UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New Series No 385 - ISSN 0346-6612
From the Department o f Pharmacology
University o f Umeå, Umeå, Sweden
Cyanide and Central Nervous System
A Study with Focus on Brain Dopamine
by
Gudrun Cassel
University o f Umeå
Umeå 1993
The drawing on the front cover:
Gunilla Guldbrand
Copyright © 1993 by Gudrun Cassel
ISBN 91-7174-824-5
Printed in Sweden by
Tidtryck
Umeå 1993
From the Department o f Pharmacology
University o f Umeå, Umeå, Sweden
Cyanide and Central Nervous System
A Study with Focus on Brain Dopamine
AKADEMISK AVHANDLING
som för avläggande av doktorsexamen i
medicinsk vetenskap vid Umeå Universitet
offentligen försvaras i
Farmakologiska institutionens föreläsningssal A 5, byggnad 6 A
fredagen den 29 oktober 1993 kl 09.15
av
Gudrun Cassel
£
o
£
AlV3
Handledare: Professor Sven-Åke Persson
Fakultetsopponent: Professor Frode Fonnum
ABSTRACT
Cyanide and Central Nervous System - a study with focus on brain dopamine.
The brain is a major target site in acute cyanide intoxication, as indicated by several
symptoms and signs. Cyanide inhibits the enzyme cytochrome oxidase. This
inhibition causes impaired oxygen utilization in all cells affected, severe metabolic
acidosis and inhibited production o f energy. In this thesis, some neurotoxic effects
o f cyanide, in particular, the effects on dopaminergic pathways were studied.
In a previous study, decreased levels of striatal dopamine and HVA were found
after severe cyanide intoxication (5-20 mg/kg i.p.). However, increased striatal
dopamine were found in rats showing convulsions after infusion o f low doses of
cyanide (0.9 mg/kg i.v.), at the optimal dose rate (the dose rate that gives the
treshold dose).
Increased striatal dopamine synthesis was observed in rats after cyanide treatment
and in vitro. Furthermore, in rat, as well as in pig striatal tissue, cyanide dosedependently increased the oxidative deamination o f 5-HT (MAO-A) and DA
(MAO-A and -B) but not that o f PEA (MAO-B). Thus cyanide affects both the
synthesis and metabolism o f dopamine.
In rats, sodium cyanide (2.0 mg/kg, i.p.) decreased the striatal dopamine D j- and
D 2 -receptor binding 1 hour after injection. Increased extracellular levels o f striatal
dopamine and homovanillic acid were also shown after cyanide ( 2.0 mg/kg; i.p.).
DOPAC and 5-HIAA were slightly decreased. This indicates an increased release
or an extracellular leakage o f dopamine due to neuronal damage caused by
cyanide. Thus the effects o f cyanide on dopamine D j- and D 2 ~receptors could in
part be due to cyanide-induced release of dopamine.
Because o f reported changes in intracellular calcium in cyanide-treated animals, the
effects o f cyanide on inositol phospholipid breakdown was studied. Cyanide
seemed not to affect the inositol phospholipid breakdown in vitro.
The effects o f cyanide on the synthesis and metabolism of brain GAB A were also
examined. A decreased activity o f both GAD and GAB A-T were found in the rat
brain tissue. The reduced activity o f GAB A-T, but not that of GAD returned to the
control value after adding PLP in the incubation media. The cyanide-produced
reduction o f GABA levels will increase the susceptibility to convulsions, and could
partly be due to GAD inhibition.
In conclusion, cyanide affects the central nervous system in a complex manner.
Some effects are probably direct. The main part, however, appears to be
secondary, e.g. hypoxia, seizures, changes in calcium levels or transmitter release
produced by cyanide.
Keyword: CNS; cyanide; dopaminergic system; convulsions; receptor binding;
tyrosine hydroxylase; monoamine oxidase; extracellular release; inositol phosphate;
GABA; GAD.
ISBN 91-7174-824-5
ABSTRACT
Cyanide and Central Nervous System - a study with focus on brain dopamine.
The brain is a major target site in acute cyanide intoxication, as indicated by several
symptoms and signs. Cyanide inhibits the enzyme cytochrome oxidase. This inhibition
causes impaired oxygen utilization in all cells affected, severe metabolic acidosis and
inhibited production o f energy. In this thesis, some neurotoxic effects of cyanide, in
particular, the effects on dopaminergic pathways were studied.
