Regulation of Extracellular Arginine Levels in the Hippocampus In Vivo
by Joanne Watts B.Sc. (Hons)
r
Thesis submitted for the degree of Doctor of Philosophy in the Faculty of Science, University of London
The School of Pharmacy University of London ProQuest Number: 10105113
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract
Nitric oxide (NO) has emerged as an ubiquitous signaling molecule in the central nervous system (CNS). NO is synthesised from molecular oxygen and the amino acid L-arginine (L-
ARG) by the enzyme NO synthase (NOS), and the availability of L-ARG has been implicated as the limiting factor for NOS activity. Previous studies have indicated that L- ARG is localised in astrocytes in vitro and that the in vitro activation of non-N-methyl-D- aspartate (NMDA) receptors, as well as the presence of peroxynitrite (ONOO ), led to the release of L-ARG. Microdialysis was therefore used in this study to investigate whether this held true in vivo. The results indicated that, while L-ARG was localised in glia in vivo and the infusion of a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) caused the release of L-ARG, this increase in extracellular L-ARG levels did not drive the NOS reaction. The increased L-ARG levels were halved by the coinfusion of CNQX but not totally inhibited. NMDA receptor activation is recognised as a major pathway of NOS activation and hence NO production and, while the results of this study concur, the results herein also suggest no need for increased L-ARG in the extracellular space as a prerequisite for NOS activation. Interesting results were also achieved using the NOS inhibitors N“- nitro-L-ARG methyl ester (L-NAME) and 7-nitroindazole (7-NI). Both inhibitors increased the basal activity of NOS and production of NO, while each drug had contrasting effects on the NMDA-stimulated response. L-NAME blocked the increased NOS activity with no effect on NO production while 7-NI had no effect on NOS but blocked the production of NO. In conclusion, the regulation of the supply of L-ARG for NOS in vivo is far more complicated than in vitro studies suggest.
11 A cknowledf^ements
Acknowledgements
There are a great number of people without whom none of this would have been possible.
Many thanks go to Professor Trevor Smart and Professor Alex Thomson for allowing me to use the facilities at the School. I would like to express my gratitude to my supervisors
Drs Brian Pearce and Peter Whitton for their help and advice, with special thanks to Brian for reading this thesis. Thanks also to the Medical Research Council and the School of
Pharmacy for financial support.
I am extremely grateful for the help and advice imparted by Dr Les Fowler, especially for his considerable expertise regarding HPLC. Thanks also to Analytix for the loan of the NOA
Many thanks also to Steve Coppard, Dave Zeraschi and Donna Howell for all their assistance with the volunteers.
I would also like to thank several people in the School who have supported me throughout and kept me amused. These include Vicky Welsh, Kshipra Desai, Jonathan Wilkinson, Raj
Tiwary, Nicola Wilson and Chris Courtice.
Thanks must also go to the staff of the computer and multimedia units for their support, especially Annie Cavanagh, Graham Florence and Paul Cuthbert.
Ill Dedication
!for 9drs 9{.Cv[. !Parton
IV Table o f Contents
Table of Contents Page
Chapter 1: General Introduction ...... 20
1.1 History of Nitric Oxide ...... 21
1.2 Synthesis of NO ...... 22
1.3 Amino acid transporters ...... 27
1.4 NOS isoenzymes ...... 29
1.4.1 Structure and fu n ctio n ...... 29
1.4.2 Calcium and calmodulin regulation ...... 31
1.4.3 Expressional regulation ...... 32
1.4.4 Enzyme activation and cellular localisation ...... 33
1.4.5 The arginine paradox and eN O S ...... 39
1.4.6 The roles of haem and tetrahydrobiopterin (BH 4 ) ...... 40 1.5 NO and signal transduction ...... 41
1.6 NOS in h ib ito rs ...... 44 1.6.1 Mechanisms of in h ib itio n ...... 44 1.6.2 The oxygenase dom ain ...... 44
1.6.3 The calmodulin binding s ite ...... 48
1.6.4 The reductase dom ain ...... 49 1.6.5 Arginine transporters ...... 49
1.6.6 Inhibitor specificity ...... 50 1.7 NO releasing com pounds ...... 53
1.7.1 General properties of NO donors ...... 53
1.7.2 Direct donors ...... 53
1.7.3 Donors requiring metabolism ...... 57
1.7.4 Bifunctional donors ...... 58
1.8 Roles of NO throughout the body ...... 58
1.8.1 The cardiovascular system ...... 58
1.8.2 Host defence ...... 60
1.8.3 The peripheral nervous system ...... 63
1.9 NO in the central nervous system ...... 64
1.9.1 Glutamatergic NO release...... 64
V Table o f Contents
1.9.2 Glutamate as a neurotransmitter ...... 65
1.9.3 The NMDA receptor ...... 67
1.9.3.1 Special features of the NMDA receptor ...... 67
1.9.3.2 Molecular biology of the NMDA receptor ...... 70
1.9.4 The AMPA receptor ...... 71
1.9.5 NO in neurotransmission ...... 73
1.10 A im s ...... 78
Chapter 2: Materials and M ethods ...... 79
2 . 1 /« v/vo microdialysis ...... 80
2.1.1 Probe Construction ...... 80
2.1.2 In vitro recoveries of amino acids ...... 82 2.1.3 Stereotaxic implantation ...... 84
2.1.4 Validity of microdialysis...... 8 8
2.1.5 Microdialysis...... 90
2.1 . 6 Anatomical verification of probe placement ...... 90 2.2 Analysis of amino acids ...... 92
2.2.1 Detection of amino acids ...... 92 2.2.2 Derivatisation of amino acids ...... 92
2.3 High performance liquid chromatography (H PLC) ...... 93
2.3.1 HPLC Apparatus ...... 93 2.3.2 Analysis...... 95
2.3.3 Mobile phase composition ...... 95
2.3.4 Identification of amino acids ...... 96
2.4 Detection of N O ...... 99
2.4.1 The Nitric Oxide Analyser (N O A ) ...... 99 2.5 In vitro s tu d ie s ...... 101 2.5.1 Tissue extraction ...... 101
2.5.2 NOS assay...... 101
2.5.3 Glial cell culture ...... 102 2.5.4 Release experiments ...... 103
2.6 Drugs and Compounds ...... 103
vi Table o f Contents
2.7 Statistical Analysis ...... 103
Chapter 3: Results ...... 104 3.1 Release of L-ARG in vitro and in vivo ...... 105 3.2 NOS activity...... 115
3.3 NO production ...... 119
3.4 NO and amino acid release ...... 121
3.5 NOS inhibition ...... 126
Chapter 4: D iscussion...... 135 4.1 Release of L-ARG in vitro and in vivo ...... 136 4.2 NOS activity...... 148
4.3 NO production ...... 154
4.4 NO and amino acid release ...... 156 4.5 NOS inhibition ...... 158
4.6 Future directions ...... 165
References...... 167
Vll ______List o f Fifjures
List of Figures Page
C h ap ter 1 : 1.1: Limulus polyphemus...... 23
1.2: Biosynthesis of nitric oxide from L-ARG and molecular oxygen ...... 24
1.3: The formation of ornithine from L-GLU ...... 25
1.4: A variation of the urea cycle which converts L-CIT back to L-ARG ensuring
a constant supply of L-ARG ...... 26
1.5: A linear model of co-factor recognition sequences within each NOS isoenzyme
and cytochrome P450 ...... 30
1.6: One of the proposed mechanisms for eNOS activation ...... 35
1.7: A schematic representation of how nNOS interacts with PSD-95 and other
proteins at the synaptic membrane ...... 36
1.8: The roles of haem and BH 4 in nNOS structure and function ...... 41 1.9: Potential targets for inhibition of synthesis or activity of NO ...... 45
1.10: A ball and stick representation of the iNOS catalytic site ...... 48 1.11: The main reactions of S-nitrosothiols ...... 56 1.12: The proposed mechanism by which activation of L-GLU receptors causes the
activation of NOS and the formation of NO ...... 74
C h ap ter 2:
2.1: A diagrammatic representation of a microdialysis probe ...... 81 2.2: An anaesthetised rat held in a Kopf stereotaxic fram e ...... 85
2.3: The dorsal and lateral aspects of the skull of a 290g male Wistar rat.... 8 6
2.4: The crown of the head during a bilateral le s io n ...... 87
2.5: A photomicrograph illustrating a typical probe placement ...... 91
2.6: The reaction of OPT-thiol with a primary amine to yield a substituted isoindole
...... 93 2.7: HPLC apparatus ...... 94
2.8: Typical HPLC traces ...... 97
2.9: Calibration curves for each amino acid ...... 98
2.10: Sievers 280 NOA ...... 100
2.11: The calibration plot for the N O A ...... 100
viii List ofFij^ures
Chapter 3:
3.1: Amino acid levels in response to the application of various drugs. 1: lO^M NMDA, 2: 10/xM tADA, 3: lOfiM AMPA, 4: 50/tM SNAP or 5: 100/xM
CNQX + 50/tM SNAP ...... 105
3.2: A representative release profile for L -A R G ...... 106
3.3: Amino acid levels in response to the application of 10/xM Hb and 50/xM SNAP
to cortical cultures ...... 107
3.4: Amino acid levels in response to the application of 10/xM L-NAME to cortical
cu ltu res...... 108
3.5: Amino acid levels in response to the application of various drugs. A: Levels of
L-ARG and L-GLU in response to the 6 min application of 10/xM L-NAME
and 50fiM SNAP for the last 2min of L-NAME application. B: Peak L-
ARG and L-GLU levels with 1: lOjuM Hb + 50/tM SNAP, 2: 10/xM L- NAME, 3: 10/xM L-NAME + 50/xM SNAP ...... 109
3.6: Amino acid levels in response to the infusion of the glial toxin PC through the
probe into the hippocampus ...... I l l
3.7: Representative L-ARG release profiles in response to glutamatergic agonists 112 3.8: Peak L-ARG levels in response to various drug treatments. 1: 100/xM NMDA,
2: 10/xM tADA, 3: 100/xM AMPA, 4: 10/xM CNQX, 5: 10/xM CNQX + 100/xM A M P A ...... 113
3.9: Representative release profiles for A: L-GLU and B: GAB A in response to the
30min infusion of glutamatergic agonists into the hippocampus of freely
moving rats ...... 114
3.10: Amino acid levels in response to the infusion of various drugs. 1: 100/xM
NMDA, 2: 10/xM tADA, 3: 100/xM AMPA ...... 115
3.11: Calibration curves for the NOS assay. A: A time course for the reaction, B:
NOS reaction product as a function of the volume of supernatant used
116
3.12: A bar graph representing the hemispheric differences between naïve animals
which had not undergone surgery and those which had had probes implanted
and aCSF infused ...... 118
ix List o f Fif^ures
3.13: Changes in NOS activity with various drug treatments. 1: 100/xM NMDA, 2: 10/xM tADA, 3: 100/xM AMPA, 4: 10/xM D-AP5 + 100/xM NMDA
...... 119
3.14: NO levels with various drug treatments. A: Representative release profile in
response to the infusion of NMDA. The open box indicates the period of
infusion. B: Peak NO levels with 1: 100/xM NMDA, 2: 10/xM D-AP5 +
100/xM NMDA, 3: 100/xM AM PA...... 120
3.15: A release profile of the NO levels in response to the 30min infusion of 5mM
SNAP ...... 122
3.16: Representative release profiles for L-ARG, L-GLU and GAB A in response to
the 30min infusion of SN A P ...... 123
3.17: Peak responses to various drug treatments. 1: 5mM SNAP, 2: 10/xM ODQ, 3: 10/xM ODQ + 5mM S N A P ...... 124
3.18: Amino acid levels in response to various drug treatments. 10/xM D-AP5 and
10/xM CNQX were coinfused for a total of 90min while SNAP was coinfused for the last 30min of antagonist infusion ...... 125
3.19: Representative L-ARG release profiles in response to NOS inhibitors
...... 127 3.20: Representative release profiles for A: L-GLU and B: G ABA in response to the infusion of NOS inhibitors ...... 128
3.21: Peak L-ARG, L-GLU and GABA responses to various drug treatments. 1:
ImM L-NAME, 2: ImM 7-NI, 3: ImM L-NAME + ImM 7-NI. ... 129
3.22: NOS activity with various drug treatments. 1: ImM L-NAME, 2: ImM 7-NI,
3: ImM L-NAME ImM 7-NI ...... 130
3.23: NOS activities with various drug treatments. 1: lOO/xM NMDA, 2: Im M L-
NAM E + 100/xM NMDA, 3: ImM 7-NI -k 100/xM NMDA, 4: Im M L-
NAME + ImM 7-NI 4- lOO/xM N M D A ...... 131
3.24: Dialysate NO levels in response to the infusion of NOS inhibitors. A:
Representative NO release profiles in response to the 90min infusion of
drugs. B: Peak NO levels with 1: ImM L-NAME, 2: Im M 7-NI, 3: Im M L-NAME + ImM 7-NI ...... 132 List of Figures
3.25: Peak dialysate NO levels with various drug treatments. 1: 100/xM NMDA, 2:
ImM L-NAME + 100/xM NMDA, 3: Im M 7-N I -t-100/xM NMDA, 4: ImM L-NAME + ImM 7-NI -k 100/xM N M D A ...... 133 Chapter 4:
4.1: An adaptation of figure 1.12 to show how L-ARG and L-ClT may be trafficked
in the CNS ...... 153
4.2: Schematic representation of how the extracellular L-GLU concentration may
govern the supply of L-A R G ...... 156
4.3: Schematic representation of the localisation of eNOS and nNOS in the
b r a in ...... 164
XI List o f tables
List of Tables Page
Chapter 1:
1.1: Properties of the best known mammalian amino acid transporters ...... 28
1.2: Properties and sites of action of some NOS inhibitors ...... 52
Chapter 2: 2.1'. In vitro recoveries for each amino acid ...... 83 2.2: Pros and cons of various in vivo techniques ...... 89
2.3: The basal levels of each amino a c id ...... 96
Xll Abbreviations Abbreviations
P-ALA P-Alanine
1400W N-(3-(Aminomethyl) benzyl) acetamide
4-AP 4-Aminopyridine 5-HT 5-Hydroxytryptamine
7-NI 7-Nitroindazole
ACh Acetylcholine
aCSF Artificial cerebrospinal fluid
AD Alzheimer’s disease
ADP Adenosine diphosphate
AL Argininosuccinate lyase
ALA Alanine
ALS Amyotrophic lateral sclerosis AMTTU 1 - Amino-S-methyl-i sothiourea
AMPA a-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid
AMPK AMP-activated protein kinase
ARDS Adult respiratory distress syndrome
AS Argininosuccinate synthetase
ASN Asparagine
ATP Adenosine triphosphate
BAEC Bovine aortic endothelial cells BBB Blood brain barrier
BCH z-Aminoe«i/obicyclo-[2, 2, l]-heptane-2-carboxylic acid
BDNF Brain-derived neurotrophic factor
BH 4 T etrah ydrobi opteri n
BK Bradykinin
Borax Sodium tetraborate dehydrate
BSA Bovine serum albumin
CaM Calmodulin
C aM K I Ca^V CaM-dependent kinase I cAM P Cyclic adenosine monophosphate
Xlll Abbreviations CAPON Carboxy-terminal PDZ ligand of nNOS
CAT Cationic amino acid transporter cGK/ PKG cGMP-dependent protein kinase cGMP Cyclic guanosine 3', 5' monophosphate
CGRP Calcitonin gene-related peptide cGS-PDE cGMP-sti mul ated PDE
CNG Cyclic nucleotide gated CNQX 6-Cyano-7-nitroquinoxaline-2,3-dione
CNS Central nervous system
COPD Chronic obstructive pulmonary disease
COX Cyclo-oxygenase
CPP 3-[(±)-2-Carboxypiperazin-4-yl] propyl-1-phosphonic acid
Cupferron N-Nitroso-N-phenylhydroxylamine ammonium salt
CYS Cysteine cyt P450 Cytochrome P450
D-AP5 D-(-)-2-Amino-5-phosphopentanoic acid D-ASP D-Aspartate
DA Dopamine
DAHP 2,4-Diamino-6-hydroxypyrimidine
DETA/ NO Diethylenetriamine-NO
DG Dentate gyrus
DHK Dihydrokainate DNIC Dinitrosyl-iron complex
DPI Diphenyleneiodonium DTI Di-2-thienyliodonium
DTNB 5, 5'-Dithiobis-2-nitrobenzoic acid
DTT Dithiothreitol
DY-9760e 3-[2-[4-(3-Chloro-2-methylphenyl)-I-piperazinyl] ethyl]-5, 6 -dimetoxy-I-
(4-imidazolmethyl)-IH-indazole dihydrochloride 3.5 hydrate
EAACI Excitatory amino acid carrier I
EAAT Excitatory amino acid transporter
EBSS Earle’s balanced salt solution
XIV Abbreviations ED Erectile dysfunction
Edg Endothelial differentiation gene EDRF Endothelium derived relaxing factor
EDTA Ethylenediaminetetraacetic acid
EOT A Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid eNOS Endothelial NOS
EPSC Excitatory postsynaptic current
EPSP Excitatory postsynaptic potential FAD Flavin adenine dinucleotide
FC Fluorocitrate
FMN Flavin adenine mononucleotide
GA Glutaminase GABA y-Aminobutyric acid
GAD Glutamic acid decarboxylase
GI Gastro-intestinal
GIT GI tract
GLAST Glutamate and aspartate transporter
GLT-1 Glutamate transporter-1
GLY Glycine
GnRH Gonadotrophin releasing hormone
GS Glutamine synthase
GSNO S -N i trosogl utathi one
GST Glutathione-S-transferase
GTN Glyceryl trinitrate/ nitroglycerin
GW273629 S-[2-[(l-Iminoethyl)-amino] ethyl]-4, 4-dioxo-L-cysteine
GW274150 S-[2-[(l-Iminoethyl)-amino] ethyl]-L-homocysteine)
H 2 0 2 Hydrogen peroxide Hb Haemoglobin
HIS Histidine
HPLC High performance liquid chromatography
Hsp90 Heat-shock protein 90
XV Abbreviations i.c.v. Intracerebroventricular
ID lodoniumdiphenyl
IFN-y Interferon-y iGLUR lonotropic L-GLU receptor
iNOS Inducible NOS
i.p. Intraperitoneal
IP 3 Inositol triphosphate IPSP Inhibitory postsynaptic potential
i.v. Intravenous
K 2 Ru(NO)C , 5 Dipotassium pentachloronitrosylruthenate
KA Kainate
L-ADMA N“, N^-Di methyl -L-ARG/ asymmetric din
L-ARG L-Arginine
L-ASP L-Aspartate L-CIT L-Citrulline
L-GLN L-Glutamine L-GLU L-Glutamate
L-NAME N“-Nitro-L-ARG methyl ester
L-NIL N^-Iminoethyl-L-lysine
L-NIO N^-Iminoethyl-L-omithine
L-NMA N“-Methyl-L-ARG
L-NNA N“-Nitro-L-ARG
L-SER L-Serine
L-VNIO N^-( 1 -Imino-3-butenyl)-L-omithine/ vinyl-
LEU Leucine
LPS Lipopolysaccharide
LTD Long term depression
LTP Long term potentiation
LY367385 2-Methyl-4-carboxyphenyl glycine
M AP kinase Mitogen activated protein kinase
MBI Mechanism based inhibitor
XVI Abbreviations MET Methionine mGLUR Metabotropic L-GLU receptor
MK-801 (5S, 10R)-(+)-5-Methyl-10,1 l-dihydro-5H-dibenzo[a, d] cyclohepten-5,10-
imine maleate/ dizocilpine MPEP 2-Methyl-6-(phenylethynyl) pyridine
Myr Myristoylation
N 2 O 3 Dinitrogen trioxide
N 2 O 4 Dinitrogen tetroxide
NA Noradrenaline
NADPH Nicotinamide-adenine dinucleotide phosphate
NANG Non-adrenergic, non-cholinergic
NBAT Neutral and basic amino acid transporter
NF-k B Nuclear factor k B NKA Neurokinin A
NMDA N-Methyl-D-aspartate nNOS Neuronal NOS NO Nitric oxide
NO^ Nitrosonium
NO Nitroxyl anion
NO 2 * Nitrogen dioxide
NO 2 Nitrite
NO 3 Nitrate
NOA NO analyser
NOG Drago complexes/ NONOate
NOSIP eNOS interacting protein
NO/ Nitrite and nitrate
NSAID Non-steroidal anti-inflammatory drug
O 2 Superoxide
ODQ 1H-[1, 2,4] Oxadiazolo [4, 3-a] quinoxalin-l-one
ONOO Peroxynitrite
OPA o-Phthaldialdehyde
xvii Abbreviations 0X1/ NO Angeli’s salt/ sodium trioxodinitrate
P Phosphorylation
PAEC Pulmonary artery endothelial cell
PAPA/ NO Propylamino-propylamine NONOate
PBITU S, S’-[l, 3-Phenylenebis-(l, 2-ethanediyl)] bisisothiourea
PDE Phosphodiesterase
PDZ PSD-95-Discs large-Zona occludens
PFK Phosphofructokinase
PHE Phenylalanine phox Phagocyte NADPH oxidase
PI3-K Phosphoinositide 3-kinase PIN Protein inhibitor of nNOS
PKA Protein kinase A
PKB Protein kinase B/ Akt PKC Protein kinase C PKG Protein kinase G
PLA 2 Phospholipase A 2 PLC Phospholipase C
PPHN Persistent pulmonary hypertension of the newborn
PSD-95 Postsynaptic density-95
PTB Phosphotyrosine binding
R-SNO S-Nitrosothiol
RNI Reactive nitrogen intermediate
ROI Reactive oxygen intermediate
ROS Reactive oxygen species
Ru(N 0 )C , 3 Trichloronitrosylruthenium
RYR Ryanodine receptor
SIP Sphingosine-1 -phosphate
SARC Sarcosine
SDS Sodium dodecyl sulphate sGC Soluble guanylate cyclase
SIN-1 3-Morpholino-sydnonimine/ linsidomine
XVlll Abbreviations SNAP S-Nitroso-N-acetyl penicillamine
SP Substance P
SULPHI/ NO Sulphite NONOate
tADA rra«5-Azetidine-2, 4-dicarboxylic acid
TAU Taurine THF Tetrahydrofuran
THR Threonine
TM Transmembrane
TN F-a Tumour necrosis factor-a trans-AŒD (±)-1 -Aminocyclopentane-rmMj-1, 3-dic: TTX Tetrodotoxin
TYR Tyrosine
VCAM-1 Vascular cell adhesion molecule-1
VGCC Voltage gated Ca^^ channel
Viagra® Sildenafil citrate VIP Vasoactive intestinal peptide y+LAT y^L amino acid transport
XIX chapter 1: Çeneral Introduction Chapter 1: General Introduction
1.1 History of Nitric Oxide There is a wealth of knowledge currently available concerning nitric oxide (NO) and its biological activity. The following is a brief history of how NO came to the forefront of
scientific research.
NO was identified in the bloodstream of humans as early as 1978 (Freeman et al, 1978) and it was subsequently shown that mammals do, in fact, synthesise nitrates, excretion being higher than dietary intake. This synthesis was proven to be independent of the gastric
and intestinal flora by a direct comparison of conventional with germfree Sprague-Dawley rats (Green et al, 1981). It was also shown that the quantity of nitrates excreted was increased during inflammation (Wagner et al, 1983) and it was subsequently discovered that activated macrophages synthesise NO which is used to destroy invading pathogens (Hibbs et al, 1987). NO was then shown to activate soluble guanylate cyclase (sGC), sGC being the enzyme responsible for the conversion of guanosine triphosphate (GTP) to cyclic guanosine 3', 5' monophosphate (cGMP), a second messenger responsible for the regulation of various cellular events (Waldman and Murad, 1987). The “receptor” for NO is the haem iron moiety of sGC, NO is thus scavenged by haemoglobin (Hb). These facts went a long way towards increasing our understanding of the hypotensive, spasmolytic and antithrombotic effects of various nitrogen based compounds, especially nitroglycerin
(Vanin, 1998).
At the same time it had been found that a number of substances, including acetylcholine
(ACh), the Ca^"^ ionophore A23187, adenosine triphosphate and diphosphate (ATP and
ADP, respectively), substance P (SP), bradykinin (BK), histamine, thrombin, 5- hydroxytryptamine (5-HT) and noradrenaline (NA), caused relaxation of isolated rabbit aortic rings in an endothelium-dependent manner (Furchgott et al, 1984). The chemical released by all these substances was termed the endothelium-dependent relaxing factor
(EDRF). The nature of this EDRF was then investigated using a variety of preparations, the most common being the “sandwich” arrangement where two rabbit aortic strips, one of which was intact while the other was denuded of endothelium, were placed intimai surface to intimai surface. The intact endothelium could therefore donate EDRF to the denuded strip, the latter acting as a detector.
21 Chapter 1: General Introduction
It was established that EDRF was an extremely labile substance with a half-life of a few seconds in oxygenated physiological salt solutions (Cocks et al, 1985). It was also found that the relaxation caused by the above agents was preceded by an increase in cGMP which was consistent with the hypothesis at the time that EDRF activates sGC, thus causing relaxation (Furchgott et al, 1984). It was also discovered that this endothelium-dependent relaxation was inhibited by both Hb and methylene blue, an inhibitor of sGC (Martin et al, 1984). Thus, it was hypothesised that EDRF was, in fact, NO (Ignarro et al, 1987; Palmer et al, 1987). It was shown that the production of EDRF and the consequent vasodilation was blocked by N“-substituted L-arginine (L-ARG) analogues which could be overcome by the addition of L-ARG (Moncada et al, 1991). These findings strongly supported the hypothesis that EDRF is indeed NO and when the EDRF released from endothelial cells was detected using the chemiluminescence reaction used to detect NO via its reaction with ozone (Downes et al, 1976) it was thus concluded that NO could be produced under physiological conditions, not simply in response to macrophage activation.
In 1988 there were a number of neurophysiological studies performed to elucidate the possible role of NO in the brain. Stimulation of cerebellar slices with excitatory amino acids, such as L-glutamate (L-GLU), led to the release of a labile mediator with a similar pharmacological profile to NO, including the rise in cGMP levels and relaxation of smooth muscle which could be blocked by N“-substituted L-ARG analogues (Garthwaite et al, 1988). As well as this, stimulation of non-adrenergic, non-cholinergic (NANG) peripheral neurons also led to the release of a labile compound with exactly the same properties
(Garthwaite, 1991). Hence, the functions of cells which produce NO are not simply limited to cytotoxicity and vasorelaxation and there is also the implication of endogenously produced NO playing a part in central and peripheral nervous system (CNS) transmission.
In the years since these early discoveries, thousands of papers have been published that try to elucidate the biological role(s) of NO and in 1992 NO was named as “molecule of the year” by Science. It is now known that NO is a small, unstable, lipophilic, potentially harmful, free radical gas which has now been recognised as a major neuronal messenger.
Due to its lipophilicity, NO can diffuse through cell membranes and affect neighbouring cells. NO has the highest diffusion coefficient of any biological molecule, higher than even
2 2 Chapter 1: General Introduction oxygen or carbon monoxide (Varner and Beckman, 1995). This suggests that NO can mediate intra-cellular events and can also communicate neuron to neuron and neuron to glia (Southam et al., 1992). NO may well be of early evolutionary origin, NO-positive nerve cells having been discovered in most vertebrate classes and some invertebrates (Olsson and Holmgren, 1997). In 1991, NO was identified in Limulus polyphemus, the American horseshoe crab shown in figure 1 . 1 , a species of early evolutionary origin itself, indicating that the NO synthesis pathway ranks among the oldest regulatory systems that have contributed to the development of animal life (Radomski et al., 1991). In fact, this simple compound (NO is the tenth smallest molecule) may well be the universal transduction system for the activation of sGC (Berrazueta et al, 1990).
Figure 1.1: Limulus polyphemus.
1.2 Synthesis of NO
NO is synthesised from L-ARG and molecular oxygen by the enzyme nitric oxide synthase
(NOS) with L-citrulline (L-CIT) and NO being produced concomitantly. The precise mechanism is unknown but the reaction involves a 5 electron oxidation of one of the guanidino-nitrogens of L-ARG (Iyengar et al, 1987; Palmer et ai, 1988). The L-ARG is first N-hydroxylated to N“-hydroxyl-L-arginine (Stuehr et al, 1991, Zembowicz et al, 1991), which remains bound to the NOS and is oxidatively cleaved to form the products (Gross and Wolin, 1995). The reaction is shown in figure 1.2.
23 Chapter I: General Introduction
N il
CÜÜ
l.-A R (i
Figure 1.2: Biosynthesis of nitric oxide from L-ARG and molecular oxygen with N“- OH-L-ARG produced as an intermediate The synthesis involves the concomitant formation of L-CIT and NO (adapted from Nathan, 1992).
The overall reaction involves the reduction of two oxygen molecules which requires a total of eight electrons. Five reducing equivalents are derived from guanidino nitrogen oxidation while the additional three are provided by co-oxidation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH; Marietta, 1993; Mayer, 1995). L-ARG is manufactured in mammals from L-GLU and L-aspartate (L-ASP). The carboxyl group of L-GLU reacts with ATP to give an acyl phosphate which is then reduced by NADPH to an aldehyde. The glutamic y-semialdehyde is then transaminated to ornithine which is fed through the urea cycle to give, eventually, L-ARG. Figure 1.3 shows the reaction as far as ornithine.
24 Chapter I: General Introduction
( I II \ ------
\ II (■ ------N il.
( )rniihinc
Figure 1.3: The formation of ornithine from L-GLU (adapted from Stryer, 1995). The ornithine is then fed through the urea cycle to form L-ARG.
The intracellular availability of L-ARG has been shown by several sources to be rate limiting for the production of NO (Hecker^/a/., 1990; Bogle et ai, 1996; Mori and Gotoh, 2000). In addition, nitrite foimation, an indicator of NO formation, is only detected in cortical neuronal cultures when astrocytes are present. This may be interpreted as demonstrating that neurons themselves do not have sufficient L-ARG for NO production
(Snyder and Bredt, 1992). Immunocytochemistry studies have indicated that L-ARG is localised in glia while L-CIT is localised in neurons in situ (Aoki etaL, 1991a; Row, 1994). This has led to the theory that L-ARG must have to be transferred to the nerve terminal, possibly via de novo synthesis of the y^cationic amino acid carrier system (see section 1.3; Vega-Agapito et ai, 1999), in order to replenish the neuronal precursor pool and to allow the formation of NO and L-CIT. Localisation of L-ASP in glia (Aoki et ai, 1991a; Row, 1994) indicates that the L-CIT then has to be transferred back to the glia to be recycled into L-ARG. Figure 1.4 shows a representation of this cycle and the enzymes involved.
25 Chapter 1: General Introduction
Arginase L-ARG
UREA
pLimaratc
NADPH NOS
AL NADP NO
L-CIT ARGININO SUCCINATE ATP ASP AMP + PPi AS
Figure 1.4: A variation of the urea cycle which converts L-CIT back to L-ARG ensuring a constant supply of L-ARG (adapted from Amt-Ramos et al, 1992). AL= argininosuccinate lyase, AS= argininosuccinate synthetase.
In support of this hypothesis, immunohistochemical studies have shown that L-CIT is localised only in neurons that also contain NOS (Amt-Ramos et al, 1992). This proposed mechanism would be analogous to the glutamate-glutamine cycle. Here, the L-GLU released from neurons is taken up by glia and converted into L-glutamine (L-GLN) by glutamine synthase (GS). The L-GLN is then released from the glia and taken up by the neurons where it is converted back into L-GLU by glutaminase (GA). Antibodies raised against the enzymes show that GS is only present in glia (Martinez-Hemandez et al, 1977) and GA only in neurons (Salganicoff et al, 1965). This is also the case for AS; immunocytochemical studies have shown that AS is expressed in astroglial cells when they are stimulated to produce NO by immunostimulants (Schmidlin and Wiesinger, 1998). In neuron-rich cultures, AS was expressed in astroglial cells without any prior treatment with immunostimulants. The L-ARG was shown to have been formed from L-CIT as opposed
26 Chapter 1: General Introduction to ornithine, as in the urea cycle. This is confirmed by the fact that the enzymes responsible for the production of L-CIT from ornithine, ornithine transcarbamylase and carbamyl phosphate synthetase 1, are absent from the brain (Amt-Ramos et al, 1992). NOS is thus the only apparent endogenous source of L-CIT. However, very little is understood as to what the signal molecule for L-ARG transfer from glia actually is and hence why the supply of substrate for NOS is limited. It has been shown by in vivo microdialysis studies that direct infusion of fluorocitrate (FC), a glial toxin (Clarke, 1991), through the probe caused increased L-ARG levels (Largo et al, 1996) and increased N O / (nitrite and nitrate) levels (Yamada et al, 1997). This suggests that a supply of L-ARG from glial cells to nitrergic neurons may regulate NO production in vivo,
1.3 Amino acid transporters
The cDNA has been identified for more than twenty mammalian amino acid transporters, all of which belong to four protein families (Palacin et al, 1998). Table 1.1 lists the properties of the best known transport systems found in the plasma membranes of mammalian cells. It has been shown that the transport of leucine (LEU) and L-GLN in neurons, synaptosomes and astrocytes is partially Na^-dependent and is inhibited by L-
ARG, characteristics typical of the y^L amino acid transport (y^LAT) system. Contrary to previous theory, L-ARG transport in human platelets has been found to be mediated via system y^L and not the classic transport system y^ (Mendes Ribeiro et al, 1999). The release of L-ARG has consequently been shown to be mediated by two different transporters, y^LATI and y^LAT2, y^LATI being localised in kidney epithelium, leucocytes and lung with y"^LAT2 being more widely distributed. In fact, it has been shown that y^LAT2 is a L-GLN/ L-ARG exchanger which could play a role in both the uptake of L-
GLN into neurons and the supply of L-ARG to certain brain cells (Broer et al, 2000). L-
ARG is also a potent substrate for the neutral and basic amino acid transporter (NBAT), a member of the b®’^ system, which has been postulated to facilitate Na^-independent transport of L-amino acids in neurons. NBAT is also present in nitrergic neurons which thus have a high requirement for L-ARG. It may be that systems y^L and NBAT are also involved in supplying L-ARG for NO synthesis (Pickel et al, 1999).
