NOVEL ORGANIC NlTRATES AS POSSIBLE NEUROPROTECTANTS IN AN hV VITRO MODEL OF S'I'ROKE IN THE RAT HPPOCAMPUS
Allison Elizabeth Clarke
A thesis submitted to the Department of Pharmacology and Toxicology in conformity with the requirements for the degree of Master of Science
Queen's University Kingston, Ontario, Canada May, 2001
Copyright O Allison Elizabeth Clarke, 2001 National Library Bibliothèque nationale ($1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 WeUingîon Street 395, nie Wellington Ottawa ON KIA ON4 OttawaON K1AON4 Canada canada
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The auîhor retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Allison Elizabeth Clarke: Novel Organic Nitrates as Possible Neuroprotectants in an In Vitro Mode1 of Stroke in the Rat Hippocampus. M.Sc. Thesis, Queen's University, Kingston, Ontario, Canada, May, 2001.
Novel organic nitrates are a group of established nitric oxide donors based on the chemical structure of glyceryl trinitrite (GTN). It has been previously suggested that nitnc oxide can potentially play a neuroprotective role in ischemia due to its ability to: inhibit ~a'+influx through the N-methyl-D-aspartate (NMDA) receptor, act as an antioxidant and increase cGMP levels in the neuron. A group of investigators examining the neuroprotective properties of the secreted fom of amyloid precursor protein (sAPPu) have discovered a protein kinase G (PKG)dependent. cGMP mediated mechanism. They have postulated that this neuroprotection is due to: activation of K' channels, inhibition of the NMDA receptor and enhancement of glucose and glutamate uptake into synaptic compartrnents. This thesis tested the hypothesis that novel organic nitrates are neuroprotective in an in vitro model of stroke possibly due to a cGMP mediated mechanism. The first objective was to establish the in vitro model of stroke with respect to testing of known neuroprotectants such as hypothermia and determining an appropriate length of insult. A half hour insult time was chosen because it caused a subrnavimal increase in lactate dehydrogenase (LDH) release. LDH release was used as a marker of ce11 viability. The induction of hypothermia during the ischemic insult completely protected the hippocarnpal slices from the in vitro iscliemic insult. The in vitro ischemic insult involved low[Oz] and low[glucose] in the incubation buffer. The second objective was to determine if the novel organic nitrates had any neuroprotective properties and if any observed neuroprotection was dependent upon cGMP generation. Three novel organic nitrates, GT-091, GT-094 and GT-310 were significantly protective against low[02] and low[glucose]. Similarly, the cGMP analogue, dibutyql cGMP was also neuroprotective in the same model suggesting that the neuroprotection observed with the novel organic nitrates may be due to a cGMP mediated mechanism. Unexpectantly, the neuroprotection provided by GT-094 could not be attenuated by CO-application of 1H- [ 1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. Additionally, GT-094 was unable to increase cGMP levels in hippocampal slices der hypoxialhypoglycemia as assessed by a cGMF radioirnrnunoassay. These results indicate that the neuroprotective mechanism of GT-094 does not involve cGMP generation. Interestingly, CAMP could mimic the neuroprotection observed with cGMP. A protein kinase A (PU) or PKG inhibitor could not attenuate the neuroprotective effects of CAMP and cGMP, respectively. In summary, these findings suggest GT-094 and the cyclic nucleotides are exerting neuroprotection by two separate and independent mechanisms. Furthemore, these results indicate that the cyclic nucleotides are acting by a pathway that does not involve PKA or PKG activation. 1 would like to take this opportunity to thank my supervisor Dr. Roland J. Boegman for al1 his guidance and insight in the completion of this thesis project. 1 would also like to thank Dr. James N. Reynolds for al1 his invaluable help. 1would like to acknowledge and thank Lihua Xue and Diane Andenon for generously providing me with some of the data contained in this thesis. Their help is greatly appreciated. Lihua Xue completed the LDH study on Sin4 chloride CO-administeredwith ODQ as well as the cresyl violet staining with the hippocampal slices treated with Sin-1 chlonde and GT-094. Diane Anderson provided me with the RIA data on GT-094 and ODQ. 1 would also like to thank the other members of the GoBang team: Dr. Jhamandas. Dr. Bennett, Dr. Thatcher, Margo Poklewska-Koziell and Adrian Nicolescu.
GoBang Therapeutics and Queen's Medical Discoveries primarily financed this research. Queen's School of Graduate Studies provided personal funding. This thesis is dedicated to my farnily. TABLE OF CONTENTS Page.. Abstract 11
S.. Acknowledgrnents 111
Table of Contents v
List of Figures vii
List of Abbreviations and Symbols ix
1 INTRODUCTION 1
1.1 S tatement of Research Problem 1 1.2 Ischemic Ce11 Damage 3
1.2.1 NMDA Receptor Antagonists 5 1.2.2 Metabotropic Glutamate Receptors 7
1.3 Nitric Oxide 8
1.3.1 Nitric Oxide Synthase Antagonists 10 1.3.2 Nitric Oxide and Neurotoxicity 11 1 -3.3 Peroxynitrite 12 1.3.4 Nitrk Oxide Production and Neuronal Outcome 12 1.3.5 Nitric Oxide and Neuroprotection 12 1.3.6 Niûic Oxide and NMDA Receptor Inhibition 14 Antioxidant Properties of Nitric Oxide 15
18
cGMP and Neurotoxicity 18 cGMP and Neuroprotection 19 cGMP and P-Arnyloid Precursor Protein 20 Glutamate and Glucose Uptake 21 Inhibition of the NMDA Receptor 2 1 Potassium Channels 22
5 Cyclic P{ucleotide Gated Ion ChanneIs 23 1.6 Phosphodiesterases and Ischemia 23 1.7 CAMP 25 1.8 Guanine Nucleotide Exchange Factors 26 1.9 Novel Organic Nitrates 27 1.10 In Vitro Mode1 of kchernia 29 1.1 1 Research Rationale, Hypothesis and Objectives
2 METHODS AND MATERIALS
2.1 Chemical Solutions 2.2 Experimental Animals 2.3 Tissue Isolation 2.4 LDH Assay 2.5 Protein Determination 2.6 cGMP Radioimmunoassay 2.7 Cresyl Violet Staining 2.8 Data Analysis
3 RESULTS
3.1 Time Course of LDH Release 3.2 Temperature and LDH Release 3.3 Conventional NO Donors 3.4 Novel Organic Nitrates 3.5 Synthetic cGMP Analogues
3 S.1 Dibutyryl cGMP and Rp-8-pCPT-cGMP
3.6 Synthetic CAMP Analogues
3.6.1 Dibutyryl CAMPand H-89
3.7 Dibutyiyl CAMP and Forskolin 3.8 ODQ 3.9 cGMP Radioirnmunoassay 3.10 Cresyl Violet Staining
4 DISCUSSION
4.1 Future Research Directions
Re ferences
Vita LIST OF FIGURES Page 1.1 Sumrnary of hypoxic/hypoglycemic injury 6
1.2 A schematic depicting NO production by NOS
1.3 Summary of the neurotoxic properties of NO
1.4 Sumrnary of the neuroprotective properties of NO
1.5 Chemical structure and proposed biotransformation of the novel organic nitrates
3.1 Rat hippocarnpal slices exposed to differing lengths of hypoxiahypoglycemia
3.2 Rat hippocampal slices exposed to hypothermie conditions
3.3 Rat hippocampal slices treated with Sin-1 chlonde
3.4 Rat hippocarnpal slices treated with NO-exhausted Sin-1 chloride
3.5 Rat hippocarnpal slices treated with GSNO
3.6 Rat hippocampal slices treated with GT-09 1
3.7 Rat hippocampal slices treated with GT-094
3.8 Rat hippocampal slices treated with GT-3 10
3.9 Hippocarnpal slices treated with 1mM 8-bromo-cGMP
3.10 Rat hippocarnpal slices treated with dibutyryl cGMP
3.1 1 Rat hippocampal slices treated with cGMP
3.12 Rat hippocarnpal slices treated with 8-pCPT-cGMP
3.13 Rat hippocampal slices treated with dibutyryl cGMP and Rp-8-pCPT-cGMP
3.14 Rat hippocarnpal slices treated with dibutyryl CAMP 3.15 Rat hippocampal slices treated with I mM 8-bromo-c AMP
3.16 Rat hippocampal slices treated with dibutyryI CAMP and H-89
3.1 7 Rat hippocampal slices treated with 50pM fonkolin and 100pM dibutyryl cGMP
3.1 8 Rat hippocampal slices treated with 50pM GT-094 and 0.5pM ODQ
3.19 Rat hippocampal slices treated with Sin- 1 chloride and ODQ
3.20 Rat hippocarnpal slices treated with ODQ
3.2 1 Concentration of cGMP in rat hippocampal slices
3.22 Rat hippocampal slices stained with cresyl violet
4.1 Proposed mechanism of action of the novel organic nitrates
4.2 Proposed mechanism of neuroprotection mediated by cGMP LIST OF ABBREVIATIONS AND SYMBOLS a alpha AP arnyloid beta peptide AMPA a-arnino-3-hydroxy-5-methyl-4-isoxazole propionate ANOVA analysis of variance P beta BSA bovine semalbumin ca2+ calcium ion CaM calmodulin CaMK II calcium-calmodulin-dependent protein kinase II CAMP cyclic adenosine 3'5 '-monophosphate CBF cerebral blood flow cGMP cyclic guanosine 3 ' 5 '-monophosphate CNS centnl nervous system CPT CAMP 8-(4-c hlorop heny1thio)-adenosine 3 ' :5 ' -cyclic- monophosphate CREB cyclic AMP-responsive element binding protein DHPG 3'5-dihydroxyphenylglycine DMSO dimethyl sulfoxide eNOS endothelial nitric oxide Epac exchange protein directly activated by cyclic AMP FAD flavin adenine dinucleotide ~e'* ferrous iron FMN flavin mononucleotide g gram GEF guanine nucleo tide exchange factor(s) GT GoBang Therapeutics GTN glyceryl trinitrite GTP guanosine triphosphate GSNO S-nitrosogIutathione H-89 N-[2-(p-bromocinnarnylamino)ethyl]-5- isoquinolinesulfonamide HNE 4-hydroxynonenal Hz02 hydrogen peroxide ICP intracranial pressure iNOS inducible nitric oxide in vitro in glass in vivo in the living body potassium ion activation constant; concentration required for half- maximal activation potassium chloride potassium phosphate, monobasic inhibition constant; concentration required for half- maximal inhbtion KREB modified Krebs-Henseleit bicarbonate solution kilogram(s) lipophilicity defined as the extrapolated capacity factor for 100% water in isocratic reversed-phase HPLC L ii tre(s) LDH lactate dehydrogenase L-NAME bf-nitro-L-arginine methyl ester M molar milligram(s) magnesium ion metabotropic glutamate receptor magnesium sulfate 8-para-chlorop henylthio-cGMP microgram(s) micrometre(s) min minute MK-80 I (+)-MK-80 1 maieate rnillilitre(s) rnillimolar. rnmo 1 millimole(s) n number of determinations N? molecular nitrogen Na' sodium ion NaCl sodium chloride NADH nicotinamide adenine dinucleotide reduced form NADPH nicotinamide adenine dinucleotide phosphate Na.iiC03 sodium bicarbonate NaOH sodium hydroxide NMDA N-methyl-D-aspartate NMDA-R N-methyl-D-aspartate receptor NNA N-a-ni tro-L-arginine nNOS neuronal niûic oxide NO nitnc oxide NOS nitric oxide synthase 02 molecular oxygen of- superoxide anion ODQ 1H-[1,2,4]oxadiazolo[4,3-alquinoxalin- 1 -one ONOO' peroxynitrite PARS poly(ADP-ribose) synthase PBS phosphate buffered saline PDE phosphodiesterase(s) negative base 10 logar-ithrn of hydrogen ion concentration PKA protein kinase A PKC protein kinase C PKG protein kinase G PLAz phospholipase Ar pmol picornole(s) PSD-95 postsynaptic density protein-95 RIA radioimrnunoassay ROS reactive oxygen species Rp-8-pCPT-cGMP (Rp)-8-@ara-chlorophenyIthio)guanosine-3 ' ,Y- cyclic rnonophosphorothioate sAPPa secreted form of amyloid precursor protein 4C3HPG s-4-carbox y-3-hydroxy-phenylgl ycine SEM standard enor of the mean sGC soluble guanylyl cyclase Sin- 1 3-morpholino-sydnonimine SOD superoxide dismutase Tm tetrahydrobiopterin VSCC voltage-dependent calcium channels XO xanthine oxidase OC degrees centigrade (Celsius) Y0 percent + plus or minus a3 registered trademark 1. rnTRODUCTION
1.1 Statement of the Research Problem
Stroke is an inhibition of blood flow to the brain usually occuring as a result of a blood clot. Each year, stroke claims the lives of 15,000 people and severely debilitates another 350,000 individuals (Dr. Tony Hakimj , Neuroscience Stroke institute, Neuroscience Seminar). During stroke glucose and oxygen are unable to gain access to the brain, which sets the stage for cellular energy store depletion and ce11 death. The consequences of stroke are magnified because many individuals misinterpret the symptoms of stroke, which include headache, nausea, diuiness, blurred vision and muscle weakness, ofien causing them to wait hours before they seek medical attention. Treatrnents have focused on removing the blood clot, either surgically or with dnigs such as tissue plasminogen activator. Although this does improve outcorne, these treatments are only effective when administered within two houn after the Srst signs of stroke. At present there are no clinically available treatments to inhibit neuronal ce11 loss after the stroke. The problem will become more acute as the baby boomers approach middle age. Novel treaments need to be explored in order to arrest this growing medical problem.
Excessive release of glutamate and overactivation of ionotropic glutamate receptos such as the N-rnethyl-D-aspartate (NMDA) receptor have been postulated to be the underlying mechanism of neuronal ce11 death due to ischemia. Calcium influx through the NMDA receptor activates nitric oxide synthase (NOS), which produces nitric oxide frorn the conversion of L-arginine to L-cimilline (see review: Yun H. et al., 1996). Several physiological processes such as vascular smooth muscle relaxation
(Huang et al., 1995), long term potentiation (Wu et al., 1997, see review: Hawkins et al., 1998), and even neurotoxicity (Almeida et al., 1998, Panahian et ai., 1996) have been associated with the production of nitric oxide. The role of nittic oxide in ischemic ce11 damage is controversial. It has been hypothesized that niûic oxide rnay act as an neurotoxin due io its ability to inhibit mitochondrial enzymes (Cassina et al.,
1996, Stadler et al., 199 1, Lizasoain et al., 1996), to cause DNA damage (2hang et al.,
1994) and to react with superoxide anion to form peroxynitrite (Endres et al., 1998).
Al1 of these events would increase cellular damage due to ischemia.
Convenely, nitric oxide has also been show to inhibit the NMDA receptor
(Manzoni et al., 1992), react with lipid radicals to form stable nitroso-compounds
(Rubbo et al., 1994) and increase the production of cGMP. These functions of nitric oxide al1 have the potential to be neuroprotective. The NMDA receptor is continuously activated in ischemic conditions due to the accumulation of glutamate in the synaptic cleR. Thus, inhibition of the NMDA receptor is expected to improve neuronal outcome following an ischemic insult. Additionally, in the reperfusion penod following ischemia, there is increased production of oxygen radicals which
leads to lipid peroxidation. Nitnc oxide's ability to react with lipid radicals to fom
stable compounds may inhibit lipid peroxidation and be beneficial in the treatment of
isc hemia.
Studies examining the role of cGMP in neuronal cell death have found
conflicting results. Some investigators have ascertained that treatment with cGMP
potentiates cell injury (Montoliu et al., 1999, Yonghong et al., 1997). Alternatively,
cGMP has also demonstrated neuroprotective properties (Moro et al.. 1998,
Garthwaite et al., 1988, Yoshioka et al., 2000, Mattson et al., 1999, Furukawa et al.,
1998, Barger et al., 1995, Furukawa et al., 1996). A group of researchers studying the
neuroprotective properties of the secreted fom of amyloid precursor protein observed
that the neuroprotection provided with the secreted form of amyloid precursor protein could be rnimicked by synthetic cGMP analogues and be attenuated by a PKG inhibitor (Mattson et al., 1999, Furukawa et al., 1998, Barger et al., 1995, Furukawa et al., 1996). They suggested that this neuroprotection is due to activation of K' channels (Fumkawa et al., 1996), inhibition of the NMDA receptor (Furukawa et al.,
1998) or increased uptake of glutamate and glucose into synaptic compartments
(Mattson et al., 1999).