In a previous study, decreased levels o f striatal dopamine and HVA were found after
severe cyanide intoxication (5-20 mg/kg i.p.). However, increased striatal dopamine
were found in rats showing convulsions after infusion of low doses of cyanide (0.9
mg/kg i.v.), at the optimal dose rate (the dose rate that gives the treshold dose).
Increased striatal dopamine synthesis was observed in rats after cyanide treatment and in
vitro. Furthermore, in rat, as well as in pig striatal tissue, cyanide dose-dependently
increased the oxidative deamination of 5-HT (MAO-A) and DA (MAO-A and -B) but
not that o f PEA (MAO-B). Thus cyanide affects both the synthesis and metabolism of
dopamine.
In rats, sodium cyanide (2.0 mg/kg, i.p.) decreased the striatal dopamine D j- and D 2 receptor binding 1 hour after injection. Increased extracellular levels o f striatal dopamine
and homo vanillic acid were also shown after cyanide (2.0 mg/kg; i.p.). DOPAC and 5HIAA were slightly decreased. This indicates an increased release or an extracellular
leakage o f dopamine due to neuronal damage caused by cyanide. Thus the effects of
cyanide on dopamine D j- and D 2 -receptors could in part be due to cyanide-induced
release o f dopamine.
Because o f reported changes in intracellular calcium in cyanide-treated animals, the
effects o f cyanide on inositol phospholipid breakdown was studied. Cyanide seemed not
to affect the inositol phospholipid breakdown in vitro.
The effects o f cyanide on the synthesis and metabolism of brain GABA were also
examined. A decreased activity o f both GAD and GAB A-T were found in the rat brain
tissue. The reduced activity of GABA-T, but not that of GAD returned to the control
value after adding PLP in the incubation media. The cyanide-produced reduction of
GABA levels will increase the susceptibility to convulsions, and could partly be due to
GAD inhibition.
In conclusion, cyanide affects the central nervous system in a complex manner. Some
effects are probably direct. The main part, however, appears to be secondary, e.g.
hypoxia, seizures, changes in calcium levels or transmitter release produced by cyanide.
Keyword: CNS; cyanide; dopaminergic system; convulsions; receptor binding; tyrosine
hydroxylase; monoamine oxidase; extracellular release; inositol phosphate; GABA;
GAD.
CONTENTS
List of papers.....................................................................................................5
Abbrevations......................................................................................................6
Introduction........................................................................................................7
Background......................................................................................................................7
Neurotoxic effects o f cyanide........................................................................................ 7
Dopaminergic system......................................................................................................9
Synthesis o f dopamine........................................................................................ 9
Storage, release and reuptake of dopamine...................................................... 10
Metabolism o f dopamine.................................................................................... 11
Regulation o f the dopamine neurons................................................................. 11
GABAergic system......................................................................................................... 13
The purpose of the thesis................................................................................. 14
Materials and methods...................................................................................... 15
Animals..............................................................................................................................15
Chemicals......................................................................................................................... 15
Radiochemicals....................................................................................................15
Other chemicals...................................................................................................15
M ethods............................................................................................................................15
Methods used to kill the animals....................................................................... 15
Selection and handling o f brain tissue............................................................... 16
HPLC analysis......................................................................................................16
Convulsive threshold estimation (Paper I ) ....................................................... 16
In vivo estimation o f dopamine synthesis. (Paper II).......................................17
Microdialysis (Paper VI).................................................................................... 17
Dopamine D l- and D2-receptor binding (Paper V ) ........................................17
Inositol phospholipid breakdown (Paper V I)................................................... 18
Assay o f TH activity (Paper III)........................................................................ 18
Assay o f MAO activity (Paper IV )....................................................................18
Assay o f GAD activity . (Paper V II)................................................................ 19
Assay o f GABA-T activity (Paper VII)............................................................19
Enzyme kinetics (Paper III, IV, VII)................................................................ 19
Protein measurement..........................................................................................19
Statistics...........................................
