, 2006; Wang & Zhao, 2010).
Chapter One: Introduction
8
Figure 1.2 Differentiation of the villi in first trimester and third trimester. Representative
cross section of a first trimester villous (A) and third trimester villous (B).
1.2.6 Placental structure at term
As gestation progresses, the fetus’ demand for nutrients increases so the placenta must
be able to meet its requirements. Average placental diameter is 22 cm, a width of 2-3
cm and an average weight of 470 g (Yetter, III, 1998). The maternal facing side of the
placenta is referred to as the basal plate and is attached to the uterine tissue. The
placenta has approximately 20 cotyledons, each with its own fetal and maternal blood
Chapter One: Introduction
9
supply. The fetal side of the placenta consists of the chorionic plate which is attached to
the embryonic membranes and umbilical cord which contains a single vein and two
arteries. On average, each umbilical cord artery provides eight terminal chorionic plate
arteries, which deliver deoxygenated blood back to the placenta while the veins carry
oxygenated blood to the fetus (Wang & Zhao, 2010). The intervillous space surrounds
the villous trees, whose terminal ends are the sites of the majority of maternal to fetal
exchange (Gude et al., 2004). The villous tree is enclosed within a structural tissue
matrix called the stroma which is produced from connective tissue cells, fibres and fetal
vessels (Kaufmann et al., 1977) (Figure 1.4C). Within the villi, the fetal blood is
brought into close proximity with the maternal circulation but the syncytiotrophoblast
ensures that there is no mixing between the blood of the two circulations (Figure 1.3 and
Figure 1.4A).
Figure 1.3 - Placental structure and circulation at term. Maternal blood enters the
intervillous space via the spiral arteries. Nutrients and gases are transported across the
syncytiotrophoblast surrounding the chorionic villi and enter the fetal circulation. Oxygenated
blood reaches the fetus through the umbilical vein and deoxygenated blood is carried back to the
placenta via the umbilical arteries. Adapted from Cornell University.
Chapter One: Introduction
10
Figure 1.4 - Structure of the placental villi at term A) The maternal facing microvillous
membrane (MVM, green showing Datura stramonium lectin staining), is in direct contact with
the maternal blood and mediates nutrient transfer to the fetus. Cell nuclei are shown in red. B)
Structure of the placental vessels (green showing Aleuria auranlia lectin staining) and nuclei in
red. The capillaries extend to the edges of the villi, minimising the diffusion distance for
nutrient and gaseous exchange. C) The villous stroma at term (green showing Pisium sativum
agglutinin staining). Images provided courtesy of Dr Rohan Lewis and Dr Suzanne Brookes.
Chapter One: Introduction
11
1.2.7 The term placental barrier
The maternal and fetal circulations are physically separated via the placental barrier
which is made up of the syncytiotrophoblast that is in direct contact with the maternal
blood, a discontinuous cytotrophoblast layer, connective tissue and the fetal
endothelium which is in direct contact with the fetal circulation. Collectively, these
layers form a barrier which stops molecules that are not freely diffusible from
exchanging between the two circulations.
The syncytiotrophoblast is the interface between the maternal and fetal circulations,
protecting the fetus from bacteria and high molecular weight drugs / toxins that are too
big to pass through the barrier (Vahakangas & Myllynen, 2009). It is also responsible
for synthesising hormones required for pregnancy and mediating the transfer of oxygen,
nutrients and waste products to and from the fetus (Ji et al., 2013). The
syncytiotrophoblast has a fetal facing basal membrane (BM) and a maternal facing
microvillous membrane (MVM). The MVM is in direct contact with the maternal blood
and maximises its surface area for nutrient exchange by being deeply covered in
microvilli (Figure 1.5) (Burton et al., 2009a).
The discontinuous cytotrophoblast layer maintains the syncytiotrophoblast throughout
gestation, allowing it to expand by replenishing cells that have undergone apoptosis
(Huppertz & Borges, 2008; Kar et al., 2007).
The connective tissue layer, containing stromal cells, separates the cytotrophoblast and
fetal endothelium. The terminal villous capillaries form a looped network of sinusoidal
dilations that are highly metabolically active (Karimu & Burton, 1995). The external
surface of these dilations, the endothelium, is in close proximity to the BM of the
syncytiotrophoblast. The endothelium of the terminal villous capillaries is similar to that
Chapter One: Introduction
12
of smooth muscle with paracellular tight junctions which provide a pathway for the
passage of water and hydrophilic solutes to cross the capillary (Leach & Firth, 1992).