27 Chapter 1: General Introduction
Table 1.1: Properties of the best known mammalian amino acid transporters (adapted from Palacin et al, 1998).
Transport system Type of amino acid/ description of transporter Distribution
Zwitterionic amino acids
Na^'^-deoendent
A: Small amino acids; highly regulated; pH sensitive; tolerates N- Widespread methyl groups; tram-inhibition associated; ALA, SER, GLN
ASC: N-methyl intolerant; tra/i5-stimulation associated; ALA, SER, CVS Widespread
N: For GLN, ASN and HIS; pH sensitive Hepatocytes
BETA (GATl-3, BGT- Transports P-ALA, TAU and GABA; Cl'-dependent Widespread 1,TAUT):
GLY: Transports GLY and SARC; Cl -dependent Widespread
IMINO (PROT?): Handles proline, hydroxyprolines and N-methylated glycines; Intestinal brush interacts with MelAB border
PHE: Handles PHE and MET Intestinal brush border
B°(NBB, ATB°): Broad specificity for zwitterionic amino acids, including branched Intestinal brush aromatic ones; accepts BCH but not MeAIB border membranes
Na^’^-indeoendent
L; Bulky side chain amino acids; (ram-stimulated; bicyclic amino Widespread acids as model substrates
Cationic amino acids
Na^'^-deDendent
B°+: Broad specificity for zwitterionic and dibasic amino acids; accepts Blastocysts, BCH but not with MeAIB brush border membranes
Na^’^-indeoendent
b+: Cation preferring; no interaction with homoserine even in presence Mouse of sodium conceptuses
y"(CAT-l,2A,2B,3, Handles cationic and zwitterionic amino acids with sodium; Widespread 4): sensitive to N-ethylmaleimide
y+L (rBAT/ 4F2hc): Handles cationic amino acids and zwitterionic amino acids with Erythrocytes, high affinity only with sodium; N-ethylmaleimide insensitive placenta
b°"(rBAT/4F2hc, Like B°* but limited by branching; BCH insensitive AsB° + NBAT):
Anionic amino acids
X\o(EAATl-5) L-GLU, D- and L-ASP reactive; potassium-dependent Widespread
X c L-CYS competes and exchanges with L-GLU Hepatocytes, fibroblasts For anionic amino acids a capital X is used when Na^-dependent. BCH: z- aminogWobicyclo-[2, 2, l]-heptane-2-carboxylic acid, MeAIB: methylaminoisobutyric acid.
28 Chapter 1: General Introduction
1.4 NOS isoenzymes 1.4.1 Structure and function
Three different isoforms of NOS have been identified and purified to homogeneity using
2\ 5 -ADP affinity chromatography. Two are constitutively expressed and Ca^^-dependent and were first isolated from endothelial cells (endothelial NOS; eNOS) and cerebellum
(neuronal NOS; nNOS) while the other is non-constitutive and Ca^^-independent and was first isolated from macrophages (inducible NOS; iNOS; Yui et al, 1991). The terms eNOS and nNOS, however, are somewhat misleading since the names imply that eNOS is only found in endothelium and nNOS only in neurons. This is not strictly true; nNOS has been found in neutrophils (Yui etal, 1991), intracardiac neurons (Hassall etal, 1992), skeletal muscle (Nakane et al, 1993) and the epithelia of lung (Liihrs et al, 2002), uterus and stomach (Schmidt etal, 1992b) while eNOS has been identified in CAl pyramidal neurons and astroglia of the hippocampus (Doyle and Slater, 1997). nNOS is also referred to as type
I NOS, iNOS as type II and eNOS as type IQ. Each isoform has unique characteristics. K„, values for L-ARG have been documented as 1.5/xM (Bredt and Snyder, 1990), 32.3/xM (Yui et al, 1991) and 2.9/xM (Pollock et ai, 1991) for nNOS, iNOS and eNOS respectively, indicating that nNOS and eNOS have a greater affinity for L-ARG than iNOS. These same reports document Vmax values of0.96,1.052 and 0.0153 /xmol product formed per min per mg of protein for nNOS, iNOS and eNOS indicating that iNOS is capable of producing more NO than eNOS or nNOS. The cDNA for each isoform has been identified using molecular cloning. The three distinct genes for each isoform have been mapped in the human genome to chromosomes 12, 17 and 7 for nNOS, iNOS and eNOS respectively (Alderton et al, 2001). NOS has a homodimeric bidomain structure and the amino acid sequences show consensus binding sites for regulatory co-factors including flavin adenine mononucleotide (FMN), flavin adenine dinucleotide (FAD; Hevel et al, 1991) and NADPH (Marietta et al, 1988), as represented in figure 1.5. The N-terminal oxygenase domain contains the binding sites for L-ARG, haem and tetrahydrobiopterin (BH 4 ) which is linked, via a calmodulin (CaM) recognition site, to the C-terminal reductase domain which contains the binding sites for FAD, FMN and NADPH.
29 Chapter I: General Introduction
Hiicm ()\\<>c‘n;ise Doiiniin RcducMiisc Doniiiiii
p Alicniiilc splice l'üM
M \ r p
C'.iM NA!3P I I ^ H cNOS
C';iM
N A ) ^ ^ e \ P450
I A4 ODiiiain
Figure 1.5: A linear model of co-factor recognition sequences within each NOS
isoenzyme and cytochrome P450 (cyt P450). The predicted sites for alternate splicing, myristoyiation (Myr), CaM binding and protein kinase A phosphorylation (?) within each NOS sequence and the transmembrane (TM) domain in the cyt P450 sequence are indicated (adapted from Bredt, 1995).
The first step of the reaction is the binding of substrate to haem. The flavins then shuttle electrons from the NADPH to the haem which catalyses the haem-dependent activation of molecular oxygen and the formation of NO in the presence of saturating concentrations of
L-ARG and BH 4 .
e e" NADPH ^ { FAD, FMN 1 ^ Haem-Fe^* (Abu-Soud and Stuehr, 1993)
In the absence of substrate, NOS can transfer electrons from NADPH to O 2 and form superoxide (Oy) and hydrogen peroxide (H 2 O 2 ; Mayer et al., 1991; Pou et ai, 1992).
30 Chapter 1: General Introduction
Substrate oxidation occurring at the haem iron is analogous to cyt P450 reductase (Bredt et al, 1991) but the NOSs are unique in that their oxidase and reductase domains are contained on a single polypeptide which was considered to be localised exclusively in the cytosol of macrophages (Marietta et al, 1988), neuroblastoma cells (Gorsky et al, 1990) and brain tissue (Knowles et al, 1989). It has now been shown in bovine aortic endothelial cells (BAECs) that NOS activity is associated with the particulate fraction (Forstermann et al, 1991). In fact, eNOS has a consensus sequence for myristoyiation at the N-terminal glycine which is necessary for post-translational acylation of two adjacent residues by palmitic acid (Marietta, 2001). This indicates that it is associated with the membrane
(Nishida and Ortiz de Montellano, 1998). It has been shown that eNOS is especially associated with plasmalemmal caveolae, membrane micro-domains that are particularly rich in sphingolipids and other signaling molecules (Igarashi and Michel, 2000). The presence of nNOS in the particulate fraction of rat cerebellum has also been demonstrated (Hiki et al, 1992). In fact, the majority of nNOS found in the brain is in the particulate fraction (Brenman and Bredt, 1997). This purified enzyme proved to have a low specific activity, concurring with the low specific activity of the soluble form of eNOS (see above), possibly indicating the loss of an essential cofactor during the purification process (Mayer, 1995).
It has now been shown that all three can exist as both cytosolic and particulate forms (Alderton et al, 2001).
1.4.2 Calcium and calmodulin regulation
Midway through the NOS sequence there is an amphipathic alpha helix domain which fits the consensus sequence for CaM binding (Bredt et al, 1991). In the brain, nNOS is activated by an increase in intracellular Ca^^, either via glutamatergic ion channels or voltage gated Ca^^ channels (VGCC), which stimulates CaM to facilitate the electron transfer within the reductase domain. In the endothelium, eNOS is activated by neurotransmitters activating metabotropic pathways to produce inositol triphosphate (IP 3 ) which mobilises internal Ca^^ stores. Although iNOS is Ca^^-independent it still has CaM recognition sites (Lowenstein et al, 1992). In fact, studies on macrophages have established that CaM is so tightly bound that it should be considered more as an enzyme subunit than a regulatory mechanism (Bredt, 1995). This high affinity CaM binding indicates that iNOS
31 Chapter 1: General Introduction will be maximally activated at the concentration of Ca^"^ present in a resting cell (Butt et al , 2000). In effect, therefore, each isoform can be said to require Ca^V CaM for activity.
1.4.3 Expressional regulation
Given that NO is not stored, released or inactivated by the conventional mechanisms recognised for a neurotransmitter it must be regulated at the level of biosynthesis, hence regulation of NOS is of great importance. iNOS is a transcriptionally regulated enzyme, its activity being regulated predominantly by endotoxins and cytokines in macrophages, hepatocytes and smooth muscle (Nathan, 1997), while eNOS and nNOS are generally considered to be constitutive. It has, however, been demonstrated that they are subject to expressional regulation (Forstermann etal, 1998). The nNOS mRNA has emerged as one of the most structurally diverse genes yet described in terms of promoter usage. The expression of the mRNA and protein is regulated at several levels: transcription, pre-mRNA splicing and translation (Wang et al, 1999). nNOS has been shown to be up-regulated by many substances and conditions in a variety of preparations including gonadotropin releasing hormone (GnRH) and cyclic adenosine monophosphate (cAMP) in pituitary gonadotrophs (Bachir et al, 2001), heregulin in cerebellar granule cells (Krainock and Murphy, 2001), formalin injection into the rat hind paw in the L4-5 dorsal horn area (Lam et al, 1996) and axotomy and hypoxia in lower brain stem motor neurons (Chang et al, 2001). Down-regulation has been induced in response to intrathecal injection of substance P(l-7) in thoracic dorsal root ganglia and the spinal cord (Kovacs et al, 2001), sepsis in human skeletal muscle (Lanone et al, 2001) and vestibular inner ear damage in the ipsilateral dentate gyrus (Zheng et al, 2001).
Likewise, several substances and conditions have proven to up-regulate eNOS: cold exposure increases eNOS expression in brown adipose tissue (Kikuchi-Utsumi et al, 2002), thermal therapy in arterial endothelium (Ikeda et al, 2 0 0 1 ) and sepsis in human skeletal muscle (Lanone etal, 2001). Down-regulation occurs in response to endotoxin in bronchial epithelium (Ermert et al, 2002) and tumour necrosis factor-cc (TNF-a) and acute myocardial infarction in neutrophils (De Frutos etal, 2001). It has also been demonstrated that some hormones, oestrodiol in particular, increase the specific mRNAs for both eNOS
32 Chapter 1: General Introduction and nNOS (Weiner et al, 1994). This would suggest that regulation of NOS expression may be one of the most important mechanisms for controlling endogenous NO levels.
1.4.4 Enzyme activation and cellular localisation
eNOS is activated in a very complex fashion by a variety of signals. eNOS has been shown to be localised in endothelial cell membranes via the formation of an inhibitory complex with caveolin-1, a principal structural protein of caveolae (Garcia-Cardena et al, 1996), which suppresses eNOS catalytic activity (Michel et al, 1997; Ju et al, 1998). It has been demonstrated that eNOS is activated by increases in intracellular Ca^^ which causes a reversible Ca^V CaM mediated dissociation of the eNOS-caveolin complex (Feron et al, 1998; Golser et al, 2000). In a similar manner it has been established that eNOS forms an inhibitory complex with the BK B2 receptor, dissociation occurring in response to ligand binding and intracellular Ca^^ increases (Ju gfa/., 1998). The newest protein shown to bind eNOS has been termed eNOS interacting protein (NOSIP), a 34 kDa protein which modulates enzyme activity by an, as yet, unknown mechanism (Dedio et al, 2001). In addition, eNOS activation can be enhanced by both the molecular chaperone heat-shock protein 90 (Hsp90; Garcia-Cardena et al, 1998) and dynamin-2, a large GTPase (Cao et al,
2001).
eNOS has also proven to become activated in an apparently Ca^^-independent fashion by shear fluid stress in vascular endothelial cells (Fleming and Busse, 1999). Recently sphingolipids have emerged as major messenger molecules (Marietta, 2001). Cell membranes are made up of phospholipids, glycolipids and cholesterol, the phospholipids being derived from either glycerol or sphingosine. Membrane caveolae have an extremely high composition of cholesterol and sphingolipids, the acylated derivatives of sphingosine
(Igarashi and Michel, 2000). A biologically active sphingolipid is sphingosine-1 -phosphate
(SIP) which has been implicated in a diverse range of biological processes. It has been shown that SIP activates members of the endothelial differentiation gene (Edg) family of
G protein linked receptors to initiate cell growth, survival, migration and morphogenesis
(Liu etal, 2000). The Edg receptor family consists of eight subtypes, Edgl- 8 , and the Edgl receptor, like eNOS, has been localised to the caveolae of cell membranes (Igarashi et al,
33 Chapter 1: General Introduction
2001). Activation of the Edgl receptor triggers the Gj pathway while overexpression induces mitogen-activated protein kinase (MAP kinase) and phospholipase A 2 (PLA 2 ; Lee et al, 1996). BAECs treated with SIP show phosphorylation of eNOS at a C-terminal L- serine (L-SER) residue, specifically L-SER**^^ of the bovine enzyme, a residue known to be phosphorylated by protein kinase Akt (protein kinase B; PKB; Michell et al, 1999), and L-SERin humans (Marietta, 2001). Figure 1.6 shows a schematic representation of this mechanism postulated for eNOS phosphorylation. This phosphorylation causes an acute increase in eNOS activity (Igarashi et al, 2001) and renders the enzyme 40% more sensitive to CaM (Michell et al, 1999). At the same time it was demonstrated that the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin did not completely inhibit the SIP induced phosphorylation of eNOS. This may be interpreted as showing that there are other kinases capable of phosphorylating the enzyme. Indeed, AMP-activated protein kinase (AMPK) has been shown to phosphorylate eNOS at L-SER(Chen et al, 1999), as have protein kinases A and G (PKA and PKG; Butt et al, 2000). The full repertoire of signaling pathways which control eNOS activation is still unknown and further research is required.
Our understanding of the mechanisms by which nNOS is regulated is beginning to increase in complexity. nNOS is known to interact with caveolin-3 in skeletal muscle, as does eNOS in cardiac myocytes (Venema et al, 1997). The N-terminal of nNOS contains a PDZ (PSD- 95-Discs large-Zona occludens) motif, a consensus sequence that mediates protein-protein interactions (Firestein and Bredt, 1999), which interacts with similar motifs in postsynaptic density-95 (PSD-95) and a related protein PSD-93. PDZ interactions localise nNOS to the postsynaptic membrane in cerebellum and to al-syntrophin, a dystrophin and caveola- associated protein, in skeletal muscle and the suprachiasmatic nucleus of rat brain
(Brenman et al, 1996; Hashida-Okumura et al, 1999; Schuh et al, 2001).
34 Chapter I: General Introduction
Edg- 1 receptor
► ( A ^
Phosphorylation
I -SI K
I \l) H N \l)l’l I^HeNOS
Figure 1.6: One of the proposed mechanisms for eNOS activation. SIP binds to the Edg-1 receptor which activates PI3-K and protein kinase Akt to phosphorylate L-SERincreasing eNOS activity (adapted from Marietta, 2001). The eNOS is shown anchored to the caveoia by one molecule of myristate and two of palmitate.
35 Chapter 1: General Introduction
NMDAR2
f ( (( (C (
PSD-Q5
nNOS Catalytic domain
I A A \ 0909 > CAPON X Figure 1.7: A schematic representation of how nNOS interacts with
PSD-95 and other proteins at the synaptic membrane (adapted from Christopherson etal., 1999).
36 Chapter 1: General Introduction
This interaction with PSD-95/ 93 places nNOS adjacent to other PSD ligands, such as the
NR2 subunit of the N-methyl-D-aspartate (NMDA) receptor (Komau et al, 1995; Christopherson et al, 1999; Husi and Grant, 2001), which may explain the efficient activation of nNOS during NMDA receptor activation (see section 1.9). nNOS has also been shown to interact with a novel protein termed CAPON (carboxy-terminal PDZ ligand of nNOS), which impedes its activation (Jaffrey etal, 1998). CAPON also has a C-terminal
PDZ domain, which enables its interaction with nNOS and binding studies have shown that it is associated with the soluble form of the enzyme, possibly implicating CAPON in the targeting of nNOS in neurons (Jaffrey et al, 2002). Figure 1.7 shows a representation of how nNOS interacts with PSD-95 and other proteins. CAPON has an N-terminal phosphotyrosine-binding (PTB) domain to which synapsins 1, II and HI bind. Synapsins are a group of neuron-specific phosphoproteins which associate with synaptic vesicle membranes and have been implicated in the control of neurotransmitter release (Murthy, 2001; Ferreira and Rapoport, 2002). There are two conserved domains in all synapsins: an N-terminal domain that has a phosphorylation site for PKA and Ca^V CaM-dependent kinase I (CaMK I) and a central domain that binds ATP (Hosaka et al, 1999). Upon both action potential stimulation and phosphorylation the synapsins dissociate from the synaptic vesicles, allowing them to move to the active zone (Sihra et al, 1989). Phosphorylation of synapsins has also been shown in response to CaMK II and IV and MAP kinases (Murthy,
2001). When the stimulation ceases the synapsins become dephosphorylated and reassociate with the vesicles (Chi et al, 2001). It is possible that nNOS, CAPON and synapsin form a ternary complex. Because synapsins are exclusively presynaptic this may indicate a mechanism by which nNOS is targeted presynaptically and is involved in neurotransmitter release (Jaffrey et al, 2002).
It has also been demonstrated that phosphofructokinase (PFK) binds to the nNOS PDZ domain in both brain and skeletal muscle. However, PFK binding had little or no effect on enzyme activity. Since PFK is cytosolic it may target nNOS to the cytosol (Firestein and Bredt, 1999). Hsp90 has been shown to facilitate nNOS activity in vivo, and in vitro studies have demonstrated the mechanism to be the augmentation of CaM binding to increase nNOS activity in a dose-dependent fashion (Song et al, 2001). A lOkDa protein found to interact and inhibit nNOS activity has been designated PIN (protein inhibitor of nNOS;
37 Chapter 1: General Introduction
Jaffrey and Snyder, 1996). Immunoblotting of various brain regions with an antibody for nNOS indicated strong expression in the cerebellum and moderate expression in other brain regions including the cerebral cortex, midbrain, hippocampus and medulla. Conversely PIN expression was shown to be highest in the cerebral cortex, midbrain, hippocampus and medulla with only weak expression in the cerebellum (Greenwood et al, 1997). This may indicate that PIN acts as an endogenous inhibitor of nNOS, possibly explaining the differences in enzyme activity between these brain regions. The inhibition of nNOS by PIN is possibly mediated by the ability of PIN to dissociate the nNOS homodimer (see section 1.4.6; Fan et al, 1998), although it has now been demonstrated that PIN can bind to nNOS with no effect on enzyme activity (Rodnguez-Crespo et al, 1998; Hemmens et al, 1998). It is possible that PIN, like CAPON, could be involved in the axonal transport of nNOS.
nNOS has been found to be phosphorylated by a number of kinases, although the physiological significance of this phosphorylation remains unclear (Bredt et al, 1992). Protein kinase C (PKC) phosphorylation has been shown to lead to a moderate increase in enzyme activity (Nakane et al, 1991) while phosphorylation by PKA had no effect (Brune and Lapetina, 1991). It has now been discovered that PKA, PKC and CaMK all phosphorylate separate L-SER residues on the nNOS protein (Bredt et al, 1992). The CaMKs have proved to phosphorylate nNOS at L-SER*"^^and the phosphorylated enzyme exhibited a decreased activity (Hayashi et al, 1999). Conversely, déphosphorylation of
SER*'^^ of nNOS by protein phosphatase 2A is responsible for reversible activation of the enzyme in neuronal cells (Komeima and Watanabe, 2001). This may indicate a regulatory control by CaMK although whether phosphorylation at L-SER*"^^ alters protein-protein interactions remains to be elucidated.
In addition to all the regulatory controls mentioned above, both of the Ca^7 CaM-dependent
NOSs have been shown to contain a 45 amino acid insert which potently inhibits CaM binding and may well serve as an autoinhibitory control element (Salerno et al, 1997). When CaM binds it displaces this inhibitory element and activates the enzyme. However, it is unclear how this inhibitory element inhibits electron transfer.
38 Chapter 1: General Introduction
1.4.5 The arginine paradox and eNOS
L-ARG is present in endothelial cells at concentrations far exceeding the values for any of the NOS isoforms, BAECs containing up to SOOfiM (Baydoun et ai, 1990) while freshly isolated endothelial cells can contain as much as 2mM (Girerd et al., 1990). Therefore NOS should always be saturated with substrate and thus should not respond to changes in extracellular L-ARG levels. However, it is known that L-ARG supplements do, in fact, enhance vascular reactivity in a mechanism resembling eNOS activation (Girerd et al, 1990; Bode-Bôgeret al, 1994; Bôgeret al, 1994; Morris et al, 2000). This phenomenon has been termed the “L-ARG paradox” and there have been many proposed explanations for this:
The intravenous infusion of L-ARG has been shown to stimulate the release of insulin, a hormone with a vasodilating capacity (Giugliano et al, 1997; Kurz and Harrison, 1997). In turn insulin has proved capable of phosphorylating eNOS via the activation of the PI3-K/ Akt pathway (Gao et al, 2002), increasing eNOS activity. It is possible that endogenous NOS inhibitors are responsible, two of the most potent being N“, N“-dimethyl-L-ARG (asymmetric dimethylarginine; L-ADMA) and N“-methyl-L-ARG (L-NMA; see section 1.6; Tsikas et al, 2000). It has been proposed that there is an exchange of intracellular inhibitors against the circulating L-ARG, the increase in L-ARG concentration promoting uptake into the cell in exchange for the endogenous inhibitor. Alternatively, the compartmentalisation of eNOS in caveolae may be the answer (Hardy and May, 2002).
Immunohistochemical studies have localised the L-ARG transporter CAT-1 in the caveolae of porcine pulmonary endothelial cells (McDonald et al, 1997). It has thus been hypothesised that there may be a unique caveolar pool of L-ARG specifically for the manufacture of NO by eNOS. In support of this hypothesis it has been shown that the enzymes responsible for the regeneration of L-ARG, AS and AL, cofractionate with caveolae (Flam et al, 2001). In effect, therefore, there may well be two separate pools of L-ARG in endothelial cells, one being independent of the extracellular L-ARG (Gloss et al, 2 0 0 0 ).
39 Chapter 1: General Introduction
1.4.6 The roles of haem and tetrahydrobiopterin
nNOS and eNOS are constitutively expressed as inactive monomers. When the intracellular
Ca^^ levels rise CaM binds and active NOS homodimers are formed. By contrast, iNOS is bound to CaM at physiological Ca^^ concentrations, forms iNOS homodimers and is thus always active (Ratovitski et al, 1999). It has been shown that nNOS monomers isolated from mammalian cells lack BH^ and haem but still contain the binding sites for NADPH and CaM, bound FAD and FMN and are still capable of catalysing electron transfer to artificial acceptors such as cytochrome c (Sennequier et al, 1999). When the haem- deficient nNOS monomer is exposed to haem it forms a homodimeric protein which readily dissociates when exposed to SDS. In this conformation nNOS can act as an NADPH oxidase and is capable of catalysing the formation of O 2 and HjOj (Pou et al, 1992). When
BH 4 binds there is another conformational change which results in a superstable SDS- resistant dimer (Klatt et al, 1996). Thus, haem seems to be necessary for dimer formation while BH 4 may be responsible for the stability of this dimer. Figure 1.8 shows a representation of this proposed mechanism for nNOS dimer formation. The situation for eNOS is a little different. In the absence of L-ARG or BH 4 , eNOS does not appear to catalyse an appreciable amount of uncoupling of oxygen reduction (List etal, 1997). In this sense eNOS resembles iNOS not nNOS. The concentration of BH 4 may dictate the product of the nNOS reaction. The two haem moieties seem to function independently of each other in respect of their susceptibility to reduction by NADPH, only one molecule being involved
(Siddhanta et al, 1996). It has been postulated that at very low BH 4 concentrations NOS produces O 2 , no BH 4 -haem binding occurring; at intermediate BH 4 concentrations NOS is capable of producing both O 2 and NO, and hence peroxynitrite (ONOO ), one BH 4 -haem molecule being formed, and at very high BH 4 concentrations NOS only produces NO, both haems binding BH 4 (Gorren and Mayer, 1998). However, the presence of L-ARG has also emerged as a factor in the formation of SDS-resistant dimers (Klatt et al, 1995), nNOS actually being more readily stabilised by L-ARG than by BH 4 (Panda et al, 2002) and NO itself has recently been shown to block NOS dimérisation (Chen et al, 2002). Whether the dimérisation of NOS is a major regulatory mechanism governing NO production is unclear and further research is required.
40 Chapter 1: General Introduction
KMN U FAD
Monomer I laem (cx loehromc e retkielasc)
H M N M HADaem
1-ADI m I FMN ! laem
Dimer (NADP! I oxidase)
aem
F A D M FMN I laem U RIF
SDS-resislant dimei (NOS)
Figure 1.8: The roles of haem and BH 4 in nNOS structure and function (adapted from Klatt et ai, 1996). Upon the binding of haem, nNOS monomers form unstable homodimers. When BH 4 binds the stability of the molecule in increased considerably.
1.5 NO and signal transduction
Our understanding of the signal transduction pathways of NO has increased greatly in the past few years (Denninger and Marietta, 1999). NO greatly enhances the GTPase activity of G;g, Gj„, and the protooncogene p21"^' (Lander et ai, 1993). The activation by NO of p21"^' has been shown to be through the S-nitrosylation of the cysteine residue CYS"^ (Lander et ai, 1997). NO has also been found to inhibit nuclear factor-KB (NF-kB) and vascular cell adhesion molecule-1 (VCAM-1) by increasing the expression of their cytoplasmic inhibitor IxBcc (Spiecker et ai, 1997; Sekkai et aL, 1998). In addition, it has been demonstrated that NO inhibits mitochondrial respiration by competition with oxygen and inhibition of cytochrome oxidase, thus modulating the respiratory rate and ATP
41 Chapter 1: General Introduction synthesis (Brown, 1995; Giulivi, 1998). NO is also capable of S-nitrosylating Hb. When oxygenated, this S-nitrosohaemoglobin causes contraction of blood vessels and a decrease in cerebral blood flow. Upon deoxygenation and the removal of NO, methaemoglobin is formed which relaxes vessels, hence improving blood flow (Stamler et aL, 1997; Gow and
Stamler, 1998). Another target for S-nitrosylation by NO is the cardiac calcium release channel, ryanodine receptor 2 (RYR2), the result being channel activation (Xu et al., 1998).
Caspases are also subject to S-nitrosylation. The caspases are a group of cysteine proteases involved in cytokine maturation and apoptotic signal transduction and execution mechanisms. NO has been shown to inhibit caspase activity and prevent apoptosis in hepatocytes (Li et al, 1997).
However, the most well-defined target of NO is sGC (Denninger and Marietta, 1999). As mentioned previously, sGC is responsible for the conversion of GTP to the second messenger cGMP. Three targets of cGMP have been characterised: cGMP-dependent protein kinases (cGK I and W PKG), cGMP-gated ion channels and phosphodiesterases (PDEs), resulting in interactions with the cAMP signaling pathway (Vaandrager and De Jonge, 1996).
cGK I has been found to phosphorylate IP 3 in smooth muscle and thereby inhibit Ca^^ release or increase Ca^^ compartmentalism. The role of cGK I in the brain remains controversial, although it has been speculated that it may be important in controlling Ca^"^ which would be important in neurotransmission (Lohmann et al, 1997). It has been shown that the expression of the mRNA for cGK II is widespread in the brain (El-Husseini et al,
1995), with low levels being expressed in the perikaryon and higher levels in the neuropil of the hippocampus (De Vente et ai, 2001). In the visual cortex cGMP has been found to have opposing effects at pre- and postsynaptic sites. Presynaptically, PKG decreases spontaneous and evoked excitatory postsynaptic currents (EPSCs) while it potentiates the postsynaptic NMDA responses (Wei etal, 2002). Stimulation of NOS, activation of sGC or increasing the concentration of cGMP depressed excitatory synaptic transmission in CAl hippocampal neurons via inhibitory effects on a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors (Lei et al, 2000). However, this effect was shown to be independent of phosphorylation by PKG.
42 Chapter 1: General Introduction
Cyclic nucleotide gated (CNG) channels were originally thought to be localised only in rod and cone photoreceptors and olfactory receptor cells (Kingston et al, 1996). The olfactory channel can be activated by both cAMP and cGMP while the photoreceptor channels are solely activated by cGMP. The CNG channels are structurally homologous to voltage-gated channels, having six transmembrane domains, despite the fact that they are ligand-gated (Zagotta and Siegelbaum, 1996). The CNG channels do not exhibit desensitisation but remain open in the presence of ligand (Bradley et al, 1997). CNG channels display an unusually high permeability to divalent cations, especially Ca^^, the permeability of the olfactory channel being greater than that of the rod channel (Kingston et al, 1996). CNG channels are expressed in the brain (Zufall et al, 1997; Strijbos et al, 1999) with hippocampal neurons expressing the mRNA for both types of channel, the olfactory channel being localised in the cell bodies and processes of CAl and CA3 pyramidal neurons and the granule neurons in the dentate gyrus (Bradley et al, 1997). It has been postulated that the cation conductance of the CNG channel contributes to the development of neurons and synaptic plasticity (Zufall et al, 1997; Kuzmiski and Mac Vicar, 2001).
The hippocampus also shows a very high expression of cGMP-stimulated PDE, especially in the pyramidal and granule cell bodies of the CAl, CA2 and CA3 areas and the dentate gyrus, the function of which is thought to be a reduction of cAMP levels in response to elevated cGMP levels (Respake et al, 1993). PDEs usually control the intracellular concentration of cyclic nucleotides by catalysing their hydrolysis causing inactivation
(Conti and Jin, 1999). All known PDEs can be separated into three groups: those hydrolysing both cAMP and cGMP (PDE2, PDE3, PDEIO and PDEl 1), those hydrolysing cAMP (PDEl, PDE4, PDE7 and PDE 8 ) and those specific for cGMP (PDE5, PDE 6 and
PDE9; Andreeva et al, 2001). The regulatory subunits of each PDE family confer particular properties; for example calmodulin binding domains (PDEl), two non-catalytic cyclic nucleotide-binding domains (PDEs 2,5 and 6 ), N-terminal membrane targeting (PDE4) or hydrophobic membrane-association (PDE3) domains, or calmodulin (PDEl)-, cyclic AMP
(PDEs 1, 3 and 4)- and cGMP (PDE5)-dependent protein kinase phosphorylation sites
(Degerman etal, 1997). PDEs regulate a diverse range of processes including learning and memory, modulation of visual transduction (PDE 6 ), olfaction, platelet aggregation (PDEs 2 and 5), aldosterone synthesis, insulin secretion and T cell activation (Soderling et al,
43 Chapter 1: General Introduction
1999). PDE9A has the highest affinity for cGMP, making it a major regulator of intracellular cGMP concentrations and its mRNA expression has been found to be strong in the hippocampus and other brain regions involved in olfaction, motor control and learning (Andreeva et al, 2001).