19
Results and discussion.................................................................................... 20
General remarks............................................................................................................... 20
Estimation o f the convulsive effect o f cyanide. Paper 1 .............................................. 20
In vitro and in vivo effects on tyrosine hydroxylase. Paper II-III.............................. 21
The effects o f cyanide on MAO. Paper IV....................................................................23
The effect o f cyanide on dopamine D l- and D2 - receptors. Paper V .......................24
Inhibition o f release or reuptake? Extracellular levels in striatum after NaCN.
Paper VI............................................................................................................................24
The effect o f NaCN on GABA synthesis and metabolism. Paper V II.......................25
General discussion........................................................................................... 27
Conclusions.......................................................................................................30
Acknowledgements...........................................................................................31
References.........................................................................................................32
LIST OF PAPERS
This thesis is based on the following papers, which will be referred to in the text by the
Roman numerals I-VII.
I.
Gudrun E. Cassel (1993) Estimation of the convulsive effect of cyanide in
rats. Submitted to Eur. J. Pharmacol.
II.
Gudrun Cassel and Sven-Åke Persson (1992) Effects of acute lethal cyanide
intoxication on central dopaminergic pathways. Pharmacol. Toxicol., 70,
148-151.
III.
Gudrun E. Cassel and Sven-Åke Persson (1993) The effect of cyanide on rat
brain tyrosine hydroxylase activity in vitro. Submitted to Arch. Toxicol.
IV.
Gudrun E. Cassel, Sven-Åke Persson and Anders Stenström (1993) Effects of
cyanide in vitro on the activity of monoamine oxidase in striatal tissue
from rat and pig. Submitted to Biochem. Pharmacol.
V.
Gudrun E. Cassel, Tom Mjömdal, Sven-Åke Persson and Emma Söderström
(1993) Effects of cyanide on the striatal dopamine receptor binding in the
rat. Eur. J. Pharmacol. In press.
VI.
Gudrun E. Cassel, Mona Koch and Gunnar Tiger (1993) The effects of
cyanide on the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxyindoleacetic acid and
inositol phospholipid breakdown in the brain. Submitted to Eur. J.
Pharmacol.
VII.
Gudrun Cassel, Lena Karlsson and Åke Sellström (1991) On the inhibition
of glutamic acid decarboxylase and y-aminobutyric acid transaminase by
sodium cyanide. Pharmacol. Toxicol., 69, 238-241.
ABBREVATIONS
3-MT
5-fflAA
5-HT
6 -MePtH 4
AADC
AOAA
ATP
Ca2+
CBF
CNS
DA
DOPAC
EDTA
GABA
GABA-T
GAD
HVA
i.p.
InsP
L-DOPA
MAO
NA
NaCl
NaCN
NEN
NSD 1015
PC12
PCA
PEA
PLP
TH
6
3-Methoxytyramine
5-Hydroxyindoleacetic acid
5-Hydroxytryptamine (serotonin)
6-Methyl-5,6 ,7,8 -tetrahydropterine dihydrochloride
L-Aromatic amino acid decarboxylase
Aminooxyacetic acid
Adenosine triphosphate
Calcium ion
Cerebral blood flow
Central nervous system
Dopamine
3,4-Dihydroxyphenylacetic acid
Ethylenediaminetetraacetic acid tetra sodium salt
y-Aminobutyric acid
y-Aminobutyric acid transaminase
Glutamic acid decarboxylase
3-Methoxy-4-hydroxyphenylacetic acid
(Homovanillic acid)
Intraperitoneal
Inositol phosphate
3,4-Dihydroxy-L-phenylalanine
Monoamine oxidase
Noradrenaline
Sodium chloride
Sodium cyanide
New England Nuclear
3-Hydroxybenzylhydrazine (HCL)
Pheochromocytoma cells
Perchloric acid
Phenylethylamine
Pyridoxal-5-phosphate (Vitamine B 6 )
Tyrosine hydroxylase
INTRODUCTION
Background
Cyanide is one o f the most rapidly acting poisons known. Acute lethal cyanide
poisoning may result from direct exposure to hydrogen cyanide or its alkali salts,
and also from biotransformation of more chemically complex cyanogens. Acute
cyanide intoxication will, in severe cases, cause symptoms such as respiratory
arrest, fatigue, unconsciousness, convulsions, tremor and ultimately death
(Egekeze and Oehme, 1980; Ballentyne, 1983; Way, 1984; D'Mello, 1987; Johnson
et a l , 1986, 1987). The death occurs after only a few minutes, while the signs and
symptoms o f cyanide poisoning appear within a few seconds after ingestion of
cyanide or inhalation o f cyanide vapours. Several symptoms o f the acute cyanide
poisoning also indicate that the brain is a major target site for cyanide (Ballantyne,
1987; Way, 1984). Acute, lethal cyanide intoxication is consistently accompanied
by high concentrations o f cyanide in the brain.