Figure 1.5 Structure of the syncytiotrophoblast at term showing the exchange barriers
involved in nutrient transfer. The maternal facing microvillous membrane (MVM) is bathed
in maternal blood and the basal membrane (BM) faces the fetal endothelium.
1.3 Endocrine roles of the placenta
Placental hormone secretion is essential for adapting maternal physiology to support the
pregnancy and to prepare for birth. In the early stages of pregnancy, the
syncytiotrophoblast secretes human chorionic gonadotropic (hCG) which promotes the
maintenance of the corpus luteum and stimulates the secretion of oestrogen and
progesterone which prevent menstruation. The fetal adrenal gland synthesises steroid
sulphate precursors which the placenta requires for oestrogen synthesis (Soucy & Luu-
The, 2000).
Placental growth hormone and lactogen are secreted by the placenta into the maternal
circulation in order to modulate intermediary metabolism and increase the supply of
glucose to the fetus (Cross & Mickelson, 2006; Freemark & Handwerger, 1989;
Handwerger & Freemark, 2000). Placental growth hormone and lactogen are thought to
Chapter One: Introduction
13
be involved in fetal growth, with the levels of these hormones positively correlating
with fetal birth weight (Handwerger & Freemark, 2000). A summary of the hormones
produced by the placenta and their functions is shown in Table 1.1.
The placenta secretes paracrine hormones that allow maternal physiology to be altered
so that gestation can progress. The secretion of growth hormone and lactogen into the
maternal circulation stimulates production of insulin like growth factors (IGF-I and
IGF-II), which the placenta is also able to synthesise. Both IGFs act through the IGF1
receptor (IGFIR), which dimerises, initiating autophosphorylation. In vitro studies also
suggest that IGFs may regulate placental development by initiating trophoblast invasion
into the decidua and myometrium, helping to remodel the spiral arteries (Harris et al.,
2011). At birth, the IGF-I knockout mouse has severe fetal growth restriction (FGR),
which indicates that placental abnormalities and mutations can directly affect fetal
growth (Liu et al., 1993).
Placental hormones can also affect the activity of nutrient transporters in the placenta.
Leptin for example upregulates SNAT1 and 2 transporter activity while high insulin
levels increase the uptake of the amino acid analogue MeAIB (Jansson et al., 2003; von
Versen-Hoynck et al., 2009).
Chapter One: Introduction
14
Table 1.1 Key hormones produced by the placenta and their functions in pregnancy.
Placental hormone
Function
(Gude et al., 2004; Murphy et al., 2006).
Oestrogen (estrone, estradiol and
estriol)
Upregulates progesterone synthesis, adapts
maternal physiology, regulates
syncytiotrophoblast function and proliferation
Progesterone
Regulation of adhesion molecules and growth
factors that prepare the endometrium for
implantation
Placental lactogen
Initiates insulin like growth factor release
Human chorionic gonadotropin (hCG)
Promotes maintenance of the corpus luteum so
that progesterone can be secreted in the first
trimester
Placental growth hormone
Acts in concert with lactogen to initiate insulin
like growth factor release. Supresses maternal
hypothalamic pituitary adrenal axis
IGF-I and IGF-II
Growth factors
Placental leptin
Trophoblast proliferation, transporter activity
Chapter One: Introduction
15
1.3.1 Developmental origins of health and disease
A large body of evidence now supports the hypothesis that susceptibility to chronic
disease in postnatal life is influenced by in-utero conditions (Gluckman & Hanson,
2004). Pioneering studies by Professor David Barker demonstrated that low birth weight
is associated with an increased prevalence of diabetes and coronary heart disease in later
life (Barker et al., 1989). Subsequent studies have shown that it is not just low birth
weight babies who are at risk of chronic diseases in later life and that there is a graded
association across the range of birth weights (Hanson & Gluckman, 2008). The
exception to this is with very high birth weight babies where risk begins to increase,
particularly where there is maternal diabetes (Persson et al., 2011). Birth weight is
likely to be a surrogate indicator for a poor intrauterine environment, which predisposes
the fetus to disease in later life.