1.6 NOS inhibitors 1.6.1 Mechanisms of inhibition
A variety of compounds have been developed for use as pharmacological and possibly clinical inhibitors of NO synthesis assisting investigation of the actions of NO. Due to the complexity and variety of NOS regulatory sites, it is possible for NOS inhibitors to interact in a number of different ways, including: 1 ) by targeting specific cofactor requirements of each isoform (CaM, BH^etc), 2) via the targeting of L-ARG transporters or the availability of L-ARG, 3) exploitation of differential uptake mechanisms and/ or particular characteristics of the cells expressing NOS, or 4) the development of agents with isoform specificity (Southan and Szabo, 1996). The full repertoire of targets for inhibitors is represented in figure 1.9.
1.6.2 The oxygenase domain
The majority of inhibitors identified so far compete with L-ARG for its binding site (Alderton et al, 2001). In fact, many analogues of L-ARG inhibit NOS, the earliest identified and most commonly used of which are the N“-substituted-L-ARG analogues, such as L-NMA and N“-nitro-L-ARG (L-NNA) and its methyl ester L-NAME (Moore and
Handy, 1997). L-NAME is an inactive prodrug which undergoes enzymatic or non- enzymatic hydrolysis to form L-NNA (Pfeiffer et al, 1996; Mayer and Andrew, 1998). The reversible effects of L-NAME may therefore be attributed to L-NNA. However, L-NNA has been shown to inhibit rat liver arginase activity, while L-NAME has not (Robertson et al,
1993).
44 Chapter 1: General Introduction
I,-ARC.
-ARC I ‘'"O V T Bll. D >
^ NADPi i Ca' 1 NO
Figure 1.9: Potential targets for inhibition of synthesis or activity of NO. A: inhibition
of uptake, B: inhibition of BH 4 synthesis, C: reduction of intracellular concentration of cofactors, D: inhibition of up-regulation, E: scavenging of extracellular NO, F: L-ARG
binding site, G: BH 4 site, H: haem site, J: CaM site, K: NADPH binding site, L+ M: FMN and FAD binding sites (adapted from Moore and Handy, 1997).
Other commonly used compounds include non-L-ARG based amino acid analogues or guanidine derivatives such as S-ethylisothiourea (Nakane et al, 1995), aminoguanidine (Wolff and Lubeskie, 1995) and L-thiocitrulline (Ware and King, 2000). It has been demonstrated that these all interact with L-GLU^^^ of bovine eNOS or L-GLU^^‘ of murine iNOS. This conserved L-GLU residue usually interacts with the guanidino group of L-ARG (Alderton etal, 2001) and is essential for substrate binding (Crane etal, 1998). In general, only the L-isomers are effective as inhibitors, although there is one exception: D-NNA has been found to be just as effective as L-NNA in inhibiting endothelium-dependent relaxation in rat aortic rings (Southan and Szabo, 1996).
45 Chapter 1: General Introduction
However, it has now been shown that many L-ARG site NOS inhibitors do not simply compete with L-ARG for its binding site but work via other additional mechanisms. It has been demonstrated that the acetamide inhibitors N^-iminoethyl-L-omithine (L-NIO) and N^- iminoethyl-L-lysine (L-NIL), GW273629 (S-[2-[(l-iminoethyl)-amino] ethyl]-4,4-dioxo-L- cysteine), GW274150 (S-[2-[(I-iminoethyl)-amino] ethyl]-L-homocysteine), as well as aminoguanidine, are all mechanism based inhibitors (MBIs) of NOS (Alderton etal,2001).
In fact, L-NMA was the first mechanistic inactivator to be identified (Bryk and Wolff,
1999). MBIs fit into the L-ARG site and are processed by NOS to form their reactive intermediates, in the case of L-NMA L-N“-hydroxy-NMA, which attack the structures of the active site, such as amino acid residues, haem and other cofactors. Upon covalent modification of the active site, the enzyme is no longer capable of catalysis (Silverman,
1988). In this way MBIs cause a time-dependent irreversible loss of enzyme activity.
Inactivation of NOS by L-N^-hydroxy-NMA is not only associated with covalent modification of the protein but also with the loss of haem from the enzyme (Olken et al, 1994; Maurer et al, 2000). It has now been demonstrated that L-NIO (Maurer et al, 2000) and L-NIL (Bryk and Wolff, 1998) cause inactivation of iNOS through the loss of the haem residue, not via the formation of reactive intermediates via N-hydroxylation (Maurer et a l,
2000).
There is a considerable body of evidence concerning inhibitors that target BH 4 . The BH 4 binding site exists in close proximity to the L-ARG binding site and the haem cofactor, as represented in figure 1.10 (Alderton et al, 2001). BH 4 synthesis can be inhibited using 2, 4-diamino-6-hydroxypyrimidine (DAHP; Stickings and Cunningham, 2002). DAHP actually inhibits GTP cyclohydrolase I, which is the rate limiting enzyme in BH 4 production. However, BH 4 is also a cofactor for several other enzymes, including tyrosine hydroxylase and tryptophan hydroxylase. Therefore, inhibition of BH 4 production would also adversely effect the production of NA, 5-HT, adrenaline and dopamine (DA; Southan and Szabo, 1996). BH 4 analogues, such as 4 -amino-BH 4 (Pfeiffer et al, 1997; Frolich et al,
1999; Werner and Wemer-Felmayer, 2002), act at the BH 4 binding site in a truly competitive manner. Other less obvious compounds also compete for the BH 4 binding site, including the antihypertensive (% 2 -^drenergic agonist clonidine (Venturini et al, 2000), the anti thyroid melanoma seeking drug 2-thiouracil (Palumbo etal, 2000) and 7-nitroindazole
46 Chapter 1: General Introduction
(7-NI; Mayer and Andrew, 1998). However, the actions of 7-NI have also been shown to be through competition with L-ARG through binding to haem (Mayer et al, 1994). Other compounds which act at both the L-ARG and BH 4 sites include N-(4-nitrophenacyl) imidazole and N-(4-nitrophenacyl)-2-methyl-imidazole. It has been demonstrated that both of these act noncompetiti vely against L-ARG but competitively versus BH 4 (Sorrenti et al.,
2 0 0 1 ), indicating allosteric interactions between these two sites.
The haem domain has also been implicated as a target for certain inhibitors, in particular imidazole. Two imidazole molecules bind to each NOS monomer, the first molecule binding the haem iron in the sixth axial position while the other hydrogen bonds with the
L-GLU^^' (Crane et al, 1997). A new class of inhibitor are the pyrimidineimidazole-based compounds discovered using combinatorial chemistry (McMillan et al., 2000). Because of the imidazole group they coordinate directly to the haem and cause displacement of the L-
ARG binding residue. In addition, part of the dimer interface becomes distorted by inhibitor binding, preventing both L-ARG and BH 4 binding and, hence, preventing dimer formation (see section 1.4.6; Blasko et al, 2002).
47 Chapter 1: General Introduction
P H E 4 7 6 *
T R P 4 6 3
Figure 1.10: A ball and stick representation of the iNOS catalytic
site with haem (grey), L-ARG (green) and BH 4 (red; adapted from Alderton et al., 2001).
1.6.3 The calmodulin binding site
There are a number of CaM antagonists including the naphthalenesulphonamides W-7 and
N-(6-aminohexyl)-5-chloro-l-nalphthalenesulphonamide,the phenothiazine trifluoperazine, imidazolium compounds such as calmidazolium (Sugimura et al, 1997) and the indole derivative 3-[2-[4-(3-chloro-2-methylphenyl)-l-piperazinyl] ethyl]-5, 6-dimetoxy-l-(4- imidazoylmethyl)-lH-indazole dihydrochloride 3.5 hydrate (DY-9760e; Fukunaga et ai, 2000). However the use of these is limited by the sheer diversity of CaM-dependent enzymes which, other than NOS, include phosphodiesterase, protein kinases and protein phosphatases.
48 Chapter 1: General Introduction
1.6.4 The reductase domain
Flavoprotein/ NADPH inhibitors include diphenyleneiodonium (DPI), di-2-thienyliodonium
(DTI) and iodoniumdiphenyl (ID), all of which have been shown to completely abolish
NOS activity (Stuehr a/., 1991). The inhibition proved to be potent, irreversible, time and temperature-dependent and independent of enzyme catalysis. Again the use of these inhibitors is limited by the number of processes flavoproteins are involved in including the electron transport chain and reactive oxygen species (ROS) production by mitochondria (Li and Trush, 1998).
1.6.5 Arginine transporters
Research suggests that the carrier proteins for system y^ transporters may be specific for the
NOS isoform that they are supplying. CAT-1 is ubiquitously expressed, except in the liver where CAT-2A is predominant (Ito and Groudine, 1997), and the colocalisation of CAT-1 with caveolae has led to the suggestion that this transporter may provide eNOS directly with substrate (Closs et al., 2000). Expression of CAT-2B appears to be low or absent in normal tissues, although induction of CAT-2B has been shown in vivo and in vitro, using RNA blot analysis, along with coinduction of iNOS and AS (Kawahara et at., 2001), leading to speculation that CAT-2B is responsible for substrate supply to this isoform (Kakuda et al, 1999). However, in situ hybridisation studies have demonstrated that CAT-2B is expressed in neurons and oligodendrocytes but not astrocytes, a distribution differing from that found in vitro (Braissant et al, 2001). The expression of CAT-3, like that of CAT-2 but unlike
CAT-1, is highly tissue specific (Ito and Groudine, 1997). CAT-3 transcripts have been localised in the brain of adult rats (Hosokawa et al, 1997) and mice, predominantly along the midbrain-thalamus-hypothalamus axis (Ito and Groudine, 1997). However, CAT-3 expression in humans is not restricted to the brain but is also found in the thymus and other peripheral tissues (Vekony et al, 2001). Little is known so far regarding the expression or function of CAT-4. It still seems likely, however, that systems y^L and NBAT are also involved in the supply of L-ARG to neurons (see section 1.3). A diverse range of compounds have been found to alter NOS activity via effects on L-ARG transporters. Allicin, a biologically active compound found in garlic, has been shown to decrease NOS
49 Chapter 1: General Introduction mRNA levels at low concentrations while high concentrations have proven to down- regulate the mRNA of CAT-2 (Schwartz etal, 2002). The naturally occurring amino acids L-homoarginine, L-lysine and L-omithine have also been shown to compete with L-ARG for transport by system y^ in a murine macrophage cell line (Bogle et al , 1992), specifically CAT-2B as expressed in Xenopus laevis oocytes (Gloss et al., 1997), as have the inhibitors L-NMA, L-NIO (Bogle et al, 1992) and L-ADMA (Gloss et al, 1997). In addition, glucocorticoids, specifically dexamethasone, have been shown to suppress L-ARG uptake by GAT-1, GAT-2A and GAT-2B (Simmons et al, 1996). These transporters, however, remain unaffected by aminoguanidine (Stühlmiller andBoje, 1995), L-NNA, L-NAME, and
L-GIT (Baydoun and Mann, 1994). Uptake of L-NNA is thought to be mediated by the neutral amino acid transport system in non-epithelial cells, probably via system b^'^ or system B® "" (Hosoya et al, 1998), or even a combination of both (Hatanaka et al, 1999).
1.6.6 Inhibitor specificity
Clinically it is advantageous to target iNOS, as opposed to eNOS, due to the adverse side effects associated with eNOS inhibition while pharmacologically it is preferable to target each individual isoform in order to elucidate their particular roles. A number of compounds are now available which show specificity towards particular isoforms. In vitro studies have indicated that 7-NI is an equally potent inhibitor of all three isoforms (Wolff et al, 1994). However, the situation in vivo is a little different where 7-NI has little effect on eNOS but potently inhibits nNOS (Moore et al, 1993). This may indicate that there are alternative drug metabolism and/ or uptake mechanisms present in vivo (Mayer and Andrew, 1998). 7-Nl should thus be considered cell-type specific as opposed to isoform specific. Other nNOS inhibitors include the isothiourea derivatives S-ethyl-N-[4-(trifluoromethyl) phenyl] isothiourea and 1 -amino-S-methyl-isothiourea (AMITU), and the non-amino acid ARL
17477. However, the latter compound is only 5 times more selective for nNOS than iNOS (Alderton et al, 2001). The most potent and nNOS selective drugs discovered so far are N^- (l-imino-3-butenyl)-L-omithine (vinyl-L-NIO; L-VNIO; Babu and Griffith, 1998) and a series of N-phenylamidines (Lowe et al, 1999).
50 Chapter 1: General Introduction
The first selective iNOS inhibitors were the bis-isothioureas and S, S’-[l, 3-phenylenebis-
(1,2-ethanediyI)] bisisothiourea (PB ITU) is 190 times more selective for iNOS over eNOS.
Selectivity was defined primarily on the basis of their potency under identical conditions in the physiological range. However, PBITU only has a 5-fold selectivity against nNOS.
This suggests that there may be significant differences between the iNOS and eNOS substrate binding sites (Alderton et al, 2001), which may be advantageous for future drug development. The MBl aminoguanidine shows a 10- to 100-fold selectivity for iNOS but its use is limited due to its effects unrelated to NOS inhibition. These include inhibition of histamine metabolism and inhibition of catalase and other copper or iron-containing enzymes (Southan and Szabo, 1996). Another MBl which has a high selectivity for iNOS is N-(3-(aminomethyl) benzyl) acetamidine (1400W) and the inhibition proved to be either very slowly reversible or irreversible (Garvey et al, 1997). The administration of a single subcutaneous dose of 1400W to rats also delayed the vascular injury attributed to lipopolysaccharide (LPS)-induced NOS. This is in contrast to both L-NIO and aminoguanidine. However, 1400W is acutely toxic at high doses which limits its therapeutic potential (Alderton et ai, 2001). Two other MBls which act in a very similar way to 1400W are the hetero-substituted analogues and homologues of L-NIL GW273629 and GW274150 (Young et al, 2000), but, unlike 1400W, they are not toxic at high doses (Alderton et al, 2001).
It has been documented that L-NNA and L-NAME are specific toward eNOS (Southan and Szabo, 1996) but, while they are 100-fold more potent than L-NMA for eNOS (Gross et al,
1990), they have also been shown to inhibit nNOS in CNS tissue with an IC 5 0 of 0.05/xM
(Lambert et al, 1991).
Several hundred compounds that target NOS are now commercially available. Table 1.2 lists an overview of those mentioned in this section but the list is far from complete.
51 Chapter 1: General Introduction
Table 1.2: Properties and sites of action of some NOS inhibitors.
Compound Site(s) of action Com m ent
1400W - Potent iNOS inhibitor
Aminoguanidine L-ARG binding site, haem? MBl, non-specific iNOS inhibitor
AMITU - nNOS specific
4-Amino BH 4 BH 4 binding site -
ARL 17477 - nNOS specific
ADMA L-ARG binding site/ L-ARG Endogenous inhibitor transporter
Calmidazolium CaM site Does not inhibit iNOS, has multiple effects
Clonidine BH 4 binding site -
DAMP BH 4 Inhibits BH 4 synthesis
DPI Reductase domain Non-specific inhibitor of all three isoforms *
DTI Reductase domain -
DY-9760C CaM site Non-specific for NOS
S-Ethylisothiourea L-ARG binding site/ haem? -
Glucocorticoids L-ARG transporters/ BH 4 Non-specific for NOS
GW273629/ GW274150 L-ARG binding site MBIs, iNOS specific
ID Reductase domain -
Imidazole Haem/ L-ARG binding Very weak inhibitor
L-NIL Loss of haem via binding to L- MBl, more specific for iNOS ARG site
L-NIO Haem/ L-ARG transporter MBl, irreversible inhibitor of iNOS in vitro
L-N'^-substituted L-ARG analogues L-ARG binding site D-isomers are usually inactive
L-NMA L-ARG transporter/ L-ARG site/ First reported MBl of NOS haem
L-NNA L-ARG binding Possibly inhibits arginase
L-NAME L-ARG binding site Undergoes degradation to yield L- NNA
7-NI Haem/ L-ARG/ BH 4 Selective for nNOS in vivo
Phenacyl imidazoles L-ARG/ BH 4 -
N-Phenylami dines - Potent nNOS inhibitors
Pyramidine imidazoles L-ARG/ haem/ BH 4 Prevent dimer formation
PBITU - Potent iNOS inhibitor
L-Thiocitrulline Haem/ L-ARG Non-selective inhibitor
2-Thiouracil BH 4 binding site -
Trifluoperazine CaM site Does not inhibit iNOS
Vinyl-NIO - nNOS specific
W-7 CaM site Non-specific for NOS
52 Chapter 1: General Introduction
1.7 NO releasing compounds 1.7.1 General properties of NO donors
Other classes of drugs used to investigate the pharmacological actions of NO are the NO donors. Clinically this class of drugs are used in several pathological conditions where a deficiency of NO has been implicated. Organic nitrate and nitrite esters, such as nitroglycerin (glyceryl trinitrate; GTN), have been used in cardiovascular therapeutics since the nineteenth century and have been used to treat angina, ischaemic heart disease, heart failure and hypertension for some time (Ignarro et ah, 2002). However, these compounds are limited by their short half-life, systemic absorption and thus potential adverse effects and drug tolerance. It is therefore beneficial to develop novel compounds to overcome these restrictions. For the development of NO-prodrugs a number of points need to be taken into consideration: 1) where the NO needs to be released, intracellular versus extracellular, 2) the kinetic properties of the compound, short-acting drugs for acute treatment or long- lasting for prevention, 3) physicochemical properties of the compound to allow either a solution for intravenous (i.v.) infusion or a long half-life to give sufficient bioavailability with oral administration, 4) the redox state of the NO species generated: NO radical (NO®/ NO*), nitrosonium (NO^) or nitroxyl anion (NO ; Stamler et al, 1992), 5) the cofactors required for NO formation including thiols, oxygen, enzymes or pH, 6) the stability of the compound towards light, heat and pH and 7) the rate at which NO is released: the release needs to be relatively slow in order to avoid toxicity (Schonafinger, 1999).
1.7.2 Direct donors
Direct NO donors spontaneously release NO. Three common members of this class of compound include NO gas, sodium nitroprusside (SNP) and sodium trioxodinitrate
(Angeli’s salt). The use of NO gas is limited to inhalation therapy for pulmonary vascular disorders due to its short half-life and its reactivity towards molecular oxygen (Ignarro et al, 2002). SNP is an inorganic complex with ferrous iron in a square bipyramid and NO bound as NO^ (Feelisch, 1998). SNP has been used for decades in the treatment of hypertension but the mechanism by which it releases NO is incompletely understood. It has now been demonstrated that SNP requires either reduction or irradiation with a light source
53 Chapter 1: General Introduction to release NO, so it would seem that the assumption that SNP releases NO spontaneously is a misconception. The use of SNP clinically is limited by the formation of cyanide upon decomposition of the complex and, hence, the development of thiocyanate toxicity. As well as this it has to be administered parenterally, usually i.v., so tolerance develops (Ignarro et al, 2002; Feelisch, 1998). Angeli’s salt decomposes to nitrite (NO 2 ) and NO". NO is yielded from the one-electron reduction of NO* (Ma et al, 1999), a reaction that is thermodynamically possible under physiological conditions (Ohshima et al, 1999). NO is generally considered to be far more toxic than NO*. The administration of Angeli’s salt to
lung fibroblast cells was found to be cytotoxic, with the co-administration of ferricyanide, responsible for the conversion of NO to NO’, affording considerable protection (Wink et al, 1998). The cytotoxicity was attributed to DNA double strand breaks, concurring with another study in plasmids where strand breaks were also induced with Angeli’s salt with protection conferred by electron acceptors (Ohshima et al, 1999). Likewise NO has also been found to increase postischaemic myocardial injury in rabbits (Ma et al, 1999), implicating NO as a major mediator of ischaemia-induced vascular toxicity (Fitzhugh and Keefer, 2000).
Diazeniumdiolates contain the [N(0)N0]'functional group (Ignarro et al, 2002) bound to a nucleophilic residue via a nitrogen atom (Feelisch, 1998). The diazeniumdiolates are also referred to as NONOates, Drago complexes or NOC compounds and many of them spontaneously decompose to NO* without redox activation at rates that are relatively insensitive to the presence of thiols or other redox-active agents (Fitzhugh and Keefer,
2000). However, the sulphite NONOate (SULPHl/ NO) is a notable exception since it forms N 2 O and sulphate, as opposed to sulphite and NO*. Furthermore, Angeli’s salt is actually an NONOate, with O 2 serving as the nucleophile (0X1/ NO). Like Angeli’s salt, propylamino-propylamine NONOate (PAPA/ NO) has been shown to form nitrosylmyoglobin from metmyoglobin, indicative of NO formation (Feelisch, 1998). The diazeniumdiolates have been shown to reverse cerebral vasospasm, to decrease pulmonary vascular pressure and to improve oxygenation in acute lung injury (Ignarro et al, 2002). Due to their variable half-lives, a range of 2s to 20h (Fitzhugh and Keefer, 2000), the NONOates are an invaluable in vitro tool. However, their first-order decomposition limits their in vivo application. There has therefore been a directed effort to design prodrugs which
54 Chapter 1: General Introduction cannot release NO until they are enzymatically converted to their active NONOate in the target tissue (Feelisch, 1998). This is the case when N-nitroso-N-phenylhydroxylamine ammonium salt (cupferron) is O-alkylated at the terminal oxygen. Release of NO would only occur by increasing the pH or by a protease-catalysed hydrolysis (Hou et al, 2000).
Other heterocyclic compounds which release NO include the mesoionic heterocycles, such as the sydnonimines and the related compounds oxatriazolium-olates and imidates, and the heterocyclic N-oxides, such as the furoxans and triazole-oxides (Schonafinger, 1999).
These groups of compounds require cofactors to release NO, the sydnonimines require oxidants while the furoxans require thiols (Ignarro et al , 2002). A representative compound of the sydnonimines is molsidomine, which is used clinically for angina pectoris.
Molsidomine is a prodrug which is converted to the active metabolite 3-morpholino- sydnonimine (SIN-1; linsidomine) by liver esterases (Feelisch, 1998; Schonafinger, 1999).
SIN-1 spontaneously decomposes at physiological pH into NO* with the concomitant formation of Oj'(Al-Sa’Doni and Ferro, 2000; Ignarro et al, 2002).
However, pharmacologically the most commonly used NO donors are the S-nitrosothiols. The S-nitrosothiols have the general formula R-SNO. They were first synthesised in 1840
(Oae and Shinhama, 1983) and have been used in biomedical research since the 1970s
(Hogg, 2000). R-SNOs are endogenous NO donors, having been identified in human and rabbit plasma, human airway lining fluid and neutrophils (Al-Sa’Doni and Ferro, 2000).
Only three R-SNOs have been purified in their solid form: S-nitroso-N-acetyl penicillamine
(SNAP), S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (Feelisch, 1998). R-SNOs undergo many biological reactions, the main of which include NO release, transnitrosation and S-thiolation (Hogg, 2000), as represented in figure 1.11.
55 Chapter 1: General Introduction
Direct effects RS' NO' RSNO+ RSI
RSI
R-SNO
Cu-Cii RSH
RSH + NO' RSSR + NO
Figure 1.11: The main reactions of S-nitrosothiols (adapted from Hogg,
2000).
The mechanism by which R-SNOs produce NO is the least well understood. Many R-SNOs in physiological solutions undergo decomposition to produce the corresponding disulphide
and NO by homolytic cleavage of the S-N bond. However, in addition to homolytic fission,
R-SNOs may also decompose heterolytically to yield NO^ or NO'. R-SNOs are therefore capable of donating NO*, NO^ and NO', depending on the physiological conditions
(Feelisch, 1998). The actions of several nitrovasodilators, including GTN and SIN-1, have now been shown to be through the formation of R-SNO intermediates (Hogg, 2000).
However, it is now thought that R-SNOs do not spontaneously release NO and it may be their direct extracellular activity which is responsible for their effects. It has also been suggested that the reduction of R-SNOs by Cu^ and other metal ions may mediate the release of NO (Al-Sa’Doni and Ferro, 2000; Hogg, 2000). However, the effects of chelating
56 Chapter 1: General Introduction agents have been shown to vary depending on the type of bioassay, the chelator chosen and the nature of the thiol (Gordge et al, 1995). The transnitrosation reactions, in which there is the transfer of bound NO from one thiol to another, are particularly important functions of R-SNOs. If the transfer of NO occurs from GSNO or SNAP, both of which are relatively stable, to a thiol such as CYS, which is abundant in vivo, the result is a relatively unstable
R-SNO. In the presence of Cu^ this complex may decompose to form NO (Al-Sa’Doni and
Ferro, 2000). R-SNOs have also been shown to undergo enzymatic, photolytic and thermal decomposition to generate NO. In fact, SNAP can also directly activate sGC in the presence of thiol depleting agents (Haj-Yehia and Benet, 1996). It is still unclear how R-SNOs produce NO in vivo but it may involve all of the above mechanisms to a greater or lesser extent.
1.7.3 Donors requiring metabolism
In 1879, Murrell reported on the benefits of GTN in the treatment of angina and all of the organic nitrates used since are prodrugs requiring enzymatic metabolism for the generation of NO (Ignarro et ai, 2002). Despite much effort, the site of enzymatic biotransformation has not been identified, although it has been postulated that two distinct sites may be involved, one of which being membrane bound (Feelisch, 1998). Two enzyme systems have been suggested to account for the bioactivation of organic nitrates: an NADPH-dependent cyt P450 system and glutathione-S-transferase (GST; Ignarro et ai, 2002). As mentioned previously this class of drugs is limited by nitrate tolerance.
NO can also be supplied by photolabile caged compounds such as trichloronitrosylruthenium (Ru (NO) 0 ,3 ) and dipotassium pentachloronitrosylruthenate
(K 2 RU (NO) C 1 5 ; Carter et al , 1997), which release NO upon a flash of ultraviolet radiation.
This type of molecule allow precise spatial, temporal and concentration control of NO for in vitro investigation (Makings and Tsien, 1994).
57 Chapter 1: General Introduction
1.7.4 Bifunctional donors
To exploit some of the beneficial effects of NO and to improve the safety profile of existing
drugs, compounds have been developed that combine an NO moiety without altering the
pharmacological profile of the parent drug. A rationale which exemplifies this is a new
class of non-steroidal anti-inflammatory drug (NSAID) which release NO, the so-called
NO-NSAIDs. NO-NSAIDs are created by the addition of a nitroxylbutyl moiety to a conventional NSAID (Fiorucci, 2001) through chemical spacers such as aliphatic, aromatic or heterocyclic chains (Burgaud et al, 2002). The major drawback of NSAID therapy is their tendency to cause gastro-intestinal (GI) toxicity. The ability of NSAIDs to cause GI bleeding and ulceration has been linked to topical damage due to their acidic nature in addition to inhibition of gastroprotective prostaglandins (Grosser and Schroder, 2000), especially cyclo-oxygenases-1 and 2 (COX-1 and 2, respectively; Sigthorsson etal, 2002). The pre-treatment of rats with SNP significantly reduced the GI injury induced by the NSAID flurbiprofen (Wallace et ai, 1994) and it is thought that NO confers this gastroprotection by acting as a smooth muscle relaxant to counteract the reduction in gastric blood flow elicited by prostaglandin inhibitors (Grosser and Schroder, 2000). The addition of an NO-releasing moiety to NSAIDs has proved to reduce their GI toxicity greatly without affecting their anti-inflammatory, anti-pyretic and analgesic properties (Wallace et al, 1995). The use of NO-NSAIDs and other NO releasing drugs has lead to the possibility of designing new drugs able to deliver NO into target sites in a sustained and controlled manner.
1.8 Roles of NO throughout the body 1.8.1 The cardiovascular system
In the vasculature, the synthesis of NO by eNOS can be activated by the shear stress exerted by the circulating blood, which leads to flow-dependent vasodilation (Adnot et al, 1995). Furchgott and Zawadski (1980) were the first to demonstrate that the vasodilation due to
ACh was down to EDRF/ NO and studies with transgenic mice lacking the eNOS gene have identified the role of NO in the maintenance of pulmonary vascular tone (Steudel et al, 1997; Fagan et al, 2000). Children with pulmonary hypertension and congenital heart
58 Chapter 1: General Introduction
defects have been found to have low NO levels (Narin et al, 2000) and the i.v. infusion of L-NMA increases pulmonary vascular resistance in normal adults (Clini and Ambrosino,
2002). However, there are considerable inter- and intra-species differences. To date constrictor responses are reported more often in pigs and humans while the only species in
which NOS inhibitors consistently cause pulmonary vasoconstriction is sheep (Hampl and
Herget, 2000). The fact that eNOS knockout mice have increased pulmonary tension may be explained by the role of NO in the development of the neonatal pulmonary circulation.
Foetal pulmonary circulation is high pressure and low flow while postnatally it needs to become low pressure high flow (Abman et ai, 1990) and endogenous NO production is essential for this. This transition will thus be impaired and may well be responsible for the hypertension observed as opposed to being the consequence of no NO production. In support of this, immunostaining studies have shown that eNOS levels are high in foetal rats, being highest around the first postnatal day, with levels at their minimum in adulthood (North et al, 1994). It has been suggested that eNOS may not be the only isoform of the enzyme involved in the maintenance of vascular tone in the lung, nNOS having been detected in lung epithelium (Asano et al, 1994; Liihrs et al, 2002) and the uncontrolled production of NO by iNOS contributes to vasodilation, hypotension and dysregulated vascular responses during states where inflammation is unregulated (see section 1.8.2; Hart,
1999). However, it is generally accepted that eNOS plays a role in vascular tone, which is illustrated by studies focusing on vascular disease. Clinically, pulmonary hypertension is defined as a condition involving increased pulmonary arterial pressure and/ or pulmonary vascular resistance. It is a syndrome common to several cardiovascular disorders including adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease
(COPD), lung fibrosis, mitral stenosis and congenital heart defects (Hampl and Herget,
2000). Autopsy or biopsy specimens from patients with primary or secondary forms of pulmonary hypertension indicated that the expression of eNOS was low or absent compared with controls while a study with COPD patients revealed that their NO-dependent vasodilation response was impaired by 50% (Hart, 1999), although other studies have found that COPD is not necessarily associated with endothelial dysfunction (Adnot et al, 1993).
Pulmonary vasoconstriction can be induced by alveolar hypoxia which makes it a useful experimental tool (Hampl and Herget, 2000). ACh and A23187 have both proved to reverse the vasoconstriction due to hypoxia, an effect that was completely inhibited by L-NMA
59 Chapter 1: General Introduction
(Adnot, 1995) demonstrating that NO is responsible for the relaxation. Hypoxic vasoconstriction in eNOS knockout mice is twice as intense and treatment with ACh followed by L-NMA has no effect. Both nNOS and iNOS knockout mice exhibit normal hypoxic vasoconstriction, suggesting that eNOS is the main isoform involved (Fagan et al,
1999). Hypoxia has been shown to down-regulate the expression of the constitutive isoforms of NOS in human endothelial cells via transcriptional and posttranscriptional mechanisms (McQuillan et ai, 1994) and this is supported in histochemical studies where NOS immunoreactivity is diminished (Giaid and Saleh, 1995). Inhalation therapy with NO gas has emerged as a useful therapy to reverse the pulmonary vasoconstriction due to hypoxia, primary pulmonary hypertension, congenital heart disease, persistent pulmonary hypertension of the newborn (PPHN) and ARDS (Zapol et a/., 1994; Adnot et al, 1995). NO therapy is advantageous because it is without systemic side effects because it is effectively scavenged by haem, although prolonged treatment can lead to toxicity including synergistic lung injury and death of lung epithelial cells (Franek et al, 2002). Studies with eNOS knockout mice have also highlighted a role for NO in the suppression of inotropic responses to P-adrenergic agonists (Huang, 2000).
1.8.2 Host defence
The innate immune responses, which include the release of antimicrobial peptides at epithelia, phagocytosis and pathogen destruction by macrophages and neutrophils, as well as the activation of the complement cascade, form the first line of defence against invading pathogens. The main mechanism by which phagocytic cells kill microorganisms is via the so-called respiratory burst activity, which incorporates the generation of highly reactive intermediates derived from O 2 , as well as intermediates derived from NO if iNOS is present and activated (Cherayil and Antos, 2001). These free radical intermediates are highly cytotoxic but they are non-specific so are also capable of causing damage to host cells. Respiratory burst activity causes the modification and, in some cases, the inactivation of the protein, lipid and nucleic acid components of the engulfed organism. Respiratory burst is mediated by phagocyte NADPH oxidase (phox) activity and, although iNOS and phox are differentially regulated, they can both be stimulated by inflammatory stimuli such as interferon-y (IFN-y). The interaction of reactive oxygen and nitrogen intermediates
60 Chapter 1: General Introduction
(ROIs and RNIs, respectively) establishes the molecular basis for synergy between respiratory burst and NO production (Fang, 1997). The term RNI refers to oxidation states and adducts of the products of NOS (Nathan and Shiloh, 2000). In addition to NO*, important related compounds include ONOO, R-SNOs, nitrogen dioxide (NO 2 *), dinitrogen trioxide (N 2 O 3 ), dinitrogen tetroxide (N 2 O 4 ) and dinitrosyl-iron complexes (DNIC; Fang,
1997). RNIs of dietary origin are used as antimicrobial agents in gastric juice and hence contribute to the innate immunity of the gastric epithelium. Macrophages have the ability to produce both NO’ and O 2 in nearly equimolar quantities and, because NO* has an extremely high affinity for the O 2 radical (Inoue et al, 2 0 0 0 ), they are therefore prolific generators of ONOO. In fact, ONOO is far more cytotoxic than either O 2 or NO* alone and it has proved to be involved in a wide range of toxic oxidative reactions including the initiation of tyrosine (TYR) nitration, lipid peroxidation, inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of the NaV ATPase pump, inactivation of membrane Na"^ channels and other oxidative modifications of proteins (Szabo and Billiar, 1999). ONOO is also a potent trigger of DNA strand breakage and has been shown to induce DNA base mutations, especially G: C T: A transversions (Juedes and Wogan, 1996). NO itself has been implicated in DNA damage although this ability has been linked to its cytotoxic nature as opposed to its genotoxicity, as investigated using the micronucleus and comet assays
(Stopperez < 2 /., 1999).