GT-015 is part of a group of novel organic nitrates, which are synthetic nitric oxide donors, based on the chernical smicture of glyceryl trinitrate (GTN). GT-O15 was neuroprotective in an in vitro model of ischemia using hippocampal brain slices and in an in vivo middle cerebral artery occlusion model. in the itz vitro model, it was
found that inhibition of guanylyl cyclase attenuated the neuroprotection of GT-0 1 5
indicating that the neuroprotection observed with GT-015 was due to generation of
cGMP (Clarke et al., 2000). Therefore it was proposed that the effects of GT-015 are
due to production of cGMP. possibly by one of the aforementioned mechanisms.
The research hypothesis explored in this thesis postulates that novel organic
nitrates, which are established nitric oxide donors, are neuroprotective in an in vitro
model of stroke due to a cGMP-PKG mediated mechanism.
1.2 Ischemic Ceii Damage
In the initial stages of ischemic ce11 loss, there is a massive decrease in
energy stores. This occurs within 1 to 2 minutes of the cessation of blood flow
(Martin et al. 1994). This enormous decrease in energy reserves leads to neuronal
damage. Low levels of ATP contribute to alterations in ion homeostasis by inhibiting
the N~'/K' ATPase and activating the K' ATPase present on the neuronal membrane.
This results in an early rise in extracellular levels of K', which in tum causes
membrane depolarization. Using intracellular recordings in striatal qiny neurons exposed to glucose and oxygen deprivation, Calabresi and colleagues (1999), found that increases in ~a'and cal' levels mirrored membrane depolarization. This produces a dramatic rise in intracellular ~a'and cal' levels which further disnipts ion
homeostasis.
Glutamate, released by reversal of Na'/ glutamate transporter and ~a"
dependent exocytosis, has been postulated to be the major player in ischemic ce11
death (Longuemare et al., 1995, Kimura et al., 1998). This process has been termed
glutamate excitotoxicity. Excessive release of glutamate overactivates several
glutamatergic receptors and in particular, the ionotropic NMDA receptor, which has
been associated with neurotoxicity. Calcium influx through the NMDA receptor leads
to a toxic accumulation of ca2' in the neuron. Ellrén and colleagues (1989)
discovered that NMDA toxicity in pyramidal cells of the immature hippocampus was
~a"dependent as accessed by rnorpholo@cal analysis and LDH release.
Additionally, this group found that NMDA toxicity was only partially ~a"dependent
in granule cells, suggesting that the requirement for ca2' in NMDA neurotoxicity
diffee among ce11 types. Another scientific group examining cytosolic ~a"levels as
determined by confocal fluorescent microscopy, has suggested that the early nse in
~a'+levels are due to NMDA activation and that the sustained levels of cal' are due
to reversa1 of the mitochondrial2~a~-~a"exchanger (Zhang et al., 1999). Regardless
of its source, toxic levels of cal' can cause a number of destmctive events.
The activation and upregulation of potentially neurotoxic enzymes such as
calpain, proteases, and nNOS by ca2' can lead to neuronal ce11 death. Calpain and
proteases aid in the degradation of intracellular proteins. The enzyme nNOS produces
nitric oxide, which can be toxic under certain circumstances. Mitochondrial injury
occun dunng ischemia as a result of lactic acidosis, increased production of reactive oxygen species and activation of ~a"regulated proteolytic enzymes such as calpain
(see review: Fiskum et al., 1997). The mitochondria normaily buffer increased ca2' levels by energy dependent sequestration. However, damage to the mitochondria caused by ischemia impairs this process making neurons more susceptible to ~a" mediated injury (Sciamanna et al., 1 992). in addition, mitochondrial respiration becomes more sensitive to Ca'' inhibition. This contributes to further energy depletion of the neuron. It has been suggested that Ca" influx can lead to either apoptosis or necrosis, depending on the energy charge of that cell. Further, Tenneti and colleagues (1998) found that caspase inhibitors were unable to arrest the disturbance in mitochondnal membrane potential that takes place after exposure to
NMDA receptor activation. This indicates that mitochondrial membrane potential detenoration may be an early event in both necrosis and apoptosis.
The cascade of events that take place in ischemia can result in apoptosis or necrosis depending on the energy charge of the cell. Necrosis, which is charactenzed by neuronal swelling and membrane breakdown, occurs when energ stores are compietely depleted. However, if cellular energy levels are adequate, apoptosis rnay occur. Membrane potential disruption of the mitochondria causes the opening of the membrane transition pore and reiease of apoptotic initiators such as cytochrome C.
Apoptosis leads to the shrinking of the cytoplasm and formation of apoptotic bodies.
Figure 1.1 depicts the events that take place in ischemia. The hypothesis that glutamate excitotoxicity is one of the key incidents in ischemia is supported by the following experiment.
1.2.1 NMDA Receptor Antagonists
An in vitro study, exarnining the neuropmtective effects of NMDA receptor
antagonists, found that a combination of NMDA and AMPA receptor antagonists Neuronal Ce11 Damage as a Result of Ischemia
1 L~issueATP Therobic Glycolysil
&w ) t~issueLactate d~issuepH Tce TN~'~Tc1-i 1 1 1 Neuronal Depolarkation 1
drivaiionctivation of non-NMDA-~
Activation of: PLA2 Xanthine Oxidase PKC CaMK II Calcineunn A Calpain I&ii activation LROS Formation Endonucleases Proteolysis I DNA DamagelFragmentatior. PARP Activation L w Energy Depletion Lipid Peroxidation L Ce11 Death
Activation of Neuroimrnune Response
Figure 1 -1: Summary of hypoxic/hypoglycemic injury. Adapted from Samdani et al., 1997. CBF indicates cerebral blood flow; VSCC, voltage-dependent calcium channels; NMDA-R, N-methyl-D-aspartate receptor, PLA2, phospholipase Az; PKC, protein kinase C; CaMK II, calcium-calmodulin-dependent protein kinase II; ROS ; PARS; poly(ADP-ribose) synthase; reactive oxygen species and [CE, interleukin- l B converting enzyme. resulted in alrnost complete protection against an in vitro ischemic insult in hippocampal brain slices. Additionally, it was discovered that omitting caZ' kom the buffer mimicked the protection that was observed with the NMDA and AMPA receptor antagonists. This indicates that ca2' influx through ionotropic glutamate receptors in ischemia may be one of the key events responsible for ischemic damage
(Anas et al., 1999).
1.2.2 ~MetabotropicGlutamate Receptors
Interestingly, unlike ionotropic glutamate receptors, it has been shown that metabotropic glutamate receptors play a protective role against excitotoxic or hypoxic/hypoglycemic injury (Bussion et al., 1995, Bruno et al.. 1995, Schroder et al.,
1999, Srnall et al., 1996, Pivi et al., 1996). Metabotropic glutamate receptors are
coupled to G-proteins. Group 1 metabotropic receptors, mGluRl and mGluR5, are
posi tively coupled to phospholipase C, whereas group 2 metabotropic receptors.
mGluR2-3, and group 3 receptors, mGIuR4-6-7-8, are negatively coupled to adenylyl
cyclase. These studies suggest that decreasing CAMP Ievels and activating PKC are
protective against ischemic injury. Smali and colleagues ( 1996), discovered that ten
minutes of oxygen and glucose deprivation in rat hippocampal slices resulted in a six
fold increase in CAMP levels and an approximately 50% decrease in PKC activity.
They also observed that pretreatment with a PKC activator significantly protected the
slices against the hypoxic/hypoglycemic insult, whereas pretreatment with an adenylyl
cyclase activator did not, indicating that PKC activity is protective against
hypoxic/hypoglycemic injury. Additionally, Bussion and colleagues ( 1999,
discovered that a selective agonist for group 2 metabotropic receptors, s-4-carboxy-3-
hydroxy-phenylglycine (4C3HPG), decreased an NMDA-induced increase in CAMP
levels. The protective effects observed with 4C3HPG following exposure to NMDA could be attenuated by the addition of 8-(4-chloropheny1thio)-adenosine 3'5'-cyclic- monophosphate (CPT CAMP) to the ce11 culture media. The authors propose that decreasing CAMP levels through activation of group 2 metabotropic receptors may be protective against excitotoxicity associated with oxygen glucose deprivation.
Activation of group 1 metabotropic recepton has also been affiliated with neuroprotection (Schroder et al., 1999, Pivi et al., 1996). Schroder and colleagues
(1999), observed that protection with the group 1 metabotropic agonist, 3,5- dihydroxyphenylglycine (DHPG),only occurred when the hippocarnpal slices were exposed to the drug pnor to the hypoxic/hypoglycemic insult. This protection could aIso be attenuated by CO-applicationwith a PKC inhibitor. This suggested that PKC might be involved in protective mechanisms in hypoxic/hypoglycemic injury, but only if activated pnor to the injury. These studies with metabotropic glutamate receptor agonists may provide ches as ro what second messenger systems are affected by ischemic damage and what needs to be conected in order to decrease impairment.
1.3 Xitric Oxide
Activation of the NMDA receptor by glutamate stimulates neuronal nitric oxide synthase (nNOS), which in tum increases nitric oxide levels in the neuron.
Nitnc oxide synthase converts L-arginine into L-ciûulline in a ~a~'1calrnodulin dependent manner in the presence of oxygen and NADPN. Representation of the
production of NO from NOS is displayed in figure 1.2. Under conditions, when the
level of L-arginine is rate-limiting NOS is able to produce superoxide anion as well as
nitric oxide (Heinzel et al., 1992). Sirnilarly, when the cofactor tetrahydrobioptenn
(TB)is absent, the toxic species hydrogen peroxide (H2O2)and superoxide anion are
also fonned by NOS (Heinzel et al., 1992; Pou et al., 1992). Therefore, in situations
such as ischemia, when substrates are lirnited, NOS is able to form superoxide anion NO Production by nNOS
HOOC NADPH FAD FMN HEME TBH? - . .6
. HOOC NADPH FAD FMN HEME TBH? - NH2
Reductase domain Oxygenase domain
Figure 1.2: A diagrüni depicting NO production by NOS. Adüpted from ladecola, 1997. Abbreviations: CaM, calrnodulin; Cyt c, cytochrome oxidase; FAD, flavin adenine dinucleotide; FMN,flaviii mononucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NOS, nitric oxide synthase; TBH, tetrahydrobiopterin. and H202,which could contribute to neurotoxicity. There are three different isoforms of niûic oxide synthase, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Both nNOS and eNOS are constitutively expressed whereas iNOS is produced by macrophages in times of irnmunological challenge (see review:
Iadecola, 1997). The NMDA receptor is directly coupled to nNOS by postsynaptic density protein -95 (PSD-95). In a study examining the role that PSD-95 may play in neurotoxicity, it was suggested that PSD-95 coupling to nNOS might be responsible for the toxicity observed with NMDA receptor activation (Sattler et al., 1999).
Suppression of PSD-95 protein in cultured cortical neurons was found to attenuate the toxicity associated with NMDA receptor activation. Additionally, it was observed that treatment of the cultured cortical neurons with nitric oxide donors restored neurotoxicity. These results indicate that nitric oxide may be a mediator of ischemic damage.
1.3.1 Nitric Oxide Synthase Antagonists
Studies examining generalized antagonism of NOS following ischemic
damage have found increases in infarct volume, no change in infarct volume or a
decrease in infarct volume (Yamamoto et al., 1992; Hamada et al., 1995; Dawson et
al., 1992; Nowicki et al., 1991; Huang et al., 1994, Panahian et al., 1996). In an
attempt to explain these conflicting results investigators have hypothesized that some
isoforms of NOS are protective while other isoforms are destructive. The use of
general NOS antagonists such as N-w-nitro-L-arginine (NNA) and p-nitro-L-
arginine methyl ester (L-NAME), which simultaneously block eNOS and nNOS,
resdted in an increase in infarct volume (Hammada et a1.,1995; Yamamoto et al.,
1992). It has been suggested that eNOS is beneficial following ischemia due to its
ability to increase collateral blood flow to the infarct area and that nNOS is detrimental because it is able to dramatically increase NO levels in the neuron
(Samdani et al., 1997). The use of knockout iriice has partially resolved this conflict.
Two studies looking at mice lacking nNOS and cerebral ischemia have found that deficient nNOS mice have significantly Iess brain damage than wild-type mice. Brain darnage was accessed by infarct volume and by qualitative grading and ce11 counting in the CA1 region of the hippocampus. No behavioral differences were observed behveen the two groups (Huang et al., 1994; Panahian et al., 1996). This further supports the notion that the negative eRects of nitnc oxide in ischemia are due to activation of nNOS and that eNOS may be beneficial in decreasing cerebral brain damage.
1.3.2 Nitric Oxide and Neurotoxicity
It has been suggested that the toxicity associated with increased nitric oxide levels is due to energy depletion (Almeida et al., 1998; Brorson et al., 1999), DNA damage (Endres et al., 1998) and oxidative stress (see review: Gross & Wolin, 1995).
Nitric oxide was also show to deplete cellular ATP levels in cultured hippocampal neurons (Brorson et al., 1999). in a ce11 culture study, it was discovered that glutamate exposure inhibited succinate-cytochrome C reductase and cytochrome C oxidase. A NMDA receptor antagonist or a NOS inhibitor couid reverse these effects,
indicating that nitric oxide is able to disrupt mitochondnal energy production
following glutamate neurotoxicity (Almeida et al., 1998). Nitric oxide is able to
inhibit complex 1, II (Stadler et al., 1991) and reveaibly inhibit cytochrome C oxidase
of the electron transport chain (Cassina and Radi, 1996). Mitochondrial aconitase of
the tricarboxylic acid cycle is also inhibited by NO (Stadler et al.. 1991). This
decreases the production of AïP, which further contributes to the energy depletion
experienced in ischemia. 1.3.3 Peroxynitrite
It has also been suggested that the neurotoxic properties of nitric oxide stems fiom its reaction with superoxide anion to form the powerful oxidant peroxynitrite.
Peroxynitrite is able to cause DNA damage, which results in the subsequent activation of poly(ADP-ribose) synthase (PARS). The ATP dependent process by which PARS repain DNA, depletes energy reserves increasing the cell's susceptibility to damage.
In an i11 vivo study conducted by Endres and colleagues (1998), there were sipificantly lower levels of PARS immunostaining in nNOS knockout mice as compared to wild-type mice. Additionally, it was observed that peroxynitrite, but not various nitic oxide donors, activated PARS irt vitro. It was also found that ce11 loss induced by exposure to peroxynitrite bi vitro could be attenuated by CO-applicationof
PARS inhibitors. This indicates that some of the toxicity associated with nitric oxide is a result of peroxynitmrite formation and activation of PARS. The neurotoxic effects of NO are sumrnarized in figure 1.3.
1.3.4 Nitric Oxide Production and Neuronal Outcome
In a clinical snidy it was found that higher levels of nitric oxide following the onset of stroke were associated with early neurological deterioration and poor outcome at three months. Levels of nitric oxide were determined by measuring the amount of nitrates and nitrites in the patient's blood in the first 24 houe following the onset of symptoms. Early neurological deterioration was defined as a fa11 of one or more points on the Canadian Stroke Scale within the first 48 hours (Castillo et al.,
2000). This clinical investigation demonstrated that nitric oxide levels are correlated with a greater neurological damage.