The placenta modulates fetal environment, regulating nutrient supply to the fetus and
waste product removal back to the mother. The maternal diet, body composition and
lifestyle may all in part contribute to the regulation of placental function which will
affect fetal growth and development (Lewis et al., 2013b). The fetal tissues undergo
periods of critical growth and if demand for nutrients is not met by the placenta, the rate
of cell division decreases resulting in disproportionate growth. Recently, animal studies
have shown that the peri-conceptional time frame is a particularly vulnerable period as
maternal peri-conceptional malnutrition results in an increased risk of chronic disease in
later life (Fleming et al., 2012).
Records kept during the Dutch Famine in World War II provide an in-depth insight into
the effects of maternal under-nutrition throughout different trimesters of gestation.
Women who had experienced the famine during the first six months of gestation had
Chapter One: Introduction
16
babies who were of normal birth weight but were more at risk of cardiovascular disease
and obesity in later life compared to women who only experienced under-nutrition at
the end of gestation (Ravelli et al., 1999; Roseboom et al., 2000). This suggests that
fetal programming is not determined by birth weight but the intrauterine environment.
Placental function will alter nutrient transfer to the fetus which will directly affect its
growth and development. It is thought that fetal growth restriction is a consequence of
placental dysfunction that leads to fetal under nutrition (Krishna & Bhalerao, 2011). If
fetal growth and development is to be optimal in order to prevent chronic diseases in
later life, understanding the mechanisms behind normal placental function is crucial.
1.4 Placental influences on fetal development
An insufficient supply of any nutrient to the fetus may become limiting for fetal growth.
This may lead to fetal growth restriction (FGR) or pre-eclampsia (Gude et al., 2004).
Contrastingly, over nutrition can result in large for gestational age babies (LGA), as
seen in some maternal diabetes cases where there is an increase in glucose transfer to
the fetus (Jansson et al., 2002).
FGR is defined as a fetus not reaching its genetically pre-determined birth weight. This
may be caused by abnormal placental function, as well as pre-existing conditions and
diseases of the mother, chromosomal defects, maternal diet and environment (e.g.
smoking, alcohol intake / drug intake) (Briana & Malamitsi-Puchner, 2009; Sram et al.,
2005).
Pre-eclampsia causes FGR and is also the world’s largest leading cause of maternal
mortality and is characterised by the development of hypertension and proteinuria
during the latter stages of pregnancy (Powe et al., 2011). The exact mechanisms and
Chapter One: Introduction
17
cause of pre-eclampsia are still unknown but abnormal placental development and
hypoxia in the intra-uterine environment is thought to be a key feature.
Maternal hypoxia is thought to reduce the abilities of the invasive cytotrophoblast and it
has been observed that there is a higher incidence of pre-eclampsia in women at high
altitude (Genbacev et al., 1996; Zamudio, 2003; Zamudio, 2007). During the first
trimester of pregnancy, levels of placental oxygen are low as the cytotrophoblast cells
block the spiral arteries. However, once maternal blood flow to the placenta is initiated,
oxygen tension increases and it is thought that this sudden change in oxygen levels
increases oxidative stress, which may also contribute to pre-eclampsia (Burton &
Jauniaux, 2004; Jaffe et al., 1997).
LGA babies are defined as having a birth weight above 4000 g or being in the 90
th
percentile or above for their gestational age (Chauhan et al., 2005; Henriksen, 2008).
LGA fetuses are associated with obstetrical and neonatal complications that present
risks for both the fetus and the mother including a higher risk of hypoxia,
hypoglycaemia and shoulder dystocia for the fetus whilst the mother is likely to have a
prolonged labour, greater chance of a caesarean as well as postpartum haemorrhage and
infections (Boyd et al., 1983; Modanlou et al., 1980).
Maternal diabetes and maternal obesity are not thought to be the sole determinants of
LGA infants as not all diabetic / obese women have LGA babies. It is likely that
placental function is also implicated as LGA requires increased placental nutrient
transfer (Leung & Lao, 2000; Lewis et al., 2013b). In the long term, LGA babies appear
to be at an increased risk of developing metabolic syndrome, diabetes, asthma and being
overweight in later life (Boney et al., 2005; Rogers, 2003).