The cytotoxicity of NO has been linked to its ability to interact with and S-nitrosylate a large variety of substances (see section 1.5). Since the early 1990s an oversimplified view of the roles of NO has been established: the low levels of NO produced by constitutive
NOS have been implicated in physiological processes while the high levels produced by iNOS were considered to be responsible for pathological processes. In 1993, however,
Griscavage et al reported that iNOS in rat alveolar macrophages can be inhibited by NO itself. This feedback inhibition has been attributed to the ability of NO to inactivate the transcriptional factor NF-kB (Togashi et al, 1997) which regulates the expression of many host defence proteins, including iNOS (Connelly et al, 2001). This theory thus creates a paradox: the low physiological levels would inhibit the induction of iNOS, making iNOS induction a rare event, which is not the case. A well established activator of iNOS is
61 Chapter 1: General Introduction bacterial LPS and studies have demonstrated that the administration of LPS plus IFN-y to human astrocytoma cells inhibits nNOS activity via phosphorylation of a TYR residue (Colasanti et al, 1997), by protein-tyrosine kinase(s) or phosphotyrosine-specific phosphatase(s; Colasanti et al, 1999), thereby creating optimum conditions for iNOS activation and resolving the apparent paradox (Colasanti and Suzuki, 2000).
Although the aetiology of a number of neurodegenerati ve and neuroinflammatory disorders is still unknown, there is increasing evidence that NO and RNIs play an important role. NO produced from iNOS in glia has been implicated in Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Huntingdon’s disease, multiple sclerosis, stroke and HTV dementia (Dawson and Dawson, 1998; Calabrese et al, 2000). Because it has been shown that rat alveolar macrophages (Miles et al, 1997) and the murine macrophage cell line RAW 264.7 (Schmidt et a l , 1992b) can produce NO from constitutive NOS, as can microglia (Kopec and Carroll, 2000), iNOS may not be the only isoform involved in pathogenesis. In fact, both eNOS and nNOS have been identified in astrocytes (Arbones et al, 1996; Colasanti et al, 1998). Evidence now suggests that, under normal conditions, NO produced by constitutive NOS in microglia actually attenuates phagocytic activity in the brain (Kopec and Carroll, 2000). NO is associated with neurite growth, plasticity and regeneration in the brain (see section 1.9.5) and inhibition of phagocytosis would be advantageous. However, as a consequence there would be reduced phagocytosis in an inflammatory environment which may allow for the buildup of debris and increased neural degeneration. This theory has been proposed as a contributory factor in the development of AD. One of the key pathological features of AD is the development of p- amyloid deposits which persist and develop into plaques. Increased levels of NO have been reported in both the neurons and astrocytes of AD brain (De la Monte and Bloch, 1997) which would effectively reduce microglial phagocytosis and contribute to the buildup of
P-amyloid (Kopec and Carroll, 2000). The continuous production of NO has thus been implicated in the development of neurodegeneration seen in chronic neuroinflammatory diseases.
Obviously, both of these theories cannot both be correct. If the answer to the paradox is correct then in neuroinflammatory situations where there is the release of inflammatory
62 Chapter 1: General Introduction
Stimuli there would be a down-regulation of constitutive NOS activity and hence no attenuation of phagocytic activity. However, the evidence for the involvement of inflammatory mediators in AD is a little controversial. Samples of cerebrospinal fluid from patients with AD showed no difference in levels of interleukins, neopterin, IFN-y and TNF- a compared to age and sex matched control patients (Engelborghs et al, 1999). This is contradicted in another study in neuroblastoma cells where TNF-a combined with IFN-y induce the formation of p-amyloid (Blasko etal, 1999). In turn, P-amyloid, in the presence of IFN-y and TNF-a, has been shown to induce NO production in a rat astrocyte cell line by increasing the expression of iNOS, although each substance alone had no effect on iNOS expression as measured by Northern blot analysis (Rossi and Bianchini, 1996). In addition, elevated levels of both iNOS and eNOS have been found in association with P-amyloid deposits in AD patients and transgenic mice expressing the human p-amyloid precursor protein, as well as in the vicinity of the lesion site in a rat model of AD (Liith et al, 2001). Given that AD is a progressive neurodegenerati ve disorder it is difficult to pinpoint the initial trigger. It is clear that the elucidation of the protective versus the toxic roles of NO will remain a contentious issue for some time.
1.8.3 The peripheral nervous system
Throughout the autonomic nervous system a major component of transmission has been established to be mediated by neither noradrenaline nor ACh, giving rise to the concept of
NANG control of autonomic neuromuscular tone. The major transmitters of NANG neurons have been identified as NO, ATP and neuropeptides such as the tachykinins SP and neurokinin A (NKA; Bennett, 1997). It has also been proposed that, other than neurons, interstitial cells and smooth muscle cells may be a source of NO for NANG transmission
(Lefebvre, 1995). Evidence for nitrergic control of smooth muscle has been obtained from many systems throughout the body. In the GI tract (GIT) there is much evidence to support the view that the inhibitory transmitter is NO with SP as the excitatory transmitter released from NANG neurons. Relaxation of the smooth muscle of the GIT followed by tonic contraction contributes to peristaltic movement and the relaxation has been attributed to NO (Ivancheva et al, 1998). The relaxation due to the stimulation of NANG nerves can be blocked by L-NNA or L-NAME in a variety of preparations including the lower
63 Chapter 1: General Introduction
oesophageal sphincter of the opossum (Tottrup et al, 1991), canine duodenal longitudinal muscle (Toda et al, 1990), proximal colon (Ward et al, 1992a), ileocolonic sphincter (Ward et al, 1992b; De Man et al, 1993), and jejunal circular muscle (Stark et al, 1991), guinea-pig gastric fundus (Desai etal, 1991), rat gastric fundus (Boeckxstaens etal, 1992;
Lefebvre era/., 1992; Li and Rand, 1990) and hamster ileum (Matsuyama era/., 1999). The
only part of the GIT where NO as the inhibitory transmitter has not been established is in
the taenia coli (Bennett, 1997).
The role of NO in the reproductive system is also well established. Erectile dysfunction
(ED) affects millions of men worldwide in varying degrees. Corpus cavemosum relaxation
is essential for penile erection, a function which is regulated by NANG, vasoactive
intestinal peptide (VIP) and possibly calcitonin gene-related peptide (CORF) containing nerves (Nehra et al, 2001). Sildenafil citrate (Viagra®) has emerged as one of the most potent oral therapeutics for ED (Ayajiki et al, 2002). Viagra® is a reversible inhibitor specific to PDE5 so it effectively prolongs the action of NO although its coadministration with nitrates has been absolutely contraindicated within the same 24-hour period (Nehra et al, 2001). The priapism resulting from Tityus serrulatus scorpion venom has also been attributed to the release of NO from NANG nerves (Teixeira et al, 2001).
Inhibitory NANG nerves may well be the only neural bronchodilator in human airways,
which has lead to the implication of NANG dysfunction in the pathogenesis of asthma. As
yet, however, no differences have been found between asthmatics and healthy controls (Van der Velden and Hulsmann, 1999). Furthermore, the amount of NO in the epithelium greatly exceeds that in airway nerves (Widdicombe, 1998). The role of NO and NANG nerves in airway smooth muscle remains to be elucidated.
1.9 NO in the central nervous system 1.9.1 Glutamatergic NO release
At the same time as amino acids were recognised as neurotransmitters, it was discovered that the application of L-GLU to rat cerebellar cortex increased the cGMP content several fold (Biggio and Guidotti, 1976). The L-GLU analogue kainate (KA) also proved effective
64 Chapter 1: General Introduction at markedly increasing cGMP concentrations in rat cerebellar slices (Schmidt et al, 1976; Foster and Roberts, 1981). At the same time it had been found that various nitrogen based compounds, including sodium azide (Kimura etal, 1975), SNP, GTN and hydroxylamine, were capable of activating sGC (Katsuki et al, 1977). Deguchi (1977) was the first to hypothesise that there was the release of an endogenous activating factor for sGC and
Garthwaite et al (1988) put all the evidence together to suggest that L-GLU acting on NMDA receptors causes the release of NO, at the time still referred to as EDRF. By 1993 there was direct evidence for the release of NO in response to NMDA receptor activation in the cerebellar cortex of freely moving rats (Luo et al, 1993). It therefore seems appropriate to give an overview of the role of L-GLU in the CNS.
1.9.2 Glutamate as a neurotransmitter
L-GLU is widely accepted as the major excitatory neurotransmitter in the brain. L-GLU is stored presynaptically in vesicles and released in response to stimulation of afferent pathways in vivo (Jasper and Koyama, 1969). Astrocytes can release L-GLU in response to molecules such as prostaglandin Ej, ATP and BK. L-GLU does not cross the blood brain barrier (BBB) so it therefore has to be manufactured de novo in the brain by astrocytes (see section 1.2; Nedergaard et al, 2002). Because L-GLU is not known to be metabolised in the synaptic cleft, L-GLU signaling is therefore governed by transporters and receptors.
Transporters are essential for the termination of synaptic transmission and keeping the extracellular levels of L-GLU below neurotoxic levels (Harada et al, 1998). So far molecular cloning has identified five structurally distinct subtypes of L-GLU transporter: glutamate transporter-1 (GLT-1), the glutamate and aspartate transporter (GLAST), excitatory amino acid carrier 1 (EAACl) and excitatory amino acid transporters 4 and 5
(EAAT4 and 5 respectively; Tanaka, 2000). GLT-1, GLAST and EAACl were originally named EAATl, 2 and 3 respectively (see table 1.1; Rao et al, 2001). Although there is active reuptake into presynaptic neurons this mechanism seems to be less important than astrocytic transport (Daikhin and Yudkoff, 2000) and GLT-1 and GLAST are found in mature glial processes while the others are neuronal transporters. EAACl and EAAT4 however are not found in axons and presynaptic terminals but in the somata and dendrites
65 Chapter 1: General Introduction of neurons. Because GLT-1 are exclusively located in glial cells (Conti and Weinberg,
1999) and because they have the highest level of expression (Danbolt, 2001) it has been hypothesised that they are responsible for the majority of the total L-GLU transport (Matute et a l , 2002). Studies with transgenic mice have illustrated that L-GLU uptake was impaired in GLT-1 knockouts compared to their wild type littermates. Many other studies have attempted to block GLT-1 by using dihydrokainate (DHK), an analogue of L-GLU, but these experiments are complicated by the fact that DHK also binds to and activates L-GLU receptors (Hamann et al, 2002).
L-GLU receptors can broadly be divided into two distinct groups, ionotropic or metabotropic. The three ionotropic L-GLU receptors (iGLURs) have been identified on the basis of their affinities for certain ligands, namely NMDA, AMPA and KA. KA receptors can be activated by both AMPA and KA but can be pharmacologically isolated using
GYK153655, a selective AMPA antagonist. However, since it has often been difficult to distinguish between the AMPA and KA subtypes, they are often referred to as non-NMDA receptors. All of the iGLURs are associated with transmembrane cation channels that, when activated, allow Na"^ influx, K^ efflux, membrane depolarisation and in some cases Ca^^ influx (Hansson et al, 2000). There are eight metabotropic L-GLU receptors (mGLURs) which have been subdivided into three groups depending on their signal transduction mechanism, amino acid homology and pharmacological profile (Nakanishi, 1992). Group
I consists of mGLURl and 5 and they are positively coupled to phospholipase C (PLC) in addition to acting on various K^ channels via a G^ or Gq protein. Activation of group I mGLURs results in phosphoinositide hydrolysis and hence the mobilisation of Ca^^ from intracellular stores (Ong et al, 1998). Groups U (mGLUR2 and 3) and IQ (mGLUR4, 6 ,7 and 8 ) are all associated with the inhibition of adenylate cyclase (Hansson et al, 2000).
Advances in methodological techniques such as immunocytochemistry, molecular biology and electrophysiology have brought new insight into glial cell physiology and demonstrated that although iGLURs are generally considered to be postsynaptic, the major macroglial cells, astrocytes and oligodendrocytes, both express various types of L-GLU receptor
(Steinhauser and Gallo, 1996). Molecular cloning has established that each iGLUR is composed of several subunits with a high homology that are encoded by at least six gene
66 Chapter 1: General Introduction
families (Dingledine et al, 1999). NMDA receptors are composed of NMDARl, NMDAR2A-D (the corresponding transcripts in the mouse and the rat are called f 1 and e 1-
4 and NRl and NR2A-D respectively; Sucher et al, 1996) and NMDAR3A (previously known as, initially %-l; Andersson etal, 2001; and then NMDA-L; Paarmann et al, 2000) and 3B (Nishi et al, 2001), AMPA receptors of GLURl-4 and KA receptors of GLUR5-7 and KAl and 2 subunits (Matute et al, 2002). Alternate splicing of iGLUR subunits increases the diversity of the receptor physiology even further. As with most ligand gated
ion channels the receptor complexes are most likely to exist as homomeric or heteromeric
units with five subunits forming the receptor channel, although some evidence exists to contradict this and suggest that there are only four subunits (Dingledine et al, 1999). Each subunit is arranged with a topology of three transmembrane domains with a reentrant
segment (Steinhauser and Gallo, 1996) with an extracellular N-terminal domain and an
intracellular C-terminus (see figure 1.7; Bettler and Mulle, 1994; Gozlan and Ben-Ari,
1995). The mGLUR proteins consist of seven transmembrane domains which is analogous
to all other G-protein linked receptors (Steinhauser and Gallo, 1996).
1.9.3 The NMDA receptor 1.9.3.1 Special features of the NMDA receptor
NMDA receptors mediate several important physiological processes, including long term potentiation (LTP) and depression (LTD), while overstimulation can trigger pathological processes that can lead to neuronal death. Regulation of NMDA receptor activity is
therefore very important in the CNS and this is achieved through several intra- and
extracellular modulatory sites. A vast array of substances modulate NMDA receptor
mediated responses including H^, Mg^"^, Ca^^, Zn^^, kinases, phosphatases and redox drugs
(Gozlan and Ben-Ari, 1995).
The NMDA receptor has some unique properties which set it apart from the other ionotropic receptor classes. In addition to allowing monovalent cations through the channel it is also highly permeable to Ca^^. This Ca^"" permeability has widespread implications, especially in terms of neurotransmission and neurotoxicity in the CNS. The fact that
NMDA receptors gate Ca^^ also has important implications in terms of the Ca^V CaM
67 Chapter 1: General Introduction activation of NOS (see section 1.4.2), activation of NMDA receptors leading to the formation of NO. Another remarkable property of the NMDA response is that it is potentiated by glycine to a high degree, an effect that is not mediated by the strychnine- sensitive glycine receptor (Johnson and Ascher, 1987). In fact, co-agonism with glycine is thought to be essential for NMDA receptor activity (Danysz and Parsons, 1998). The EC 5 0 for glycine at this site has been documented as 670nM and amongst the analogues of glycine that were as effective at eliciting a response were D-SER and D-alanine (Kleckner and Dingledine, 1988). It has now been shown that glia are capable of converting L-SER to D-SER using the novel enzyme SER racemase and that D-SER has, in fact, a higher affinity for the “glycine site” than glycine itself. It is now thought that D-SER may be the endogenous ligand for the NMDA receptor, not glycine (Wolosker et al, 2002). However, NMDA receptors would be constantly activated if the extracellular L-GLU and glycine concentrations were the only regulatory factors. The NMDA class of receptor is also unique in that it is jointly controlled by agonist binding as well as the neuron’s membrane potential. This voltage dependence stems from Mg^^ ions which, at physiological concentrations and resting membrane potentials, effectively block the open ion channel
(Sharma and Stevens, 1996). Replacement of an asparagine residue on the reentrant segment of the el subunit with a L-GLN residue strongly reduces the Mg^^ block but also affects the Ca^^ permeability (Sakurada et al, 1993). In addition, the NMDA receptor is extremely sensitive to the presence of ions, with selective inhibition occurring at an IC 5 0 close to physiological pH, indicating that the receptors are not fully active under normal conditions (Traynelis and Cull-Candy, 1990). Endogenous polyamines, such as spermine and spermidine, have also been shown to regulate NMDA receptor activity, although the effects are complex. Electrophysiological studies have demonstrated that polyamines enhance NMDA receptor currents by increasing their channel opening frequency or by increasing the receptors affinity for glycine while it has also been found that they reduce
NMDA currents by inducing a voltage-dependent reduction of single channel amplitudes and/ or by producing an open channel block (Rock and Macdonald, 1995). Uncompetitive antagonists act on the activated receptor as opposed to the receptor at rest and, in addition to Mg^^ ions and cytoplasmic polyamines, a number of other compounds are known to enter the open channel to cause blockade. These include phencyclidine and MK-801, which are clinically limited by their neuropsychotic and pathological side effects, and
68 Chapter 1: General Introduction
dextromethorphan and the anaesthetic ketamine, which are more tolerable. The onset of the
block elicited by these agents is use-dependent and, once bound inside the channel, the blocker can be trapped by closure of the channel so that recovery from these agents is
generally slow (Dingledine et al, 1999). The classical and most widely used competitive antagonists of NMDA receptors are the phosphono derivatives of short chain amino acids
such as D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), although several new
compounds have been developed. Zn^^ has been found to block the NMDA current in a
noncompetitive and voltage-independent manner (Liu and Zhang, 2000). In addition to all
of the above unique regulatory controls, NMDA receptors are also governed by redox
modulation. This is not a specific property of NMDA receptors but is common to several
other ionotropic receptors. Disulphide reducing agents, such as dithiothreitol (DTT),
enhance NMDA-evoked currents while thiol oxidising agents, such as 5, 5'-dithiobis-2-
nitrobenzoic acid (DTNB), cause a reduction (Tang and Aizenman, 1993). This modulation
is dependent on conserved CYS residues but, unlike the other ionotropic receptors, these CYS residues are not associated with ligand recognition but with glycine co-agonism. As well as this, excitatory amino acid receptors contain an extra set of CYS residues which
only form a functional disulphide bridge in NMDA receptors composed of NRl and either
NR2B, NR2C or NR2D subunits while neither receptors containing NRl and NR2A
subunits nor AMPA or KA receptors have this property (Gozlan and Ben-Ari, 1995). Other
endogenous redox modulators include other thiol and disulphide reagents, ascorbic acid (Majewska et al, 1990) and free radicals (Aizenman et al, 1990), particular attention focusing on NO, which has been shown to decrease NMDA receptor activity (Lei et al, 1992).
Phoshphorylation of NMDA receptors is now emerging as an important regulatory mechanism. Electrophysiological studies have demonstrated that application of phosphatase inhibitors enhances the stimulated synaptic currents in adult rat dentate gyrus granule cells
(Lieberman and Mody, 1994) while protein phosphatases 1 and 2A decrease the open probability of the NMDA channels in inside-out patches (Wang et al, 1994). It has been found that NMDA receptors are both SER-threonine (THR) and TYR phosphorylated and that PKC directly phosphorylates the receptor in CAl pyramidal neurons of the hippocampus causing enhancement of receptor currents. Thus, G-protein coupled receptors
69 Chapter 1: General Introduction
that activate non-receptor tyrosine kinases and indirectly activate PKC will enhance NMDA activity (Lu et al., 1999). The enhancement of NMDA activity by insulin has been shown to be through tyrosine kinase activation (see section 1.4.5; Chen and Leonard, 1996).
Activation of NMDA receptors themselves results in a signaling cascade. Because there is
an influx of Ca^^ when the ion channel opens and because Ca^^-permeable AMPA receptors
(see section 1.9.4) have proved to activate MAP kinase through a PI3-K-dependent
mechanism, it is now thought that NMDA receptor stimulation can activate this pathway (Perkington et al., 2002). A study in cortical neuronal cultures has recently confirmed that PKB activity is tightly regulated by synaptic activity and is coupled to NMDA receptor
activation (Sutton and Chandler, 2002). This may, in turn, represent a pathway in which
NMDA activation upregulates eNOS activity (see section 1.4.4).
1.9.3.2 Molecular biology of the NMDA receptor
The cloning of each of the receptor subunits has allowed investigators to explore the characteristics of each subunit when they are expressed in Xenopus oocytes using recombinant technology and in situ hybridisation studies have allowed the elucidation of their distribution in the brain. NR 1 mRNA is ubiquitously distributed in the brain (Mori and
Mishina, 1995) and evidence suggests that functional NMDA receptors exist as heteromeric
assemblies composed of multiple NRl subunits combined with at least one type of NR2.
The NR3 subunits do not form functional receptors alone but may co-assemble with the
NRl/ NR2 complexes. The expression of the subunits changes depending upon
developmental stage with NR2B and D subunits predominating in the neonatal brain to be
supplemented or replaced by NR2A, or even NR2C subunits in some brain areas, in
adulthood (Cull-Candy et al, 2001). In contrast to the NRl subunit the NR2 subunits display distinct regional patterns. The expression of the NR2A subunit mRNA is widespread in the brain and the highest levels are found in the cerebral cortex, the hippocampal formation and cerebellar granule cells. The NR2B transcript is expressed mainly in the forebrain with higher levels present in the cerebral cortex, the hippocampal formation, the septum, the caudate putamen, the olfactory bulb and the thalamus. NR2C mRNA transcripts are localised mainly in the cerebellum, predominantly in the granule
70 Chapter 1: General Introduction cells, with lower expression in the olfactory bulb and the thalamus. Low levels of NR2D subunit mRNA are detected in the thalamus, the brainstem and the olfactory bulb (Mori and Mishina, 1995; Ozawa et al, 1998). The function of NR3A subunits is incompletely understood and, similar to both the NR2B and D subunits, they are predominantly expressed during early development (Eriksson et al, 2002). NR3A subunits are unable to reach the cell surface unless they are coexpressed with NRl subunits (Pérez-Otano et al,
2002). Unlike the other subunits the NR3A subunit inhibits the receptor ion channel in a novel manner which has suggested a role for it in synaptogenesis (Sucher et al, 1995). The expression of the subunit peaks at postnatal day 8 (Sasaki et al, 2002) and has been found in all areas of the isocortex, areas of the amygdaloid nuclei and selective layers and nuclei of the hippocampus, thalamus, hypothalamus, brainstem and spinal cord (Ciabarra et al, 1995; Wong et al, 2002). The NR3B mRNA is expressed ubiquitously throughout the brain, suggesting that it is a common constituent of NMDA receptor complexes, with the highest levels being present in the CAl and CA3 regions and the dentate gyrus of the hippocampus and in the granule cell layer of the cerebellar cortex (Andersson et al, 2001). In the adult hippocampus, therefore, it is possible that NMDA receptors are composed of a combination of NRl, NR2A and NR3B subunits (Matsuda et al, 2002). When NR3A or 3B coassemble with only NRl subunits they actually form excitatory glycine receptors that are unaffected by L-GLU or NMDA and inhibited by D-SER (Chatterton et al, 2002), implying that an NR2A subunit must be included in the native neuronal NMDA receptor.
The postsynaptic expression of receptors incorporating an NR2A subunit would indicate a mechanism by which nNOS is colocalised with NMDA receptors (see section 1.4.4). The inclusion of an NR2A subunit would also suggest that the receptor would not be affected by redox modulation or down-regulated by NO (see section 1.9.3.1). There is general consensus that L-GLU molecules bind to the NR2 subunits while glycine binds to NRl (Cull-Candy etal, 2001). Coimmunoprecipitation analysis has revealed that coexpression of NR3B reduces the Ca^^ permeability of L-GLU induced currents in Xenopus oocytes expressing NRla, a splice variant of NRl, and NR2A subunits. Although all three subunits are expressed in the hippocampus, NMDA receptor complexes containing just N Rla and
NR2A subunits may still be formed and, due to its ability to reduce Ca^^ permeability,
NR3B expression has been implicated in protection against L-GLU induced toxicity (Matsuda et al, 2002).
71 Chapter 1: General Introduction
1.9.4 The AMPA receptor
AMPA receptors are characterised by their rapid desensitisation upon the application of
AMPA or L-GLU (Fletcher and Lodge, 1996) and, again, the subunit composition of the receptor confers unique properties on the ion channel. Important splice variants of the four subunits are the so-called “flip” and “flop” configurations (Sommer et al, 1990). The flop isoforms confer faster gating kinetics in receptors composed of GLUR2, 3 or 4 subunits than their flip counterparts (Lomeli et al, 1994). The permeability of the ion channel to Ca^^ is also dependent on the subunit composition of the receptor. Channels formed by any combination of GLURl, 3 or 4 have a high Ca^^ permeability and both inward and outward rectification in their agonist-evoked currents, while the incorporation of GLUR2 confers a linear current-voltage relationship and low Ca^^ permeability (Steinhauser and Gallo,
1996). The molecular basis for this change in Ca^^ permeability has been elucidated using site-directed mutagenesis coupled with functional analysis of recombinant receptors. When the GLUR2 subunit is incorporated into the receptor there is a change in a single amino acid in the channel forming region of the receptor protein. This amino acid is the result of an RNA editing mechanism that is responsible for the conversion of the GAG (L-GLN/ Q) codon, which is present in GLURl, 3 and 4 transcripts, into the CGG (L-ARG/ R) codon found in mature GLUR2 mRNAs (Bettler and Mulle, 1995). This results in an L-ARG residue in the GLUR2 subunit where there is otherwise a L-GLN. This so-called Q-R site is found on the reentrant segment of the subunit and it is analogous to the area conferring both the Ca^^ permeability and the Mg^^ block in NMDA receptors (see section 1.9.3.1). In addition, the Q-R site influences the single-channel conductance and the sensitivity of the channel to block by polyamine spider toxins, such as philanthotoxin, and endogenous internal polyamines (Isa et al, 1996). The most commonly used competitive antagonist of non-NMDA receptors is 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX).
The subunit stoichiometry of AMPA receptors, unlike other receptors, is thought not to be fixed. Evidence suggests that the native receptors exist not as mixtures of the two extreme types, either containing or lacking GLUR2, but as a graded spectrum with variable numbers of GLUR2 subunits (Washburn et al, 1997). Although the receptors can exist with any number of GLUR2s, a single GLUR2 subunit is sufficient to disrupt the Ca^^ permeability
72 Chapter 1: General Introduction
(Geiger et al, 1995). Both Ca^^-permeable and Ca^^-impermeable AMPA receptors are expressed in vivo. In the hippocampus, Ca^^-permeable AMPA receptors are found exclusively at synapses from mossy fibres while Ca^^-impermeable AMPA receptors mediate fast synaptic transmission in CA3 pyramidal neurons. In fact, the majority of the
Ca^^-permeable receptors are found on GABAergic (y-amino butyric acid; G ABA) intemeurons within the stratum lucidum, the termination zone of mossy fibre axons, within the CA3 region (Toth and McBain, 1998; Bolshakov and Buldakova, 2001). These intemeurons show a long-term depolarisation, thought to be a major mechanism underlying
LTP, which requires the activation of Ca^^-permeable AMPA receptors and the influx of
Ca^^ (Ross and Soltesz, 2001).
Again, phosphorylation of residues on the receptor protein has emerged as an important regulatory mechanism. Of particular relevance are the residues L-SER*^‘ and L-SER®'^^ on the GLURl subunit which can be phosphorylated by CaMK W PKC and PKA respectively (Castellani era/., 2001). LTP induction causes phosphorylation of L-SER*^*, which, in turn, doubles the single channel conductance of homomeric GLURl AMPA receptors (Derkach et al, 1999). LTD induction decreases the phosphorylation state of L-SER*"^^ which, ordinarily, is phosphorylated at resting potentials (Lee etal, 1998). Phosphorylation of this residue increases the “open time” of the channel (Banke et al, 2000).
1.9.5 NO in neurotransmission
As introduced in sections 1.9.3.1 and 1.9.4, the production of NO in neurons is triggered by the actions of L-GLU on AMPA and NMDA receptors. Activation of postsynaptic
AMPA receptors causes the membrane to become depolarised which removes the Mg^^ block from the NMDA receptors, opening the channel and allowing the influx of Ca^^. This
Ca^^ then interacts and binds with CaM and activates NOS to produce NO from L-ARG.
The NO then activates sGC (see section 1.5) in the postsynaptic neuron via binding to the haem iron moiety of the enzyme. Because NO is a freely diffusible gas it is also capable of retrograde signaling to the presynaptic terminal and surrounding glia. Given this diffusibility of NO its true sphere of influence is very difficult to determine. Although
NMDA receptors and NOS are tightly coupled they are not the sole mechanism by which
73 Chapter 1: General Introduction
NOS may become activated. In addition to VGCCs, the fact that some AMPA receptors are Ca^^-permeable (see section 1.9.4) and mGLURs of group I mobilise intracellular Ca^^ stores (see section 1.9.2) implicates all of these receptors as possible activators of NOS. However, because nNOS has a PDZ domain and because the NR2 subunit is a PSD ligand this would certainly suggest that NMDA receptor activation and the production of NO from nNOS are tightly coupled in vivo (see figure 1.7). A representation of how L-GLU receptor activation is thought to lead to the production of NO is shown in figure 1.12.
I'KI SY\ \l'l l( 11 U\ll\ M WIR'K Y I’ko ci ss
l.-C'l I
1)1 M)KIII NADP \ ^l.-ARCi
NADPII
Figure 1.12: The proposed mechanism by which activation of L-
GLU receptors causes the activation of NOS and the formation of NO (adapted from Garth wai te, 1991). N= NMDA, A= AMPA.
74 Chapter 1: General Introduction
This coupling with NMDA receptors thus implicates NO in a number of physiological and pathological processes in the CNS such as synaptic plasticity, neurotransmitter release and excitotoxicity. Bliss and L 0 mo (1973) were the first to theorise that the strengthening of synaptic connections, the phenomenon known as LTP, is important in memory formation and learning. The synaptic plasticity and memory hypothesis states that “activity-dependent synaptic plasticity is induced at appropriate synapses during memory formation and is both necessary and sufficient for the information storage underlying the type of memory mediated by the brain area in which that plasticity is observed” (Martin et ai, 2000).
The major part of the brain implicated in memory formation is the hippocampus, hence research has focused in this region. Brief (milliseconds) tetanic stimulation of synaptic afferents in the hippocampus produces long lasting (hours) changes in synaptic strength.
Induction of this LTP has been shown to require the activation of NMDA receptors and the subsequent influx of Ca^"^ while the maintenance of LTP requires, in part, a change in presynaptic function (Williams, 1996). Because NO can diffuse out of the postsynaptic terminal and affect presynaptic neurons it would seem an ideal candidate for the retrograde messenger required for LTP. In fact, NO has proved to increase terminal action potential duration in ciliary ganglia while NOS inhibition markedly decreases LTP in the same preparation (Bennett, 1994). However, studies with nNOS knockout mice showed that synaptic plasticity measured in brain slices from these animals was unimpaired and the mice showed no sign of learning deficits. This confounded the issue until it was shown that hippocampal pyramidal neurons also express eNOS (O’Dell etal., 1994). However, eNOS knockout mice also show no impairment of LTP or memory (Son et ai, 1996).
Upregulation of the alternate constitutive NOS isoform may explain these apparently conflicting results since there is only an influence on LTP when both eNOS and nNOS are lacking, only then demonstrating the importance of NO in plasticity (Hdlscher, 1997). NOS inhibition has proved to prevent memory formation in young chicks (Hdlscher and Rose,
1992) and to impair both spatial learning in a radial arm maze test and olfactory memory in a social avoidance learning test (Bdhme et al, 1993). A vast array of enzymes and receptors have now been implicated in the induction and consolidation of LTP including
Ca^^-dependent lipases, kinases and proteases, iGLURs (Lynch, 1998) as well as mOLURs (Hdlscher et al, 1999). Although it is clear that the influx of Ca^^ is essential it remains
75 Chapter 1: General Introduction unknown how the structural changes required for long-term memory formation actually occur.