1.3.5 Nitric Oxide and Neuroprotection
Alternatively, nitric oxide has been found to be neuroprotective in several models of neurotoxicity (Fernandez-Tome et al., 1999; Zhang et al., 1994; Vidwans et al., 1999). In a middle cerebral artery occlusion (MCA) mode1 of ischemia, infusion of nitric oxide donors following injury decreased infarct volume and improved cerebral blood flow and EEG amplitude (Zhang et al., 1994). Using an irr vitro mode1 of neuronal injury, Femandez-Tome and colleagues ( 1999) demonstrated that nitric oxide donors could protect against hydrogen peroxide (HzOz)induced damage. The protection afforded by the nitric oxide donor could be attenuated with ODQ, a puanylyl cyclase inhibitor, and Rp-8-pCPT-cGMP, a protein kinase G inhibitor. This suggests that the neuroprotective effects of nitric oxide donors may be due to a cGMP mechanism. Vidwans and colleagues ( 1999) observed that various nitric oxide donors could decrease ca2' accumulation and NMDA neurotoxicity in a cortical ce11 culture system. Since NMDA receptor antagonists could mimic the actions of the nitric oxide donors, they suggested that the nitric oxide donors might be inhibiting the NMDA receptor. They also proposed that the NO donating character of the NO donor may determine whether that agent is neuroprotective.
1.3.6 Nitric Onde and NMDA Receptor Inhibition
It has been postulated that NO may have neuroprotective properties due to its
ability to donate a nitrosonium ion to the NMDA receptor and downregulate its
activity (Lipton et al., 1993). Lipton and colleagues (1993) hypothesized that the
neurotoxic/neuroprotective properties of nitnc oxide are dependent upon the redox
environment of the neuron. Conditions that favor the formation of a nitrosonium ion
(NO+) Iead to S-nitrosylation reactions and have the potential to be protective.
Donation of a nitrosonium ion to the redox modulatory site of the NMDA receptor by
a S-nitrosylation reaction inhibits ~a"80w through the receptor. A nitrosonium ion- thiol group reaction at the redox modulatory site downregulates the NMDA receptor, which blocks caZCinflux into the neuron. Altematively, conditions that favor the formation of peroxynitrite from nitric oxide and superoxide anion can be toxic to the neuron. Peroxynitrite causes DNA damage and further depletes energy stores by activating PARS as discussed in section 1.2.3. Studies investigating the properties of
NO donors have demonstrated that NO is able to inhibit the NMDA receptor. The NO donor, 3-morpholino-sydnonimine (Sin-1), was able to inhibit NMDA receptor activation and increases in intracellular ca2' in a ceIl culture system (Mazoni et al.,
1992). incubation of Sin- 1 with hemoglobin blocked the effects of Sin-1, indicating that the actions of Sin-1 were due to nitric oxide generation and inhibition of the
NMDA receptor. It has also been shown that the NO donor, nitroglycerin, is able to inhibit the NMDA receptor. It has been suggested that nitroglycerin reacts with thiol groups present on the redox modulatory site to produce nitric oxide, which results in the formation of a disulfide bond and subsequent downregulation of the NMDA receptor (Lipton et al., 1993; Lei et al., 1992).
Nitric oxide is also able to block the NMDA receptor at an alternative site.
This is suggested by the observation that including metal ion-chelators in the extracellular media could attenuate the actions of nitric oxide. Thus the inhibition of the NMDA receptor at this alternative site by nitric oxide requires divalent ions (Fagni et al., 1995). In surnmary, nitic oxide may be able to inhibit ~a"flow through the
NMDA receptor by acting at the redox modulatory site or by interacting with divalent
ions at an alternative site. Both of these actions can potentially limit ischemic damage
due to stroke by inhibiting ca2' influx through the NMDA receptor.
1.3.7 Antioltidant Properties of Nitric Oxide It has been posnilated that antioxidants are beneficial in the reperfûsion period following ischemic injury due to their ability to interact with oxygen free radicals to forrn stable compounds. It has been hypothesized that nifric oxide may be able to act as an antioxidant based on its physio-chemical properties. Nitric oxide is a nitrogen- centered fiee radical and is extremely reactive in the cell. Rauhala and coIIeagues
(1998) proposed that nitric oxide is able to react with peroxyl lipid radical produced from lipid peroxidation to fom a stable nitroso-compound (LOO + NO + LOO-
NO). It has also been demonstrated by Kanner and colleagues (1991) that nitric oxide can inhibit the initiation reaction of lipid peroxidation by reacting with ferrous iron.
Ferrous iron interacts with hydrogen peroxide in the Fenton reaction to initiate lipid peroxidation. This is particularly relevant in the reperfusion period of ischemia when lipid peroxidation is taking place at an increased rate.
Studies both in vivo and in vitro have shown that nitric oxide is able to inhibit lipid peroxidation (Rauhala et al., 1998; Rubbo et al., 1994; Rauhala et al., 1996;
Kanner et al., 1991). Using both techniques it was observed that S-nitrosothiol was able to protect dopaminergic neurons against oxidative stress. The in vivo mode1 consisted of measunng fluorescent products of lipid peroxidation in brain hornogenates of anirnals that were previously infùsed with ferrous citrate, with or without S-nitrosoglutathione (GSNO). In the substantia nigra, GSNO and NO were able to significantly decrease lipid peroxidation. This effect could not be mimicked by photodegraded GSNO, GSH or GSSG indicating that the inhibition of lipid peroxidation was due to production of NO (Rauhala et al., 1996). Additionally, it was shown that GSNO was able to significantly decrease lipid peroxidation in brain homogenates exposed to ferrous citrate as indicated by fluorescent end products.
They suggested that this effect was mediated by the ability of nitric oxide to interact with peroxyl lipid radicals (Rauhala et al., 1998). By measuring hydroxyl radical generation as an indication of lipid peroxidation, it was observed that nitnc oxide- myoglobin was able to inhibit the formation of ferryl myoglobin, which initiates lipid peroxidation. The production of ferryl myoglobin results fiom a reaction between meûnyoglobin and oxymyoglobin with hydrogen peroxide. These results demonstrate that nihic oxide may be able to inhibit the initiation of lipid peroxidation since it hinders the formation of ferryl myoglobin. (Kamer et al., 199 1).
In a liposomal rn~del,it was found that nitric oxide was able to either increase or decrease lipid peroxidation depending on its concentration. It was demonstrated that at concentrations of nitric oxide equimolar to that of Oi', XO-dependent lipid peroxidation was stimulated in a liposome model. However, when the rate of nilric oxide production exceeded that of Or'-, lipid peroxidation was inhibited. It was also demonstrated by mass spectrornetry that nitric oxide was able to form nitrito-, nitro-, nitrosoperoxo-, andlor nitrated lipid oxidation adducts which are termination products of lipid peroxidation (Rubbo et al., 1994). This study rnight help explain the conflicting neuroprotective/neurotoxic effects observed with nitric oxide. This theory suggests that if the concentration of nitric oxide is lower than that of hydroxyl radicals, nitric oxide increases ischemic injury by stimulating lipid peroxidation.
Nitic oxide could also exacerbate ischemic injury by interacting with superoxide anion and forming the powerful oxidant peroxynitrite. Superoxide anion concentrations are increased in ischemic injury due to uncoupling of the electron transport chain present in the mitochondria. Altematively, if nitric oxide was present at concentrations exceeding that of hydroxyl radicals, it could terminate the propagation of lipid peroxidation by forming stable nitroso-compounds. This study demonstrates that nitnc oxide may be beneficial in the treatrnent of ischemic injury due to its ability to inhibit lipid peroxidation, if present at the proper concentration.
These findings also indicate that increasing the concentration of NO may be rnost beneficial during the reperfùsion period when lipid peroxidahon is occurring at an increased rate.
1.4 cGMP
Nitric oxide is also able io influence second messenger pathways by interacting with the heme moiety present on guanylyl cyclase, which stimulates its activity. Guanylyl cyclase produces cGMP fiom GTP. Like nitric oxide, the role of cGMP in ischemia is controveaial. Some investigators have found that cGMP contributes to neuronal ce11 death (Li et al., 1997; Montoliu et al., 1999), while others have observed neuroprotective effects of cGMP against glutamate excitotoxicity and beta amyloid toxicity (Keller et al., 1998; Garthwaite et al., 1988; Furukawa et al.,
1996; Barger et al., 1995; Fumkawa et al., 1998; Mattson et al., 1999; Yoshioka et al.,
2000; Moro et al., 1998).
1.4.1 cGMP and Neurotoxicity
Two studies examining glutamate excitotoxicity in ce11 culture systems have conciuded that cGMP potentiates ce11 death. One group found that membrane permeable analogues of cGMP increased ce11 death in cortical and hippocampal neurons exposed to glutamate. This effect could be attenuated by soluble guanylyl cyclase inhibition. They postulated that the neurotoxic properties of cGMP involved activation of a ~a"ion channel, since inhibition of soluble guanylyl cyclase arneliorated increases in ca2' levels and cGMP analogues elevated ~a"levels (Li et al., 1997). Interestingly, another goup of scientists observed that increasing
intracellular levels of cGMP induced ce11 death whereas extracellular elevations in
cGMP were neuroprotective against glutamate excitotoxicity. However, the mechanisms involved in this neuroprotective pathway mediated by extracellular cGMP are unknown. This may partly resolve the neuroprotective/neurotoxic controversy surrounding cGMP. Perhaps the localization of cGMP is important in determining whether cGMP is protective or toxic to the neuron (Montoliu et al.,
1999).
1.4.2 cGMP and Neuroprotection
Altematively, a number of studies have found cGMP to be neuroprotective
against a vanety of neuronal insults (Moro et al., 1998. Garthwaite et al.. 1988, Keller
et al., 1998, Yoshioka et al., 2000). In one study, it was observed that 1H-
[1,2,4]oxadiarolo[4,3,-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, dose-
dependently increased cell death in primary cortical neurons exposed to Sin-1 in the
presence of superoxide dismutase. Sin4 is toxic in these conditions due to enhanced
production of Ht02 mediated by the enzyme superoxide dismutase. Superoxide
dismutase forms H202and molecular oxygen from two superoxide anions and two H+.
The cGMP analogue, 8-bromo-cGklP, was able to reverse the toxicity associated with
ODQ. Therefore it was concluded that cGMP plays a neuroprotective role in H202
toxicity (Moro et al., 1998).
Similarly, in a study examining the role of cGMP in excitotoxicity, it was
found that guanylyl c yclase activators, phoqhodiesterase inhibitors and synthetic
cGMP analogues were neuroprotective. Conversely, inhibiting guanylyl cyclase with
vanous guanylyl cyclase inhibitors was neurodestntctive. Since the toxicity
associated with guanylyl cyclase inhibition resernbled that observed with oxygen
radical generatos, it was proposed that cGMP might be able to limit oxidative damage
(Garthwaite et al., 1988). Oxygen radicals are also able to activate guanylyl cyclase.
Therefore, it was hypothesùed that cGMP may be acting in a negative feedback fashion by protecting neurons against oxidative stress (Mitta1 et al., 1982). It was suggested that this protection afforded by cGMP is due to direct scavenging of lipid radicals or transcription of endogenous proteins involved in oxygen radical inhibition.
Cyciic nucleotides were also shown to significantly decrease ce11 loss in PC6 cells and cultured hippocampal neurons exposed to 4-hydroxynonenal (HNE). HNE increases free radical formation, thus promohng lipid peroxidation. The results from this study indicate that cyclic nucleotides are able to inhibit lipid peroxidation (Keller et al.,
1998). The findings fiom these studies suggest that cGMP may be able to protect neurons fiom oxidative stress.
Yoshioka and colleagues (2000) have proposed a PKG-cGMP mediated mechanism of neuroprotection. They found that synthetic cyclic nucleotides and phosphodiesterase inhibitors increased cGMP concentrations and significantly decreased kainate induced cal' influx in oligodendroglial-like cells (OLC). In
addition, they discovered that an activator of PKG, 8-(4-ch1orophenylthioI)-
guanosine-3',5'-monophosphate, could protect the oligodendrogIial ceil from
excitotoxicty in a similar marner. Western blot analysis revealed that PKG IP was
translated in OLCs exposed to a PKG activator or protein phosphatase 1 and 2A
inhibitors (Yoshioka et al., 2000). These results indicate that a PKG mechanism may
be responsible for decreasing ca2+influx mediated by kainate receptor activation.
This would protect neurons from excitotoxicity.
1.4.3 cGMP and &AmyIoid Precursor Protein
Investigatoa researching the neuroprotec tive properties of the secreted fom of
amyloid precursor protein (sAPPa) have suggested a PKG mediated mechanism of
neuroprotection (Mattson et al., 1999; Furukawa et al.. 1997; Barger et al., 1995;
Funikawa et al., 1996). The neuroprotection observed in these studies could be mimicked by synthetic cGMP analogues and be inhibited by a PKG inhibitor, suggesting that cGMP and PKG are involved in the underlying mechanism of neuroprotection afforded by sAPPa.
1.4.4 Glutamate and Glucose Uptake
It has been proposed that a cGMPlPKG mechanism enhances glutamate and glucose uptake into synaptic compartments. Glucose and glutamate transport was
impaired in cortical synaptosomes exposed to ~e'- and amyloid P-peptide (AP)
(Mattson et al., 1999). This could be beneficial in ischemia when excessive amounts
of glutamate are being released into the neuronal synapse. If glutamate is taken up
into synaptic compartrnents this would decrease the levels of glutamate present in the
synapse and perhaps prevent excitotoxicity. Glutamate excitotoxicity has been
suggested to be the main mechanism involved in neuronal ceil loss in
isc hemic/reperfùsion injury.
1.4.5 Inhibition of the WAReceptor
It has also been suggested that cGMP mediated activation of PKG rnay inhibit
the NMDA receptor. It was discovered that sAPPa and cyclic nucleotide analogues
were able to decrease ca2' levels in cultured hippocampal and cortical neurons after
exposure to glutamate. A PKG inhibitor could block this effect indicating that the
decrease in intracellular ca2' levels observed with cGMP and sAPPa were due to
PKG activation (Barger et al., 1995). Additionally, it was found that sAPPa and
cGMP analogues could attenuate glutamate and NMDA currents produced in cultured
hippocampal neurons. Data was obtained from whole ce11 patchîlamp recordings.
Protein kinase G inhibitors reversed the effects of sAPPa and cGMP analogues on
NMDA and glutamate induced currents. Further, they found that a phosphatase
inhibitor, okadaic acid, also blocked the effects of sAPPa and cGMP analogues. It was proposed that PKG may upregulate or activate a phosphatase that would dephosphorylate the NMDA receptor, decreasing its open probability (Furukawa et al., 1997). The notion that cGMP may be able to decrease currents generated from the NMDA receptor indicates that cGMP may be part of an endogenous mechanism to downregulate the NMDA receptor when it becomes excessively active. The NMDA receptor increases the production of NO, which activates guanylyl cyclase and subsequently increases the level of cGMP in the neuron. Thecefore, cGMP may be able to inhibit the NMDA receptor in an attempt to control its activation.
Manipulating this endogenous system by increasing cGMP may be protective in disorders caused by glutamate excitotoxicity such as ischemia.
1.4.6 Potassium Channels
Using whole ce11 perforated patch and single channel patch-clamp techniques it was observed that sAPPa hyperpolarized hippocampal neurons by activating K' charnels. This effect could be mimicked by cGMP analogues and blocked by PKG inhibitors. It was found that phosphatase inhibitors could attenuate the hyperpolanzation observed with sAPPa (Funikawa et al., 1996). Thus APPa is able to activate K' channels and hyperpolarize the membrane by means of a dephosphorylation reaction. It was also found that sAPPa is able to decrease ~a"
levels in the neuron. Hyperpolarization of the neuron causes a decline in ca2+. These
results indicate that augmenting cGMP levels may be protective in ischemia due to its
ability to offset increases in ca2' levels.
In summary, shidies have shom that activation of PKG can protect neurons by
three mechanisrns: increased uptake of glucose and glutamate into synaptosomes and
activation of a phosphatase which downregulates the NMDA receptor or activates K'
channels. Al1 of these events have the potential to be protective in ischemia where ion homeostasis and glutamate regulation are disrupted. The potential neuroprotective mechanisrns of nitric oxide are illustrated in figure 1 -4.
1.5 Cyclic Nucleotide Activated Ion ChanneIs
There are alternative mechanisrns by which cGMP may be exerting its effects in the neuron. Cyclic GMP may be activating cyclic nucleohde activated ion charnels or inhibiting phosphodiesterases (PDEs). Cyclic nucleotide activated ion channels are a group of recently characterized ion channels present in most tissues. They are activated by the cyclic nucleotides, cGMP and CAMP, and allow passage of Na', K' and ca2' into the neuron. These channels depolarize the neuronal membrane, which lead to increases in cytosolic ca2' (Dzeja et al., 1999). However, it is unlikely that the neuroprotective effects observed with cGMP are due to activation of cyclic nucleotide activated ion channels since activation of these channels increase ca2+levels in the cell.