Chapter One: Introduction
18
1.5 Mechanisms of placental transport
The supply of nutrients to and the removal of waste products from the developing fetus
is necessary if optimal fetal growth and development is to occur (Cleal et al., 2011;
Marconi & Paolini, 2008). The placental syncytiotrophoblast separates the fetal and
maternal circulations. Thus, in order for substances to pass from the maternal to fetal
circulation or vice versa, they must be transported across both the MVM and BM of the
syncytiotrophoblast. Most nutrients, including glucose and amino acids, require
membrane transporters, as they are either charged or too large to cross the membrane by
diffusion. Membrane transporters are key to the transfer of nutrients across the placenta
to the fetus, although some molecules can be transferred by the paracellular route
(Figure 1.6).
Once a solute has been transferred across the BM it must then cross the fetal capillary
endothelium. The endothelium is not thought to be a barrier to glucose and amino acids
as placental capillaries have a similar permeability to that of skeletal muscle, with
paracellular clefts that are not occluded, unlike those in the brain (Leach & Firth, 1992).
However, molecules such as non-esterified fatty acids are unlikely to be transferred into
the fetal circulation via paracellular diffusion and will require other transport
mechanisms.
1.5.1 Active transport
Active transport involves the movement of a solute across the plasma membrane,
against a concentration or electrochemical gradient. If a substance is present in a higher
concentration in the fetal blood, then it is likely that the substance is transported by
active transport, which is divided into two classes; primary active transport and
secondary active transport. Primary active transport is carried out by four types of
Chapter One: Introduction
19
transporter; the P-type ATPase, the F-ATPase (mitochondrial membranes), the V-
ATPase and the ATP binding cassette (ABC) transporters (Lodish et al, 2000). These
transporters all mediate the transport of a solute, which is directly coupled to the
hydrolysis of ATP. The P-type ATPase family includes the Na
+
- K
+
-ATPase pump
which helps the cell to maintain low concentrations of sodium and high concentrations
of potassium, helping to maintain resting membrane potential (Palmgren & Nissen,
2011). The F-ATPase family of transporters are found in the inner membranes of the
mitochondria where energy harnessed from the transport of protons from high to low
concentration is used to phosphorylate ADP into ATP (Kinosita, Jr. et al., 2004). The
V-type ATPase family couple ATP hydrolysis to proton influx into organelles such as
lysosomes (Beyenbach & Wieczorek, 2006). The ABC transporters mediate the
transport of a variety of substrates, including drugs, xenobiotics, lipids and sterols, by
coupling to the hydrolysis of ATP (Ni & Mao, 2011).
1.5.1.1 Primary active transporters in human placenta
In the human placenta, two examples of expressed primary active transporters are the
Na
+
- K
+
ATPase pump and the ABC transporters. The Na
+
- K
+
ATPase pump utilises
ATP in order to drive the efflux of three sodium ions and the influx of two potassium
ions, providing the cell with a high concentration of potassium and a low concentration
of sodium (Ogawa, 2009). This mechanism not only helps to maintain resting
membrane potential but also assists transport. When sodium ions are effluxed from the
cell, this provides a driving force for the secondary active transporters which are
described below.
In the human placenta, there are three other main groups of ABC transporter: P
glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and the multidrug
Chapter One: Introduction
20
resistance proteins (ABCC1, ABCC2, ABCC3). The apical expression of the majority
of the ABC transporters in the placenta suggests that their function is to protect the fetus
from the detrimental effects that many drugs and xenobiotics have on fetal development
by driving the efflux of these substances back into the maternal circulation if they enter
the placenta (Ni & Mao, 2011). This would be particularly beneficial if the mother is
taking medication for pregnancy-induced conditions.
1.5.1.2 Secondary active transport
Secondary active transport involves a transporter utilising the energy stored in an
existing concentration gradient. Ions such as sodium (Na
+
) are transported into the cell,
down their concentration gradient, which increases entropy and provides energy for the
transporter to also transport a solute at the same time (Figure 1.6). The majority of
nutrient transfer across the syncytiotrophoblast is mediated by the solute carrier family
(SLC) superfamily (Ni & Mao, 2011; Staud et al., 2012). The SLC series of transporters
is made up of 52 families and currently 386 genes have been identified (Schlessinger et
al., 2013). These genes encode transporters which mediate facilitated diffusion and
secondary active transport by either functioning as co-transporters or antiporters and can
be divided into accumulative transporters, exchangers and facilitated transporters
(Fotiadis et al., 2013; Halestrap, 2013; Kanai et al., 2013) Symporters, such as the
excitatory amino acid transporters (EAAT), transport the solute and the sodium ion in
the same direction while exchangers mediate the transport of the solute and ion in
opposite directions (Alberts et al., 2002; Lodish et al., 2000).