The main target enzyme for the action o f cyanide is most probably cytochrome c
oxidase, the terminal oxidase o f the respiratory chain. Cyanide interacts with the
ferric ion o f cytochrome a3 . The inhibition o f cytochrome c oxidase activity will
inhibit the mitochondrial electron transport system and produces a cytotoxic
hypoxia in presence of normal haemoglobin oxygenation. Studies on the inhibition
of cytochrome c oxidase activity in various organs following cyanide intoxication
show correlation between the severity of the intoxication and the degree of
inhibition o f cytochrome c oxidase (Albaum et a l , 1946; Isom & Way, 1976; Isom
et a l, 1982). Cyanide, however, also affects the activity of a number o f other
enzymes, e.g. decarboxylases and transaminases, (Dixon & Webb, 1958;
Solomonson, 1982). The mechanisms for these effects include combination with
functionally essential metal ions; formation of cyanohydrins; elimination of sulphur
as thiocyanate; addition to the Schiff base aldimine with formation of aminonitrile
(Hansen and Dekker, 1976).
There appear also to be regional differences in how cyanide inhibits the enzymes or
in the bioavailability o f the antidotes. For instance, animals given the antidotes,
sodium nitrite and thiosulfate, appeared to have recovered the cytochrome c
oxidase o f the liver but not that o f the brain (Isom & Way, 1976; Isom et al.,
1982).
Neurotoxic effects of cyanide
The nervous system is considered particulary susceptible to the toxic actions of
cyanide, because o f its limited anaerobic metabolism, low energy reserves, and high
energy demands (Brierley et ah, 1976; Funata et a l, 1984; Johnson et a l, 1986,
1987; Yamamoto, 1990). The vulnerability of the brain to cyanide is partly due to
relatively low concentrations of cyanide-metabolizing enzymes in the central
nervous system (CNS) (Mimori et a l, 1984). The inhibition of cytochrome c
oxidase is, with high probability, the molecular basis of the central effects produced
by cyanide. But direct effects o f cyanide cannot be excluded. Whatever the
mechanisms, cyanide will cause impaired oxygen utilisation in all cells affected and
produce severe metabolic acidosis (Yamamoto & Yamamoto, 1977). The
inhibition o f the brain cytochrome c oxidase by cyanide results in a neuronal
cytotoxic hypoxia. In previous investigations, where the effects o f cyanide were
not directly studied, various conditions o f hypoxia have been shown to affect a
number o f neuroactive substances such as neurotransmitters and cyclic nucleotides
(Davis & Carlsson, 1973; Shimada et al., 1974; Gibson et al., 1978; 1981 a & b;
Freeman et al., 1986; Folbergrovâ et al., 1981). In conditions with insufficient
energy production in the brain, convulsions frequently occur (Folbergrovâ et al.,
1981; Nakanishi et al., 1991; Shukla et al., 1989). This is also the case during
severe cyanide intoxication (Ballentyne, 1983; Yamamoto, 1990). It has, however,
not been clarified whether the convulsions are a direct effect o f cyanide or
secondary to the induced cytotoxic hypoxia.
Acute administration o f sodium cyanide (NaCN; 5-20 mg/kg i.p.) dramatically
decreased the striatal levels o f dopamine (DA) and homovanillic acid (HVA) in the
rat (Persson et al., 1985). However, 3,4-dihydroxyphenylacetic acid (DOPAC)
levels was not significantly changed. Thus, NaCN produced rapid and regional
changes in central dopaminergic pathways. Maduh et al. (1988) found a calciumdependent release o f norepinephrine and DA from cyanide-treated PC 12 cells.
Furthermore, a dose-dependent release o f catecholamines from these cells has also
been reported (Kanthasamy et al., 1990). Cyanide also produced a marked increase
in plasma catecholamines by stimulating the sympathoadrenal system (Kanthasamy
et al., 1991). A transient and remarkable increase in striatal DA release was
observed after application o f 2 mM NaCN through a brain microdialysis membrane
(Kiuchi et al., 1992).