1.6 Phosphodiesterases and Ischemia
Phosphodiesterase inhibitors increase the levels of cGMP and cAMP by blocking the breakdown of cGMP and cAMP into ScGMP and YcAMP. Studies have shown that post-ischemic treatrnent with a PDE inhibitor rnay be neuroprotective through its ability to increase the concentration of CAMP in the neuron. in one of
these studies, the dnig roliprarn was exarnined in a four-vesse1 occlusion mode1 of
ischemia. Rolipram is an inhibitor of PDEr, a phosphodiesterase specific for the breakdown of CAMP, thus it increases the Ievels of cAMP in the neuron. This agent
has been used in clinical irials as an antidepressant (Bertolino et al., 1988). In this
study, roliprarn was administered six hours after a 20 minute ischernic insult and was
continued once daily for seven days. Neuronal damage was accessed four weeks
following injury by counting the number of su~vingneurons in the hippocampus and Neuroprotective Properties of NO
1 Nitric Oxide 1
1 Inhibit the NMDA 1 lncrease Production of Receptor. I cGMP
-Donation of a nitrosoniuni -Interaction wiih lipid -cGMP-mediated protection ion at the redox niodulatory radicals to fonii stable from oxidative stress. site ai the NMDA receptor. nitroso-conipounds. -A cGMP-PKG mechanisni: -Interaction with divülent -1nteract with ferrous a) Enhniice glucose/glutamate ions and inhibition of the ions to iiiliibit the uptake into synaptic compartments. NMDA receptor at an initiation of lipid b) Inhibit the NMDA receptor. alternative site. peroxidatioii. c) Activate Ktchannels.
Figure 1.4: Summary of the neuroprotective properties of NO. Abbreviations: PKG, protein kinase G;NMDA, N-methyl-D-aspartate. striatum. It was found that post-ischemic treatment with rolipram could significantly increase neuronal survival in the hippocampus and striatum (Block et al., 1997).
Furthemore, it was demonstrated that rolipram could improve leaming and memory after cerebral ischemia. Leaming and memory outcomes were accessed by a 3-panel runway paradigm. Post-ischemic treatment with rolipram could significantly improve behavioural outcome following four-vesse1 occlusion (Imanishi et al., 1997). These results suggest that CAMP may play a neuroprotective role in ischemic injury.
1.7 CAMP
It has been suggested that cAMP may play a role in neuronal survival. It was found that, in the absence of tropic factors. spiny motor neurons were able to survive in conditions of elevated CAMP levels in a ce11 culture system (Hanson et al., 1998).
AdditionaIIy, it was observed that decreased CAMP binding occurred in regions of the hippocampus, such as the CA1 region, that are susceptible to ischemic damage.
Cyclic AMP binding was determined by measunng the levels of radiolabelled cAMP present in the hippocampal slices (Tanaka et al., 2000). These studies indicate that
CAMPmay be involved in mechanisms associated with neuroprotection.
Cyclic AMP may be exerting its effects through the activation of the cyclic
AMP-responsive element binding protein (CREB). Phosphorylation of CREB at serM3causes CREB to become active. A number of protein kinases such as protein kinase A (PM) and ~a~'/calmodulin-dependent protein kinases are able to phosphorylate CREB. It has been suggested that activation of CREB can be neuroprotective (Tanaka et al., 2000; Walton et al., 1999; Hu et al., 1999). It was observed that there was a greater amount of phospho-CREB in the dentate granule cells in the hippocampus following 15 minutes of ischemia than in the CA1 pyramidal cells (Hu et al., 1999). Because dentate granule cells are more resistant to ischemia, the increased levels of phospho-CREB in the dentate granule cells may be part of a neuroprotective mechanism. In a cell culture system, using PCL2 cells, it was found that CREB phosphorylation increased ce11 survival (Walton et al., 1999). Further, there was more CREB phosphorylation in neurons that showed no histological signs of damage, suggesting that CREB phosphorylation may protect neurons from ischemia. A voltage-sensitive ca2'/Na' channel blocker could not attenuate CREB phosphorylation, indicating that CREB was phosphorylated by PKA as opposed to
~a~'lcalrnodu1independent kinases which are dependent upon Ca" for their activation
(Tanaka et al., 2000).
Altematively, it was observed that there was no potentiation of ischemic injury in CREB knockout mice (Hata et al., 1998). It is possible that this lack of potentiation may be due to compensatory mechanisms in the CREB knockout mice.
Therefore one cannot conclusively declare that CREB phosphorylation does not play an important role in ischemic injury. The evidence thus far supports the hypothesis that CREB phosphorylation is protective in ischemia. The reason for this is that
CREB phosphorylation is responsible for the transcription of a number of genes such as bcl-2. Mc11 and bdif (Bonni et al., 1999; Riccio et al., 1999; Walton et al., 2000), which may play neuroprotective roles against isc hemic injury.
1.8 Guanine Nucleotide Exchange Factors
A novel CAMPmediated mechanism that is independent of PKA activation has been recently discussed in the literature. The mechanism involved guanine nucleotide
exchange factors (GEFs). GEFs increase the dissociation of GDP from small
GTPase's such as Rap 1 to allow the binding of GTP, which is subsequently
hydrolyzed. An exchange factor, Epac, is directly activated by CAMP in a manner
similar to that of PKA. Epac interacts with the guanine nucleotide binding protein, Rap-1 (see review: Zwartlauis et al., 1999). The precise role of GEFs remains unclear, however, they have been suggested to be involved in cell proliferation
(Altschuler et al., 1998), ce11 differation (York et al., 1998) and in ceIl cycle control.
It will be interesting to discover their precise tunction and determine whether they play a significant role in ischemia.
1.9 Novel Organic Nitrates
Novel organic nitrates are a goup of established NO donors that are denved
fiom the chemical structure of glyceryl trinitrite (GTN). GTN has previously been
show to decrease neurotoxicity in both in vitro and in vivo rodent models (Sathi et
al., 1993, Lei et al., 1992). Treatment with GTN for 36h before and 48h after an
ischemic insult significantly decreased infarct size as calculated fiom MR images.
The isc hemic insult was induced by photothrombosis or by bilateral carotid ligation.
In the in vivo studies, blood pressure dropped initially afler application of GTN and
renirned to normal within 90 minutes (Sathi et al., 1993). GTN was also protective
against NMDA excitotoxicity in a brain slice rnodel (Lei at al., 1992). These studies
demonstrate that GRI has the potential to be therapeutically useful in the treatment of
ischemia.
in place of the rhird nitrate present in GTN, novel organic nitrates have a
substituted phenol group attached to the glyceryl backbone by a disulfide bond.
Evidence suggests that novel organic nitrates spontaneously release nitric oxide in the
presence of thiol groups (Zavonn et al., 2001). The chemical structure of GTN and
the novel organic nitrates, as well as the proposed biotransformation, of the novel
organic nitrates are depicted in figure 1S.
A major disadvantage of using nitric oxide donors in the in vivo treatment of
ischemia is the vasodilation that occurs. This generalized vasodilation would decrease Novel Organic Nitrates
- ONO, GTN Novel Organic Nitrates
S- S- Ph S- S- Ph OH ONO, PhSH (aq.) +NO pH 7.4 ONO, EONO,
+ PhSSPh + Other Products Figure 1.5: Chemical structures of GTN as compared to the novel organic nitrates. Proposed biotransformation of the novel organic nitrates. Adapted from Zavorin et al., 200 1. Abbreviations: Ph, phenol; NO, nitric oxide. blood flow to the brain and perhaps exacerbate injury. However, the vasodilatory effects of the novel organic nitrates are ten times less potent than that of GTN
(Bennett et al., 2000). Thus, these agents would be more efficacious clinically than
GTN as they do not produce generalized relaxation of blood vessels, which would decrease blood perfusion of the brain.
GT-015,a novel organic nitrate, was neuroprotective in both in vivo and in vitro models of neurotoxicity (Clarke et al.. 2000). The neuroprotection obsemed with GT-015 could be attenuated by the CO-applicationof ODG, a guanylyl cyclase
inhibitor, indicating that the effects of GT-015 were cGMP dependent. Another novel organic nitrate, GT-715, was able to improve spatial leaming in scopolamine-impaired male rats. Leaming was measured using the Moms water maze. GT-715 was also observed to have a greater activation potential for soluble guanylyl cyclase (sGC) in
hippocampal homogenate in the presence of 1mM L-cysteine than GTN as assayed by
a cGMP radioimmunoassay. Because GT-715 had a high activation potential for sGC.
it was proposed that GT-715 improves leaming by a cGMP-mediated mechanism
(Smith et al., 2000).
1.10 In Vitro iModel of Ischemia
An in vitro model consisting of hippocarnpal brain slices was chosen for the
evaluation of organic nitrates due to the fact that agents could be investigated quickly
and easily. There are a number of inherent advantages in using this model. The
extemal environment of the neuron is controlled by the investigator, which facilitates
manipulations such as drug administration and changes in temperature. Further, it is
ideal for a mechanistic study as extemal influences are minirnized. Slices frorn the
same animal can be used for various manipulations, which is advantageous, as they al1
possess the same expenmental history. Anesthetics do not have to be used for tissue preparation and brain slices maintain some integrity. A major disadvantage of in vitro models, however, is the extent that they represent the in vivo situation. Therefore, any discovenes that are made in vitro should also be replicated in an in vivo mode1 (Schurr et al., 1986).
The rrisynaptic circuit is maintained in the hippocampas brain slice, which makes it an ideal tissue preparation. The circuitry in the hippocampus is highly glutarnatergic that is important because ischemic damage is associated with glutamate excitotoxicity. Additionally, the hippocarnpus has been implicated in learning and memory, which are two functions that are adversely affected by ischecis.
1.1 1 Research Rationale, Hypothesis and Objectives
Nitric oxide could potentially play a neuroprotective role in ischemic injury by inhibiting ~a"flow through the NMDA receptor (Lipton et al., 1993), by acting as an antioxidant (Rauhala et al., 1998 & 1996; Rubbo et al., 1994; Kanner et al., 1991) and by increasing production of cGMP. Investigators studying sAPPa have proposed a cGMPIPKG mediated mechanisrn of neuroprotection. They suggested that this neuroprotection is due to inhibition of NMDA receptor (Funikawa et al., 1997; Barger et al., 1995), enhanced uptake of glucose and glutamate (Mattson et al., 1999) and activation of K+ channels (Furukawa et al., 1996). Ln addition, GT-015, a novel nitric oxide donor, displayed neuroprotective properties in both in vivo and in vitro models
of ischemia. It was hypothesized that this neuroprotection was cGMP dependent since
it could be attenuated by a guanyIy1 cyclase inhibitor in vitro (Clarke et al., 2000).
The goal of this thesis was to test the hypothesis that novel organic nitrates,
which are established nitric oxide donors, are neuroprotective in an in vitro stroke
mode1 by a cGMP-PKG mediated mechanism. in order to test this hypothesis the
following objectives were established: 1) To define the in vitro model of ischemia with respect to duration of ischemia and neuroprotection with hypothemia.
2) To evaluate several novel organic nitrates in the in vitro model of ischemia for neuroprotective properties. To determine if any observed neuroprotection is due to generation of cGMP by examining whether the neuroprotection could be mimicked by synthetic cGMP analogues or be attenuated by CO-applicationof ODQ or PKG inhibitors. 2. MATERIALS AND METHODS
2.1 ChemicaI Solutions
Sodium chloride (NaCl), potassium chloride (KCI), potassium phosphate
(KH2POr), calcium chloride (CaCI?), magnesium sulfate (MgS04), sodium bicarbonate (NaHC03), glucose, sucrose, NADH, pymvate, dibutyryl cGMP, dibutyryl cAMP,8-bromo-cGMP and dimethyl sui foxide (DMSO) were al1 purchased fiom Sigma Chemical Co. (St. Louis, MO). 8-bromo-cGMP, H-89, ODQ and Sin4 chloride were obtained fiom Tocris (Ballwin, MO). 8-pCPT-cGMP and Rp-8-pCPT- cGMP were purchased fiom Calbiochem (San Diego, Califomia). The reagents and the standard, bovine semm albumin (BSA) for the dye-binding assay were purchased from Bio-Rad Labotatories (Mississauga, Ont.). Forskolin and 8-bromo-CAMP were generously given to us fiom Dr.Maurice, a professor fiom the Deparûnent of
Pharmacology and Toxicolo~at Queen's University. GSNO was produced and generously given to us From Dr. Gin, a postdoctoral student from the Department of
Pharmacology and Toxicology at Queen's University. Came1 hair fine paint brushes were purchased fiom Wallacks (Kingston. Ont.). The fiozen tissue embedding media, the superfiost microscope slides, Hemo-De and acetic acid were purchased fiom
Fisher Scientific (Nepean, Ont.). Ethanol was obtained fiom Commercial Alcohols
Inc. (Brampton, Ont.). Al1 the novel organic nitrates were synthesized in the
Department of Chemistry at Queen's University (Kingston, Ont.). Al1 aqueous solutions were made with deionized water purchased from Aquaterra Corporation
(Mississauga,Ont.).
2.2 Experimental Animais
Adult male Sprague-Dawley rats weighng beween 225-250% were purchased from Charles River Canada inc. (St. Constant, Quebec). Anirnals were housed in pairs with free access to food and water and were exposed to a 12hrs lighvdark cycle.
The Queen's University Animal Care Cornmittee approved the experimental protocol.
The animals were cared for according to the pnnciples and guidelines of the Canadian
Council on Animal Care.
2.3 Tissue Isolation
Male Sprague-DawIey rats were euthanized, without prior anesthetic, by decapitation. The brain was then quickly excised and placed in ice-cold sucrose substituted Krebs' ( 1 18mM sucrose, 4.7 mM KCI, 1.2 mM KH2PO~,1.3 mM CaClt,
1.2 mM MgSOr, 25mM NaHCO], and lOmM glucose). The hippocampus was dissected on an ice filled petri dish and transverse slices of 400pm were made with a
Mcnwain Tissue Chopper. Slices were separated in a petri dish filled with sucrose substituted Kreb's solution with came1 hair fine paint brushes. The slices were allowed to equilibrate with the Kreb's solution ( 1 18mM NaCl, 4.7mM KCl, 1 .ZmM
KH2PO4, 1 -3mM CaClr, 1.2 mM MgSO~,25rnMNaHC03 and 1OmM glucose) for 1h pnor to the expenment. The slices were placed on a strainer in a beaker with standard
Kreb's solution and bubbled 95% 02/5%N2. The slices were separated into a control group and a low[02]/low[glucose] group. The slices that were part of the control group were subdivided into smaller groups of 3-4 slices each and were placed into vials with 2mL of standard Kreb's solution and bubbled 95% 02/5% Nz. These vials were placed in a water bath heated to 37OC. Al1 of the remaining slices undenvent an in vitro ischemic insult. ïhey were placed in a beaker with Kreb's solution which had glucose substituied with equimolar sucrose to maintain proper osmolarity and bubbled
95% Nd 5% O2 for 1/2 h. A half hour insult time was chosen because it was previously shown to induce a submaximal increase in lactate dehydrogenase (LDH) release. This will be discussed in more detail in section 3.1. Slices were then subdivided into groups of 3-4 slices and divided into control and treatrnent groups.
Treatment groups received the specified concentration of dmg immediately following the low[Oz]Aow[glucose] insult. The organic nitrates, Rp-8-pCPT-cGMP, 8-pCPT- cGMP, H-89 and ODQ were dissolved in dirnethyl sulfoxide (DMSO). Sin-1 chloride, GSNO, cGMP, dibutyryl CAMP, the synthetic cGMP analogues dibutyryl cGMP and 8-bromo-cGMP were dissolved in deionized water. Drug vehicle in equal concentrations was also included in the control groups. Slices were then placed into vials with 2mL standard Kreb's solution, bubbled 95% 02/5%Nz and drug or vehicle
for a 4h reperfusion penod. These vials were also placed in a water bath prewarmed to 37°C.