1.5.1.3 Exchangers
Exchangers are integral membrane proteins that mediate the transport of one solute
along its electrochemical gradient while transporting another solute against the
Chapter One: Introduction
21
electrochemical gradient. For exchangers to function there must be substrates on either
side of the membrane before transport is able to occur. If one of the substrates is also a
substrate for a facilitated transporter, this may produce a transmembrane gradient which
could drive the activity of the exchanger by secondary active transport (Lodish, 2000).
1.5.2 Facilitated transport
Facilitated transport is a passive process whereby solutes, such as glucose, are able to be
transported across the membrane by binding to trans-membrane integral transporter
proteins. When a particular solute binds to a transporter, the protein undergoes a series
of conformational changes that results in the transport of the solute from one side of the
membrane to the other. Once the solute has been released, the transporter undergoes
another conformational change, reverting back to its original state, ready to bind another
substrate.
Facilitated diffusion can also be mediated by ion channels which form a hydrophilic
pore across the membrane that is narrow, highly selective and is able to open and close.
Unlike transporters, ion channels do not directly interact with the substrate (Alberts et
al., 2002). Rather, the solute, usually inorganic ions, passes through the channel when in
its open state. For channels that are not constitutively open, channel opening can be
initiated in one of three ways; a change in membrane potential (voltage-gated), the
binding of a ligand (ligand-gated) and through mechanical stress (mechanical-gated)
(Alberts et al., 2002).
Chapter One: Introduction
22
Figure 1.6 - Mechanisms of nutrient transfer across the syncytiotrophoblast. Gases such as
oxygen and carbon dioxide are freely diffusible but larger, hydrophobic molecules require
active transport or facilitated diffusion to be transferred from one side of the membrane to the
other.
1.5.3 Passive diffusion
Passive diffusion can be paracellular whereby a substance diffuses through the gaps
between cells or transcellular in which a substance diffuses through the cell, down its
concentration gradient. A paracellular route across the syncytiotrophoblast exists but its
nature is debated due to the apparently continuous nature of the syncytiotrophoblast
layer. There is evidence that the syncytiotrophoblast has fibrinoid filled denudations
which provide a paracellular route for low molecular weight substances (Brownbill et
al., 2000). It is also hypothesised that paracellular routes are formed under pressure via
transtrophoblastic channels but it is not clear if these channels form one connecting
route from the basal membrane to the microvillous membrane (Kertschanska et al.,
1994; Sibley et al., 1981; Stulc et al., 1969). The ability of a substance to diffuse
passively is determined by its size and lipid solubility, with small, hydrophilic
substances diffusing more easily than larger, hydrophobic ones (Alberts et al., 2002;
Tieleman, 2006). Molecules such as oxygen and carbon dioxide are transported across
Chapter One: Introduction
23
the placenta in this manner. It is possible that the paracellular route also provides a route
through which xenobiotics reach the fetus.
1.6 Placental transport
Both the MVM and BM of the syncytiotrophoblast are equipped with these different
types of transporters that allow the transfer of different amino acids, therapeutic drugs
and toxins from one circulation to the other (Lewis et al., 2011). The combined action
of these transporters allows the syncytiotrophoblast to control its uptake and efflux of
critical molecules and compounds required for fetal growth and development as well as
placental growth and function.
Accumulative transporters mediate the influx of amino acids into the placenta from the
maternal and fetal circulations and this can occur against a concentration gradient
(Noorlander et al., 2004). EAAT1, CAT2b, CAT3, CAT4
and SNAT1, SNAT2 and
SNAT4 transporters mediate the influx of amino acids into the placenta (Figure 1.7).