The hippocampus is diffusely innervated from several sources. There is dense cholinergic innervation from the septal nucleus, noradrenergic innervation from the locus coeruleus, serotonergic innervation from the raphe nuclei of the midbrain and dopaminergic innervation from the ventral tegmental area of Tsai. NO is known to modulate the release of several neurotransmitters including ACh (Ohkuma et al, 1995), catecholamines (Montague et al, 1994; Bubgon et al, 1994; Segieth et al, 2000), L-GLU (Sorkin, 1993; Segieth et al, 1995) and GABA (Kuriyama and Ohkuma, 1995; Getting et al, 1996), 5-HT (Segieth et al, 2001), histamine (Prast et al, 1997; Horie et al, 2000; Wan et al, 2001) and adenosine (Bon and Garthwaite, 2002) and, for the most part, NO increases their release, although concentration-dependent effects are often observed, with low concentrations promoting and high concentrations decreasing release. In the hippocampus this effect seems to be dependent on both cGMP and Ca^^. High concentrations of NO can also cause energy depletion and non-exocytotic L-GLU outflow (Prast and Philippu, 2001).
The mechanism by which NO increases monoamine levels is via the inhibition of reuptake by affecting their transporters, although at least some of the effects of NO on neurotransmitter release may be mediated by ONOO (Ohkuma et al, 1995; Haskew et al, 2002). This would be an example of non-synaptic interactions altering transmitter release.
Because of the relationship between NO and glutamatergic transmission this may also indicate a mechanism by which glutamatergic and monoaminergic neurons communicate
(Kiss and Vizi, 2001).
NO has been implicated in neurotoxicity with excitotoxic cell death being characterised by the overstimulation of NMDA receptors and the activation of NOS. As discussed in section
1.8.2, derangements in glutamatergic transmission and the overproduction of NO have been implicated in several neurodegenerative disorders, including stroke and epilepsy, and it is likely that this neurotoxicity is mediated by ONOO (Dawson and Dawson, 1995).
However, it has now been demonstrated that the toxicity induced by the application of NMDA is dependent on the availability of L-ARG. At low L-ARG concentrations nNOS favours the production of NO and O2 (Heinzel et al, 1992) and hence ONOO (Beckman
76 Chapter 1: General Introduction and Koppenol, 1996) while higher L-ARG concentrations prevent the production of so that the enzyme produces NO alone (Grima et al, 2001). This would be analogous to the proposed situation with BH 4 (see section 1.4.6) and may also implicate the concentration of L-ARG as important in terms of neurotransmitter release. It has also been demonstrated that ONOO itself stimulates the release of L-ARG from cultured rat astrocytes, an effect which appeared to be mediated by L-ARG transporter activation (Vega-Agapito et ai, 1999), specifically system y^ (Vega Agapito et al, 2002). In this way glia would play a protective role by supplying extra L-ARG to prevent any further generation of neurotoxic
ONOO. This would be a good example of the proposed cross-talk between neurons and glia which is now considered important in the maintenance of normal brain function.
In the majority of brain regions the synapses are entirely surrounded by astrocyte processes.
This is the case in the CAl region of the hippocampus, where glial cells have been identified within 1/xm of the dendritic spine synapses (Rothstein etal, 1994). As mentioned in section 1.2, abundant evidence suggests that L-ARG is localised in glial cells (Aoki et al, 1991a; Aoki et al, 1993; Pow, 1994; Kharazia et al, 1997). The fact that the infusion of the glial toxin FC increases the levels of several glial derived substances, such as taurine (TAU), as well as L-ARG, in the extracellular space (Yamada et al, 1997) certainly indicates that L-ARG is stored in glia. It has also been demonstrated that AMP A receptors are not simply postsynaptic but that glial cells also express non-NMDA receptors (Gallo and Ghiani, 2000; Verkhratsky and Steinhauser, 2000). It has been postulated that activation of these non-NMDA receptors on glia causes the release of L-ARG for the supply of substrate to neurons for the production of NO (Grima et al, 1997). The exact mechanism of this release is still unknown although there are a number of suggestions including reversal of uptake, vesicular release or “transmembrane leak”. So far, however, all the evidence gathered suggesting that non-NMDA receptor activation is the signal for L-ARG release has been obtained via in vitro techniques and there is little evidence for this being the case in vivo. Now that the concept of bidirectional signaling between neurons and glia is becoming accepted as important in terms of brain function (for review see Carmignoto,
2 0 0 0 ), it is important to elucidate the situation in vivo.
77 Chapter 1: General Introduction
1.10 Aims
W hile in vitro models such as brain slices, synaptosomal preparations and tissue cultures have been used extensively in the past to investigate the release and actions of NO and L- GLU in the CNS, it is impossible to recreate the true environment of the brain in vitro. To this end in vivo microdialysis was employed to sample the amino acid and NO levels in the extracellular space of the ventral hippocampus in response to various drug treatments.
The overall aim of this study was to investigate the signal(s) responsible for regulating L- ARG supply in vivo. Of particular importance in this regard were attempts to address the following interrelated questions:
1) Is L-GLU receptor activation, both NMDA and non-NMDA subtypes, the sole requirement for L-ARG release into the extracellular space?
2) Is this response influenced in any way by the prevailing extracellular concentrations of both L-GLU and NO?
78 Cfiapter 2: Materials andMctfiods Chapter 2: Materials and Methods
2.1 In vivo microdialvsis 2.1.1 Probe Construction
Concentric microdialysis probes were constructed in the laboratory as follows:
a) Two pieces of fused silica capillary tubing (Scientific Glass Engineering, U.K.) were cut to 25mm using a razor blade. One piece was inserted into a 15mm cannula (240,0.52mm
O.D.; Tomlinson Tube & Instrument Ltd., U.K.) so that it extended 5mm from either end
(the inlet) and the other was inserted so that 15mm extended from the top (the outlet). The silica was then glued to the cannula using Araldite® rapid setting epoxy resin. b) When the glue was dry the outlet silica was cut to 7mm and non sterile fine bore polythene tubing (pplO tubing, 0.28mm I.D., 0.61mm O.D.; Portex Ltd., U.K.) was then fitted over the top of both pieces of silica, ensuring that the inlet was shorter than the outlet to allow distinction to be made between them. The tubing was then secured with more epoxy resin. c) When the epoxy resin was dry the entire join was covered with dental acrylic (Duralay,
Reliance Dental Mfg. Co.).
d) The inlet was trimmed to 3mm and a dialysis membrane (cuprophan capillary membrane, type FI 8 200, 0.2mm O.D., 10 KD molecular weight cut off; Gambro, GFB9 dialysis membrane, Hechingen, Germany) was slipped over the end and pushed up the cannula until resistance was met. The membrane was glued to the cannula with a tiny drop of epoxy resin. When the glue was dry, the membrane was cut to 1mm past the end of the fused silica and the end plugged by another tiny drop of epoxy resin. When this glue was dry the probe was then ready for use.
Figure 2.1 shows a schematic representation of a constructed probe.
80 Chapter 2: Materials and Methods
Oiiltlow
In flo w Pol>lhene tubing (ppIO)
Dental acn lic
Fused silica
Epo\\ resin (Araldite' ) 15 mm
Dial) sis membrane
Figure 2.1: A diagrammatic representation of a microdialysis probe (not to scale). The red arrows indicate the direction of flow.
81 Chapter 2: Materials and Methods
2.1.2 In vitro recoveries of amino acids
In order to determine how much variability could be introduced into a microdialysis experiment due to probe to probe variation (possibly due to differences in probe construction such as different quantities of epoxy resin on the dialysis membrane), the ability of the probes to recover known concentrations of amino acids was tested in vitro. This was done as follows: Dialysis probes were suspended in a standard solution of the amino acids of interest (each at a concentration of 50/xM). The amino acids were dissolved in artificial cerebrospinal fluid (aCSF; composition 2.5mM KCl, 125mM NaCl, l.lSmM
MgClj, 1.26mM CaCl2 ). The inlet of the probe was connected to a 500/il Hamilton syringe via a length of pplO tubing and the outlet was connected to a collecting vial. The probe was then infused with aCSF at a flow rate of 0.8/i 1/ min using a Harvard apparatus infusion pump. Following a 60 min equilibration time, six 30 min samples were collected. These samples were then analysed for their amino acid content using high performance liquid chromatography (HPLC) with fluorometric detection. The % relative recovery of amino acids from the bathing medium could then be calculated using the equation given by
Benveniste (1989).
Recovery,., „.,„ = C„„,/C,,,
where is the concentration in the outflow from the probe and is the concentration in the bathing medium. Estimation of the true interstitial concentration can be made from the relationship:
= recovery,,,,,,.
where is the true interstitial concentration and is the concentration in the outflow in vivo. The in vitro % recoveries for each amino acid are shown in table 2.1.
8 2 Chapter 2: Materials and Methods
Table 2.1: In vitro recoveries for each amino acid.
Amino acid % Recovery
L-ASP 30.7 ± 3.0
L-GLU 25.4 ± 1 .5
L-SER 47.4 ± 3.5
L-GLN 17.7 ± 1.3
L-CIT 15.4 ± 0 .9
L-ARG 10.5 ± 0 .6
TAU 24.8 ± 3.2
GABA 19.5 ± 0 .8
Results are expressed as means ± s.e.m. from 4 determinations.
However, for these relationships to be valid it would have to be accepted that conditions in vitro correlate with conditions in vivo and this is clearly not the case- it is impossible to reproduce the metabolic pathways present in vivo in an in vitro environment. In vivo recovery is less than that in vitro due to the presence of cell membranes, giving rise to tortuosity- a prolongation of the diffusion pathway resulting in reduced mass transport.
Other factors affecting recovery of substances include perfusion speed, the higher the flow rate the lower the recovery, length of membrane available for dialysis, recovery being proportional to length, and temperature, the higher the temperature the better the recovery.
Substances may also interact with the dialysis membrane and it has been noted that recovery of acidic amino acids is higher than that of their basic counterparts (Tossman and
Ungerstedt, 1986).
83 Chapter 2: Materials and Methods
2.1.3 Stereotaxic implantation
All animal experiments were performed in accordance with the guidelines of the Animals
(Scientific Procedures) Act, 1986. Male Wistarrats (250-300g; Bantin and Kingman, U.K.) were anaesthetised using isofluorane and secured in a stereotaxic frame with blunt ear bars
(David Kopf, U.S.A.), as shown in figure 2.2. The incisor bar was set according to the size of the animal and it was ensured that the skull was flat. A medial incision was made down the length of the skin covering the skull and the skin and muscle were retracted so that there was direct access to the surface of the skull. It was then possible to visualise Bregma, as seen in figure 2.3, and, from there, using the co-ordinates from the Atlas of Paxinos and Watson (1982), to measure to the ventral hippocampus and position a dental drill (tungsten carbide burr tip, 2mm), held by a micro-manipulator, above it. Here, a small burr hole was drilled to expose the dura mater which was pierced using the tip of a sterile hypodermic needle before probe implantation. Two more burr holes were drilled away from the initial hole and small steel screws (size lOB, Clerkenwell Screw Co., U.K.) were inserted so that approximately half of the shaft was embedded in the skull. Probes were implanted unilaterally into the brain without the prior insertion of guide cannulae. The probe was mounted in a holder which was attached to the micro-manipulator so that the probe could then be lowered to the required depth. The surgery is illustrated in figure 2.4. The probe was secured using dental cement which also covered the two anchor screws, helping the adherence of the cement to the skull surface. The cement was also built up around the shaft of the probe to improve durability. When the cement was set the rat was removed from the stereotaxic frame and left to recover overnight. Food and water were available ad libitum.
84 Cfiapter 2: Materials and Methods
\'ci licul JcljusliiK'n
vilci'.il uiljustincn . I’ ltihc lio klc r (iiriK'cl a s i d e ) ni/incd iis'ulc)
\- I’ iKlJiislnicnl
Incisor hai adjiislincii
Figure 2.2: An anaesthetised rat held in a Kopf stereotaxic frame (adapted from Krinke, 2000).
85 Chapter 2: Materials and Methods
2 ^)0 " M u le \\ i>lui () ()|„|„ InlciuuruiIiik
ll.dmiii
Inleruuiul line
Figure 2.3: The dorsal and lateral aspects of the skull of a 290g male Wistar rat (adapted from Paxinos and Watson, 1982). The position of Bregma can clearly be seen. The red dots indicate the position of the ventral hippocampus from the dorsal and lateral views.
8 6 Chapter 2: Materials and Methods
C l010(1 tic clip
lull' Ixif (
I'ohc hoklci
Figure 2.4: The crown of the head during a bilateral lesion. A: the skull is exposed and the hippocampus located and marked. B: the holes are drilled. C: the screws are put in place and the probe is lowered to the correct depth. Everything is then secured with dental cement (adapted from Krinke, 2000).
87 Chapter 2: Materials and Methods
The stereotaxic co-ordinates for the ventral hippocampus are as follows:
5.0mm Posterior (relative to Bregma) 5.0mm Lateral (relative to midline) 7.5mm Ventral (relative to the surface of the dura mater)
2.1.4 Validity of microdialysis
Many techniques have been developed over the years to sample the biochemical composition of the brain extracellular space in vivo. The first technique to be developed was the cortical cup in 1953 (Macintosh and Oborin, 1953), followed by the push-pull cannula in 1961 (Gaddum, 1961) and microelectrodes. Microdialysis was introduced in 1966 (Bito et ai, 1966) and the methodology has been refined over the years. It has been shown that the microdialysis technique accurately reflects events within the blood brain barrier and that there is no damage to the small vessels in the barrier which would lead to an increased contribution to the extracellular space from the blood stream. This is particularly important when investigating amino acid levels since they are far more concentrated in the blood stream than they are in the extracellular space of the brain
(Tossman and Ungerstedt, 1986). Another major advantage of this method is that samples are obtained from awake, freely moving animals so the effect of the anaesthetic does not have to be taken into account. Table 2.2 shows the advantages and disadvantages of the various in vivo techniques. It can be seen that, for microdialysis, the advantages far outweigh the disadvantages.
88 Chapter 2: Materials and Methods
Table 2.2: Pros and cons of various in vivo techniques (Reproduced from Benveniste, 1989).
Advantages Disadvantages
In situ
Ion-selective 1. All brain regions can be examined 1. Only detects ions
microelectrode
2. Time resolution < ls
3. Tip of electrode <4/rm
Carbon fibre 1. All brain regions can be examined 1. Only detects oxidizable compounds
microelectrode
2. Can be used in awake animals 2. Selectivity bad without previous HPLC
a n alysis
3. Time resolution <1 min 3. Drainage
4. Tip of electrode <300/xm 4. Some electrodes have short working life in
vivo
Ex situ
Cortical cup 1. No tissue penetration 1. Time resolution >10 min
2. Can be used in awake animals 2. Only cortex can be analysed
3. Drainage
4. Deproteinization before HPLC
5. Enzymatic degradation of eollected
e om p ou n d s
Push-pull cannula 1. All brain regions can be examined 1. Time resolution >10 min
2. Can be used in awake animals 2. Drainage
3. BBB intact following implantation 3. Deproteinization before HPLC
4. Enzymatic degradation of collected
co m p o u n d s
5. Tissue trauma following implantation
6. o.d. >100/im
Microdialysis probe 1. All brain regions can be examined 1. Time resolution >5 min
2. Can be used in awake animals 2. Drainage
3. BBB intact following implantation
4. Minute tissue trauma within the first 2
days
5. No need for deproteinization
6. No enzymatic degradation
89 Chapter 2: Materials and Methods
2.1.5 Microdialysis
In all dialysis experiments rats were housed in purpose built perspex cages throughout and all experiments were performed using freely moving animals that showed no sign of stress following surgery. After 20h of recovery following anaesthesia and surgery, microdialysis was performed. A length of pplO tubing was attached to the inlet tubing of the probe. The pplO had been flushed with aCSF and it was connected at the opposite end to a 500^1
Hamilton microsyringe mounted on a micro-infusion pump (Harvard Apparatus, syringe infusion pump 22). The aCSF was flushed through the probe to expel any air bubbles. The outlet tubing of the probe was then connected to a length of FEP tubing (dead volume
O.ljLtl/ cm^ Carnegie Medicin, Sweden) which was fed into a collecting vial. The inlet tubing and the collecting vial were both weighted with Blu-tack™ in order to stop the rats from being able to get at the tubing and gnaw on it. Once it had been established that the probe was working there was a 60min equilibration time in which any sample collected was discarded. As the aCSF flowed through the probe the neurotransmitters in the extracellular space surrounding the membrane flowed down their concentration gradient and into the probe. Upon the start of perfusion transmitter levels in the brain drop (Benveniste, 1989).
This 60min thus serves as an equilibration period to allow transmitter levels to return to basal. In this way it was possible to collect dialysate samples and monitor the changes in neurotransmitter levels in response to drug treatment. Twelve samples over 30min periods would normally be collected and there were usually four basal samples before the various drug treatments. The infusion rate was O.S/tl per min.
2.1.6 Anatomical verification of probe placement
Microdialysis experiments were concluded by verifying the location of the probe. The animals were sacrificed and their brains removed and frozen over dry ice. Coronal brain sections were cut on a cryostat, fixed in ice-cold paraformaldehyde, stained with haematoxylin and eosin and mounted on gelatin-coated slides. Verification of probe placement was made by visualisation of the probe tract. A typical probe placement is shown in figure 2.5. The tract of the probe can clearly be seen entering the hippocampus.
90 Chapter 2: Materials and Methods
"W,
m
" m m m m #
###
Figure 2.5: A photomicrograph illustrating a typical probe placement. The arrow shows the entry of the probe shaft into the hippocampus. The area for diffusion extends 4mm below this (magnification xl5). Specific areas of the hippocampus are indicated (DG= dentate gyrus).
91 Chapter 2: Materials and Methods
2.2 Analysis of amino acids 2.2.1 Detection of amino acids
The samples collected were analysed for their amino acid content using gradient reverse
phase HPLC with fluorometric detection (Lindroth and Mopper, 1979). Reverse phase
chromatography allows solutes to elute in order of decreasing polarity (increasing
hydrophobicity) and increasing the non-polar component of the mobile phase via the
gradient system decreases the retention times. Thus it was possible to detect relatively acidic (L-ASP, L-GLU) through to non-polar, basic amino acids (GABA) in a 20min run time.
2.2.2 Derivatisation of amino acids
Since amino acids do not fluoresce naturally, it was necessary to introduce a reagent that would label the amine group with a fluorescent moiety. For this, o-phthaldialdehyde (OPA) was introduced in the presence of a thiol-reducing agent ( 2 -mercaptoethanol) to form a new heterocyclic species, a substituted isoindole. This was the source of the fluorescent activity of the OPT-amino acid derivative. The reaction of OPT-thiol with an amino acid, or any primary amine, is shown in figure 2.6. The OPT-thiol reagent was prepared as follows:
27mg of OPA was dissolved in 500/xl of absolute ethanol and 5ml of O.IM sodium tetraborate dehydrate (Borax) was added. This was mixed well and 50fi\ of 2- mercaptoethanol was added in a fume cupboard. This solution was mixed thoroughly and, due to the photosensitive nature of the reagent, stored in a darkened vial at 4°C. The reagent should be made up at least 24 h before use to allow aging to occur and it can be kept for several weeks with periodic additions of 2 0 /xl 2 -mercaptoethanol to maintain the yield of derivative.
92 Chapter 2: Materials and Methods
CHO NHi— CH— R
CHO COOH
N— CH — R
COOl
Figure 2.6: The reaction of OPT-thiol with a primary amine to yield a substituted isoindole.
2.3 High performance liquid chromatography (HPLC) 2.3.1 HPLC Apparatus
The apparatus used included: Two Gilson solvent delivery pumps (model 302), aDynamax
C i 8 reverse phase column (5/tm ODS particles, 10cm x 4.6mm, Anachem) with column heater (30°C; Anachem), a 5/tm C,; guard column (Anachem), a Gilson refrigerated auto injector (model 231) equipped with a Rheodyne injection valve, a 502 contact module and a Gilson data master (model 620) which acted as an interface to a Viglen PC system. The
PC was equipped with Gilson 712 chromatography software with an inbuilt gradient manager. Detection was achieved with a Gilson fluorometer (model 121). Figure 2.7 shows the apparatus used.
93 Chapter 2: Materials and Methods
M.inoiiKlrn. IK ik iiiiic nunliil»;
IlkTiiiiuiauLiioi
111111)11
UlU>ll1)C(ihi|
K
Figure 2.1 \ HPLC apparatus.
94 Chapter 2: Materials and Methods
2.3.2 Analysis
The two pumps, pump A for mobile phase and pump B for methanol, pre-mixed the
solvents before elution on the column. Both solutions were filtered through 0.2/im
nitrocellulose membrane filters and degassed through a vacuum pump before use. The
sample injector mixed 15/il of the OPT-thiol reagent with 15/il of dialysate. The solutions
(30/d) were left to derivatise for 3 min and were then injected onto the column through a Rheodyne with a 20fi\ sample loop. The rack which held the samples was cooled to 4°C by a Grant refrigeration unit with a Harvard thermocirculator pump. The software on the PC,
synchronised with the autosampler and the 502 contact module, controlled the composition of the mobile phase as each run proceeded. Each run ended with a 3min wash in which pure methanol was pumped through. As the sample eluted from the column it passed through the fluorometer (360nm excitation filter; 455nm emission filter) which, in turn, passed the signal to the PC which integrated the peaks and calculated the peak areas.
2.3.3 Mobile phase composition
The mobile phase was made up as follows: A O.IM solution of sodium acetate (CHjCOONa) was prepared by dissolving 13.6Ig of salt in 900ml of HPLC grade water.
The pH was adjusted to 6.95 and the solution was diluted to 11. 900ml of the solution was mixed with 100ml of HPLC grade methanol and the mixture was filtered through a 0.2/xm nitrocellulose membrane filter (Anachem). 975ml of the solution was then mixed with 25ml
HPLC grade tetrahydrofuran (THF). Acetate mobile phase was used, as opposed to a phosphate buffer, to prevent salt precipitation in the pump head.
95 Chapter 2: Materials and Methods
2.3.4 Identification of amino acids
The amino acids in dialysates were identified via a direct comparison with a standard
solution containing the amino acids of interest. Figure 2.8 shows examples of a 25/tM
standard solution and a typical dialysate. The column was calibrated for each of the amino
acids and the calibration curves are shown in figure 2.9. These curves were not used to
calculate results; the results were calculated as a percentage of the basal level, calculated
from the first four “basal” values. It was, however, useful to ensure that the column was
calibrated to ensure that quantitative results would be achieved. The basal levels of each
amino acid are shown in table 2.3.
Table 2.3: The basal levels of each amino acid.
Amino acid pmoles per 15^1 dialysate (n=32)
L-ASP 29.7 ±4.2
L-GLU 31.8 ±2.9
L-SER 60.3 ± 7.6
L-GLN 486.9 ± 33.3
L-CIT 17.3 ±3.4
L-ARG 51.9 ±8.3
TAU 42.3 ± 3.2
GABA I5 .I ± 2 .5
96 Chapter 2: Materials and Methods
A:
00
0.00 5.00 10.00 15.00 I imc (min)
B:
00
0.00 5.000.00 15.00 20.00
Figure 2.8: Typical HPLC traces. A: a 25jnM standard solution and B: an actual
dialysate sample. 1: L-ASP, 2: L-GLU, 3: L-SER, 4: L-GLN, 5: L-CIT, 6: L-ARG, 7: TAU and 8: GABA. The black line on A represents the gradient used.
97 Chapter 2: Materials and Methods
400000-1 400000-1
• I 300000-
Z 200000 - Z 200000-
< 100000- < 100000-
0 25 50 100 12575 150 0 2550 75100 125 150 pmolcs on cokimn pmoks on column
500000-1 750000-1
g 400000-
500000- & 300000- I 200000- < 250000- 100000-
0 25 50100 12575 0 100 200 300 400 500 600 pmoles on cokimn pmoles on cokimn
300000-1 400000-1
a 300000- 20 0 0 0 0 -
! Z 200000- ^ 100000- < 100000-
0 10 20 3040 50 60 70 0 25 50 75 100 125 pmoles on column pmoles on column
300000-1 400000-1
300000- I - f 200000 i f 200000- B 100000- < 100000-
0 50 100 150 200 0 50 100 150 200 250 pmoles on cokimn pmoles on column
Figure 2.9: Calibration curves for each amino acid.
98 Chapter 2: Materials and Methods
2.4 Detection of NO 2.4.1 The Nitric Oxide Analyser (NOA)
The samples from dialysis experiments were analysed for their NO content using a Sievers
280 NO Analyser (NOA) as shown in figure 2.10. Due to its short half-life, any NO in the
samples would have degraded rapidly to form NO 2 . Using chemical reducing agents (50mg
INa in 1ml HjO and 4ml glacial acetic acid) this NO 2 was converted back into NO by the
NOA. This NO then reacted with ozone in a gas-phase chemiluminescent reaction:
NO + O 3 - NO 2 * + O 2
NO 2 * - NO 2 + hv
The excited nitrogen dioxide emits in the red and near-infrared region of the spectrum and was detected by a thermoelectrically cooled red-sensitive photomultiplier tube. The software provided with the NOA, the Liquid Program, provided complete system control and calculated the concentrations in the sample automatically. The system was calibrated using various concentrations of sodium nitrite, the calibration curve is shown in figure 2 . 1 1 .
Again, results were calculated as a percentage of the basal level but the calibration curve was used by the software program to calculate the concentrations. The system was capable of measuring concentrations in the low nM range in sample volumes as low as 1/xl.
Ordinarily 5/il were injected.
99 Chapter 2: Materials and Methods
j is if S S S ?
Figure 2.10: Sievers 280 NOA.
7500n
5000-
2500-
1 0 0 200 300
p m o le s N O Figure 2.11: The calibration plot for the NOA.
100 Chapter 2: Materials and Methods
2.5 In vitro studies 2.5.1 Tissue extraction
Male Wistar rats had probes implanted and microdialysis was performed as described in section 2.1.5. Animals were decapitated and their hippocampi dissected out, freeze-clamped in liquid nitrogen and stored at -80°C until use. Tissues were extracted at 0-4°C by homogenisation (with a Ystral homogeniser) in 1ml of buffer (320mM sucrose, 50mMTris, ImMEDTA, ImM DL-dithiothreitol, 100/xg/ ml phenylmethylsulphonyl fluoride, lOfig/ ml leupeptin, lOjig/ ml soybean trypsin inhibitor, and 2/ig/ ml aprotinin, pH 7.0). The homogenates were centrifuged at lOOOOg for lOmin and the supernatants were used in the assay described below.
2.5.2 NOS assay
NOS activity was measured according to the method of Salter et al. (1991) which quantitates the conversion of L-[U-"^C] ARG to L-'^^C-CIT. 45/xl of tissue extract was added to a 10ml glass tube pre-warmed to 37°C containing 250/il of buffer (60mM L-valine, nOfiM NADPH, 1.2mM L-CIT, 24/xM L-ARG, L-[U-*^C] ARG [150000 dpm ^0.1/iCi],
1.2mM MgClj and 0.24mM CaCl 2 in 50mM potassium phosphate at pH 7.2). Samples were incubated at 37°C for lOmin before termination of the reaction by the addition of 2ml of
1:1 v/v H 2 O/ Dowex-50W (200-400,8% cross-linked Na^-form). The Na^ form of Dowex- 50 was prepared by washing the H^ form of the resin in 4 volumes of IM NaOH and then washing with water until the pH was less than 7.5. 2ml of H 2 O was added to the resin incubate mix, left to settle for lOmin and 1 ml of supernatant was removed and examined for the presence of L-^^C-CIT by liquid-scintillation counting. The Ca^^-dependent NOS activity was determined through a comparison of the incubation in the buffer mentioned above and an incubation buffer also containing ImMEGTA. The Ca^^-independent activity was determined similarly by comparison between the incubation with ImMEGTA and an incubation containing ImM EGTA and ImM L-NAME.
1 0 1 Chapter 2: Materials and Methods
2.5.3 Glial cell culture
Mixed glial cell cultures from neonatal rat cerebral cortex were prepared according to the method of Dutton et al. (1981). Following decapitation of 1 to 2 day old rat pups, the cerebral cortices were removed, thoroughly cleaned of meninges and vasculature with fine forceps and placed in a few drops of sterile disaggregation medium of the following composition: 14mM glucose, 3mg/ ml bovine serum albumin (BSA), 1.5mM MgSO^ in
Ca^^- and Mg^^-free Earle’s Balanced Salt Solution (EBSS, Gibco). The tissue was then coarsely chopped with a sterile scalpel and transferred to a flask containing 250/xg/ ml trypsin in 10ml of disaggregation medium and placed in a shaking water bath at 37°C for
15min. Ten ml of a solution containing 192jLtg/ ml soybean trypsin inhibitor, 6.4^g/ ml
DNase and 240jLtM MgSO^ in disaggregation medium was then added and the resulting suspension transferred to 50ml sterile plastic tubes and centrifuged at lOOg for 5s (Denley, BS 400) to sediment cell bodies. The supernatant was removed and the cell pellet resuspended in a few drops of a solution containing 1 .2 mg/ ml soybean trypsin inhibitor, 40/xg/ ml DNase and 1.5mM MgSO^ in disaggregation medium. The tissue was then mechanically dissociated by gentle trituration through a 1.5mm diameter stainless steel cannula attached to a 5ml sterile syringe and the cells allowed to settle. The supernatant was removed and transferred to a 15ml sterile plastic tube. This step was then repeated. The final cell suspension obtained was underlaid with a 4% (w/v) BSA solution in disaggregation medium and the intact cells were pelleted through the BSA underlay by centrifugation at lOOg for 5min. The supernatant, which contained cell debris, was removed and the cell pellet resuspended in a small volume of growth medium of the following composition: Minimum Essential Medium with Earle’s Salts (L-GLN free, Gibco) supplemented with 10% v/v foetal calf serum, 2mM L-GLN, 33mM D-glucose and antibiotic/ antimycotic solution with 10 OOOunits penicillin, lOmg streptomycin and 25/xg/ ml amphotericin B (1% v/v. Sigma). The cell suspension was appropriately diluted in growth medium to give a seeding density of 100 000 cells per well, then seeded onto 50jLig/ ml poly-D-lysine coated 6 -well (35mm diameter) plates (Nunc). The cells were maintained for 14days in vitro in a humidified atmosphere containing 5% CO 2 in air at 37°C. Growth medium was changed every 7days.
1 0 2 Chapter 2: Materials and Methods
2.5.4 Release experiments
Cultures grown on 35mm dishes were washed three times with buffer composed of 116mM
NaCl, 26mM NaHCOj, ImM NaH 2 P 0 4 , 1.5mM MgSO^, 5mM KCl, 1.3mM CaClj and
20mM glucose, gassed with 5% CO 2 in O 2 . The cultures were then incubated at 37°C in 5%
CO 2 / air atmosphere in buffer for 60min prior to starting the experiment. Cultures were then incubated in 2 ml volumes of buffer for ten consecutive 2 min periods, the buffer being collected after each incubation period and replaced with fresh buffer. The first five samples were collected to establish the basal level of each amino acid. Drug additions were made for 2 or 6 min before returning to drug-free buffer for the remainder of the experiment. All of the incubations were carried out at room temperature. All samples were analysed by HPLC with fluorometric detection according to sections 2.2 and 2.3.
2.6 Drugs and Compounds
All compounds were obtained from Sigma, U.K., where available or Fluka, U.K., while drugs were obtained from Tocris Cookson Ltd., U.K.
2.7 Statistical Analysis
Statistical analysis was performed on the data using either the Mann-Whitney U test or one way ANOVA and results were considered significant if P values were equal to or less than
0.05. The Mann-Whitney U test, also known as the rank sum test, is a non-parametric test used for the comparison of two unpaired sets of data, such as the case with the NOS assay, where the assumption was made that the data had an unequal distribution and were transformed, making a t-test inappropriate. One way ANOVA tests whether the mean (or median) of a variable differs among three or more groups, in this case the 6 rats in the control group versus the 6 rats in the treatment groups.
103 Cfiaptcr 3: ‘K ^ u tts Chapter 3: Results 3.1 Release of L-ARG in vitro and in vivo
Initial studies were carried out to determine whether L-GLU receptor activation is the only stimulus capable of eliciting L-ARG release from cultured cells. To this end, cultured cortical glia were challenged with various glutamatergic agonists as well as the NO donor
SNAP and the release of endogenous L-ARG, L-GLU and L-GLN into the bathing medium was determined. Figure 3.1 shows the peak levels achieved after the application of the various drugs. The application of neither NMD A nor rra« 5 -azetidine- 2 ,4-dicarboxylic acid
(tADA), a selective group I mGLUR agonist, caused any change in L-ARG or L-GLU levels although they both caused significant decreases in L-GLN; NMD A and tADA decreased L-GLN levels to 59± 6 and 40± 6 % of basal, respectively. L-ARG levels were increased with both AMPA and SNAP. In fact, SNAP elicited increases in both L-ARG and
L-GLU, effects which were blocked by the coadministration of CNQX.