2.4 LDH Assay
At the end of the 4h reperfusion penod, l.lmL of the incubation buffer was
assayed for LDH release. LDH release was used as a measure of ce11 viability. LDH
is a cytosolic enzyme involved in glycolysis that is released when the celiular
membrane is damaged. The amount of LDH was determined by incubating the
samples with 2OOpL of 1 AlmM NADH for 5-10 minutes at 25°C. Adding ZOOPL of
11.5mM pyruvate to start the reaction then accessed LDH activity. Phosphate buffer
(O. I M) was used to make the aqueous solutions of NADH and pynivate. NADH was
measured on a Beckrnan 500 spectrorneter with a winUV enzyme kinetics program at
an absorbance of 340nm. The decline of NADH that took place over a 1 minute
period was used as an indication of the amount of LDH present. LDH converts
pynivate into lactate using one equivalent of NADH. Therefore, by measunng the
decline of NADH one can calculate the amount of LDH available. The results were
standardized to protein content of each vial.
2.6 Protein Determination Following the removal of 1. l mL of the incubation buffer for LDH analysis, the remainder of the incubation buffer was discarded. In order to digest the tissue, ImL of 1N sodium hydroxide (NaOH) was added to the slices. The amount of protein was determined spectrophotometricly using bovine semm albumin as the standard in a protein dye binding assay based on the Bradford method (Bio-Rad Laboratones, inc.,
Mississauga, Ont.).
2.7 cGMP Radioimmunoassay
After the hypoxic/hypoglycemic insult the slices were equilibrated for 1 Smin, prior to the administration of the therapeutic agents. The supematant was discarded.
The slices were centnfuged and then frozen in liquid nitrogen 3 minutes afier the administration of drug or vehicle. The slices were hornogenized with 600pL of 6%
TCA and were centrifbged at 2200 rpm for 20 minutes. The supematant was frozen for subsequent radioimmunoassay (RIA) analysis. The pellet was digested in 1mL of
NaOH for later protein detemination.
On the day of the RIA analysis, 50pL of IN hydrogen chloride (HCL) was added to the slices. Water saturated diethyl ether was then used to extract the TCA from the slices. After the KAextmction. 5OpL of IN NaOH was added to neutralize the HCL as well as 50pL 1M sodium acetate pH 4. The samples were acetylated with
20pL triethylamine and lOpL acidic anhydride to enhance sensitivity.
A series of standards were made using cGMP concentrations in the fentamol range. Radiolabelled cGMP (I%GMP) was added to the samples, standards, non- specific binding, total and blank tubes. Antibody specific to cGMP was added to the sarnples, standards and blank tubes. The following day, gamma globulin was added to every tube except the total tube and 100% isopropyl alcohol was added to every tube.
The gamma globulin was used to precipitate out the antibody-radiolabelled cGMP complexes from solution. Al1 tubes were centrifbged at 2000 rpm for ljmin. to separate the gamma globulin-antibody-radiolabelled cGMP complexes from the isopropyl alcohol. The isopropyl alcohol was poured out and the samples were read using a BeckrnanB gamma counter.
The levels of cGMP were deterrnined fiom the cGMP standard curve that was prepared. The detection of cGMP was based on the cornpetitive aspect of the binding of cGMP and radiolabelled cGMP to the antibody. This depicts an inverse relationship benveen the amount of cGMP present and the gamma counts detected.
2.8 Cresyl Violet Staining
After 1 .1 mL of the incubation bu ffer was taken From the hippocampal slices for
LDH analysis, a 4% (w/v) paraformaldehyde solution was added to the slices. The following day the slices were placed in a 20% (wh) sucrose solution for another 24h.
At this time the slices were kozen ont0 a chuck using fiozen tissue embedding media.
The slices were then cut into 40nm sections using a Richert-Jung Cryocut 1800. The
40nm slices were immediately placed onto microscope slides. The tissue was first deparaffinized by placing slices in a glass container containing Hemo-De for 2
consecutive periods of 5 minutes. The tissue was then rehydrated using a graded
ethanol series: 100% rthanol for 2 periods of 5 minutes, 95% ethanol for 2 periods of
5 minutes, 80% ethanol for 1 period of 2 minutes, 70% ethanol for 1 period of 2
minutes, 50% ethanol for 1 period of 2 minutes and distilled water for 1 period of 5
minutes. The slices were then stained with the cresyl violet stain (0.5g cresyl violet,
300mL of distilled water, 30mL of t .OM Na acetate, 170rnL of 1.OM acetic acid) for 1
to 2 minutes. The slices were then destained in distilled water for 5 minutes. Placing
sIices in 70% ethanol for 1 minute, 95% ethanoVacetic acid for 1 to 2 minutes, 95%
ethanol for 1 minute and 100% ethanol for 2 periods of 2 minutes dehydrated the tissue. The slices were then clanfied by placing them in Hemo-De for 2 minutes and covealiped. The slides were viewed using a microscope.
2.9 Data Analysis
Al1 the data are presented as group means f SEM. Data from the LDH release are expressed as enzyme units per milligram protein. An enzyme unit is defined as the amount of LDH that is able to reduce Ipol of pymvate to lpmol of lactate in one minute. The data obtained hom the cGMP radioimmunoassay is expressed as picomol cGMP per milligram protein. Because slices from the same animal were exposed to al1 conditions, a repeated-measures one-way Anova was used to determine if any of the groups were statistically different (Px0.05). A Banlett's test for heterogeneity of variance was completed pnor to the Anova. A Newman-Keuls post hoc test was then used to determine which groups were statistically different. 3. RESULTS
3.1 Time Course of LDH Release
LDH release was examined as a function of time. This data is presented in figure
3.1. Differing lengths of low[O~]/low[glucose],1 jmin., 30min, JSmin, and 60 min. were analyzed for LDH release. A LDH time course was done in order to determine an appropriate insult tirne. It was observed that increasing the duration of the
Iow[Oz]/low[glucose] insult linearly increased the arnount of LDH release up to
15minutes. AAer this, LDH release plateaued. The 30min. insult time was chosen for subsequent expenments because it produced a submavimal increase in LDH release.
3.2 Temperature and LDH Release
Decreasing the temperature dunng conditions of low[02]/Iow[glucose] was used
as a positive control. These results are presented in figure 3.2. Lowing the temperature
has previously been show to be neuroprotective in in vitro and in vivo ischemic models
(Tanimoto et al., 1987; Barone et al., 1997). It has been suggested that hypothemia is
protective due to its ability to lower the metabolic rate and conserve energy stores.
Hypothemia has also been used as a clinical treatment in people who suffered severe
ischemic stroke of the middle cerebral artery. Within 14 hours of the stroke, the induction
of hypothermia for 18-72 hours with cooling blankets and cold washings took place. It
was found that hypothermia decreased intracranial pressure (ICP) and improved sumival
from the stroke. nie only major side effect was the occurrence of pneumonia upon
rewarming. This indicates that hypothemia might be usehl clinically (Schwab et al.,
1998). In our study, the temperature was lowered to 30°C during the
low[02]/low[glucose] insult and the reperfûsion penod to produce a hypothermie state in Rat Hippocampal Slices Exposed to Differing Lengths of Low[02]/Low[Glucose]
I I I I O 15 30 45 60 Length of lschemia (minutes)
Figure 3.1 : Hippocampal slices were exposed to varyi ng lengths of low[02]/low[glucose], n= 12. Rat Hippocampal Slices Exposed to Hypotherrnic Conditions
Figure 3.2: lschemia @ 30*C refers to a 30min. ischemic insult and 4h reperfusion period at 30*C. Groups with different letters are significantly different from one another, n=5 for each group. the hippocampal brain slices. Lowering the temperature completely attenuated rises in
LDH release fiom hypoxia and hypoglycemia. LDH release fiom the slices exposed to hypothermia was the same as slices that had not undergone any insult. Hypothermia provided approximately 96% protection from ischemia. This result established that the in vitro mode1 of ischemia was efficient since it replicated what others have previously documented (Tanimoto et al., 1987; Barone et al., 1997; Schwab et al., 1998).
3.3 Conventional NO Donors
in order to determine whether NO has the potential to be protective in the treatrnent of ischemia, NO donors were administered at the beginning of the reperfusion period following a low[Oz]ilow[glucose] insult. These results are presented in figures
3.3-3.5. The NO donon Sin-1 chloride and GSNO were chosen due to their stability and relatively long half-lives in solution. There was some concem with Sin-1 chloride as it has been shown to spontaneously release superoxide anion as well as NO (Feelisch et al.,
1989). This may increase the formation of peroxynitnte, which is associated with neurotoxicity. For this reason, a second NO donor, GSNO was also examined. It was fond that Sin4 chlonde significantly protected the hippocampal brain slices from ischemia in a dose dependent rnanner. Maximum effects were observed at a concentration of 250pM. Sin-1 chloride afforded approximately 60% protection as compared to controls (Fig. 3.3). To verify that the results observed with Sin-1 chloride were due ro NO generation and not to any metabolites, NO exhausted Sin4 chloride was also studied. It has previously been show that a solution of Sin- 1 chloride stored at room temperature for more than 12 hours no longer releases NO (DrAeynolds: persona1 communication). Therefore, Sin4 chloride that had been in solution at room temperature Rat Hippocampal Slices Treated Wth Sin-1 Chloride
Figure 3.3: Groups with different letters are significantly different from one another. The number of experiments repeated is indicated in ( ). for at lest 12 hours was used. NO exhausted Sin4 chlonde had no significant effect on
LDH release (Fig. 3.4). These results indicate that the protective effects observed with
Sin-1 chloride are due to NO generation. The NO donor, GSNO, also significantly decreased LDH release from hypoxic/hypoglycemic slices. hterestingly, lOOpM of
GSNO was unable to protect the brain slices against ischemia, yet 50pM of GSNO did
(Fig. 3.5). The results obtained with GSNO differ from Sin4 chlonde in that greater concentrations of GSNO did not provide any neuroprotection.
3.4 Novel Organic Nitrates
Several organic nitrates were studied for neuroprotective effects. The following agents were examined, GT-091, GT-094 and GT-310. The chemical structure of these agents is similar to GTN. However, they differ from GTN in that they contain a substituted phenol group in place of the third nitrate. These results are presented in figures 3.6-3.8. Al1 dmgs were tested at an initial concentration of ZOOpM since this dose of the novel organic nitrates has previously been show to be neuroprotective in in vitro studies (Clarke et al., 2000). Toxicity was observed in the slices when the DMSO concentration was greater than 0.1%. For this reason al1 experiments where DMSO had to be used, the DMSO concentration was limited to less than or equal to 0.1%. The same arnount of DMSO was also included in the control groups to exclude the possibility that the DMSO was masking any neuroprotective effects of the agents being studied.
The novel organic nitrate, GT-091, produced maximal neuroprotective effects at al1 doses examined (Fig. 3.6). The GT-09 1 group was not significantly different from the control group, which did not undergo an ischemic insult. hterestingly, the LDH release observed with the GT-091 group was lower than that of the control group, which Rat Hippocampal Slices Treated With NO-Exhausted Sin4 Chloride
Figure 3.4: Groups with different letters are significantly different from one another. Sin-1-NO Ex stands for Sin-1 chloride that is NO exhausted. Sin-1 chloride was administered at a concentration of 250uM, n=3 for al1 groups. Rat Hippocampal Slices Treated With GSNO
control ischemia
Figure 3.5: Groups with different letters are significantly different from one another, ~~0.05,n=3 for al1 groups. Rat Hippocampal Slices Treated With GT-091
control ischemia 5OPM 10OPM 20OPM
Figure 3.6: Groups with different letters are significantly different from one another, pc0.05, n=3 for al1 groups. theoretically should have shown no darnage. This suggests that GT-091 can protect against some of the darnage that occurs as a result of tissue isolation and brain slice preparation.
GT-094 was also able to significantly decrease LDH release From the low[Oz]/low[glucose] control (Fig. 3.7). This effect was dose dependent with maximum effects being observed at 50pM. GT-094 was unable to decrease LDH levels to control values, yet GT-094 was still 83% protective as compared to controis.
GT-3 10 was synthesized as a congener of GTN and Trolau, an antioxidant. This was done in order to combine any neuroprotective effects of NO with that of an antioxidant. Treatment with GT-310 significantly protected the hippocampal brain slices from ischemia by approxirnately 55%. Maximum effects were observed at a concentration of 100pM and the effects of GT-310 were dose-dependent (Fig. 3.8).
3.5 Synthetic cGMP Analogues
Previous studies have shown that the novel organic nitrate, GT-015 is able to increase cGMP. In order to elucidate if this was how the novel organic nitrates were causing neuroprotection, a nurnber of synthetic cGMP analogues were analyzed for neuroprotective properties. The goal of these expenments was to determine whether the synthetic cGMP analogues could mimic the neuroprotection observed with the novel organic nitrates. Three different synthetic analogues, 8-bromo-cGMP, dibutyryl cGMP and 8-pCPT-cGMP were studied. They were chosen due to their differences in cell membrane permeability and phosphodiesterase sensitivity. It was undesirable to miss any neuroprotective actions of cGMP due to susceptibility to breakdown by Rat Hippocampal Slices Treated With GT-094
control ischemia 5OPM
Figure 3.7: Groups with different letters are significantly different from one another, pc0.05, n=3 for al1 groups. Rat Hippocampal Slices Treated With GT3lO
control hypoxia 25pM 5OPM 100CiM
Figure 3.8: Groups with different letters are significantly different from one another, pc0.01, n=5 for al1 groups. phosphodiesterases or an inability to cross the membrane. These results are presented in
figures 3.9-3.12.
8-Brorno-cGMP is a synthetic cGMP analogue that is more resistant to breakdown by PDEs than cGMP. Furthemore, 8-bromo-cGMP has a greater activation potential for
PKG than cGMP. ùiitially, 8-bromo-cGMP appeared to completely protect the
hippocarnpal brain slices from ischemic injury. However, it was discovered that this
apparent protection as exhibited through a reduction in LDH activity was actually due to a
direct inhibition of the LDH enzyme itself. This was observed when 8-bromo-cGMP was
purchased From Sigma as opposed to Tocns. 8-Bromo-cGMP purchased from Sigma did
not produce any significant neuroprotective effects (Fig. 3.9). it was later determined that
the neuroprotective effects observed with 8-bromo-cGMP were due to direct inhibition of
the LDH enzyme. Al1 the dmgs used in the preparation of this thesis were subsequently
tested for any interference with the LDH assay.
Dibutyryl cGMP is a membrane penneable synthetic cGMP analogue. It was
observed that dibutyryl cGMP was able to significantly protect the brain slices from
low[Oz]/low[glucose] in a dose dependent manner. This partial protection was observed
to be approximately 37% as compared to controls (Fig. 3.10). These results demonstrate
that cGMP may be involved in a neuroprotective mechanisrn against ischemic injury.
Cyclic GMP was also exarnined. Since cGMP is unable to pass the ce11
membrane, it was used to determine whether dibutyryl cGMP is involved in an
intracellular or extracellular rnechanisrn of neuroprotective. No signi ficant
neuroprotection was observed with cGW (Fig. 3.1 1). This indicates that the effects
observed with dibutyryl cGMP are due to an intracellular mechanism. Hippocampal Slices Treated with 1mM 8-BromocGMP
Figuire 3.9: A. 8-bromo- cGMP was purchased from Tocris ~uoksonInc. B. &bromcGMP was purchased from Sigma-Aldrich Co. Groups with different letters are significantly different from one another. lhe number of experiments repeated are indicated in ( ). Rat Hippocampal Slices Treated With Dibutyryl cGMP
Figure 3.10: Groups ~4thdifferent letters are significantly different from one another, pc0.05. The number of experiments repeated are indicated in ( ). Rat Hippocampal Slices Treated With cGMP
control ischemia
Figure 3.11: Groups with different letters are significantly different from one another, ~~0.05,n=3 for al1 groups. 8-pCPT-cGMP was analyzed for neuroprotective properties. This cGMP analogue differs fiom 8-bromo-cGMP and dibutyryl cGMP in that it is highly resistant to breakdown by PDEs. It is also membrane penneable and a potent activator of PKG. 8- pCPT-cGMP was unable to significantly protect the hippocampal slices from low[Oz]/low[glucose] (Fig. 3.12). This suggests that PKG activation is not involved in a neuroprotec tive mec hanism.