The accumulative transporters create transmembrane amino acid gradients which can
drive the uptake of other amino acids by the amino acid exchangers. Amino acid
exchangers (ASCT1, ASCT2, LAT1, LAT2, y
+
LAT1 and y
+
LAT2) on the BM / MVM
mediate the influx of a specific amino acid in exchange for the efflux of another amino
acid into the fetal / maternal circulation (Cariappa et al., 2003; Cleal et al., 2011; Furesz
et al., 1995; Ramadan et al., 2006). There must be amino acid substrates for both influx
and efflux before exchange can occur and as a result, exchangers are unable to change
the net concentration of amino acids (Cleal & Lewis, 2008).
The facilitated transporters, TAT1, LAT3 and LAT4, are expressed on the BM of the
syncytiotrophoblast and drive the efflux of specific amino acids into the fetal circulation
(Cleal et al., 2011). These transporters are critical for determining the quantity of amino
Chapter One: Introduction
24
acids that the fetus receives, providing net transport of alanine, tyrosine, phenylalanine,
isoleucine and valine into the fetal circulation for growth and development (Cleal et al.,
2011). The accumulative transporters establish amino acid gradients which drives the
efflux of amino acids through the facilitated transporters.
Figure 1.7 Amino acid transporter localisation on the MVM and BM of the placental
syncytiotrophoblast (Cleal et al., 2011; Cleal & Lewis, 2008).
The placenta not only modulates the exchange of nutrients between the mother and
developing fetus but also protects the fetus from the detrimental effects of waste
products and xenobiotics by mediating the transfer of these substances back into the
maternal circulation across both the BM and MVM. Drugs and xenobiotics need to be
transported into the cell in order to have their effects but molecular weight and lipid
solubility affect the ability of a substance to cross the lipid bilayer. Many drugs and
xenobiotics have been shown to cause birth defects. Thalidomide for example, was
taken by pregnant women in the 1960s to stop morning sickness but was shown to be a
teratogen as babies were born with malformation of the limbs (Matthews & McCoy,
2003). As a result, transporters are required to mediate transfer back to the maternal
Chapter One: Introduction
25
circulation (Figure 1.8). On the BM, xenobiotic uptake from the fetal circulation into the
placenta is likely to be mediated by the OAT (SLC22) and OATP (SLCO) exchangers,
which transport a wide variation of drug metabolites and xenobiotics (Helix et al., 2003;
Ugele et al., 2008). Once back into the placenta, the ABC transporters are likely to work
in conjunction with the OAT/OATP transporters to drive the efflux of xenobiotics back
into the maternal circulation (Koepsell, 2013; Vahakangas & Myllynen, 2009).
Figure 1.8 - Localisation of the SLC and ABC transporters on the BM and MVM of the
syncytiotrophoblast. The ABC transporters utilise ATP to actively transport toxic substances
from the fetus and placenta back into the maternal circulation whilst the secondary active SLC
transporters mediate nutrient transport to the fetus.
1.7 Regulation of membrane transporters in the placenta
Factors such as nutrient concentration gradients, metabolism and placental blood flow
alter nutrient transport to the fetus. There is now evidence to suggest that the activity of
nutrient transporters in the syncytiotrophoblast is a rate limiting factor in the supply of
nutrients to the fetus as altered regulation has been associated with pathological
pregnancies. In FGR, SNAT1/SNAT2 and LAT1/LAT2 transporters are down regulated
but the glucose transporters are unaffected (Jansson et al., 1993). In contrast, glucose
Chapter One: Introduction
26
transporters are up regulated in mothers with type one diabetes but not in mothers with
gestational diabetes, suggesting that babies exposed to high glucose in early gestation
will develop high glucose levels later in life compared to those from mothers with
gestational diabetes (Jansson et al., 2002). As fetal growth relies upon nutrient transfer,
it is important to understand the mechanisms that regulate the placental nutrient
transporters if the mechanisms underlying fetal programming are also to be understood
(Jones et al., 2007).
Growth factors such as IGF-I and IGF-II are also known to influence transporter
function with IGF-I stimulating an increase in SNAT2 activity (Fang et al., 2006; Karl,
1995). The circulating levels of IGFs in the maternal circulation of diabetic mothers are
higher and this may also explain why some diabetic mothers have larger babies.