400 n L-ARG ESSa L-GLU 300- 7 7 7 m L-GLN
200 -
-M l l A M Drug treatment
Figure 3.1: Amino acid levels in response to the application of various drugs.
Cultured cortical glia were exposed to 1: 10/xM NMDA, 2: lO/iM tADA, 3: lO/iM
AMPA, 4: 50/iM SNAP or 5: 100/xM CNQX + 50jLtM SNAP in the bathing medium for
2min and the responses monitored ( 6 : Control). Results are expressed as means ± s.e.m.
(nk 3); levels which are significantly different from controls (P< 0.05) are marked *.
105 Chapter 3: Results
The results in figure 3.1 represent the peak amounts of amino acids present in the samples, as opposed to release profiles from each individual experiment. A representative release profile from one experiment is shown in figure 3.2. This is a release profile for L-ARG in response to the application of 50jLtM SNAP to cultured glia. The values at, in this case,
12min would be taken, averaged and the standard error calculated. It is these values which are plotted on the bar graphs throughout this section. Figure 3.2 demonstrates that the response to the application of SNAP is rapid in onset, the levels increasing almost fourfold, and that it returns to basal relatively quickly upon the removal of the drug from the bathing medium. The resting concentrations of L-ARG and L-GLU in the bathing medium were 19.5± 1.0 and 37± 1/xM (n= 5), respectively, while the in vivo basal levels for each amino acid are listed in table 2.3.
400-1 50pM SNAP No drug 300- pretreatment
I 200- PQ
100-
-f— —I 12 16 20 Time (min)
Figure 3.2: A representative release profile for L-ARG. Cultured cortical glia were
exposed to 50/xM SNAP for 2min. The open box represents the period of exposure
106 Chapter 3: Results
To confirm that the SNAP evoked response in cortical cultures was, in fact, due to NO, the responses were investigated using the NO scavenger Hb. Figure 3.3 represents the responses of both L-ARG and L-GLU to SNAP in the presence of Hb. The Hb was applied to the bathing medium for 6 min in total and the SNAP was coadministered during the last
2min of Hb application. Hb itself had little effect on either amino acid and the graph clearly shows that the coapplication of SNAP in the presence of Hb had a much reduced effect on
L-ARG and L-GLU levels, also see figure 3.5.
To determine whether NO may play a role in the tonic control of L-ARG and L-GLU in cortical glial cultures, NOS was inhibited using L-NAME. Figure 3.4 shows that the levels of both L-ARG and L-GLU were decreased when L-NAME was applied to cortical cultures for 6 min. The levels fell when L-NAME was applied and remained decreased compared with pretreatment levels for the rest of the experiment.
200 n L-ARG L-GLU 150- 1
1 100 - PQ
50-
0 4 8 12 16 20 Time (min)
Figure 3.3: Amino acid levels in response to the application of lO^M Hb and 50/iM
SNAP to cortical cultures. Cortical glia were exposed to Hb for 6 min (open box) and
SNAP for the last 2min of Hb application (shaded box) and the amino acid levels
determined.
107 Chapter 3: Results
150-1 L-ARG L-GLU
g 100- 1
0 4 8 12 16 20 Time (min)
Figure 3.4: Amino acid levels in response to the application of lOfiM L-NAME to
cortical cultures. Cortical glia were exposed to L-NAME for 6 min in total and the amino acid levels in the bathing medium determined. The open box indicates the period of exposure.
Having established that the application of an NO donor to cortical cultured glia increased
L-ARG and L-GLU levels and that blockade of NO production by L-NAME caused decreased levels in the bathing medium, it was then pertinent to investigate whether the coapplication of the NO donor SNAP at the same time as L-NAME would have any effect.
Figure 3.5 shows the results as well as a summary of the cultured glia data. Again L-NAME was applied for a total of 6 min and SNAP was coadministered for the last 2min of L-
NAME treatment. The application of L-NAME alone yet again caused decreased L-ARG and L-GLU levels while the coapplication of SNAP brought the levels back to the basal level, although SNAP in the presence of L-NAME did not cause the levels to be increased to the same extent as that found with the donor alone (see figure 3.2).
108 Chapter 3: Results
A:
200-1 L-ARG L-GLU
I I 100-
0 4 8 12 16 20 Time (min)
B:
200 n L-ARG L-GLU
1 100 -
& I 2 3 Drug treatment
Figure 3.5: Amino acid levels in response to the application of various drugs. A:
Levels of L-ARG and L-GLU in response to the 6 min application of 10/iM L-NAME
(open box) and 50/xM SNAP for the last 2min of L-NAME application (shaded box). B:
Peak L-ARG and L-GLU levels with 1: 10/xM Hb + 50/xM SNAP, 2: 10/xM L-NAME,
3: lOjLtM L-NAM E -+- 50/xM SNAP (4: Control). Results are expressed as means ± s.e.m.
(n= 4); levels which are significantly different from controls (P< 0.05) are marked *.109 Chapter 3: Results
Having confirmed that glia are a source of L-ARG in vitro, it was necessary to elucidate whether this was the case in vivo. FC is selectively taken up by glia (Kassel et al., 1992) where it acts as a suicide substrate for aconitase (Clarke, 1991), hence inhibiting the urea cycle and disrupting metabolism (Swanson and Graham, 1994). There is now evidence that
FC is a MBI (see section 1. 6 ) of aconitase with the formation of the intermediate fluoro-cw- aconitate followed by the addition of OH and the loss of F to form 4-hydro\y-trans- aconitate which binds tightly to the enzyme (Lauble et al., 1996). In vivo microdialysis studies had previously demonstrated that the infusion of FC caused increased extracellular L-ARG levels (Largo et al, 1996), implying that glia are, in fact, a source of L-ARG in vivo. Here, in vivo microdialysis was used to determine whether the infusion of FC would, in fact, elicit the release of L-ARG in the hippocampus. The FC was infused for 3h.
Figure 3.6 shows that the levels of both L-ARG and L-GLU showed a gradual increase with the infusion of FC, which reached a maximum after 90min. This level was maintained until the end of the experiment. Only when the levels had reached their maximum were they significantly different from the control values, which were obtained from rats which had aCSF (vehicle) infused and their amino acid levels monitored. The GAB A levels remained constant throughout the experiment but began to show a small yet significant rise only after
3h of FC infusion. In contrast, TAU levels were significantly increased immediately after the infusion had begun and rose to approximately sevenfold after the first hour. After this time the levels began to fall back towards basal but remained significantly increased until the end of the experiment, at which point they were still increased approximately threefold.
The levels of L-GLN remained constant for the first hour of the FC infusion, after which the levels started to decline. After 2.5h of infusion the extracellular levels were significantly decreased, a response which showed no tendency to recover.
1 1 0 Chapter 3: Results
300-, 400-, lOO/iM FC lOOjiM FC Vehicle Vehicle 300- S 200-
200-
100- 100-
120 180 240 300 120 180 240 300 Time (min) Time (min)
GABA
200-,
100-
120 180 240 300 Time (min)
TAU UGLN lOOOi 200-, 100/jM FC - O - lOO/tM FC Vehicle —it— Vehicle 750-
500- 100-
250-
120 180 240 300 120 180 240 300 Time (min) Time (min)
Figure 3.6: Amino acid levels in response to the infusion of the glial toxin FC through the probe into the hippocampus. The open boxes indicate the period of infusion. Results are expressed as means ± s.e.m. (n= 5); levels which are significantly different from controls (P< 0.05) are marked *. The FC was prepared according to the method of Paulsen et al. (1987) which involves its preparation from its Ba^^ salt: after precipitation of the Ba^^ with Na 2 S 0 4 the pH was adjusted to 7.2-T.3.
Ill Chapter 3: Results
Microdialysis experiments were also undertaken to determine whether L-ARG levels in vivo were influenced by L-GLU receptor activation. Grima et al. (1997) hypothesised that the activation of non-NMDA receptors on glia is responsible for the release of L-ARG.
Representative L-ARG release profiles for each of the agonists are shown in figure 3.7 while figure 3.8 shows the peak levels achieved. It is clear that the only glutamatergic agonist that elicited an increase in the L-ARG levels was AMPA. The L-ARG levels were significantly increased after the infusion had begun and remained increased for another
30min, at which point they returned to basal levels. All agonists were infused for a total of
30min and antagonists, when infused alone, were infused for a total of 90min; agonists were coinfused for the last 30min of the antagonist infusion when coinfusion was investigated. Figure 3.8 illustrates that the increase elicited by AMPA was not completely blocked by the coinfusion of CNQX. AMPA alone increased levels fourfold while the coinfusion of AMPA with CNQX still elevated L-ARG levels to approximately 200%.
300n lOOpM NMDA lOpM tADA lOOpM AMPA U 200 - ia Vehicle
PQI
^ 100-
0 60 120 180 240 300 Time (min)
Figure 3.7: Representative L-ARG release profiles. The glutamatergic agonists were
infused through the probe for 30min. The open box indicates the period of infusion.
1 1 2 Chapter 3: Results
500n
4 0 0 -
K I- 300 H # 200 -
100 -
0 1 i 2 I 3 4 Drug treatment
Figure 3.8: Peak L-ARG levels In response to the various drug treatments. Freely moving rats with hippocampal probes had agonists infused for 30min, antagonists for 90min and coinfusion occurred over the last 30min of antagonist infusion. 1: 100/iM NMDA, 2: lOfiM tADA, 3 : 100/tM AMPA, 4 : 10/xM CNQX, 5: lO^M CNQX + 100/xM
AMPA, 6 : Vehicle. Results are expressed as means ± s.e.m. (nk 6 ); levels which are
significantly different from controls (P< 0.05) are marked *.
The effects of glutamatergic agonists on the basal levels of L-GLU and GABA were also investigated. Figure 3.9 displays the representative release profiles for L-GLU and GABA while figure 3.10 shows the peak responses for L-GLU, GABA, L-CIT and L-GLN. It can be seen that only NMDA caused an appreciable release of L-GLU, levels increasing twofold during the drug treatment and returning to basal an hour after the end of infusion, with little effect on GABA. NMDA also caused decreased L-GLN in the extracellular space. The levels of GABA remained unchanged by each iGLUR agonist but agonism of group I mGLURs caused a time delayed decrease. tADA, like NMDA, also caused a significant decrease in the levels of L-GLN. AMPA had little effect on any of these amino acids. L-CIT remained unaffected by any of the drug treatments, levels staying at basal throughout.
113 Chapter 3: Results
A:
250n lOO^iM NMDA lO^iM tADA 200 - lOOfiM AMPA ^ 150- Vehicle
sg 100-
50-
0 60 120 180240 300 Time (min)
B:
lOOfiM NMDA lO^lM tADA lOOfiM AMPA ^ 100- Vehicle
PQI ^ 50-
0 60 120 180 240 300 Time (min)
Figure 3.9: Representative release profiles for A: L-GLU and B: GABA in response to the 30min infusion of glutamatergic agonists into the hippocampus of freely moving rats. The open boxes indicate the period of infusion.
114 Chapter 3: Results
250n ^ ■ I^ G L U GABA 200 - ^ZZm'lrCJl n u n L-GLN Î 150- i 100-
50-
0 I I 2 3 II Drug treatment
Figure 3.10: Amino acid levels in response to the infusion of various drugs. 1: 100/xM NMDA, 2: lOfiM tADA, 3: 100/aM AMPA, 4: Vehicle. Results are expressed as means
± s.e.m. (nk 6 ); levels which are significantly different from controls (P^ 0.05) are
marked *.
3.2 NOS activitv Given that NMDA activates NOS, hence promoting the formation of NO, and that AMPA causes an increase in L-ARG levels in vivo, it was pertinent to investigate the action of these drugs on the activity of NOS. To this end a NOS assay was developed based on the method of Salter et a/. (1991; see section 2.5.2). The documented incubation time (lOmin) and volume of tissue homogenate supernatant (ISfil) needed to be validated. To test the incubation time, 18/d of supernatant was incubated at 37°C for various periods of time and tested forL-^^^C-CIT formation. Optimal activity was indeed reached after lOmin (see figure
3.11 A). To optimise the activities of all three isoforms of NOS, both Ca^^-dependent and independent, the incubations (lOmin) were repeated using various volumes of supernatant.
The results are depicted in figure 3.1 IB. Because 45/d of supernatant gave the highest values for Ca^^-independent NOS this volume was used throughout, although the Ca^+- dependent NOS activity has been concentrated on in this study.
115 Chapter 3: Results
A;
150n Ca^^-dependent Ca^"^- independent
100-
50-
Time (min)
B:
40 n O Ca^^-dependent ^ Ca^^-independent 30-
20 -
10-
A r~ "T~ —f— — 1 10 20 30 40 50 Vol supernatant (pi)
Figure 3.11: Calibration curves for the NOS assay. A: A time course for the reaction,
B: NOS reaction product as a function of the volume of supernatant used. The results provide the justification behind using 45pl supernatant and a lOmin incubation time.
116 Chapter 3: Results
When probes were implanted they were always inserted into the left hippocampus. Both hippocampi could then be tested for NOS activity such that the right hippocampus could then act as the control for the left hippocampus from the same animal. It became apparent, however, that NOS activity measured in animals receiving dialysis with aCSF was lower in the left implanted side than in the contralateral “control” hippocampus with values of
99± 24 versus 159± 31 pmoles/ min/ g for left and right (n= 7), respectively being obtained.
Even when naïve animals which had not undergone surgery were tested the hemispheric difference was still apparent, the values obtained were 63± 9 versus 85± 22 pmole/ min/ g
(n= 7), which are both significantly lower than those of implanted animals. Figure 3.12 is a bar graph which represents these hemispheric differences in both naïve animals and those which had undergone surgery and had aCSF infused. However, there was quite a large variation between individual animals. For example, there was a range of between 43.1 and 213.8 pmole/ min/ g in the left hippocampus of implanted, aCSF infused animals, while on the right the values ranged from 42.0 to 313.1 pmole/ min/ g. There was not such a large variation in the naïve animals with ranges of 39.5-105.1 and 37.9-205.5 pmole/ min/ g for the left and right hippocampi, respectively. As a consequence the ratio between the left and right hippocampus from each animal was taken and a mean value determined. When the inter-animal variation was taken into account using this method the difference between the left and right hippocampi of naïve animals was not as apparent, the ratio being close to 1
(0.93± 0.17), while the ratio for implanted animals was still almost half (0.68± 0.14). In every experiment, therefore, each individual animal was taken into account and the values for the left hippocampus were divided by the left/ right (L/ R) ratio for implanted animals.
This was done so that any potential change as a consequence of drug treatment would not be masked by the higher levels in the contralateral “control” hippocampus.
117 Chapter 3: Results
Figure 3.12: A bar graph representing the hemispheric differences between naïve animals which had not undergone surgery and those which had had probes implanted and aCSF infused (NL: naïve left, NR: naïve right, AL: aCSF infused left, AR: aCSF infused right) It is apparent that in all cases the NOS activity is consistently
lower in the left hippocampus although the values are generally lower in naïve animals
compared to implanted aCSF infused animals. Results are expressed as means ± s.e.m.
(n= 7).
The effect of the infusion of glutamatergic agonists on NOS activity was investigated and the results are represented in figure 3.13. Only NMDA caused an increase in NOS activity which was completely blocked by the coinfusion of D-AP5. Even though NMDA caused increased L-GLU with no increase in L-ARG there was still increased NOS activity. It can also be seen that although AMPA caused an increase in L-ARG this appeared to be insufficient to activate NOS alone as it had no effect on NOS activity in drug-treated tissue.
118 Chapter 3: Results
Drug treatm ent
Figure 3.13: Changes in NOS activity with various drug treatments.Animals had drugs infused into the left hippocampus and were culled when the infusion was over and
their hippocampi removed. The hippocampi were then tested for NOS activity. 1 :100/xM
NMDA, 2: 10/xM tADA, 3: 100/tM AMPA, 4: 10/tM D-AP5 + 100/tM NMDA, 5: Vehicle. Results are expressed as means ± s.e.m. (n= 7); levels which are significantly
different from controls (P< 0.05) are marked *.
3.3 NO production
The effect of glutamatergic agonists on the production of NO (measured as nitrate) was also investigated using a NO analyser. Basal NO levels obtained from control aCSF infused animals were 153.7± 21.2nM (n= 38). A representative release profile for the changes in
NO levels in response to the infusion of NMDA is shown in figure 3.14, so too are the peak levels reached with each drug treatment. The NO results correlated with the NOS data achieved in this study; only NMDA caused an increase in the production of NO, an effect which, again, was blocked by D-AP5, while AMPA had no effect on NO production at all.
When NMDA was infused the NO levels increased immediately and 90min after the infusion had ceased the levels had returned to basal.
119 Chapter 3: Results
A:
500n lOOpiM NMDA Vehicle 400-
- 300- 1 S 200-
100 -
0 60 1 2 0 180 240 300 Time (min)
B:
4 0 0 -
Drug treatment
Figure 3.14: NO levels with various drug treatments. Microdialysis samples were
tested for their NO content using a NO analyser. A: Representative release profile in
response to the infusion of NMDA. The open box indicates the period of infusion. B:
Peak NO levels with 1: 100/xM NMDA, 2: 10/xM D-AP5 + 100/xM NMDA, 3: lOOfiM
AMPA, 4: Vehicle. Results are expressed as means ± s.e.m. (nk 4); levels which are
significantly different from controls (P< 0.05) are marked *. 120 Chapter 3: Results
In summary, the results so far suggest:
• Glia may be a source of L-ARG both in vitro and in vivo. • SNAP evoked the release of L-ARG and L-GLU in vitro, an effect that was reversed by the coapplication of CNQX as well as by Hb.
• L-NAME decreased L-ARG and L-GLU levels while the coapplication of SNAP
returned levels to basal values.
• AMPA evoked the release of L-ARG from glia in vitro and increased the extracellular concentration of L-ARG in vivo. • Although AMPA caused an increase in the extracellular L-ARG levels this alone was insufficient to activate NOS and cause the production of NO in vivo. • NMDA receptor activation increased the levels of L-GLU, activated NOS and caused the production of NO with no increased release of L-ARG in vivo.
3.4 NO and amino acid release
The question of whether substantial increases in the concentration of NO in the extracellular space altered the release of L-ARG, L-GLU or GABA in vivo was also investigated. To this end SNAP was infused and the amino acid levels determined. The NO levels were determined when 5mM SNAP was infused and figure 3.15 shows the results.
When the infusion started there was a massive increase in the amount of NO in the dialysates, with levels increasing to 100000% of basal. When the infusion ceased the levels started to diminish but only very slowly, and were still increased threefold at the end of the experiment. This effectively demonstrated that the infusion of SNAP resulted in the presence of NO in the extracellular space. When the actual concentration of NO in the extracellular space was calculated it was found to be in the /tM range, indicating that the recovery through the probe was relatively poor, only 6.7%.
1 2 1 Chapter 3: Results
lOOOOOOn 5mM SNAP Vehicle g - 100004
PQ1
100 -
—r-
60 1 2 0 180 240 300 Time (min)
Figure 3.15: A release profile of the NO levels in response to the 30min infusion of 5mM SNAP. Dialysates were tested for their NO content using a NO analyser. Note that the y-axis is logarithmic. The open box indicates the period of infusion. Results are
expressed as means ± s.e.m. (n= 3); levels which are significantly different from controls
(P< 0.05) are marked *.
Once it had been established that the infusion of SNAP elicited an elevation of NO concentration in the extracellular space, the effect this might have on L-ARG, L-GLU or
GABA levels in the extracellular space was investigated. Figures 3.16 and 3.17 show the results of these experiments. The infusion of SNAP resulted in an initial rise in L-GLU levels followed by a decrease 2h after the infusion had ceased. GABA levels were decreased and remained diminished for the entire experiment. L-ARG levels remained unchanged.
1 2 2 Chapter 3: Results
200-, L-ARG L-GLU GABA I
1 100 - PQ
0 60 1 2 0 180 240 300 Time (min)
Figure 3.16: Representative release profiles for L-ARG, L-GLU and GABA in response to the 30min infusion of SNAP. Dialysates were tested for their amino acid
content. The open box denotes the period of infusion.
To investigate whether the effects of SNAP on the GABA and L-GLU levels were mediated by cGMP, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-l-one (ODQ), a potent and selective inhibitor of NO-sensitive sGC, was coinfused with SNAP. The ODQ was infused for 90min and when SNAP was coinfused it was administered during the final 30min of ODQ infusion. Figure 3.17 shows the results achieved. The infusion of ODQ alone had no effect on L-ARG, L-GLU or GABA but the coinfusion of ODQ with SNAP blocked both the increase in L-GLU and the decrease in GABA seen with SNAP treatment alone.
123 Chapter 3: Results
300n L-ARG ESS3 L-GLU EZZ3GABA
200 - 1g 100- 111 2 3 Drug treatment
Figure 3.17: Peak responses to various drug treatments. 1: 5mM SNAP, 2: 10/xM ODQ, 3: 10/tM ODQ + 5mM SNAP, 4: Vehicle. SNAP alone was infused for 30min,
ODQ for 90min and SNAP was coinfused for the last 30min of ODQ infusion. Dialysates were tested for their amino acid content. Results are expressed as means ± s.e.m. (n= 5);
levels which are significantly different from controls (P< 0.05) are marked *.
To investigate whether the release of L-ARG might be under the control of tonic L-GLU release, glutamatergic antagonists were used. Both D-AP5 and CNQX were coinfused for a total of 90min and SNAP was also coinfused for the last 30min of antagonist coadministration. Figure 3.18 shows the combined data for L-ARG, L-GLU, GABA and
L-CIT levels in response to the drug treatments.
124 Chapter 3: Results
IrABG UUU 30Oi
SÎ20O m
100-
lOO-
0 60 120 180 240 300 0 60 120 180 300 lliTeCnii) Tlme(mn)
GAEA LOT
2 0 0 ] 30Oi - O - 10mMI>/P5 + ■1CMMD/V5 + 10HMCNÇK+ 10HMC1NQX+ SitMSNAP SiiMSNAP - 6 - \ t h c k -Nfchfck
I 100-
0 60 120 180 240 TO 0 60 120 240 300 Tiire(mn) Tmne(nti)
Figure 3.18: Amino acid levels in response to various drug treatments. 10/xM D-AP5 and lOjLtM CNQX were coinfused for a total of 90min (open box) while SNAP was coinfused for the last 30min of antagonist infusion (shaded box). Results are expressed as means ± s.e.m. (n= 5); levels which are significantly different from control values (P<
0.05) are marked *.
125 Chapter 3: Results
The infusion of D-AP5 and CNQX did not alter the amount of L-ARG in the extracellular space. When SNAP was also coinfused the levels started to rise and peaked at 250% of basal 30min after the infusion had stopped. The levels then started to fall but had not returned to basal by the end of the experiment. Interestingly the infusion of D-AP5 plus
CNQX caused a large significant increase in L-GLU levels. When SNAP was also coinfused there was a further increase which, again, peaked 30min after the infusion had ceased. The L-GLU levels then dropped and had returned to basal by the end of the experiment. The effect of SNAP alone (see figures 3.16 and 3.17) seems to be superimposed on top of the effect of D-AP5 combined with CNQX. The levels of GAB A remained unchanged by any of the drug treatments; coinfusion of iGLUR antagonists blocked the decreased levels of GAB A seen with SNAP alone. The L-CIT levels remained unchanged by the infusion of D-AP5 and CNQX but, similar to the L-ARG levels, they began to rise when SNAP was coinfused, reaching a peak 30min after the infusion had finished before falling to basal values towards the end of the experiment.
3.5 NOS inhibition
The effect of inhibiting NOS activity was investigated using both L-NAME and 7-Nl (see table 1.2). The inhibitors were infused for a total of 90min. Figure 3.19 depicts the release profiles for L ARG in response to the infusion of the inhibitors. The infusion of L-NAME resulted in increased L-ARG levels. These rose steadily throughout the drug treatment and when the infusion stopped they began to return towards basal, although the levels were still increased at the end of the experiment. In contrast, the infusion of 7-Nl resulted in decreased L-ARG levels. During the infusion the levels started to decrease and the levels remained diminished for the remainder of the experiment. When these two inhibitors were infused together the individual effects of each drug were cancelled out and the L-ARG levels remained similar to basal values.
126 Chapter 3: Results
300-1 ImM L-NAME lmM7-NI ImM L-NAME 4- lmM7-M ^ 200- Vehicle
100-
0 60 120 180 240 300 Time(min)
Figure 3.19: Representative L-ARG release profiles in response to NOS inhibitors. Drugs were infused for 90min and the dialysates tested for their L-ARG content. The open
box represents the period of infusion.
The L-GLU and G ABA levels were also determined in response to the infusion of NOS inhibitors. Figure 3.20 shows the representative release profiles for L-GLU and GAB A. The infusion of L-NAME caused increased L-GLU levels. There was a steady rise in L-
GLU during the drug treatment which reached a maximum at 400%. As soon as the infusion ceased the levels started to fall and had returned to basal after an hour. 7-NI had little effect on the L-GLU levels and there was no change in extracellular L-GLU when the two inhibitors were infused together. 7-NI caused a decrease in the levels of GAB A, an effect that did not return to basal once the infusion had stopped. The coinfusion of both inhibitors had little effect on the levels of GAB A. Figure 3.21 summarises the L-ARG, L-
GLU and G ABA data achieved in response to the NOS inhibitors.
127 Chapter 3: Results
400n ImM L-NAME lmM7-NI 300- ImMLrNAME-J- lmM7-M Vdiicte I 200 -
100-
0 60120 180 240 300 Time (min)
B:
ImM L-NAME lmM7-NI ImMLrNAME-k lmM7-M > 100- i0 ) Vdiicle ^ 50-
0 60 120 180 240 300 Time (min)
Figure 3.20: Representative release profiles for A: L-GLU and B: GAB A in response
to the infusion of NOS inhibitors. Drugs were infused for 90min and the dialysates were
tested for their amino acid content. The open boxes indicate the periods of infusion.
128 Chapter 3: Results
300n ■ ■ ■ L-ARG L-GLU ^ 2 3 G A B A (U> 200 cS
100 - II ill illi III 2 3 Drug treatment
Figure 3.21: Peak responses to various drug treatments. 1: ImM L-NAME, 2: ImM 7-NI, 3: Im M L-NAME + ImM 7-NI, 4: Vehicle. The inhibitors were infused for 90min
and the amino acid content of the dialysates was determined. Results are expressed as means ± s.e.m. (n= 5); levels which are significantly different from controls (P< 0.05) are
marked *.
The effect of the inhibitors on NOS activity in vivo was also examined. Figure 3.22 shows the results achieved. Both L-NAME and 7-NI actually increased NOS activity to 306± 55% and 312± 101% of basal, respectively, which was unexpected. When a combination of the two inhibitors together was infused no change in activity was obtained compared with control (130 ± 14%).
129 Chapter 3: Results
5 - I 4 - a3 - >
2 - Ù0 I g1 - X I 2 3 Drug treatment
Figure 3.22: NOS activity with various drug treatments. 1:Im M L-NAME, 2: ImM 7-NI, 3: ImM L-NAME + ImM 7-NI, 4: Vehicle. Freely moving rats had the inhibitors infused for 90min, at which point they were culled and their hippocampi removed. Each
hippocampus was tested for NOS activity. Results are expressed as means ± s.e.m. (n=
5); levels which are significantly different from controls (P< 0.05) are marked *.
Because both of the inhibitors elicited increases in NOS activity it was decided to investigate whether the drugs were effective at preventing the increase in NOS activity elicited by NMDA. The inhibitors were infused for 90min in total and the NMDA was coinfused for the last 30min. The results are depicted in figure 3.23. It can be seen that only
L-NAME was effective in blocking NMDA-stimulated NOS activity whilst 7-NI was without effect. In agreement with the effect of the coinfusion of both inhibitors on NOS activity, when they were coinfused with NMDA the effect of the agonist was reversed.
130 Chapter 3: Results
J
Drug treatment
Figure 3.23: NOS activities with various drug treatments. 1: lOO/xM NMDA, 2: ImM L-NAME + lOOfiM NMDA, 3: ImM 7-NI + 100/tM NMDA, 4: Im M L-NAME + ImM 7-NI 4- 100/xM NMDA, 5: Vehicle. Freely moving rats had NMDA infused for 30min or
inhibitors infused for 90min with NMDA coinfused for the last 30min. When the
infusions were over the animals were culled and their hippocampi removed. The
hippocampi were assayed for their NOS activity. Results are expressed as means ± s.e.m.
(n= 5); levels which are significantly different from controls (P< 0.05) are marked *,
while those values which are significantly different from NMDA alone are marked **.
Since the infusion of NOS inhibitors caused an increase in NOS activity, their effects on
NO production was also assessed. Figure 3.24 shows the results. Clearly both L-NAME and
7-NI elicited increased production of NO. The infusion of L-NAME led to increased NO production which peaked at 300% after Ih of drug treatment. When the infusion was stopped the levels started to return towards basal. A similar pattern was seen with 7-NI although the levels peaked slightly later than with L-NAME. The coinfusion of both inhibitors together resulted in NO concentrations falling below basal levels.
131 Chapter 3: Results
400i ImM IGNAME ImM 7-NI 300- ImM IGNAME + 7-NI 1 Vehicle
200 - 1CQ
100-
0 60 120 180 240 300 Time (min)
B:
750n
> 500 I I 250- I
2 3 Drug treatm ent
Figure 3.24: Dialysate NO levels in response to the infusion of NOS inhibitors. A:
Representative NO release profiles in response to the 90min infusion of drugs. The open
box indicates the period of infusion. B: Peak NO levels with 1: ImM L-NAME, 2: Im M
7-NI, 3: ImM L-NAME + ImM 7-NI, 4: Vehicle. Results are expressed as means ± s.e.m.
(n= 6 ); levels which are significantly different from controls (P< 0.05) are marked *.
132 Chapter S: Results
Given that only L-NAME was capable of blocking the increase in NOS activity elicited by
NMDA, the abilities of the inhibitors to block the increased NO production elicited by
NMDA was also explored. It can be seen from figure 3.25 that the NO levels were still increased when L-NAME and NMDA were coinfused. This time, however, the coinfusion of 7-NI was found to block the increase in NO caused by NMDA. Similar to the results obtained with NMDA and NOS activity, coinfusion of both inhibitors plus NMDA resulted in no increased NO production.
500-1
400- I- 300H ^ 200- a I I 100- I I 0 2 3 Drug treatment
Figure 3.25: Peak dialysate NO levels with various drug treatments. 1:IOO/- 1 M
NMDA, 2: ImM L-NAME 4- lOO/xM NMDA, 3: ImM 7-NI + 100/iM NMDA, 4: Im M
L-NAME 4- Im M 7-NI 4- 100/xM NMDA, 5: Vehicle. NMDA was infused for 30min
while the inhibitors were infused for 90min with coinfusion of NMDA during the last 30min. Results are expressed as means ± s.e.m. (n^ 5); levels which are significantly
different from controls (P< 0.05) are marked *. I 3 3 Chapter 3: Results
The in vivo results so far indicate:
• The infusion of SNAP led to increased NO in the extracellular space which caused
increased L-GLU and decreased GAB A with no effect on L-ARG.
• The coinfusion of ODQ with SNAP blocked the increase in L-GLU and the
decrease in GAB A seen with SNAP alone, implying that these responses to SNAP
were mediated by sGC/ cGMP.
• The coinfusion of D-AP5 and CNQX caused increased L-GLU with no effect on L-
ARG, GAB A or L-CIT.
• The coinfusion of D-AP5 and CNQX as well as SNAP caused increases in both L-
ARG and L-CIT, blocked the decrease in GAB A seen with SNAP alone but did not
block the increase in L-GLU.
• L-NAME led to increased L-ARG while 7-NI decreased L-ARG levels. Both effects were nullified by the coinfusion of both inhibitors together.
• L-NAME caused increased L-GLU with no effect on GAB A while 7-NI caused decreased GAB A with no effect on L-GLU. Again the coinfusion of both inhibitors
nullified the individual effects. • Both L-NAME and 7-NI increased NOS activity while their coinfusion had little
effect.
• Only L-NAME blocked the increased NOS activity resulting from the infusion of
NMDA.
• Both L-NAME and 7-NI increased the production of NO when infused alone while
their coinfusion resulted in the levels dropping below basal.
• Only 7-NI blocked the increased NO production observed with the infusion of
NMDA.
134 chapter 4: (Discussion Chapter 4: Discussion
4.1 Release of L-ARG in vitro and in vivo
The release of L-ARG into the extracellular space has been reported in response to both electrical stimulation of the white matter of the cerebellum and to natural stimulation of somatosensory afferents in the ventrobasal thalamus in vivo. Both 2Hz and 5Hz stimulation of white matter have been shown to cause an increase in L-ARG levels (Hansel et al., 1992), indicating that L-ARG can be released upon synaptic activation (Do et at., 1996) and that the extracellular concentration of L-ARG may be a factor in determining NO synthesis.