3.5.1 Dibutyryl cGMP and Rp-8-pCPT-cGMP
A PKG inhibitor, Rp-8-pCPT-cGMP, was CO-appliedwith dibutyryl cGMP to the hippocampal slices at the beginning of the reperfusion period in order to determine whether the protection observed with dibutyryl cGMP was PKG dependent. These results are shown in figure 3.13. Rp-8-pCPT-cGMP (7.5pM) was unable to attenuate the protection observed with dibutyryl cGMP. This concentration of Rp-8-pCPT-cGMP was previously show to inhibit PKG activation (Vaandrager et al., 1997). Rp-8-pCPT-cGMP has a K, of 0.5pM for PKG and a K, of 8.3pM For PKA (Butt et al., 1994). Thus at a concentration of 7.5pM, Rp-8-pCPT-cGMP should completely inhibit PKG and slightly inhibit PKA showing moderate selectivity for PKG. Therefore it was concluded that the effects of dibutyyl cGMP were not due to a PKG mediated mechanism.
3.6 Synthetic CAMP Analogues
An alternative mechanisrn of cGMP could involve inhibition of phosphodiesterases, which would increase the intracellular levels of cGMP and CAMP. In order to determine if increasing the CAMP levels would be protective against ischemic injury the synthetic CAMP analogue, dibutyryl CAMP was Rat Hippocampal Slices Treated Wth 8-p-CPT-cGMP
control ischemia 40OPM 20OPM 10OPM
Figure 3.12: Groups with different letters are significantly different from one another, pc0.05, n=3 for al1 groups. Rat Hippocampal Slices Treated Wth Dibutyryl cGMP and Rp-8-pCPT-cGM P
control ischemia
Figure 3.13: DB stands for di butyryl cGMP (100pM) and Rp stands for Rp-8-pCPT-cGMP (7.5pM). Groups with different letters are significantly different from one another, ~~0.05,n=3 for al1 groups. examined. Concentrations similar to that of dibutyryl cGMP were used. These results are shown in figure 3.14. Dibutyryl CAMP dose-dependently protected the slices against ischemic damage. Maximum protection of 58% as compared to controls was observed at
200pM demonstrating that CAMPrnay also be involved in a neuroprotective mechanism.
The synthetic CAMP analogue, 8-bromo-CAMP was also studied for neuroprotective properties. These results are presented in figure 3.15. No significant protection was observed with 8-bromo-CAMP. Similar to the synthetic cGMP analogues, these differing results may be due to the distinctive properties of the two synthetic analogues. 8-bromo-CAMP may be more susceptible to breakdown by phosphodiesterases or it may be less able to diffûse across the ce11 membrane as compared to dibutyryl CAMP.
3.6.1 Dibutyryl CAMPand H-89
In order to determine if the neuroprotective effects of dibutyryl CAMP were due to
PKA activation, H-89, a potent PKA inhibitor, was CO-appliedwith dibutyryl CAMPat the beginning of the reperfusion period. At a concentration of 7.5pM, PKA should have been effectively inhibited in our expenmental mode1 since H-89 has a K, of 0.048 f 0.008pM
(Chijiwa et al., 1990). These results are presented in figure 3.16. It was observed that H-
89 was unable to attenuate the effects of dibutyryl CAMP. H-89 did not have any significant protective effects against low[02]/low[glucose] on its own. These results propose that a PUmediated mechanism of neuroprotection is not responsible for the effects of CAMP. ln addition, these results demonstrate that the neuroprotective properties of cGMP are not due to PKA activation.
3.7 Dibutyryl cGMP and Forskoiin LDH Release Fold lncrease above Control (Eulmg protein) Rat Hippocampal Slices Treated With 8-Bromo-CAMP
control ischemia
Figure 3.15: Groups with different letters are significantly different from one another, ~~0.05,n=4 for al1 groups. Rat Hippocampal Slices Treated With Dibutyryl cAMP and H-89
Figure 3.16: DcAMP stands for di butyryl cAMP (50pM), H-89 is present at 7.5pM. Groups with different letters are significantly different from one another, p<0.05, n=3 for al1 groups. in order to more clearly define the relationship between CAMP and cGMP, dibutyryl cGMP and fonkolin were studied in combination against oxygen/glucose deprivation. Forskolin is a potent activator of adenylyl cyclase and increases the concentration of CAMP in the cell. If CAMP and cGMP are acting through two separate mechanisms, the effects of raising the intracellular levels of CAMP and cGMP would be expected to be additive. Similarly, the effect of raising the intracellular levels of CAMP and cGMP would be potentiated, if the two cyclic nucleotides were causing neuroprotection by the same mechanism. These results are presented in figure 3.17. It was observed that dibutyryl cGMP and fonkolin significantly decreased LDH release from the low[Oz]/low[glucose] control group both independently and together. However, the protective effects of the two agents on their own and together were not significantly different fiom one another. No potentiation or additive actions were seen with the administration of these two agents. This may be due to the dose of dibutyryl cGMP and forskolin that were studied. These doses may already be producing maximum neuroprotective effects. If maximal doses are being used then no potentiation of effects will be obsetved. However, if the two agents were acting by different mechanisms then additive effects would still be expected to occur. Therefore these results would indicate that cGMP and CAMP are acting through a cornmon mechanism.
3.8 ODQ
It is hypothesized that the effects of GT-094 are due to activation of guanylyl cyclase and generation of cGMP. If the effects of GT-094 are dependent upon cGMP production a guanylyl cyclase inhibitor, ODQ, should be able to attenuate the effects of
GT-094. GT-094 was CO-appliedwith ODQ (0.5pM) at the start of the reperfusion Rat Hippocampal Slices Treated with 50p M Forskolin and 100pM Dibutyryl cGMP
control ischemia For.
Figure 3.17: For. stands for forsko in and DB stands for d butyryl cGMP. Groups with different letters are signi Ficantly different from one another, pc0.05, n=4 for al groups. period. These results are presented in figure 3.18. It was observed that ODQ at this concentration was unable to block the neuroprotective effects of GT-094. Additionally,
ODQ did not have any significant effects on LDH release at this concentration. Thus the effects of GT-094 are not dependent upon cGMP generation.
To determine if the effects of Sin-l chloride (250pM) were dependent upon cGMP production, ODQ (0.5pM) was CO-appliedwith Sin-l chloride at the beginning of the reperfusion period. These results are presented in figure 3.19. Similar to GT-094,
ODQ could not block the neuroprotective effects of Sin-1 chloride. This indicates that cGMP does not play a role in the neuroprotective mechanism provided by nitric oxide.
hterestingly, it was also determined that ODQ on its own was able to significantly decrease LDH release from the low[O~]/low[glucose]control group. This effect was dose dependent with approximately 32% protection being observed at a concentration of
10pM. The effects of ODQ were ameliorated at a concentration o10.5pM. These results
are presented in figure 3.20. This suggests that ODQ is involved in a neuroprotective
mechanism. ODQ is able to inhibit guanylyl cyclase by interacting with a heme moiety
on that enzyme (Feelisch et al.. 1999). Therefore ODQ may be inhibiting a
neurodestructive enzyme or a toxic pathway by reacting with heme moieties.
3.9 cGMP Radioimmunoassay
Previous studies have stated that ODQ is able to inhibit guanylyl cyclase at a
concentration of 0.5uM (Tseng et al., 2000). Some NO donon are much more potent
than othen and require a higher concentration of ODQ to inhibit the activation of
guanylyl cyclase. A cGh4P radioirnrnunoassay was performed to verifj that ODQ at a
concentration of 1pM was able to inhibit guanylyl cyclase activation mediated by GT-094 LDH Release (EUlmg prote in)
control
ischemia
GT-094
GT-094 + ODQ ci' CD V) ODQ Rat Hippocam al Slices Treated Wth 2! 10pMSin-1 Chloride and 0.5pM ODQ
Figure 3.19: Groups with different letters are significantly different from one another, pc0.05, n=3. Sin-1 stands for Sin-1 chloride. Rat Hippocampal Slices Treated With ODQ
50-
40-
30-
20 -
IO-
Figure 3.20: Relative percent protection as compared to the controls in that particular experiment. The number of experiments repeated are indicated in ( ). (50pM). These results are presented in figure 3.21. The cGMP levels observed in the control grooups were similar to previous findings in our laboratory (Clarke et al., 2000).
Interestingly, GT-094 was unable to significantly increase cGMP to levels obtained in the control group. The ischemic insult significantly decreased cGMP levels as compared to the control group. Treatment with GT-094, GT-094 and ODQ did not significantly alter cGMP levels from that observed in the low[0~]/low[glucose]group. These results indicate that the effects of GT-094 are not due to cGMP generation since GT-094 is unable to increase cGMP levels,
3.1 1 Cresyl Violet Staining
Hippocarnpal slices treated with GT-094 (100pM) and Sin- 1 chloride (250pM)were
stained to veriS, the LDH release data. Photomicrographs of the hippocampal slices are
displayed in figure 3.22. Sirnilar to the LDH release findings, 30 minutes of
oxygen/glucose deprivation causes marked neuronal degeneration in the CA 1 region of
the hippocampus. The CA1 region of the hippocampus was examined because it is the
area most vulnerable to ischemic damage. There was less neuronal darnage in
hippocarnpal slices treated with GT-094 and Sin- 1 chloride. These results are comparable
to the LDH release data. Concentration of cGMP in Rat Hippocampa Slices
Figure 3.21: GT-094 was administered at a concentration of 50pM and ODQ was administered at a concentration of 1PM. Groups with different letters are significantly different from one another, p<0.05.
1.0 DISCUSSION
'I The purpose of this study was to examine the mechanisms behind preçiouslv observed neuroprotection with novel orsanic nitrates (Clarke et al.. 2000). In order to address this research problem an in \~omodel of stroke. usine hippocarnpal brain slices. was chosen. The III irrro model has several advantages over the ir~Wo model. .ln i)r i*it~.oniodel is ideal for a mechanistic study as al1 esternal svnaptic connections are severed. In addition. the external environment. which is under the control of the investirrator. can be easily manipiilated. In order to be exposed to the brain slices. dmgs are simplv included in the bathin? solution Only the membrane permeability of the particular agent and the thickness of the slice need to be considered. Funher. unlike cell culture systems. the trisynaptic circuit is maintained in brain slices preserving communicati«n between ditrerent areas of the hippocampus. .A disadvantage of usins Irr wro models is the extent to which they replicate the corresponding 11, win situation.
Although this is a major limitation. especially when interpretins data. the information provided from itr i~oesperiments can be invaluable when t-ing to elucidate physiolo@cal processes. An iil \?/ru model was thourht to be more appropriate for the purposes of this study. which was to determine if novel organic nitrates demonstrate neuroprotective propenies by their ability to increase levels of cGMP.
In order to ascertain an appropriate lenpth of insult. a time course of osygen/glucose deprivation. in relation to LDH releasr was completed. .A half hour ischemic insult produced a sub-maximal increase in LDH release and was used for ail subsequent experiments. It has been documented that 5-7 minutes of an in rim ischemic insult is suficient to cause damage io the CA1 pyramidal cells of the hippocampus.
Lon-er insuit times were required to cause sirnilar damase to the dentate granule cells of the hippocampus. It has been observed that 30 minutes results in damage to vnaptic transmission. protein synthesis. maintenance of ATP levels. cytoskeletal integrity and neuronal morpholosy (see review: Lipton. 1999). This is similar to our findings where 15 minutes of iit vitro ischemia caused an increase in LDH release. probably retlecting CAl pvramidal cell death. and 30 minutes caused a sub-maximal increase in LDH release.
Hvpothemia has been found previouslv to act as a neuroprotectant in il) ilitro and iii riia expenments (Tanimoto et al.. 1987). A clinical trial has been completed which exarnined the potential of hypothermia in the treatment of stroke. It was found that hypothermia could decrease neurolo-,ical damage followiny stroke. however. some patients developed pneumonia upon rewarming and this may limit the clinical usetùlness of hypothermia
(Schwab et al.. 1998) It has been hypothesized that the neuroprotective effects of hvpothermia are due to its ability to decrease the metabolic rate and energ requirements of the tissue This reduction in metabolic rate heips conserve energy stores. which are massivelv depleted as a result of stroke (Ha-erdal et al.. 1975). In our studies. hypothermia was used as a posirive control to validate the ut iwtv model of ischemia.
Lowering the tempernt~ire aforded complete protection a~ainstlow[02]/low[glucose].
Since the previously documented neuroprotective etfects of hypothermia could be replicated in our model. this su-gested that the model was appropriate for studyins asents in ischemia. Similar to the esperiment conducted bv Tanimoto and colleagues ( 1987). hypothermia was initiated dut-in~the ischemic insult Interestin&. when hypothermia was induced in the reperfusion period. no neuroprotective effects were observed. This suggests that hypot hermia is protecrive againsi earlv irreversible damase caused bu
O-xygen/@ucosedeprivation. The second objective of this research project was to determine if established nitric oxide donors were neuroprotective against oxyged~lucosedeptivation. Two widely used nitic oxide donors. Sin- l chloride and S-nitroso_elutathione (GSNO). were administered dunng the reperfùsion period to determine whether nitric oxide has any neuroprotective putential against ischemic injury. Hippocampal slices treated with Sin-1 chloride at the beginnins of the reperhsion prriod were signiticantly protected from a low[O:]/low[glucose] insult in a dose dependent manner In addition. NO-exhausted Sin-
I chlonde could not replicate the previously observed eîFects of Sin-1 chlonde. This indicates that the actions of Sin-l çhloride are due to VO generation These tindin-s replicate previous studirs. which have found Sin-l chloride to be protective in neuronal injury Sin- 1 chlonde significantly decreased infarct volume caused by btCh occlusion
~vhenadministered up to 60 minutes afier the insult (Zhang et al.. 1994) hother study found that treatment with Sin4 chloride concurrently rvith NMDA protected murine mised cortical cell cultures against NhIDh neiirotosicity The investisators suggested that this neuroprntection \vas due to inhibition of NMD.4 induced ~a'currents (Vidwans et al.. 1999).
In contrast. soine cell culture studies have found that Sin4 can produce neuronal tosicity Neuronal death caused by Sin-l rsposure \vas potentiated in the presence of superoside dismutase in cortical cultures. indicatina that the neurotoxic rffects observed with Sin- 1 were a result of hvdrogen peroxide formation. This group of investisators also observed that Sin- 1. up to concentrations of 500pXI. had no efect on neuronal viability
Interest in& application of synt het ic cGhP anaIoeues could reverse the neurotosicity observed with Sin- 1 (Moro et ai.. 1998). This study demonstrates that the neurotosic etfects of Sin4 are associated with hydrogen peroxide formation and sugests that the neuroprotective effects were due to 'IO mediated cGlMP production. .Another studv camed out in ceIl culture reported that the neurotoxic etiects with Sin-1 occur at concentrations equal to or greater than ImiM (Lipton et al.. 1993). In addition. in Chinese hamster V79 lung fibroblasts. ImM Sin-l potentiated cytotosicity induced by hydroren peroide (Wink et al . 1996). These tindings are contrary to what was observed in our esperiments. The touicity associated with Sin- l chloride reported above occurred at concentrations higher than i00p11 In our study. Sin- l chloride was onlv used at concentrations less than 500uXI. The diferent results observed with Sin- l chloride may be a consequrnce of the concentration ~itilized Prrhaps at higher concentrations such as
Imkl there is a qeatrr probability of perouynitrite torrnation due to increased superoxide anion production. In our study. only the beneficial etfects were observed with Sin4 chloride as indicated bv a decrease in LDH release
.A second nitric oside donor. GS60 \vas used to ver@ that the neuroprotective efkcts observed with Sin-1 chloride werr dur to \O seneration GSNO is a congener of nitric oside and glutathione that spontanrously releases nitric oside. Singh and colleagues ( 1999) demonstrated that GSNO released nitric oxide in a linear fashion up to
6h in a liposomal mode1 with SOD and hydrogen peroside in Chelex-treated phosphate buff'er This suggests that GSNO will release nitric oside for the duration of the repertiision period making it an ideal agent for our esperiments GSNO protected the hippocampal slices hm hvpoxic/hypoglycemic inju~at a concent ration of SOp M.