The mammalian target of rapamycin (mTOR) signalling pathway is thought to be an
important regulator of leucine transport via LAT1/LAT2, a regulation which is
markedly decreased in FGR (Roos et al., 2007). mTOR is a serine/threonine kinase and
acts as a nutrient sensor, monitoring the conditions in the cell’s environment and
bringing about an appropriate response. Once activated, mTOR causes a signalling
cascade that involves the phosphorylation of various kinases, which ultimately affects
protein transcription and translation (Asnaghi et al., 2004). In the syncytiotrophoblast,
mTOR signalling is highly active and inhibition leads to a reduction in leucine transport.
This suggests that in FGR, mTOR signalling is down regulated resulting in reduced
LAT1/LAT2 activity (Jones et al., 2007).
These studies suggest that a combination of placental and maternal factors determine
placental transporter regulation and that an alteration in these factors lead to an altered
Chapter One: Introduction
27
supply of nutrients to the fetus, contributing to FGR and other pathological pregnancy
conditions.
1.8 Placental glutamate
Glutamate is not thought to be transferred across the placenta into the fetal circulation;
rather there is net uptake from both the maternal and fetal circulations (Cetin et al.,
2005; Day et al., 2013). Glutamate efflux from the placenta cannot be explained by
current models of placental amino acid transport (Cleal et al., 2011). This is consistent
with the fact that within the placenta, the levels of intracellular glutamate are
remarkably high (5 mmol/l) when compared to the average levels of most other amino
acids (Phillips et al., 1978).
The high levels of glutamate are known to be important for metabolism, providing
glutamine for the developing fetus and α-ketoglutarate which enters the Kreb’s cycle
(Day, 2012). However, it is now also known that there is an unexplained glutamate
efflux route, primarily into the maternal circulation (Day, 2012). It is not only unclear
how this happens but it also unknown what the function of glutamate efflux from the
placenta is. Glutamate efflux from either side of the syncytiotrophoblast is likely to be
taken back up by the highly accumulative EAAT1 and EAAT3 transporters, which have
a K
m
of 18-28 µmol/l for glutamate (Arriza et al., 1994; Broer, 2002). It is unclear why
the placenta generates a transmembrane glutamate gradient, only for efflux to occur
before re-uptake completes the short circuit.
In other organs such as the liver and brain, glutamate in its anionic form is a substrate
for other transport mechanisms but little is known about their expression in the placenta
Chapter One: Introduction
28
(Blondeau, 2002; Sato et al., 2000). It is therefore possible that there are unidentified
glutamate transporters within the human syncytiotrophoblast.
The Maxi chloride channel is permeable to glutamate as well as other anions. The Maxi
chloride channel is an outwardly rectifying anion channel whose molecular identity is
unknown. However, it can be identified on the basis of activity in many tissues
including the MVM of the placental syncytiotrophoblast (Vallejos & Riquelme, 2007).
The Maxi chloride channel is involved in regulatory volume decrease which is initiated
when a cell undergoes an osmotic challenge (Helix et al., 2003). The channel opens,
allowing the efflux of glutamate and chloride ions and the efflux of water then follows,
restoring cell volume (Musante et al., 1999).
As regulatory volume decrease involves changing the chloride and potassium
concentrations in the cell, the electrochemical gradient is altered which is known to be a
determinant of amino acid transport across the MVM (Sibley et al., 1998). As a result,
the efflux of glutamate through the Maxi chloride channel may indirectly alter amino
acid transport into the placenta. Table 1.2 illustrates the other known human glutamate
transporters and channels.
Chapter One: Introduction
29
Table 1.2 - Transporters and channels known to mediate glutamate efflux.
Transporter
Mechanism
Expression in
placenta
Tissue
localisation
External K
m
for
glutamate
xCT
(SLC7A11)
Exchanger
No (Mann et al.,
2003)
Brain, liver,
kidney, eye,
stomach
48.1 µmol/l
(Hosoya et al.,
2002)
AGT-1
(SLC7A13)
Exchanger
No (Matsuo et
al., 2002)
Kidney
22.9 µmol/l
(Sarthy et al.,
2005)
OAT2
(SLC22A7)
Exchanger
No (Fork et al.,
2011)
Liver and kidney
1.2 mmol/l
Fork et al.,
2011)
ASCT2
(SLC1A5)
Exchanger
Yes (Johnson &
Smith, 1988)
Kidney, large
intestine, lung,
placenta
1630 µmol/l
(Utsunomiya-
Tate et al., 1996)
Maxi chloride
Channel
Yes (Riquelme,
2006)
Ubiquitous
Unknown
VRAC
Channel
Unknown
Ubiquitous
Unknown
1.8.1 Roles of placental glutamate
In the isolated perfused placenta, the high level of intracellular glutamate is thought to
have a variety of roles, including metabolism into glutamine and Kreb’s cycle
intermediate alpha-ketoglutarate as well as having a role in synthesis of the major
antioxidant glutathione and acting as an osmolyte (Musante et al., 1999; Newsholme et
al., 2003; Noorlander et al., 2004).