However, L-ARG levels can be as high as 2mM in freshly isolated endothelial cells while L-ARG supplements increase NOS activity, giving rise to the so-called L-ARG paradox
(see section 1.4.5). Why the supply of substrate for NOS is limited is unknown. The ventrobasal thalamic responses to natural stimulation of sensory afferents were mediated by iGLURs as well as the release of L-ARG, supporting the theory that glutamatergic synapses are often linked to NO production (see section 1.9). In addition, the application of exogenous L-ARG on sensory thalamic neurons has proved to facilitate sensory synaptic transmission (Do et at., 1994), supporting a role for NO in transmission. As introduced in section 1.2, previous studies have suggested that L-ARG is localised in glia in vitro and in vivo. Aoki et al. (1991a) used anti-L-ARG antibodies to show that L-ARG-like immunoreacti vity in the rat brain and spinal cord was concentrated mainly in the astrocytes.
They also used this technique to show that free L-ARG in the peripheral nervous system is concentrated in the satellite and supporting cells, cells which are analogous to glia in the
CNS (Aoki et al., 1991b). Also using immunocytochemical methods. Row (1994) demonstrated that the immunoreacti vity for both L-ASP, one of the substrates for the recycling of L-CIT back to L-ARG (see figure 1.4), and L-ARG was absent from the neuronal elements but was abundant in several glia-like cells in the rat neurohypophysis. The work of Grima et al. (1997) has shown that the application of L-GLU, AMPA or KA to cerebellar slices significantly increases [^H]-L-ARG levels in the bathing medium, with similar results achieved using astrocytic cultures. The increases in L-ARG elicited by both
AMPA and KA could be effectively blocked using CNQX. In neither preparation was
NMDA effective although the mGLUR agonist (±)-1 -aminocyclopentane-fmMj-1, 3- dicarboxylic acid (rra«5 -ACPD), a non-selective agonist for both groups I and H, produced a delayed increase in L-ARG levels. Grima et al. (1998) also demonstrated that the
136 Chapter 4: Discussion increased cGMP levels evoked by NMDA in cerebellar slices were blocked by both CNQX and D-AP5, which the authors postulate is indicative of L-ARG transfer due to glial non-
NMDA receptor activation. Another molecule that has been documented as causing the release of L-ARG from astrocytes is ONOO (see section 1.9.3). Vega-Agapito etal. (1999) have reported that the application of ONOO significantly stimulates L-ARG release from astrocytes in primary culture, an effect which was concentration-dependent. In order to confirm or refute these studies, glutamatergic agonists and an NO donor were applied to cortical glial cultures and the amino acid levels monitored. The application of both AMPA and SNAP caused increases in L-ARG in the bathing medium (see figure 3.1), effects which were blocked completely by CNQX. In contrast, both NMDA and tADA were ineffective. Given that Grima et al. (1997) found that trans-ACPD caused a delayed increase in L-ARG levels and that tADA was ineffective in this study, it can be hypothesised that the effect seen by Grima and coworkers was due to activation of group n mGLURs. The effects of SNAP were blocked by the coapplication of Hb, indicating that the observed responses were mediated by the formation of NO and not an indirect effect of penicillamine. In the light of previous work (Grima et al, 1997), blockade of the AMPA response by CNQX was expected, but the fact that the effect of SNAP was also blocked by
CNQX implies that the SNAP was exerting its effect via an AMPA-dependent mechanism; several studies have documented that NO release in the CAl region and molecular layers of the hippocampus enhances AMPA receptor sensitivity (O’Dell et al, 1991; Bôhme et al, 1991; Zhuo etal, I993',lgaetal, 1993) while the pretreatment of forebrain, especially cerebral cortex and hippocampus, or cerebellar sections of rat brain with NO donors has also been found to increase [^H]-AMPA binding via increasing affinity, an effect that was independent of cGMP in forebrain areas, possibly indicative of a direct action of NO (Dev and Morris, 1994). However, the effect of SNAP could equally have been due to the formation of ONOO in the bathing medium if O 2 were also present (see section 1.8.2).
Nevertheless, the results of this study confirm that L-ARG can be released from glia in response to AMPA receptor activation.
The data presented here show that the application of SNAP to cortical glia caused a significant increase in L-GLU in the bathing medium, NMDA, AMPA and tADA, however, elicited no change. Again, coapplication of both CNQX and Hb prevented the increases in
137 Chapter 4: Discussion
L-GLU. Taken together with the L-ARG results, it can be hypothesised that the application of SNAP leads to the generation of NO which causes the release of L-GLU, possibly via reversal of GLT-1 (see section 1.9.2), which is then free to activate any glutamatergic receptors present. The blockade by CNQX is suggestive of AMPA receptor activation which, in turn, would cause the release of L-ARG. This would be indicative of a positive feedback mechanism with the presence of NO causing the release of more substrate for its own production, although CNQX blocked the increased L-GLU seen with SNAP. The lack of effect of NMDA may be explained by the lack of NMDA receptors on glia, although this is still a matter of debate (Seifert and Steinhaiiser, 2001). Schipke et al. (2001) have demonstrated that astrocytes of the mouse neocortex express functional NMDA receptors while Conti et al. (1999) have shown that NRl and NR2A/ B immunoreactivity is consistently found in distal astrocytic processes in human cerebral cortex. However, it appears that the majority of ionotropic L-GLU receptors found on glia are of the non- NMDA AMPA subtype (Seifert and Steinhaiiser, 2001). It was also noted that both tADA and NMDA significantly decreased L-GLN release, although why this occurred is unresolved. Group I mGLURs are expressed on glia but only the mGLURS receptor protein is found in hippocampal astroglial cultures (Janssens and Lesage, 2001). The lack of effect of NMDA and tADA on either L-GLU or L-ARG levels make the changes in L-GLN difficult to explain.
A number of other in vitro studies have investigated the release of L-GLU in response to L-GLU receptor activation. The release of [^H]-L-GLU has been reported in response to
NMDA in synaptosomal preparations from guinea pig cerebral cortex, which proved to have a direct correlation with cortical NO production (Montague et al, 1994). However, superfusion of rat hippocampal synaptosomes with SNP failed to augment the K^-evoked release of L-GLU, although release was enhanced in hippocampal slices (Kamisaki et al.,
1994). It was hypothesised that NO may act indirectly on nerve terminals via interactions with glia and short neurons, which would be in agreement with this study. The production of endogenous NO in response to NMDA-mediated NOS activation has also been shown to increase the basal release of both L-GLN and L-GLU from immature chick retina (lentile et al, 1996) and L-GLU from slices of guinea pig dentate gyrus (Nei et al, 1996). The effect of NO on L-GLU release in some studies has been shown to depend on the
138 Chapter 4: Discussion concentration of the NO donor used. At concentrations <0.3mM, the NO donor hydroxylamine was found to decrease the Ca^^-dependent L-GLU release evoked by 4-
aminopyridine (4-AP) in rat hippocampal synaptosomes, while concentrations >0.3mM drastically increased basal L-GLU release via reversal of the L-GLU carrier (Sequeira et al., 1997). Another study using rat hippocampal synaptosomes showed that AMPA stimulated, while KA dose-dependently decreased, L-GLU release and that KA also depressed glutamatergic synaptic transmission (Chittajallu et ai, 1996). Conflicting results were achieved by Duarte et al. (1996) who found that KA caused a dose-dependent increase in L-GLU release from cultured chick retina cells in low Ca^^ medium, while AMPA was without effect. However, the majority of these studies involved brain slices or synaptosomal preparations and, as such, involved the presence of neuronal elements, not just glia. Bal- Price et al. (2002) investigated the effect of NO on astrocytic L-GLU release and found, in agreement with this study, that the application of either NO itself or diethylenetriamine-NO
(DETA/NO) resulted in rapid L-GLU release in a vesicular, Ca^^-dependent manner. Other substances which have been shown to cause the release of L-GLU from astrocytes include ATP (Jeremic et al., 2001), brain-derived neurotrophic factor (BDNF; Numakawa et al., 2001) and BK (Jeftinija et al., 1996). It would seem that NO causes the release of L-GLU from astrocytes which could then activate non-NMDA receptors to cause the release of L-
ARG.
The question of whether NO exerts tonic control over L-ARG and L-GLU release from glia was addressed using the Ca^^-dependent NOS inhibitor L-NAME. Application of L-NAME decreased the levels of both amino acids in the bathing medium, an effect which was reversed by the coadministration of SNAP (see figures 3.4 and 3.5). This would certainly be suggestive of tonic nitrergic control over the release of these amino acids from glia in vitro. However, the levels of L-ARG and L-GLU were unaffected by the application of Hb, Why Hb should be ineffective while L-NAME decreases levels is unclear. The evidence presented so far suggests that glia are a source of L-ARG, which can be released upon
AMPA receptor activation as well as in response to the presence of NO in a positive feedback fashion. The concentration of NO in the vicinity of glia may or may not control the concentration of both L-GLU and L-ARG in vitro. From the conflicting data achieved in studies using synaptosomes or slice preparations it is evident that the situation in vitro
139 Chapter 4: Discussion is complex, although the conditions in vivo are sure to be more complicated. Nonetheless it was important to try and elucidate the situation in intact brain tissue.
In order to investigate whether glia are a source of L-ARG in vivo, microdialysis into the ventral hippocampus of freely moving rats coupled with the infusion of the glial toxin FC was used. The lowering of extracellular levels of L-GLN, which is synthesised exclusively in glia (see section 1.2; Martinez-Hemandez et al., 1977), is generally considered a biochemical marker for glial dysfunction (Largo et al, 1996). The inhibition of aconitase has been shown to lead to both an accumulation of citrate and a reduction in the formation of L-GLN (Fonnum et al, 1997). The intrastriatal administration of FC to rats caused a 95% reduction in L-GLN formation (Hassel et al, 1992) while FC has also been documented as reducing L-GLN production by 65% in primary astrocyte cultures coupled with a 30% reduction in the uptake of L-GLU (Swanson and Graham, 1994), probably attributable to the lack of uptake by GLT-1 (see section 1.9.2). This was the case in this study (see figure 3.6) where the levels of L-GLN decreased significantly with a corresponding increase in L-GLU. T AU is also stored in astrocytes (Walz and Allen, 1987) and the administration of FC has previously proven to increase the levels of TAU in the extracellular space (Largo et al, 1996). In this study the levels of TAU increase by as much as 7 times the basal level. Together, the results achieved for L-GLN, L-GLU and TAU indicate that FC did indeed disrupt glial metabolism. In 1987, Paulsen postulated that high doses of FC may not only be toxic to glia but also to neurons and the ability of FC to cause neuronal injury was investigated by Largo et al (1996). They found that the levels of
GAB A, glycine and L-GLU showed a steep increase only after 4h of administration which they postulated to be indicative of a breakdown in neuronal integrity. GAB A is synthesised in neurons from L-GLU (Honegger and Richelson, 1979) by the action of glutamic acid decarboxylase (GAD; Chan-Palay et al, 1979). An increase in GAB A in the extracellular space would therefore indicate a possible loss of neuronal integrity. The results in this study concur with the results of Largo et al (1996) in one respect; the levels of G ABA stay at approximately basal level until the very end of the experiment, after 3h of infusion, when there is a small yet significant increase, although the L-GLU levels were increased as soon as the infusion began. The lack of uptake by GLT-1, however, could well be responsible for this. Figure 3.6 also shows that the levels of L-ARG increased significantly during the
140 Chapter 4: Discussion infusion of FC. This may be interpreted as being due to the loss of glial integrity and the subsequent leakage of L-ARG into the extracellular space. It would certainly seem likely, from both the in vitro and in vivo results achieved so far, that L-ARG is indeed localised in the glia. Yamada and Nabeshima (1997a) found that the infusion of both L-ARG andPC resulted in increased NO metabolites (NO/) in the extracellular space, which would indicate that the extracellular L-ARG levels may dictate the activity of NOS.
The signal for the release of L-ARG from glia in vivo was also investigated and the first rationale involved the infusion of various glutamatergic agonists. The infusion of neither
NMDA nor tADA affected L-ARG levels while AMPA significantly increased levels fourfold (see figure 3.8). However, levels were still doubled when AMPA was coinfused with CNQX. Infusion of CNQX alone had no effect. This would suggest that, as well as having a direct receptor mediated effect, AMPA also caused a response via non-AMPA receptor mediated means. However, there is little in the literature concerning any effects of AMPA which are not mediated by the AMPA subtype of L-GLU receptor. The concentration of CNQX is then called into question. The concentration used in this study was 10/tM, while the pA 2 value for CNQX against AMPA receptors has been documented as 5.78 ± 0.06 in immature rat spinal cord (Pook et ai, 1993). Given that pA 2 = -log,Q K^, where is the concentration required to occupy 50% of the receptors, the Kg= 1.7 ±
0.9/xM. Another binding study in rat cortex membranes revealed the presence of two [^H]- AMPA binding sites, one with a Kg of 5.49nM, the other with a Kg of 52nM (Giberti et al,
1991). Displacement of [^H]-AMPA binding by CNQX had an IC 5 0 of 272nM. A concentration of lOjuM CNQX should, therefore, have been sufficient to block the actions of AMPA at its receptor in the hippocampus. The recovery of the antagonist through the probe may explain the lack of inhibition, recovery generally being fairly poor (see table
2.1). However, the recovery would have to be a meagre 2.72% to achieve a concentration of 272nM in the extracellular space. If the of CNQX for AMPA receptors in the hippocampus is similar to that found in rat spinal cord then a recovery of below 17% would have resulted in a concentration less than the K^, resulting in less than 50% receptor occupation. Nevertheless, the CNQX was infused for 60min before coinfusion with AMPA, which should have been sufficient time for the CNQX to block the receptors.
141 Chapter 4: Discussion
As well as cholinergic, noradrenergic and serotonergic innervation, the hippocampus has glutamatergic and GABAergic pathways (see section 1.9.5). The glutamatergic pathways and cells form the trisynaptic loop; the major afferents from the entorhinal cortex synapse onto granule cells, the principal cells of the dentate gyrus. The granule cells send their axons (mossy fibres) to the proximal dendrites of CA3 pyramidal cells. In turn these cells project to the dendritic region of CAl pyramidal cells via Schaffer collaterals. The principal extrinsic projections arise from the CAl pyramidal cells and terminate in the entorhinal cortex and subiculum, completing the cortico-hippocampo-cortical loop (Freund and
Buzsaki, 1996; Vizi and Kiss, 1998). G ABA is the major inhibitory transmitter in the brain and control of synaptic plasticity and network activity patterns of principal cells in the hippocampus is a fundamental role of inhibitory neurons (Acsady et al, 1996). Neuroanatomical studies have indicated that virtually all hippocampal intemeurons are GABAergic and that they comprise approximately 10% of the total neuron population (Vizi and Kiss, 1998). The majority of hippocampal intemeurons make synaptic contact with principal cells, although a subgroup specifically innervates other intemeurons (Acsady et al, 1996).
The in vivo effect of the glutamatergic agonists on L-ARG and its effects on neurotransmitter release, specifically L-GLU and GAB A, was investigated. There are several mechanisms by which a neuron can synaptically modulate its own output. Firstly, the axon can establish autaptic contacts, synapses which are typically formed on its own somato-dendritic compartment. A more common mechanism involves receptors located at the axon terminal. Upon the entry of Ca^^, transmitter is released which activates auto-, hetero- and homoreceptors as well as receptors on the postsynaptic terminal. For the most part the activation of autoreceptors inhibits transmitter release, although they can enhance release in some cases. Finally, a neuron may regulate its firing via the activation of autoreceptors located on the soma and dendrites. These receptors are metabotropic and mediate slow autoinhibition (Salin et al, 2001).
Evidence exists to suggest that when transmitter release is enhanced at hippocampal mossy fibre synapses, the concentration of L-GLU rises and its clearance is delayed. This, in turn, allows the L-GLU to diffuse away from the synapse to activate presynaptic inhibitory
142 Chapter 4: Discussion mGLURs. At normal levels of L-GLU release, during low-frequency activation, the presynaptic receptors do not become activated but when the L-GLU concentration is increased, either by higher frequency activity or by the blockade of uptake, they become activated and lead to a reduction of transmitter release (Scanziani et al ., 1997). This would be an example of the use-dependent activation of presynaptic mGLURs representing a negative feedback mechanism for controlling the strength of synaptic transmission.
It has also been postulated that AMPA autoreceptors on the presynaptic terminal may play a role in neurotransmitter release. In 1994, Barnes et al. determined the effect of AMPA on the Ca^^-sensitive tetrodotoxin (TTX)-insensitive K^-stimulated -L-GLU release from rat hippocampal synaptosomes. They found that the presence of cyclothiazide, a drug that blocks AMPA receptor desensitisation, was necessary for the application of AMPA to cause a dose-dependent increase in [^H]-L-GLU release, although there was no effect on the basal release. This was not the case when Zhou et al. (1995) repeated the experiment; neither NMDA nor AMPA affected the K^-evoked release of L-GLU from Schaffer collateral terminals, although application of AMPA in the presence of diazoxide, a drug similar to cyclothiazide, decreased the release of L-ASP. NMDA proved to enhance the release of L- ASP, an effect which was blocked by D-AP5, as did CNQX and AMPA alone. [^H]-D-ASP has been widely used as a non-metabolised marker for neuronally released endogenous amino acids in several in vitro preparations (Balcar and Johnston, 1972; Davies and Johnston, 1976). In fact, a close relationship between [^H]-D-ASP output and the release of [^H]-L-GLU synthesised de novo from [^H]-L-GLN has been demonstrated in both striatal and cerebrocortical slices (Palmer and Reiter, 1994; Lombardi etal., 1996). [^H]-D-
ASP has proven to label both the L-GLU transmitter and the cytoplasmic pools in glutamatergic neurons (Palaiologos et al, 1989). Patel and Croucher (1997) also investigated the effect of AMPA on transmitter release in rat forebrain slices and they achieved similar results to Barnes and coworkers. AMPA enhanced the electrically stimulated release of [^H]-D-ASP in a dose-dependent fashion with no effect on basal efflux. Although the results of Zhou et al. (1995) may indicate the possibility of selective regulation of neuronal L-GLU or L-ASP, the majority of the evidence points to the AMPA receptor mediated response being activity-dependent. Similarly, it has been demonstrated that L-ARG is capable of eliciting a concentration-dependent increase in [^H]-D-ASP from
143 Chapter 4: Discussion
Striatal slices, while solutions of NO gas were capable of stimulating L-GLU release (Reiser et al, 1999). The authors postulated that this could be indicative of a direct presynaptic action of NO on [^H]-D-ASP and a possible modulation of L-GLU release by the availability of L-ARG. West and Galloway (1997) used in vivo microdialysis into the striatum to show that the infusion of N“-OH-L-ARG, the intermediate in NO formation (see figure 1.2), significantly increases extracellular L-GLU. Because AMPA caused a significant increase in extracellular L-ARG levels it would be expected that the L-GLU levels would be increased.
The effect of AMPA on the release of [^H]-GABA from superfused rat striatal slices has also been investigated. The application of AMPA increased basal [^H]-GABA outflow, a response which was potentiated by cyclothiazide (Harsing etal ., 2000). However, an in vivo study has shown that the intracerebroventricular (i.c.v.) administration of AMPA had no effect on GAB A (Giovannini et ai, 1998). Another in vivo study has been performed using reverse microdialysis in the striatum of freely moving rats. Preloading nerve terminals with -L-GLU and ["^C]-GABA and the subsequent infusion of AMPA increased [^H]-L- GLU and ["‘C]-GABA levels to 142± 6.5% and 166.8± 7.7% of basal, respectively. These responses were unaffected by the coinfusion of cyclothiazide (Patel et ai, 2001).
The effect of NMDA on the release of [^H]-GABA has also been investigated. Weiss
(1990) found that the application of NMDA to purified populations of striatal neurons in primary culture increased [^H]-GABA release, although Sherman et ai (1992) found that exposure of synaptosomes to 10/xM NMDA had no effect on GAB A, but they also found that this dose of NMDA caused the spontaneous release of L-GLU. These results are contradicted by a study in rat cerebellar cultures which found that NMDA had no effect at all on any neuroactive amino acids (Rogers et ai, 1990). However, an in vivo microdialysis study by Young and Bradford (1993) found that the infusion of NMDA elicited increases in both ["‘CJ-GABA and [^H]-L-GLU from preloaded striatal neurons, which was effectively blocked by the NMDA antagonist 3-[(±)-2-carboxypiperazin-4-yl] propyl-1- phosphonic acid (GPP). Another more recent in vivo microdialysis study effectively demonstrated that the infusion of NMDA into the corpus striatum had no effect on G ABA but caused a fourfold increase in L-GLU. The increase in L-GLU was blocked by both TTX
144 Chapter 4: Discussion and the competitive mGLUR 1 antagonist 2-methyl-4-carboxyphenyl glycine (LY367385), but not by the noncompetitive mOLURS antagonist 2-methyl-6-(phenylethynyl) pyridine
(MPEP). The levels of GAB A were only significantly elevated upon coinfusion of NMDA with LY367385 alone or combined with TTX (Battaglia et ai, 2001). However, it has also been hypothesised that NMDA receptors may play a presynaptic role in synaptic plasticity through the formation of NO. When Sequeira et al. (2001) investigated, they found that NMDA dose-dependently inhibited the release of L-GLU evoked by 4-AP in synaptosomes from CA3 and CAl fields of whole hippocampus but not from the dentate gyrus, an effect which could be reversed by both D-AP5 and L-NNA. The role of NO as a modulator of L-
GLU, L-ASP and GAB A in the nucleus accumbens was also investigated by Kraus and Prast (2002). They found that the infusion of PAPA/ NO through push-pull cannulae enhanced the basal release of all three, suggesting that NO may represent a release- enhancing neuromodulator in this brain region. They postulated that the release of excitatory amino acids leads, via stimulation of L-GLU receptors, to a subsequent release of GAB A from GABAergic intemeurons. Conversely, Kendrick et al. (1996) showed that reducing striatal NO levels using L-NNA potentiated Ca^^-dependent transmitter release in response to NMDA while increasing them reduced it. They conclude that this is consistent with a neuroprotective role for NO. The effect of NO on transmitter release may vary depending on the brain region in question. Alternatively, the effect of NO may depend on its concentration. Segieth et al. (1995) found that 500jLtM SNAP decreased hippocampal dialysate L-GLU and L-ASP while 1 and 2mM concentrations increased levels.
Because group I mGLUR activation results in phosphoinositide hydrolysis and the mobilisation of Ca^"^ from intracellular stores (see section 1.9.2) and given the possibility of NOS forming a ternary complex with CAPON and synapsin in the presynaptic terminal
(see section 1.4.4) implicates mGLUR activation in the control of neurotransmitter release.
A recent study using whole-cell current clamp recordings of the rat auditory cortex has investigated the role of mGLURs in the regulation of AMPA/ KA, NMDA and G ABA receptor-mediated transmission. It was demonstrated that the fast excitatory postsynaptic potentials (EPSPs; AMPA/ KA), slow EPSPs (NMDA) and the inhibitory postsynaptic potentials (IPSPs; GABA^, GABAg) elicited in pyramidal neurons were reduced in the presence of trans-ACPD (Bandrowski et al, 2001). These effects, however, could have
145 Chapter 4: Discussion resulted from group II mGLUR activation. tADA is a group I agonist, so it acts at both mGLUR 1 and mGLUR5. These receptors often colocalise in the same neurons but because both receptors can couple to the same effector systems the purpose of this coexpression remains unclear (Poisik et al, 2003). In the hippocampus, however, the situation is a little different. Immunocytochemistry studies have revealed that CA3 cells possess both mGLUR 1 and mGLUR5 while CAl cells only possess mGLUR5 and that, when found at synapses, immunoparticles were always found outside the postsynaptic membrane specialisations (Lujan et ai, 1996). The authors speculate that the perisynaptic position of postsynaptic mGLURs, which appears to be a general feature of glutamatergic synaptic organisation, may apply to other G-protein coupled receptors. Some authors have found that group I mGLUR activation results in increased neuronal excitability. Lanneau et al. (2002) demonstrated electrophysiologically that agonism of both mGLUR I and mGLUR5 was important in increasing excitability by increasing synchrony between cells and driving correlated network activity in rat hippocampal slices, although these effects seemed to be independent of inhibitory synaptic tone due to GAB A^ receptor-mediated synaptic inputs. The authors point out that the precise patterning of activity which took place was complex and depended upon both the existing pattern of ongoing network activity prior to mGLUR activation and the relative extent of activation of each mGLUR subtype. Mori and Gerber (2002) investigated the synaptically evoked inhibitory responses in CA3 pyramidal cells mediated by mGLURs expressed somatodendritically by intemeurons. They showed that postsynaptic mGLUR I and mGLUR5 cooperatively mediated slow feedback inhibition. It was postulated that this mechanism may allow intemeurons to monitor the activity of neighbouring principal cells to adapt inhibitory tone to the state of the network.
In the current study the only glutamatergic agonist to affect L-GLU levels was NMDA, which caused a significant increase. The levels of G ABA were only affected by tADA, which caused a time delayed decrease (see figures 3.9 and 3.10). However, the increase in
L-GLU did not result in a corresponding increase in L-ARG, which would be expected if the L-GLU was free to activate glial AMPA receptors, nor did it cause a decrease in GAB A due to the activation of group I mGLURs. A study in nNOS knockout mice revealed that the infusion of ImM NMDA into the hippocampus still resulted in increased L-GLU, indicating that the mechanism is independent of nNOS activation (Kano et al ., 1998). There
146 Chapter 4: Discussion was no change in the L-GLN levels upon the infusion of NMDA. Sundstrom et al. (1995) performed in vivo microdialysis into the spinal cord of rats to demonstrate that the infusion of 5mM NMDA evoked a ninefold increase in the levels of L-GLU which resulted in a concentration of approximately 250/iM outside the dialysis membrane. There was no change in the extracellular levels of L-GLN which, in addition to this experiment, was in agreement with another study (Lehmann et al, 1983). The authors concluded that there is not necessarily a strict relationship between the release of L-GLU and the extracellular L-
GLN levels. The L-GLU levels were increased twofold in this study which, according to table 2.3, would have resulted in 63.6pmoles in 15/tl of dialysate, equivalent to a concentration of 4.24/xM. According to table 2.1, however, the recovery of L-GLU through the dialysis membrane was 25.4± 1.5%. Taking this into account the extracellular concentration of L-GLU was increased to 16.7± 0.064/xM. The concentration of L-GLU reached outside of the cleft after synaptic release is critical in determining the extent of extrasynaptic receptor activation (Dzubay and Jahr, 1999). In hippocampal cultures the concentration of L-GLU in the synaptic cleft following release decays from l-3mM to tens of fiM within several ms due to rapid diffusion out of the cleft and buffering by transporters (Bergles et ai, 1997). The for NMDA displaceable -L-GLU binding has been documented as 251± 4nM in the cerebral cortex of newborn piglets with Kg values for [^H]- KA binding calculated as 13± 2nM in the same preparation (McGowan et ai, 2002). In guinea-pig brain stem the high affinity AMPA receptors, which comprise 50-70% of the total receptor population, bind [^H]-AMPA with a Kg in the 5-20nM range while the remaining receptors, which are thought to be postsynaptic, bind [^H]-AMPA with lower affinity, exhibiting a Kg in the 500-1000nM range (Sunejaer < 2 /., 2000). This would indicate that a concentration of L-GLU in the low [iM range is sufficient to potentially activate all
L GLU receptors present. However, this would also suggest that the basal level of L-GLU
(8.4/xM) is also sufficient to activate all glutamatergic receptors at all times, which is unlikely. A basal L-GLU level of 8.4/xM is in agreement with other studies (Skilling et al, 1988; Sundstrom et al, 1995). The lack of effect on mGLURs could possibly have been due to the time frame of the experiment, although the decrease in G ABA levels with tADA was seen an hour after the infusion had ceased and the NMDA experiment continued for
240min after the infusion, sufficient time for a response to have occurred. Given that electrophysiological studies have shown that synaptic release of L-GLU causes the
147 Chapter 4: Discussion concentration of L-GLU to increase to l-3mM, it is possible that the level of L-GLU required in the extracellular space needs to exceed a certain level to overcome diffusion and the effects of buffering mechanisms, explaining the lack of effects seen. It was hypothesised from the in vitro experiments herein that the increased L-GLU resulting from the application of SNAP activated glial AMPA receptors causing an increase in L-ARG. This, however, does not appear to be the case in vivo, where the L-GLU resulting from NMDA receptor activation seemed to have little effect on either AMPA or group I mGLURs, although the buffering mechanisms present in vivo might differ from in vitro preparations.
The lack of effect of AMPA on L-GLU or G ABA, despite increasing L-ARG levels, may be due to the rapid desensitisation of the receptor (see section 1.9.4). However, this study investigated the effect of AMPA on the basal release of L-GLU and GAB A, while most of the literature to date suggests that the AMPA mediated response is activity-dependent.
Not only did tADA decrease GAB A levels but it also decreased L-GLN levels. As mentioned previously, a decrease in L-GLN may be indicative of glial dysfunction.
However, if this were the case then the levels of L-GLU, L-ARG and TAU would all have been increased with a slow to develop increase in GAB A at the very end of the experiment as seen with FC. The infusion of tADA had no effect on L-ARG, L-GLU or TAU but caused a significant decrease in GAB A, suggesting an alternative reason for the decrease in extracellular L-GLN levels. The delayed reduction in the levels of G ABA may be indicative of slow feedback inhibition although the decrease in L-GLN itself may be responsible, indirectly, for the fall in GAB A levels. According to the L-GLU-L-GLN cycle, the L-GLN synthesised in glia from L-GLU needs to be transferred to the neurons to be turned back into L-GLU. Given that GAB A is synthesised from L-GLU in neurons, the decrease in L-GLN may, therefore, result in decreased G ABA synthesis.
None of the drug treatments had any effect on L-CIT levels. The intrathecal administration of NMDA has been shown previously to increase L-GLU, NO and L-CIT levels, effects which could be blocked with L-NAME (Sorkin, 1993). The lack of effects on L-CIT by any of the glutamatergic agonists could, therefore, indicate a lack of effect on both NOS activity and NO production.
148 Chapter 4; Discussion
4.2 NOS_açtivîtx
In order to establish whether the L-GLU agonists were affecting NOS activity the NOS assay developed by Salter et al. (1987) was implemented. It became apparent, however, that there were distinct hemispheric differences, the activity of NOS being lower in the left implanted side in comparison to the right control contralateral hippocampus (see figure
3.12). This distinction was absent from naïve animals when each individual animal was taken into consideration, so the diminished NOS activity in the ipsilateral hippocampus was most probably a direct result of the surgical procedure. Few other studies have investigated whether there are any differences between the activity of NOS in the left and right hemispheres of the brain as a result of surgery. In fact, most studies have focused on obtaining information about the localisation of NOS in different brain regions using a variety of techniques, including NADPH diaphorase, anti-NOS antibodies and -L-NNA binding, which are not quantitative, in numerous species, including rats (Kidd etal., 1995; Rao and Butterworth, 1996), mutant mice (Huang et al., 1993; Kara et al, 1996), marmosets (Gerlach et al, 1995), humans (Blum-Degen et al, 1999), carp (Conte and Ottaviani, 1998), goldfish and brown trout (Virgili et al, 2001). Salter et al (1995) have performed the only other study investigating the hemispheric differences in NOS activity, although they focused on the inhibition of NOS. They found that the lateral ventricular administration of L-NAME or L-NNA resulted in reduced NOS levels in the ipsilateral brain areas in comparison to the contralateral regions. They did not investigate, however, whether the surgical procedure alone altered NOS activity. Given that our study found that the implantation procedure itself resulted in reduced levels, it is possible that the i.c.v. administration procedure employed by Salter et al (1995) also resulted in lowered levels in the ipsilateral brain regions. The predicted response to surgery would be the activation of iNOS due to damage caused by the implant procedure. However, this was not the case, with a decrease in NOS activity being observed.
Once the NOS assay had been established the activities of NOS in response to the glutamatergic agonists could be investigated. The only agonist to increase NOS activity was
NMDA, an effect which was blocked by 10/xM D-AP5 (see figure 3.13). A concentration of lOfiM D-AP5 was sufficient to block the effect of lOOfiM NMDA given that the affinity
149 Chapter 4: Discussion
of D-AP5 is 5-10 times higher than NMDA in receptor binding studies (Olverman et ai, 1984; Monahan and Michel, 1987). East and Garthwaite (1991) found that exposure of
hippocampal slices to NMDA resulted in dose-dependent elevations of cGMP levels, while
Vallebuona and Raiteri (1994) and Luo and Vincent (1994) found that the infusion of
NMDA into the hippocampus also resulted in dose-dependent increases in extracellular cGMP. In 1995, Bhardwaj et al investigated the effect of NMDA on NOS activity using in vivo microdialysis with bilateral probes in pentobarbital anaesthetised animals. The infusion of aCSF containing ["^C]-L-ARG allowed them to examine the recovery of [’"^C]-
L-Crr in the dialysates. They found that the infusion of ImMNMDA into the hippocampus enhanced NOS activity. In 1997 they repeated the experiment to investigate the action of
non-NMDA receptors. They demonstrated that ImM AMPA also led to increased NOS
activity. The infusion of both MK-801 and CNQX reduced basal L-CIT recovery, indicating that tonic activity of both NMDA and AMPA receptors may contribute to NO production.