Interestin& at the higher dose of 1OOpM. GSNO did not decrease LDH release from that of the hyposic/hvpo~lvcemiccontrol This is contra? to what kvas obsenred with
Sin- l chlonde. which produced dose dependent neuroprotective effect s wit h maximum neuroprotection being observed at 50pM. .-\ possible reason for these codicting results is that the nitric oxide donating abilitv differs between these two agents. GSNO may be a more potent nitric oside donor with tosic concentrations of nitric oxide being obtained at
1 OOpM. With escessive concentrations of nitric oxide being produced at 1 OOpM. there will be an increased probability of an interaction with superoxide anion and tosic effects associated with the formation of perosynitrite Thus. these results support the hypothesis that nitric oside has the potential to be neuroprotecti\~eif it is present in the appropriate concentration range These findings also demonsrrate tliat nitric oside has the potential to be neuroprotective adainst ischemic injun when adrninistered at the bqinning of the repertùsion period
Novel organic nitrates are a group of established nitric oside donors derived from the chemical structure of GTS GTN has previously been found to be neuroprotective in
/il wtnand in tri wostudies t Sathi et al . le9.3. Lei et al . 1992) GTN is cumposed of a
by novrl brrlyceryl backbone with three nitrate yroups attached ester bonds The orsanic nitrates difer in that rhes contain a substituted phenol group attached bv a disulfide bond in place of the third nitrate The unique propenies of the novel organic nitrates are thought to anse form this third group In some cases. this third group has been synthesized to act as an antioxidant
Three novel orsanic nitrates were analyzed for neuroprotect ive propenies. GT-
09 1. GT-094 and GT-3 l O The novel organic nitrates were dissolved in DMSO. The
DMSO concentrations were equal to or less than O 1 O to avoid tosicity DMSO was also included in the control and ischemic sarnples to control for the effects of DMSO on LDH release The protectivr effects of GT-094 and GT-3 10 were dose dependent. Equal protection tbas observed with GT-09 1 ar al1 concentrations attempted. The relative degree of neuroprotection observed with the novel organic nitrates are as follows GT-
09 1 -GT-O94:GT-3 10.
-Alterations in the chemical formulation mav account for the different etfects observed with the novel oreanic nitrates since changes in the '10 donating- ability or the potency of the compound will alter its neuroprotective propenies. -411 of the novel organic nitrates were tested at an original concentration of 200pX1 GT-O15 \+as previously found to be neuroprotective at 100p51. which is why this concentration was chosen for subseqiienr esperiments uith the novel organic nitrates (Clarke et al.. 2000)
Additionally. GT-3 10 \vas svnthesized with an alpha rocopherol goup. which is the pan of vitamin E that is associated ivith its antiosidant tttfects Therefore the nruroprotective propenies obsened with GT-3 1 O ma? be due to its ability to behave as an antiosidant. as opposed t« a cGMP mrdiated mechanisni
Previous studies with GT-(1I S and GT-7 15 have sugested that the novel orsanic nitrates esen their neuroprotective etfect s t hrough a cGhlP mediated mechanism since the neuroprotective etfects of GT-O 1 5 could be attenuated by the co-administration of
ODQ. a guanylyl cyclase inhibitor Funhermore it was observed that GT-O l i was able to increase cGMP levels in ischemic hippocampal tissue as assayed bu a cGMP radioimrnunoassay ( Clarke et al.. 1000) This provides hnher evidence that the neuroprotective efects of GT-O15 are dependrnt upon the production of cGMP In addition. GT-7 1 5 improved task-acquisition in scopolamine-treated male rats in the
Morris water maze This was found to be associated with a geater activation potential for soluble ganylyl cyclase than GTN. Thesr findings support the notion that novel organic nitrates can improve neuronal recovery following an insult by a cGMP mediated mechanism ( Smith et al.. 2000). In order to determine whether the neuroprotection observed with the novel organic nitrates was due to cGMP seneration. a number of synthetic cGMP analogues were rxplored. If the etfects observed with the novei O-anic nitrates could be mimicked by the synthetic cGMP analogues this would suggest that the novel organic nitrates are acting throush cGMP Three synthetic cGMP analoyues. dibuty-1 cGMP. 8-bromo- cGhlP. S-pCPT-cGhIP and cGNP itself were studied These agents were chosen due to ditferences in membrane permeability and susceptibilitv to phosphodiesterases Since cGMP is unable to pass the cell membrane. this agent was administered to determine whether the efects obsened with the synthetic cGMP analogues were due to an intracellular or extracellular mechanisni Signitiçant neuruprotecti~~eetiects wre obsemed oniy witli the membrane permeable dibutyryl cGXlP The etfrcts of dibuirylyl cGhlP were dose dependent with inasiniuin neuroprotection occurring at 5OuM This indicates that the nruroprotective etfects were due to an intracellular mechanism as opposed to estracellular
Our obsenbations uith dibutvd- - cGSlP. which point to a cGh1P mechanism of ncuroprotecrion. are challen-ed by the rsperiments carried out with S-bromo-cGhIP and
S-pCPT-cGblP No neiiroprotection was observed with 8-bromo-cGMP or 8-pCPT- cGhIP Thesr results ma! be due to the different physio-chrmical propenies of these agents. S-Bromo-cGhIP is relatively resistant to breakdown by PDEs. It is also moderatelv se1ectit.e tor PKG activation with Ki, values of I O-26nM hr PKG-Iu. 2 1 O-
1000nM for PKG-IP and 3niM for PKG II. The major limitation with S-bromo-cGMP is that it is not very membrane permeable: it is only 2.5 fold more permeable than cGMP
Therefore. it is possible that 8-bromo-cGiW did not sain access to the cell's interior. Like S-bromo-cGMP. S-pCPT-cGW is a potent activator of PKG and is resistant to hvdrolvsis by PDEs The K., values for 8-pCPT-cGMP are comprisable to that of S- bromo-cGMP. however. unlike 8-bromo-cGhlP. S-pCPT-cGMP is 56 fold more membrane permeable than cGMP This would su-sest that S-pCPT-cGMP is able to enter the interior of the neuron Cnlike the other tuo asents. dibutyyl cGhlP is si~sceptibleto breakdown bu PDEs and is a poor activator of PKG Dibutvnl cGhlP is also brokrn down t» buty-yl-substituted cyclic nucleotides that ma' have etiects on signal transduction pathways Siinilar to S-pCPT-cGhIP. dibutvryl cGMP is 62 fold more membrane penneable than cGhlP (see review Scliuede et al.. 2000)
In conclusion. the lack of neuroprotective rtfects observed with S-bromo-cGMP ma- be due to its inability to cross tlir neuronal membrane Our finding su-gest rhat the neuroprotective properties of dibutvryl cGhlP are a result of an intracellular mechanism. as the- coiild not be replicated by cGMP Sincr S-pCPT-cGhP and dibutvrvl- - cGMP are borh able to cross the neiironal membrane. diferences in the physio-cltemical properties of these agents ma! provide dues as to the neuroprotective pathway mediated by dibutyl cGMP The major ditference betn ern these twagents is that S-pCPT-cGMP is a potent activator of PKG Thus the results from Our study would indicate that activation of PKG does not plav a role in rhc neuroprotective mechanism mediated by dibutyryi
LGMP However. tùnher studies wouid have to be done to determine if this is the case
In addition. esperinients could have bren camed out to ensure that the effects of dibutyl cGMP are not due to butyrate metabolites.
A number of studies esaminin- the neuroprotective role of the secreted tom of amyloid precursor protein (SAPPU) have postulated a PKG mediated mechanism of neuroprotection. The neuroprotection aforded bv SAPPU could be mimicked by synthetic cGMP analogues and be attenuated bv a PKG inhibitor (Mattson et al.. 1999.
Barger et al.. 1995- Funikawa et al.. 1998. Funikawa et al. 1996). tt has been susrested that this neuroprotection is due to activation of K' channeis. which leads to a decrease in intracellular ~a'(Funikawa et al.. 19%). This would be beneficial in ischemia when the neurons are ovrrloaded with ~a'It Iias also been demonstrated that SAPPU is able to inhibit the NMDA receptor b? a cGhlWPKG mechanism which bloclis C'a:' influx into the neuron (Funikaw et al . 1 V)S. Baryr et al . 1995) Previoiis studies have hypothesized that Nb1D.A antagmists may be iiseful in the treatment of ischemia becausr rhe NMD.4 recrptor is overactivated in h~~po~ivhypu~lycernicconditions (Arias et al..
19W Schurr et al . ICIW Theretbre. a cGMP/PKG rnediated rnechanism of NhlD.4 receptor inhibition Iias the potentiai to br nruroprotective apainst ischemic injury Finally it has been reponrd that SAPPU is respcinsible Ior inçrrased uptake of glucose and
-dutaniate into synaptic companments and that this response is mediated by a cGMP!PKG mechanism ( Jlattson et al.. 1 999) This can potentially be neuroproiective in ischemia since glucose levels are drplaed and glutamate acciimulates in the synaptic cleti.
In summary. these studies indicate that cGMP mav plav a protective role in ischemia by several PKG mediated mechanisms its ability to activate K channels
(Furukawa et al . 1996). its ability to inhibit the Nb1D.A receptor (Furuliawa et al.. 1998.
Barger et al.. 1995): and its ability to increase glucose and glutamate uptake into synaptis compartments (Mattson et al. 1999) .Al1 of these mechanisms have the potential to decrease injury caused by oygen/glucose deprivation. -4 PKG inhibitor. Rp-8-pCPT-cGhIP was CO-administeredwith dibutvryl cGMP in order to determine if the effects of diblityr-l cGMP were dependent upon PKG activation. Treatment with Rp-8-pCPT-cGhIP did not attenuate rhe protective effects that were observed with dibuty-yl cGhlP There were no signiticant differences between treatment with dibutyryl cGhlP and treatment witti dibutvryl cGMP and Rp-8-pCPT- cGXlP against neiironal dainage çaused by osygervglucose deprivation. Thus the neuroprotective etfects of dibutyyl cGhlP are not dependent upon PKG activation.
This is in contrast to the finding from the sAPPn studies and çould be due to a diffèrent esperimentai model iised in t heir stiidies The observations made in t heir rsperiments were from synaptosonies (Jlattson et al . 1999) and hippocampal cell culture systems ( Bargrr et al . 19%. Funikau.a et al . 1996 R: I WS
Since it has been proposed that the neuroprotective etiects of the novel organic nitrates and Sin- l chloride are dependent iipon cGXlP generation (Xloro et al -1998). GT-
094. was CO-adrninisteredwith the guanylyl cyclase inliibitor. ODQ The neuroprotection attorded bu GT-094. could not be attenuated bv the CO-administrationof ODQ This would indicate that the neiiroprotective propenies of GT-094 are not dependent iipon production of cGMP Therefore. GT-094 could be actins bv an alternative mechanism such as inhibition of the yL'X[D.A recrptor or bu acting as an antiosidant. In addition.
ODQ \vas not able to attenuate the protective etfects of Sin-1 chloride. indicating that the ability of Sin4 chloride to increase survival of brain slices exposed to low[02]/lo\v[~lucose]is not dependent upon cGbP seneration. Sin- l chlonde ma! be providina neuroprotection by one of the mechanisms mentioned above for GT-O94 To detemine if ODQ was etfectively inhibiting guanvlvl cyclase activation and if
GT-094 was increasing cGMP concentrations in the slices. a radioimmunoassay for cGMP \vas completed. It was obsened that GT-094 was unable to signiticantly increase cGMP concentrat ions in slices exposed to osygemglucose deprivation. In addition. ODQ did not fiirther decrease cGhIP concentrations froin the low[02]/low[glucose] controis.
This may be diie to the hct that yanylyl cylase is not being activated by GT-094 It is surprisins that GT-O94 !vas unable to inçrease çGMP levels above that of the lo~v[02]/low[glitcose]contr~ils since it has previousiy been su-eested that the novel orsanic nitrates are protectivr bu a cGhlP mrdiated rnrtchanism Therefore. the novel orsaiiic nitrates ma- act by ditferent pathways dependin2 upon their specitic physio- cliemical propenies Xeuroprotection atforded bu nitric oside donors rnay be due to their ability to inhibit the Sh1D.A rrceptor (Lei a al . I9V) or to act as antiosidants (Rubbo et al . 1994) Since the rfects of GT-094 are n«t due to cGMP generation this tvould indicate that GT-0W nia- be acting through an alternative mechanism such as inhibition of the NMDA rrceptor or scavenging of lipid free radicals.
Even rhoiigh GT-091 ma" br eserting its neuroprotective etTects by an alternative inechanism. our results indicate that cGW plays a rule in protecrins neurons tioni ischemia. In suppon of our tindings. a number of researchers have published data that support the concept that the soluble giianylyl cyclaseicGhlP pathway is involved in
neuroprotectioii. It was obsen-ed that giianylyl cyclase inhibitors increased neuronal cell death in cerebellar slices esposed to escitatoc amino acids. The effects of the guanylyl
cvclase inhibit ors coiild be reversed by administration of synthetic cGMP analo,wes or
phosphodiesterase inhibitors. It was susgested that cGMP ma? be part of a protective
mechanism agiainst oqen free radicals since the tosicity observed rvith yanylyl cyclase inhibitors was similar to that of oxvsen free radical generatins enzyme systerns such as -ducose osidase and sanithine oxidase. Glucose osidase and xanthine oxidase increase production of the oxidants. hydrogen peroside and superoxide anion. Furthemore. the addition of fier radical scavenging enzymes. such as catalase and the reduction of the partial pressure of osygen dccreased the neurotouicity observed with guanylyl cyclase inhibitors (Ganhwaite et al . 1988) In addition. another study demonstrated that cyclic nucleotides could protect neuronal PC6 crlls from lipid perosidation (Keller et al . 1998)
Thrse investisations indicate that cGMP can potentially decrease neuronal injury associated wi t h free radical ~enrratiun
It was recently discovered that ÇGMP is able to inhibit .AMP..\ ~eneratedcurrents by -3S00 using whole-cell recordings in hippocampal neurons The inhibiton. actions of cGMP coiild not hr niiniickrd by rstraçrllular perfiision of cGhlP. indicatins that the ettects of cGhfP were a result of an intracell~ilarmechanism. .AiCIP.A inhibtion was not dependent upon PKG activation (Lei et al . 2000) Similarly. in our study. dibutyryl cGhlP protected the hippocampal neiirons bu XOofrom an /Ir ~roischemic insult This protection appears to be due to an intracellular pathivay as our results sould not be replicated by the membrane impermeable cGMP Thrse resuirs suggest that cGMP ma!. be esertino neuroprotective properties in our mode1 bv inhibition of the -4MP.A receptor
The second messenger. cGhlP. is also able to nctivate cyclic nucleotide gated
(CNG) cation channels ( Dzeja et al.. 1999) and inhibit PDEs (Maurice Rr Haslam. 1990)
The recently characterized CNG channeis allow the entry of cations upon activation by
intracellular cyclic nucleotides. The! were originally discovered in olfactory and
photoreceptor sensory neurons and were hypothesîzed to play an important role in
auditory and visual tùnctions however. the- have been found in other tissues. such as the CA 1-3 hippocampal neurons (see re\;ie\v: Biel et al . 1999). Intuitively. it would not appear that CNG channels would be protective in ischemia since thev cause increases in intracellular ions. Excessive Ca" accumulation has been su-gested to be a major mediator of neuronal injury. Therefore. tünher increases in Ca' by activation of a CNG channel would more likely potentiate neuronal darnage. as opposed to being protective in ischemia.
Cyclic nucleotides are also able to inhibit PDEs. which are responsible for the breakdown of cGhlP and c.AW in the cell Recent studies with rolipram. a PDE inhibitor. have rrponed decreases in injury associated with ischemia (Block et al.. 1997.