Glutamate is also known to be a substrate for glutathione synthesis. Glutathione is a
tripeptide comprising glutamate, cysteine and glycine and has many functions in many
living organisms but acts primarily as an antioxidant by interacting with reactive oxygen
species (Lushchak, 2012). Reactive oxygen species are produced naturally as a by-
product of oxidative phosphorylation. In a narrow concentration range, reactive oxygen
Chapter One: Introduction
30
species are known to act as messengers in signalling cascades (Fisher, 2009; Linley et
al., 2012; Rhee, 1999). However, at higher concentrations, these molecules are highly
reactive and unstable, resulting in damage to DNA, proteins and vasculature and
inducing apoptosis (Figure 1.9). During gestation, oxidative stress in the placenta
increases due to increased exposure to oxygen, as well as an increase in oxidative
phosphorylation, to keep the fetus supplied with glucose and amino acids. As a result,
the synthesis and supply of antioxidants to the placenta is thought to be important in
maintaining placental function by preventing structural damage to the vasculature
(Myatt & Cui, 2004).
Glutamate is central to nitrogen metabolism, being transaminated and deaminated to
allow synthesis of other amino acids and metabolites that both the placenta and the fetus
need for growth and development (Newsholme et al., 2003). The major products of
glutamate metabolism in the isolated human perfused placenta are glutamine, the Kreb’s
cycle intermediate alpha-ketoglutarate and proline (Day et al., 2013). Similar findings
have been observed in the sheep placenta and other species (Bloxam et al., 1981; Chung
et al., 1998).
Chapter One: Introduction
31
Figure 1.9 The involvement of glutathione (GSH) with reactive oxygen species. Reactive
oxygen species interact with GSH forming GSSG (oxidised glutathione). Glutathione radicals
(GS●) combine to form GSSG. The active reduced form (GSH) is then reformed by the action
of glutathione reductase (GR). A dot indicates production of a free radical and an unpaired
electron.
The placenta metabolises amino acids into ketoacids which can then enter the Kreb’s
cycle. During this process, ammonia is produced. The placenta does not have a urea
cycle to remove ammonia but the metabolism of glutamate into glutamine by glutamine
synthetase requires the addition of ammonia. Glutamine and its ammonia group can
then be transported into the maternal circulation, preventing a build-up of toxic
ammonia within the placenta (Battaglia, 2000; Jozwik et al., 2009).
As both glutamine and glutamate have important roles in the placenta, cycling between
the two molecules is essential for optimal growth and development of the fetus as well
as placental function (Battaglia, 2000; Moores, Jr. et al., 1994) (Figure 1.10).
Glutamine is considered to be a conditionally essential amino acid as many tissues rely
on glutamine utilisation for normal function, with glutamine having roles in purine and
pyrimidine synthesis, protein synthesis and amino sugar synthesis (Boza et al., 2000;
Cory & Cory, 2006; Lacey & Wilmore, 1990).
Chapter One: Introduction
32
Figure 1.10 - The metabolic fate of glutamate within the human placenta.
It is currently unclear whether the levels of placental glutamate are altered in
pathological pregnancies. Studies have shown that the maternal serum of pre-eclamptic
women contains higher levels of glutamate (Odibo et al., 2011; Teran et al., 2012). In
FGR, no differences have been found in the expression and distribution of the EAAT
transporters in FGR placentas compared to control (Noorlander et al., 2004). However,
the average umbilical venous plasma glutamate concentration is significantly higher in
pregnancies complicated by gestational diabetes suggesting that glutamate transport is
altered (Cetin et al., 2005).
Chapter One: Introduction
33
1.9 Aim
It is unknown how glutamate efflux from the placenta occurs and