The increased L-CIT with NMDA was partially inhibited by CNQX while the increased L-
C n with AMPA was partially blocked by MK-801. They postulated that this indicates a multi synaptic amplification of NO production with iGLUR activation, although the
anaesthetic itself may have altered the neurotransmitter levels. Many anaesthetics have powerful and poorly understood biological properties. This becomes particularly relevant
when considering brain function or neurochemistry. In fact, several general anaesthetics have actions on ion channels, neurotransmitter receptors and extracellular concentrations (Rozza et al, 2000). Inhalation anaesthetics, such as halothane or isoflurane, inhibit the NO-guanylate cyclase signaling pathway in the vascular endothelium (Zuo et al, 1996) while other volatile anaesthetics interact with NMDA receptors (Martin et al, 1995). However, pentobarbital anaesthesia has recently been shown to have little effect on extracellular amino acid levels (Rozza et al, 2000).
Kiedrowski et al (1992) used the formation of [^H]-L-CIT from [^H]-L-ARG as an indicator of NOS activity in primary cultures of cerebellar granule cells and found that
NMDA stimulated a rapid Ca^^-dependent conversion. Hammer et al (1993) performed in vivo microdialysis to show that NMDA activation led to the production of OH in the striatum of freely moving rats which could be blocked by inhibitors of both NOS and PKC. Since NMDA increased NOS activity, an increase in L-CIT levels would be expected. The
150 Chapter 4: Discussion lack of an effect on L-CIT is, therefore, difficult to consolidate, unless the extracellular L-
CIT levels are a poor indicator of NOS activity in vivo.
Although AMPA receptor activation caused the release of L-ARG into the extracellular space, it would seem that this alone was insufficient to activate NOS. AMPA elicited a fourfold increase in L-ARG in the extracellular space, equivalent to a concentration of approximately 130/xM, much higher than the values for any of the isoforms of NOS (see section 1.4.1). The inability of the increased L-ARG to result in the activation of NOS may lie in the compartmentalisation of NOS and L-ARG. There may be a situation in the brain, analogous to the situation in endothelial cells, where eNOS is compartmentalised in caveolae. This hypothesis has been put forward to explain the so-called “arginine paradox”
(see section 1.4.5) and may go some way towards explaining the ineffectiveness of extra
L-ARG in the extracellular space. There may be a separate pool of L-ARG in the postsynaptic terminal that contains only a low concentration of L-ARG but a sufficient amount to act as substrate for NOS, that only needs replenishing once used. The concentration in this pool would have to be low if the immunocytochemical studies that localise L-ARG to glia are correct. Glia are most likely the sites of manufacture of L-ARG.
This would mean that L-ARG may not be the limiting factor for NO synthesis, which has recently been demonstrated in human alveolar macrophages (Muijsers et al., 2001).
Immunocyto- and histochemical analyses have demonstrated that caveolin- 1 , a principal protein component of caveolae organelles (see section 1.4.4) is expressed primarily in brain endothelial cells while caveolin-3 is expressed mainly in astrocytes (Ikezu et al., 1998), although a previous study demonstrated that the detergent-insoluble complexes isolated from astrocytes are composed of caveolin-1 a (Cameron et al, 1997). The existence of a neuron-specific form of caveolin is still a matter of debate (Masserini et al, 1999). It has been postulated that nNOS is localised in GABAergic intemeurons whereas the eNOS isoform is localised in pyramidal neurons in the hippocampus (Dinerman et al, 1994; O ’Dell et al, 1994). This may be pertinent in determining the localisation of caveolae, in particular since eNOS colocalises with caveolin-1 in endothelial cells. Another recent study has shown that L-ARG transport determines, at least in part, NO production under both basal and stimulated conditions in intact cultured BAECs (Hardy and May, 2002). The values of high affinity L-ARG transport systems range between 100 and 150jnM (Wu and
151 Chapter 4: Discussion
Morris, 1998), while low affinity transport has been documented with a of 5.9mM (Hosoya et ai, 1998). Because the concentration of L-ARG was increased to 130/xM with no effect on NOS this may indicate that there is only low affinity uptake into the separate compartment. As mentioned in section 1.3, the transport of L-ARG most likely occurs via system y^LAT2 in glia and system y^ in neurons (Wiesinger, 2001). Figure 4.1 depicts a representation of the proposed hypothesis for the compartmentalisation of L-ARG. L-CIT trafficking by system L is depicted (see table 1.1), as is thought to be the case in neurons
(Schmidlin et al, 2000).Yamada and Nabeshima (1997a), however, infused L-ARG into the cerebellum and measured the levels of NO/. A concentration of ImM, but not O.lmM, resulted in increased levels in the dialysates. Perhaps the recovery of L-ARG through their probes was higher than 1 0 % (see table 2 . 1 ), resulting in extracellular concentrations higher than 100/xM. However, when low affinity transport is considered a much higher concentration of L-ARG would have had to be infused. Nevertheless, the infusion of L- ARG resulted in increased NO/ levels, indicative of activation of NOS. Perhaps the situation in the cerebellum is different somehow from the situation in the hippocampus.
The hypothesis of compartmentalisation is not completely unprecedented. The urea cycle enzymes have been shown to be spatially organised within cells in such a way that intermediates can be efficiently transferred between them. The channeling of both L-ARG between AL and arginase and argininosuccinate between AS and ALhas been demonstrated
(Cheung et al, 1989). This supramolecular organisation has been termed a “metabolon” (Watford, 1989), although it is totally unexplored whether such a situation exists in brain cells or whether the L-CIT-NQ cycle could constitute a metabolon of its own (Wiesinger,
2001).
152 Chapter 4: Discussion
ITUMINAI
.-AR(J
.-ARC.
NO
NADP I -ARC,
Figure 4.1: An adaptation of figure 1.12 to show how L-ARG and L-
CIT may be trafficked in the CNS. A small concentration of L-ARG may
exist in neurons to act as the substrate for NOS. N= NMDA, A= AMPA, L= L type amino acid transporter, y^LAT2= y^L amino acid transporter 2.
The ability of NMDA to evoke the release of L-GLU has been shown in some studies to be dependent on the activation of NOS. Bogdanov and Wurtman (1997) found that the striatal infusion of NMDA resulted in concentration-dependent increases in L-GLU which could be blocked by L-NAME. An in vitro study using primary cultures of cerebellar granule cells demonstrated that the application of NMDA resulted in increased L-GLU, [Ca“^]j, NO and cGMP. The increases in L-GLU and [Ca^^Jj were only partially blocked by L-NMA (Oh et a i, 1999). Matsuo et al. (2001) have reported similar effects in the nucleus tractus Solaris. They also found that NO/ were increased with NMDA with a blockade by L-NAME, although studies with transgenic mice have shown that the increased L-GLU is independent of nNOS (Kano et a i, 1998). However, when Yamada and Nabeshima (1997b) calculated
153 Chapter 4: Discussion the levels of total N O / in dialysates as a measure of NOS activity, they found that NMDA, AMPA and trans-ACFD all increased NO/ in the cerebellum, although only the NMDA mediated response was reversed by NOS inhibition. They postulated that AMPA and trans- ACPD may be capable of increasing NO production through a NOS independent mechanism. The ability of the glutamatergic agonists to cause NO production in the ventral hippocampus was investigated.
4.3 NO production
Garthwaite et al. (1988) were the first to postulate that NMDA, acting postsynaptically, activates NOS to produce NO. They drew this conclusion from results obtained from cerebellar cells where the application of NMDA caused the release of a diffusible messenger, which was strikingly similar to EDRF in nature, that was responsible for the increased levels of cGMP. The cGMP content has since been used as an indirect measure of NO activity. It has now been demonstrated that the application of both trans-ACPD and AMPA to cerebellar slices from adult rats leads to increases in the cGMP content (Okada,
1992). The production of NO by NMDA has also been implicated in the increased L-GLU levels seen in the guinea-pig hippocampus. The application of 1-200/iM NMDA increased the release of L-GLU from the dentate gyrus in a concentration-dependent manner, a response which was inhibited by the application of L-NNA, with recovery by the addition of L-ARG. This, however, was not the case in the CAl region where there was a similar concentration-dependent increase in L-GLU which remained unaffected by L-NNA (Nei et al, 1996). The area investigated herein, the ventral hippocampus, corresponds mainly to the CA3 region (see figure 2.5).
In the study reported here the results of the NO experiment correlated with the results of the
NOS assay (see figure 3.14). As expected NMDA elicited an increase in NO production while AMPA was ineffective. The effect of NMDA, again, was blocked by D-AP5. From these results it can be hypothesised that NMDA receptor activation causes, as well as an increase in L-GLU, NOS activation and the production of NO. Given that the infusion of
NMDA did not elicit an increase in L-ARG in the extracellular space while NO production was increased threefold adds weight to the theory that there may be a separate pool of L-
154 Chapter 4: Discussion
ARG which is independent of the concentration in the extracellular space. Evidence suggests that NMDA receptor activation causes the release of L-GLU in a concentration- dependent manner. Coupled with the fact that when L-GLU is released presynaptically it can reach concentrations of l-3mM it may be that only a large concentration of L-GLU is sufficient to overcome any clearance mechanisms present. It may be, therefore, that the concentration of L-GLU governs the requirements for L-ARG and that the concentration needs to be high. Hence, only a large concentration of NMDA infused into the hippocampus would elicit a large enough increase in L-GLU to activate glial AMPA receptors to cause the release of L-ARG, although high concentrations of NMDA (10- lOOmM) have also been linked to excitotoxicity (Vanicky et ai, 1998). The prolonged NMDA receptor activation would cause enhanced NOS activity and an increased demand for L-ARG. Because of this increased demand the low concentration in the separate pool would need to be replenished. The L-ARG released upon glial AMPA receptor activation could then be used for this. Figure 4.2 shows a representation of this theory. It has been established that Na^-dependent transport of amino acids (including L-GLN, L-ARG and L-
GLU; see table 1.1) displays “adaptive regulation” where upregulation is exhibited during amino acid deprivation and downregulation occurs during amino acid supplementation (Taylor et al, 1996). A high concentration of L-GLU would, therefore, cause downregulation of GLT-1 and allow activation of AMPA receptors to cause L-ARG release. The concentration of L-GLN may also affect the transport of L-ARG. L-GLN has been shown to inhibit eNOS in BAECs (Wu et al, 2001) while system y^LAT2 has been demonstrated to mediate L-ARG efflux in exchange with L-GLN (Broer et al, 2000).
Downregulation of GLT-1 would effectively stop the production of L-GLN from L-GLU.
In order to maintain the glial levels of L-GLN perhaps system y^LAT2 attempts to compensate and indirectly results in the release of L-ARG in the process. However, for this to be the case whenever L-ARG was increased in the extracellular space there would have been a concomitant decrease in the L-GLN levels. This was not the case (results not shown). The basal levels of L-GLN in the extracellular space are exceedingly high
(190/iM), essentially making it more difficult to detect any changes. It is possible, however, that the intracellular concentration of L-GLN may be more important in governing the activity of y^LAT2. Alternatively, the concentration of NO may govern the release of L- ARG in vivo and this was investigated using the NO donor SNAP.
155 Chapter 4: Discussion
I UMIN \l
Hull L-AR(i C'a
NO -AR(i O,
1)1 \I)R I I NADP
NADPII
Figure 4.2: Schematic representation of how the extracellular L-GLU
concentration may govern the supply of L-ARG. The solid lines represent the situation at low L-GLU levels while the broken lines represent the situation at higher concentrations. N= NMDA, A= AMPA,
GLT-1= L-GLU transporter 1, L= L type amino acid transporter, y^LAT2= y^L amino acid transporter.
4.4 NO and amino acid release
The infusion of 5mM SNAP resulted in a large increase (/xM) in the levels of NO in the extracellular space (see figure 3.15). Although this represents a relatively poor recovery through the dialysis membrane, levels were still increased to 100000% of basal. The levels of NO remained increased for the rest of the experiment, even after the infusion had ceased. Unless the presence of SNAP in the extracellular space was prolonged somehow this may indicate that a large concentration of NO has a positive feedback effect on its own
156 Chapter 4: Discussion production. However, there is more evidence for feedback inhibition of NOS by NO,
Feedback inhibition of iNOS in rat and mouse macrophage cell lines (Griscavage et al, 1993; Assreuy et al, 1993), eNOS in vascular endothelial cells (Buga et al 1993; Ravichandran et al, 1995) as well as constitutive NOS from rat cerebellum (Rogers and Ignarro, 1992) has been demonstrated using a variety of NO donors. On the other hand, evidence is emerging for a positive feedback role of NO on eNOS gene expression.
Yuhanna et al (1999) demonstrated that exogenous application of NO to pulmonary artery endothelial cells (PAECs) resulted in increased eNOS protein expression and increased
NOS activity. Similar results were achieved by Chen gf a/. (2001). Nevertheless, alterations in gene expression are unlikely to emerge within the time frame of the experiments used in this study. Although the t , / 2 of NO is a few seconds under physiological conditions, it is not known whether the product of SNAP breakdown is NO*, NO^ or NO (see section
1.7.2). It is possible that supraphysiological concentrations of NO^ or NO take time to clear.
NO has been shown to modulate the release of several neurotransmitters (see section 1.9.5). The increased levels of NO resulted in decreased GAB A levels, had no effect on L-ARG while there was a biphasic effect on L-GLU (see figure 3.16). The effects on G ABA and L-GLU were blocked using lOfiM ODQ (see figure 3.17) indicating that the responses were mediated by cGMP. A biphasic effect on L-GLU levels is not unprecedented. In agreement with this study, Kraus and Prast (2002) found that PAPA/ NO resulted in biphasic L-GLU release in the nucleus accumbens. NO donors have resulted in increased L-GLU levels in several studies (Lawrence and Jarrott, 1993; Guevara-Guzman et al, 1994; Horn et al, 1994; Segovia et al, 1994; Ohta et al, 1996; Trabace and Kendrick, 2000), although the majority of these studies also found that G ABA levels were increased. The increased L-
GLU levels have been attributed to carrier mediated release associated with an inhibition of mitochondrial respiration (McNaught and Brown, 1998), the decreased levels to the inhibition of exocytotic processes (Sequeira et al, 1997). GAB A levels were decreased as soon as the NO levels were increased, this is contrary to the majority of the literature but may be indicative of a more excitatory role for NO in the brain.
In order to investigate whether tonic glutamatergic agoni sm affected the release of L-ARG,
157 Chapter 4: Discussion
L-GLU or G ABA, D-AP5 and CNQX were coinfused into the hippocampus. When the antagonists were infused there was no effect on L-ARG, L-CIT or G ABA levels, but there was a threefold increase in L-GLU (see figure 3.18). This increase in L-GLU was insufficient to elicit effects on mGLURs since there was no decrease in GABA levels.
However, upon the coinfusion of SNAP the L-GLU levels increased further to 400% of basal. When SNAP was coinfused there was also a significant increase in L-ARG with a similar increase in L-CIT. The release of L-ARG must have been independent of AMPA receptors, although AMPA itself was capable of eliciting an increase in L-ARG even in the presence of CNQX (see figure 3.8). GABA levels remained unchanged throughout, indicating that the decreased levels seen with SNAP alone were mediated by an effect of cGMP on ionotropic L-GLU receptors. The only documented effect of cGMP on ionotropic
L-GLU receptors is its ability to depress the responses of AMPA receptors and to inhibit EPSCs in hippocampal neurons, an effect that was independent of phosphorylation (Lei et ai, 2000). Inhibition of EPSCs would be expected to result in either a decrease in L-GLU or an increase in GABA. This was not the case here, however, where GABA levels were decreased and L-GLU levels were increased with the infusion of SNAP, the converse of what would be expected. It could also be hypothesised that depression of AMPA responses would decrease the extracellular L-ARG levels, if indeed tonic AMPA receptor activation controls L-ARG levels. Here, SNAP did not have an effect on L-ARG.
4.5 NOS inhibition
The effect of inhibiting NOS was investigated using L-NAME and 7-NI. Infusion of L-
NAME resulted in a twofold increase in L-GLU with a corresponding increase in L-ARG but had no effect on GABA. Another study documents the effect of L-NAME on L-ARG levels where direct intrastriatal infusion of ImM L-NAME also proved to increase L-ARG release and decrease L-CIT (Ohta et al, 1994). The authors postulated that this was suggestive of suppressed NOS activity due to the lack of formation of L-CIT from L-ARG, although this does not conform with the proposed theory that extracellular L-ARG does not govern the NOS reaction and that extracellular L-CIT may be a poor measure of NOS activity. 7-NI had no effect on L-GLU but caused decreased L-ARG and GABA. Any effect that the NOS inhibitors may have on L-ARG transport will be addressed later in this
158 Chapter 4: Discussion section. L-ARG is involved in several metabolic processes in the brain including the biosynthesis of GAB A via ornithine (and thence L-GLU; Pow and Crook, 1997). It follows, therefore, that the GABAergic intemeurons of the hippocampus have a high requirement for L-ARG. The decreased L-ARG with 7-NI may, therefore, have resulted in the decreased
GABA levels. However, if this were the case then the converse would be true: increased
L-ARG would result in increased GABA, which did not occur with the increased L-ARG levels with L-NAME. At low L-ARG concentrations NOS favours the production of H 2 O 2 ,
O 2 and NO, and hence O N O O . L-ARG has a documented IC 5 0 of I I/iM for the inhibition of H 2 O 2 production by purified brain NOS (Heinzel et ai, 1992). The reduction in L-ARG levels to 50% of basal, equivalent to approximately I 6 /iM (see tables 2.1 and 2.3), resulting from 7-NI treatment could still therefore result in NOS producing ONOO and not NO. However, ONOO would not have shown up in the chemiluminescence reaction used in these experiments; a chemiluminescence reaction with luminol would have been required
(Tarpey and Fridovich, 2001). This also disagrees with the proposed lack of dependence of NOS on the extracellular L-ARG levels.
When both inhibitors were infused together there was no effect on any of the amino acids
(see figure 3.21). The contrasts in the amino acid levels could well be due to the different mechanisms of action of each inhibitor. According to section 1.6.2 and table 1.2, L-NAME competes for L-ARG at its binding site while 7-NI has been found to have effects on haem,
L-ARG and BH 4 binding. The action of L-NAME is non-specific with regards to the Ca^^- dependent isoforms (Virgili et al, 1999) while, although 7-NI is non-specific in vitro, it is specific for nNOS in vivo, with an IC 5 0 of I.5/>tM for the hippocampus in vitro (MacKenzie et al., 1994). At high concentrations, however, it is possible that both isoforms are inhibited (Kano et at., 1998), although, given the low recovery through the probe, 7-NI is likely more specific for nNOS in the experiments herein.
Both of the inhibitors alone resulted in increased NOS activity (see figure 3.22) and increased NO production (see figure 3.24) while the coinfusion of both inhibitors together had no effect on NOS but resulted in a reduction of NO production. The development of
NOS knockout mice (designated nNOS -/-, eNOS -/- and iNOS -/-) may provide a clue as to the reason for this. Mechanisms to compensate for the deleted genes exist in mutant
159 Chapter 4: Discussion
mice, where upregulation of the alternate constitutive isoform substitutes for the other
(Huang and Fishman, 1996). nNOS -/- animals displayed normal LTP induced by weak intensity tetanic stimulation and immunocytochemical studies indicated that eNOS was expressed in CAl neurons (O’Dell etai, 1994). However, eNOS -/- animals also displayed normal LTP. LTP was only abolished in the stratum radiatum of CAl in nNOS and eNOS double-knockout animals, although LTP was still unaffected in the stratum oriens (Son et al, 1996). Because 7-NI is specific for nNOS in vivo and because the infusion of 7-NI resulted in an increase in NOS activity and NO production it could be that, while upregulation at the level of gene expression is unlikely, the activity of eNOS was upregulated. This theory does not hold for L-NAME treatment, however, given that L-
NAME is supposedly non-specific for the Ca^^-dependent isoforms, although Miller et al (1996) found that the administration of L-NAME in drinking water increased intestinal
NO 2 levels in rats, which the authors postulated was due to the induction of iNOS gene expression.
The contrasting effects of each inhibitor could be due to the different subcellular localisation of each NOS isoform. As mentioned previously, it has been suggested that nNOS is localised in GABAergic intemeurons whereas the eNOS isoform is localised in pyramidal neurons in the hippocampus (Dinerman et al, 1994; O’Dell et al, 1994). Glutamatergic synapses are found between Schaffer collaterals and eNOS containing cells as well as within local connections between pyramidal outputs and nNOS containing
GABAergic intemeurons (Kano et al, 1998). L-NAME is supposedly non-specific for the constitutive isoforms and, as such, should have resulted in total inhibition, which was not the case. However, the i.c.v. injection of L-NAME failed to block the induction of LTP in the dentate gyrus, measured as the change in the slope of the early rising phase of the field
EPSP. Failure to block LTP occurred following both a strong and a weak tetanus
(Bannerman et al, 1994). The authors postulated that this offers no support to the role of
NOS in the induction of the synaptic component of LTP. They also found, however, that
L-NAME resulted in a small transient increase in the baseline slope of the field EPSP. If
L-NAME was not actually causing the inhibition of NOS and actually increasing NOS activity and hence NO production then these results do conform with the theory of the involvement of NO in LTP. The systemic administration of L-NAME or 7-NI has been
160 Chapter 4: Discussion shown in several studies to decrease NOS activity in the brain. Both i.v. (ladecola et ai, 1994; Traystman et al, 1995) and intraperitoneal (i.p.; Desvignes et al, 1997; Ayers et al, 1997; Chen et al, 1998) administration have been shown to decrease NOS activity in various brain regions. Ayers et al (1997) also investigated the i.c.v. administration of L-
NAME and found that NOS activity was abolished 90min following injection. The fact that Salter et al (1995) found that the i.c.v. injection of L-NAME decreased NOS levels in ipsilateral brain areas was postulated in section 4.2 to have been due to not taking the effect of the surgical procedure into account. However, a study by Greenberg et al (1997) investigated the distribution of L-NNA following its i.c.v. administration and found that it only distributed to areas in close proximity to the ventricle, even 8 h after administration.
The authors concluded that the i.c.v. administration of NOS inhibitors may be ineffective if global inhibition of NOS is required. A ImM concentration of L-NAME has proved to inhibit NOS activity in all brain areas in vitro (Ayers et al, 1997), although this is apparently not the case in vivo.
L-NAME is thought to undergo enzymatic or non-enzymatic hydrolysis to form L-NNA (see section 1.6.2). In fact, the rat has been shown capable of hydrolysing L-NAME very rapidly leading to substantial quantities of L-NNA (Brouillet et al, 1995). While the transport of L-ARG in neurons is associated with both high and low affinity processes, L-
NNA transport is characterised by a single kinetic process with a value of 0.35mM (Hosoya et al, 1998). Both L-NNA and L-NAME are poor substrates for the cationic system y^ and it has been shown that a neutral amino acid transporter (system L) accounts for their uptake in macrophages (Baydoun and Mann, 1994; Schmidt et al, 1994), neurons (Schmidt et al, 1995) and endothelial cells (Schmidt et al, 1993) while system B®’^ is responsible in rabbit conjunctiva (Hosoya et al, 1998) and brush border membranes (Hatanaka et a l, 1999). If L-NAME is transported by system L in neurons and glia it would compete with L-CIT but would not affect transport of L-ARG. If this were the case then L-
NAME would be transported predominantly into astrocytes and not into neurons, unless the transporters became reversed. Reversal of Na^-dependent transporters has been documented in response to depolarisation (Szatkowski et a l, 1990) so, theoretically, L-NAME could be taken up by neurons in a use-dependent manner. However, under basal conditions the predominant uptake of L-NAME is likely to occur into glia, although the contrasting amino
161 Chapter 4: Discussion acid levels between the in vitro and in vivo experiments (decreased and increased, respectively) would contraindicate this and may be indicative of alternative breakdown and/ or uptake mechanisms in vivo. Perhaps in vivo L-NAME has an effect on GLT-1 equivalent to increasing the L-GLU concentration above threshold levels. Downregulation of GLT-1 would result in both increased L-GLU and L-ARG in the extracellular space. The results achieved using cortical glia would contradict this, however, given that L-NAME caused decreased L-ARG levels in the culture medium. Plus this theory does not explain why NOS activity was increased.
There is some evidence that NOS inhibitors may cause the production of NO non- enzymatically. Moroz et al. (1998) demonstrated using electron paramagnetic resonance and spin-trapping techniques that the production of NO can be driven by NOS inhibitors in central nervous and peripheral tissues of the gastropod molluscs Aplysia califomica and Pleurobranchea califomica. The authors postulated that the inhibitors could produce nitrites which have been shown to cause significant, non-enzymatic formation of NO in the ischaemic heart (Zweier gf a/., 1995), the GIT (Duncan era/., 1995; McKnight era/., 1997) and skin (Weller et a/., 1996). The reduction of nitrites by L-ascorbate, thiols or NADPH would result in the production of NO. The levels of L-ascorbate in the extracellular space can be as high as 0.5mM (Miele and Fillenz, 1996) with astrocytes containing k7mM
(Siushansian and Wilson, 1995). Plasma concentrations of L-ascorbate have been documented as 10-100/xM, which led Grünewald (1993) to hypothesise a two-stage uptake mechanism from blood to CSF and from CSF to neuropil. Moroz et al. (1998) found that L-ascorbate was the most potent reactant cofactor for potentiating NO release from NOS inhibitors. L-GLU induces the efflux of ascorbate into the extracellular space, an effect which can be eliminated using L-GLU reuptake blockers (Cammackgfa/., 1991). L-NAME caused an increase in the L-GLU levels in vivo which could equally have caused the efflux of ascorbate. However, L-NAME and 7-NI both increased the activity of NOS itself to result in NO production in the hippocampus, indicating a direct enzymatic effect.
Greenberg et al. (1995) evaluated the effects of the in vivo and in vitro administration of L-NAME, D-NAME and L-NNA and clearly demonstrated increased levels of RNIs when measured either with chemiluminescence or with the modified Griess reaction. Although this could provide an explanation for the NO results it still does not explain, however, why
162 Chapter 4: Discussion the NOS activity was also increased. The effects of the inhibitors on basal NOS activity and
NO production are exceedingly difficult to consolidate.
Because NMDA has been shown previously (Luo and Vincent, 1994) and in this study (see figures 3.13 and 3.14) to increase NOS activity and the production of NO, the ability of the inhibitors to block these effects was investigated. The basal levels of NO are generally too low to measure in the majority of assays so most studies stimulate NO production using
NMDA. L-NAME treatment blocked the increased NOS activity but did not prevent the increased levels of NO (see figure 3.23). The converse was true with 7-NI, where NOS activity was increased but there was no change in the NO level (see figure 3.25). Together
NOS activity remained at basal while NO production was decreased. This could indicate that L-NAME does indeed result in the non-enzymatic formation of NO, given the increase in NO with no change in NOS, while 7-NI could result in the formation of ONOO , given the increase in NOS without increasing NO. However, it could have more to do with the localisation of each constitutive isoform. The localisation of nNOS to GABAergic intemeurons would suggest that these neurons express functional NMDA receptors, due to the association of NR2 and nNOS with PSD-95 via PDZ domains (see section 1.4.4). In fact, Bernardo (1995) has demonstrated that L-GLU activation of slow IPSPs can involve significant interaction with NMDA receptors on inhibitory cells in CAl pyramidal neurons.
A possible arrangement of excitatory and inhibitory neurons in the hippocampus is represented in figure 4.3. The infusion of NMDA could therefore activate both excitatory and inhibitory neurons in the hippocampus to produce NO. Because each of the inhibitors only seems to affect one element of the NMDA response, whilst in combination the NMDA response was completely abolished, this may be indicative of alternate uptake and/ or metabolic pathways when the inhibitors are administered directly into the hippocampus. In fact, the in vivo specificity of 7-NI may be attributable to its selective uptake into intemeurons, although there is little in the literature conceming how 7-NI enters cells. The differential localisation may also offer up an explanation for the different effects of each inhibitor on the amino acid levels. 7-NI decreases GABA while L-NAME increases L-GLU with each inhibitor having contrasting effects on L-ARG, 7-NI decreasing levels and L-
NAME increasing levels, respectively. The reason(s) for the altemative effects remains to be elucidated.
163 Chapter 4: Discussion
I \( 11 M oin (il 1 r.Wl M l R(iU I'RI S Y \ \l'l K I I K \ll\.\l
\S I ROC VI I C’A PON ‘K< )( SS
L-ARG S\n;ipsin ^ <:i I Q
INI IIMIK )IQ- 1,-GI I NMDA (lAP \l k(.K IN 11 RM I IR( ) \
m G I.U K I 5
I -C l I
Figure 4.3: A schematic representation of the localisation of eNOS and nNOS in
the brain. nNOS is shown associated to the NR2 subunit via PSD-95 in GABAergic intemeurons and with CAPON and synapsin in the presynaptic terminal, while eNOS is shown myristoylated to the postsynaptic membrane. iNOS is predominantly cytosolic and expressed in glia, although evidence is emerging for the localisation of
eNOS and nNOS in glia.
While the majority of studies document that iNOS is the major isoform present in glia, there is some evidence for both nNOS and eNOS expression in astrocytes. Togashi et al. ( 1997) have reported that nNOS is the predominant isoform constituti vely expressed in glia and that the NO derived from nNOS is responsible for the inhibition of NF-kB (see section 1.5). The glial localisation of nNOS has been confirmed by Kawakami et al. (1998) and Caggiano and Krai g (1998). Upregulation of nNOS expression has also been found in the
164 Chapter 4: Discussion
Spinal cord and subcortical white matter of ALS animals (Anneser et ah, 2001) and the hippocampus and entorhinal cortex of AD patients (Simic et al, 2000). Upregulation of eNOS has been demonstrated in reactive astrocytes in virally infected regions of mouse brain (Bama etal, 1996). The i.p. injection of LPS has been shown to induce eNOS mRNA in rat brain astrocytes with an increase in eNOS protein (Iwase et al, 2000). Compensatory mechanisms may therefore be responsible for the actions of the inhibitors; induction of iNOS in response to L-NAME and upregulation of eNOS expression in response to 7-NI.
Because the levels of Ca^^-independent NOS activity measured with the NOS assay were generally so low it was not feasible to attain reliable results. It is, therefore, not possible to say for certain that it was iNOS responsible for the increased NO levels. However, when both inhibitors were coinfused NOS activity remained at basal while NO levels were decreased. This would contradict a role for iNOS induction in response to L-NAME treatment. Why the inhibitors have converse effects on the NMDA response in vivo is unclear but the assumption that L-NAME and 7-NI inhibit NOS in vivo is an oversimplification. The localisation of nNOS and eNOS in glia would again support the theory of compartmentalisation of L-ARG and NOS- otherwise the eNOS and nNOS in glia would be activated at all times.
4.6 Future directions
To summarise, we have shown that, while L-ARG is present in high concentrations in glia in vivo, the extracellular concentration does not necessarily drive the NOS reaction. Overall, the L-GLU concentration may govern both the activity of NOS and the release of L-ARG but only when the concentration reaches above threshold to overcome transport and diffusion. This could be investigated further via the direct infusion of large concentrations of L-GLU, although the excitotoxic nature of L-GLU may limit this to in vitro studies.
Large concentrations of NO increased L-GLU and decreased GABA, indicating a possible role for NO in the promotion of excitatory transmission. It is clear that the control of transmission in vivo is exceedingly complex and, while microdialysis is an invaluable tool for sampling the extracellular space (see table 2.2), it may be wrong to assume that the levels of substances here accurately reflects the true intracellular situation in the neurons and glia. The localisation of L-ARG in caveolae in the brain clearly requires more attention
165 Chapter 4: Discussion although care must also be taken when relating in vitro findings with the situation in vivo. The paradoxical effects of the NOS inhibitors on NO production should be investigated.
Given the wide use of microdialysis, few studies have employed it as a means of delivering these drugs, although Yamada and Nabeshima (1997b) infused ImM L-NMA directly into the rat cerebellum which resulted in decreased NOS activity. The uptake of NOS inhibitors in vivo could be examined to elucidate whether there really are differential uptake mechanisms present in vivo which could account for their differing activities. An attractive prospect would be the coupling of microdialysis with transgenic mice, although this would be complicated by the requirement for full anaesthesia throughout the experiment.
Alternatively, “knock-down” animals could be used, where the expression of one isoform of the enzyme could be prevented via the administration of anti sense or mismatch oligonucleotides. This would bypass the problem of developmental upregulation of the alternate isoform. Another approach would be to perform microdialysis into a brain region which only expresses one NOS isoform, such as the cerebellum, which only expresses nNOS, which would rule both eNOS and iNOS out of the equation.
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