[manishi et al.. 1097). Rolipram inhibits PDEJ, which is responsible for the breakdown of cAX1P Therefore. treatinrnt ivith rolipram increases the concentration of c.-\MP in the nruron. Ir aas hund thpost-ischemic rreatrnrrit ivith rolipram decreased deticits of working memory in rats caused by four-vesse1 occliision. Learning and memory were accessed bu a 3-panel ninway paradigm (Imanishi et al.. 1997). Thus increasinp c.-\XIP atirr an ischemic insult can be beneticial in treatin- the behavioral manifestations of cerebral ischemia. It was also obsensed that treatrnent with rolipram up to six hours atier the ischemic insult decreased ce11 loss in the CA 1 region of the hippocampus and the striatum. The ischemic insult was induced by tour-vesse1 occlusion for 20 minutes
(Block et al.. 1997). This is clinically relevant. since the majority of patients do not seek rnedical attention until afier the stroke has occurred. At this time the only available agents that are etfective in decreasins neuronal damape after ischemia are tissue plasminogen activators (TP.4). Therefore novei therapeutics designed to increase may be eficacious in improving neuronal outcome following stroke. This hypot hesis was tùnher examined bu investigating the possible neuroprotective propenies of the synthetic CAMP analogues. 8-bromo-CAMP and dibutyryl CAMP Similar to the synthetic cGhIP analogues. 8-bromo-CAMP did not protect the hippocampal neurons from neuronal injury However. dibuty-l CAMP was able to sig~ificantlpdecrease LDH release tiom the low[Ol]/low[glucose] slices in a dose dependent manner Dibutyryl CAMP mimicked the neuroprotection obsened with dibiitynj cGhlP. indicatins that c.UIP ma! be involved in a neuroprotective mechanism
Because dibuty-yl cGhlP protected the hippocampal slices from osy-eniglucose deprivation and this wns not dependent upon PKG activation. it was hvpothesized that dibutvnl- - cGXlP may be inhibitinp PDE; uliich is a cGMP regulated PDE responsible for
CAMP breakdow This would increase the c.A;LIP concentration in the neuron. Thus. the neuroprotection atiorded by a cGhIP analo-iie may actually be mediated through
CAMP
If the neuroprotective etfects of dibutyryl cGhlP were due ta the inhibition of
PDE; and subsequrnt elrvati«n of c.NP concentrations. t tien forskolin might increase
the abiiit!: of dibut--1 cGMP to protect hippocampal slices from hyposia/hypogiycrmia.
If the etfects of dibutvryl cGXIP were potentiated by forskolin. this rvould indicate that
cGhlP and c.\XIP were producinr!- neuruprotective rKects by the same niechanism.
However. if the eKects of dibutyryl cGhfP were additive with that of forskolin. this woiild
suegest that cGhlP and CAMP \iere acting b'. t~koseparate and independent mechanisms
Forskolin is a direct activator of adenylyl cyclase and was chosen because it is able to
eficiently increase intracellular cXMP levels I Awad et al.. 1983 ). Equal neuroprotection
was observed against lo~~~[Ol]ilow[glucose]in hippocampal slices esposed to dibutyql
cGW or forskolin or the two agents together. These tindinss suggest that dibutvn-1- - cGMP and forskolin are producing maximum protection individually by a common pathway and also indicate that cGhZP and cXiMP ma? be acting by the same mechanism
However. additional studies would have to be done to determine conclusivelv if cGMP and CAMP are acting bv a murual pathwav
Work by Walton et al (1999). Hu et al (1999) and Hanson et al ( 1998) support the idea that c.lXIP may be a mediator of neuronal suri-ivai Embryonic spinal motor ncurons were able to sunive fm- 1 week in the absence of tropic factors when the levels of c..\MP were elevated. Cyclic AhlP activates protrin kinase A I PKA). which is able to phosphorplate the cylic responsivr element binding protein ICREB). a transcription tàctor CREB promotes the transcription of a numbrr of proteins upon its phospliorylation. Two studies Iwe suggested that CREB phosphorylntion leads to the transcription of proteins involveci in neuroprotection One study demonstrated that increased levels of CREB çoiild inhibit apoptosis The- proposed that CREB might play
a neuroprotective role aeainst apoptotic cell death (Walton et al.. 1999) Fiinherrnore. it
\vas hund that follotving cerebral ischeniia in the rat. neuronal populations that are more
resistant to injury such as the dentate ganule çells of the hippocampus. had higher levels
of phospho-CREB ( Hii et ai.. 1999).
Thus. \ve administered a PKA inhibitor. H-89 with dibutvryl c.AW at the
be~inningof the repertùsion period. to determine if the eRects of dibuty-yl CAMP were
dependent upon PKA activation. H-89 did not attenuate the neuroprotective etrects of
dibutvwl- - c.LVP nor did it evhibit neuroprotective propenies on its own. demonstratinp
that PKA is not involved in a neurotosic cascade.
Because a PK.4 or PKG inhibitor did not block the actions of cGMP and CAMP.
this suagests that the neuroprotective properties of cGhW and crLMP are independent of protein kinase activation. However. a CAMP-dependent mechanism. which does not require PKA activation was described recently in the literature. Guanine nucleotide exchange factors (GEF) are a goup of proteins that activate GTPase's by causing the exchange of GDF with GTP (sec review Zwankruis and Bos. 1999). One GEF. Epac. is activated by CAMP in a manner similar to that of PKA. Epac stimulates the activity of a
Rapl GTPase (Rooij et al. lïW3) Rapl has been irnplicated in a number of cellular processes such as crll proliferation ~Altschuleret al . 1998) and cell differentiation (York et al.. 1998) .\lthough the Rap l pathwü). has not been clearly definrd. the GEF's represent a non-PKA mediated CAMP dependent mechanism Since protein kinase inhibitors could not atteniiate the protective rtfects of dibutyryl c.WP and dibutyyl cGMP. it is possible that cyclic nucleotides are acting ttirough a novel mechanism such as
GEF activation
In addition. c.\.JfP ma' be providins neiiriiprotrction by increasing adenosinr concentrations. It has been supgested that adenosinr provides neuroprotection by inhibitin- $tamate release and by otfsetting C'a' accumulation bv hyperpolarization of the neuron ( ser reviw Scliiiben et al . 1997) Bot11 of these actions could potentiall! protect neurons from ischeinic damage Augmenting adenosine receptor activation was found to be protective in two iii wri models of ischeniia (Von Lubitz et al. 1999. Halle et al.. 1907). .A selective adenosine Ai receptor ayonist. protected neurons in the stratum pyramidale of the gerbil in a bilateral carotid occuliision model. su-gestin- that adenosine plays a neuroprotective role in ischemia (\.'on Lubitz et al.. 1999) Enhancing adenosinr
.AI receptor binding in neonatal rats was also found to be protective against hyposia- ischemia as rxhibited throush a decrease in the rveieht loss of the lefi cerebral hemisphere
(Halle et al.. 1097). These findings swgest ihat adennsine receptor activation is protective in ischemic injury This pathway represents another potential CAMP mediated.
PKX-independent neuroprotective mechanism
In summary. the resiilts from this study sugpest that the cyclic niicleotides and novel organic nitrates are esening their neuroprotective etiects bv two independent mechanisms ODQ was ina able to attenuate the etfects observed with GT-094 In addition. GT-O94 coiild not increase cGW levels significantly above the low[OZ]/low[glucosr]controls These findinss indicate that the neiiroprotective effects of
GT-094 are not due to cGhlP generation .Altrrnativrly, GT-094 may be providing neuroprotection by inhibiting rit her lipid perovidation tir the NJ1D.A receptor
Interestingly. ODQ on its own prodciced dose dependent neuroprotection. The ne~iroprotectiwrtfects of ODQ LLere not additive nith or potentiatrd b> GT-OW -4s disçussed previously. this nia? be due t« the fact ttiat ODQ and GT-094 are actins bu a common mechanisin and their effects ma'. br masiinal resiilting in no potentiation of the response Therefore. determining hou ODQ is protectin~the hippocampal neurons ma? provide clues as to how GT-(IL)-! is esening its neuroprotection. ODQ inhibits puanylyl cyclase by interactins wirh its hrme nioiety (Tseng et al. 1000) One study has demonstrated that the rffects of ODQ are not specifc They found that ODQ wsable to inhibit bioactivation of GTS and SSP as wll as nitric oside synthase in aonic homogenates (Feelisch et al . 1999). while another group obsemed that ODQ was able to interact with heme goups present in mvoglobin (We-ener et al. 1999). These studies stron-lu sugaest that ODQ is able to react with other iron cornpleses in the ceIl besides that of G-cyclase. Therefore. ODQ ma' be esening its neuroprotective etfects by interacting with heme groups. which would inhibit the initiation of lipid perosidation. Since lipid peroxidation occurs at an increased rate durin? the reperfusion penod. ODQ may be protective by its abilitv to hinder the initiation ot'lipid peroxidation.
Similari';. CiT-O94 ma- also be able to interfere with the propagation of lipid peroxidation in the neuron. It has been denionstrated previously that nitric oxide is able to interact witli lipid radicals to form stable nitroso-compounds which block the series of rertctions involved in lipid peroxidation (Riibbo et al . 199-4) Uitric oside is also able to interact with tèrrous cornpleses. which w«dd inhibit the initiation of lipid perosidation
(Kanner et al . 109 1 ) Xitric oside donors have been bund to be protective in botli ri1 iwo and 111 \n*ostiidies against iron-indiicrd lipid perosidation (Railhala et al . 1996 %
I9W) Thrse studies indicate that asents. such as GT-094. which are established nitriç oside donors. are able t« arrcst lipid perinidation by formin? stable nitroso-compounds or bu interacting with ferrous coinpieses inhibitin: their ability to initiate lipid perosidation
The novrl organic nitrates are brins administrred during the repertùsion period
when lipid perosidation is takinp place It is ivrll rstabiished that the rnetabolic stress
that occiirs as a result of ischemia sets the stage for production of superoside anion and
initiation of lipid perosidation durin2 the rcpertiision period The concentrations of
superoside anion are increased during the repertùsion period bl; purine metabolism (sec
review- hlcCord. 19S7). prostaglandin synthesis ( Kukreja et al.. 1986). and respiration
(see review Freeman et al.. 1982). Hydrosyl radical can also be produced from the
Fenton reaction. a reaction betiveen hydrogen peroside and ferrous iron containing
proteins Thrse species are able to initiate lipid perosidation (see review Watson ri al..
1989) Nitric oside is able to inhibit lipid perosidation by forming stable nitroso-
compounds or by interacting with ~e-compounds (Rubbo et a!.. 1994: Kanner et al..
199 1). ODQ is also able to interact with ~r"compounds thus hinderino the initiation of lipid perosidation. Therefore it is possible that GT-O94 and ODQ are demonstratinr their neuroprotective properties throiigh a common ability to act as antiovidants
Nitric oside is able to inhibit C'a' flow through the MD..\receptor. This takes place by donation of a nitrosoniiim ion frorn iiitric oside to the redos modulatory site on the NMDA receptor forming a disulfide bridge. This results in decreased ca2' tlow through the WID.4 receptor (Lipton et al . 10% Becnuse a substantial arnount of the . injury caused by ischemia is a result OF ercessive Ca' levels. it is plausible that the novel organic nitrates are eserting their neuroprotective eKects by restrictina ~a"intlow throiish the SVDA receptor .A st~idythat rsaminrd the rtfects of SXID.U.AMP.-! receptor antagonism on an III i*rn-Oischemic insult administered these agents during the insult (Arias et al . 1999) The rtr utro isctiemic insiilt consisted of 30 minutes of hypogiycernia ccinciirrent with anosia diiring tlir Iast i-IO minutes of hypoglycernia.
However. the novel organic nitrates were al1 administered during the rrpertùsion pei-iod when lipid peroxidation is occurrins .At this rime point. it ma! be too late to arresr neuronal damage caused by XMDX receptor activation Therefore the novel orsanic nitrates are more likely to esen their neuroprotecti\.e rtieçts by acting as antiosidanrs
In conclusion. the novel orsanic nitrates. GT-094. GT-3 10 and GT-09 1 eshibited neuroprotection in an iir ivlti.o mode1 of stroke. The neuroprotective properties of GT-094 were not dependent upon cGMP activation. The fact that the effects of GT-094 could be mimicked by ODQ sugpests that GT-094 may be acting as an antiosidant in our model
The cyclic nucleotides. cAMP and cGhfP were also neuroprotective in the same iir i~ta model of stroke Neither PKA nor PKG activation mediated the neuroprotection afforded by c.&VP or cGW respectivelv. Funhermore. the protective efects of cGW could be mimicked bv cAiiIP. suggestin- a common mechanism of neuroprotection. These findinps suggest that the neuroprotection obsened with the novel or-anic nitrates and the cvclic nucleotides are a result of two separate and independent rnechanisms. Funher studies need to be conducted to clearly detine the pathways involved in the neuroprotective actions of the nowl orgmis nitrates and the cyclic nucleotides .A summat-y of the proposed mechanisms of neuroprotection of the novel organic nitrates and cyclic nucleotides are presentrd in figiires 4 1 and 1 2 respectively
The results iibtained rhus far suggest that nitnc ovide donors çan decreasr the injury associated with stroke when administered post-ischemia This novel treatment should be funher esplored in ordrr to masimizr its potential as a much needed tlierapeutic in strokr
4.1 Future Resewcti Directions
Based on the findings of this thrsis projeçt. research should be conducted iii the tollowin,(r areas
(a) it is not iincciininon hr the 111 \wo siti~ationtu differ from that of the r/r \*rw
III order tu veri5 that the eKects t liat were obsewed rtr rrrm also take place ur
iwj. these dmgs \bill also have ro be studied in an rir \~i~jmodel The middle
cerebral anen occlusion (SICA) model is a ivtrll established rtr model for
analvzin! ischemic injury Tlierefore the neuroprotective efects of the novel
organic nitrates should alsri be st~idiedin a SICA model
(b) it iras sug-ested that the novel organic nitrates are esening their
neuroprotective propenies by acting as antiosidants. These agents should be
studied in models of osidaiive stress to determine if the? have any antiosidant
propenies. There are a nurnber of spectrophotometnc assavs available that
could be used to address this problem. Proposed Neuroprotective Pathway Mediated by Novel Organic Nitrates
- -- ~Adrnitio~of~ove~ Organic Nitrate 1
Biotransformed to NO in the presence of thiol groups.
Inhibition of lipid peroxidation Donation of a nitrosonium ion by scavenging Iipid radicals to to the redox regulatory site present form stable ni troso-compounds on the NMDA receptor inhibiting or interacting with ferrous Ca3 influx into the neuron. compounds inhibiting initiation of lipid peroxidation.
Neuroprotection against
Figure 4.1 : Proposed mechanism of action of the novel organic nitrates. Proposed Neuroprotective Pathway Mediated by Cyclic Nucleotides
Increase in intracellular cGMP 1 1
Inhibition of the Inhibit lipid AMPA receptor peroxidation
Increase iiitracellular lncrease CAMP [Adenosine]
Block Ca2+influx 1 Activation of Epac, a lnhibit NMDA-R, into the neuron 1 CAMPspecific GEP. I hyperpolarize the neuron.
Transcription of neuroprotective proteins
Neuroprotection against ischemic injury
Figure 4.2: Proposed mechanism of neuroproiection mediated by cGMP. Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole; PDE,, phosphodiesterase 3; Epac, exchange protein directly activated by CAMP; GEF, guanine nucleotide exchange factor; NMDA-R, N-rnethyl-D- aspartate receptor. (c) Nitric oxide has also been sugeested to be able to inhibit ~a'-tlow through the
NMD.4 receptor (Manzoni et al.. 1991. Lipton et al.. 1993). In particular. the
nitric oside donor. GTS has previously been shown to be able to inhibit ~a"
intlus caused bv NMDA receptor activation (Lei et al.. 1992). To address this
issiir electrophysiologicd studies should be done to determine if the novel
or-anic nitrates are able to inhibit ~a'tlow into the neuron as a result of
Nh1D.A receptor activation. C«rn-ersely. if the novel orsanic nitrates do not
7 iiitliience Ca- intlus rhis would provide hnher evidencr that the no4 orsanic
nitrates are actin- by an alternative inechanisiii.
(d) The results froni tliis thesis sugpt iliat the çvclic nuclrotides are rsening thrir
neuroprotrctive etfects independently of protein kinase activation. To
determine potential transcriptional targets of cyclic nucleotide treatnient. a
cDh.4 iiiiçroassay clip could be done \rith hippocampal tissue from a treatrd
and a control animal This would provide mechanistic dues as to how the
qclic nucleiitides are producing nruroprotection. This procedure ivould
identi% potential targets of the qclic nucleotides. which could thrn be tùnher
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