Tursky and Sajter (1962) showed that potassium cyanide inhibited the pyridoxal-5phosphate (PLP)-requiring enzymes glutamic acid decarboxylase (GAD) and yaminobutyric acid transaminase (GABA-T) in the rat brain. Increased amount o f
glutamic acid in cerebellum, striatum and hippocampus was seen after
administration o f NaCN (5-10 mg/kg; i.p.). But higher dose (20 mg/kg i.p.)
decreased the levels o f glutamic acid and y-aminobutyric acid (GABA) (Persson et
al., 1985). In addition, animals have been shown to have a significantly increased
acetylcholine esterase activity in cerebral cortex, hippocampus and midbrain in
acute cyanide intoxication (Owasoyo & Iramain, 1980).
Cyanide-induced accumulation o f calcium (Ca2+) within nervous tissue has been
correlated with convulsion and tremor (Johnson et a l, 1986; Yamamoto, 1990).
Most likely the complex interplay between Ca2+-accumulation and convulsions
also involves the release o f neurotransmitters (Nachshen & Sanches-Armass,
1987). Johnson et al. (1986) also suggest that calcium channel blocking agents
may be useful in limiting the severity o f centrally-mediated symptoms o f acute
cyanide poisoning. Furthermore, they proposed that Ca2+ fonctions as a
toxicogenic second messenger following cyanide-induced inhibition o f adenosine
triphosphate (ATP) production (Johnson et al., 1987).
Maduh et al. (1990) showed that diltiazem, which blocks calcium channels at the
cell surface, prevented cyanide-induced mitochondrial swelling. Cyanide-stimulated
increase in cytosolic Ca2+ appears to activate neurotransmitter secretion as
8
evidenced by the granula depletion and reversal o f this phenomenon by diltiazem.
These observations have important implications in cyanide toxicity, since the
functional correlate would be release o f central transmitters producing excessive
CNS firing.
Marked increase (200 %) in cerebral blood flow (CBF) resulting in substantial
increase in brain O2 delivery was seen after NaCN injection (2 mg/kg; i.v.) (Lee et
al., 1988 a&b). Pitt et al., (1979) and Russek et al., (1963) also found increased
CBF after administration o f cyanide. Permanent neurological damage by cyanide
stems from inhibition o f cellular metabolism and is preceded by changes in cell
morphology (Brierley et al., 1976; Ashton et al., 1981; Funata et al., 1984).
Changes in cell morphology are therefore important indications of neuronal lesions
A calcium channel blocker prevented cyanide-induced morphological changes in
PC 12 cells. These changes were probably mediated by an influx of extracellular
calcium. A proposed mechanism o f structural damage resulting from disruption of
cell metabolism suggests that adenosine triphosphate (ATP) depletion inhibits
active membrane transport of ions, producing altered ionic homeostasis and
accumulation o f intracellular sodium and water (Schwertschlag et al., 1986).
Furthermore, severe cyanide intoxication is known to cause symptoms and signs
similar to Parkinsons disease as well as leisons in the basal ganglia (Schwab &
England, 1968; Finelli, 1981; Utti et al., 1985; Carella et al., 1988; Messing &
Storch, 1988; Rosenberg et a l, 1989; Grandas et al., 1989). Whether such leisons
can be considered specific for cyanide (directly or indirectly) is not clear.
Dopaminergic system
Synthesis of dopamine
The catecholamines are synthesized from the aromatic amino acid L-tyrosine
(Figure 1). L-tyrosine is transported across the blood-brain barrier by an active
transporter shared by all large neutral amino acids (Pardridge, 1977).
Subsequently, tyrosine is taken up into the DA neuron by an active mechanism.
The suggestion that L-tyrosine is converted in a sequence to 3,4-dihydroxy-Lphenylalanine (L-DOPA), DA, noradrenaline (NA) and adrenaline by enzymes was
confirmed in vitro in the adrenal medulla and in adrenergic nerves by Goodall &
Kirshner (1957, 1958). This work was preceded by in vivo studies showing
adrenaline formation from L-tyrosine (Gurin & Delluva, 1947) and also from LDOPA and DA (Udenfriend & Wyngaarden, 1956). Tyrosine hydroxylase (TH)
catalyzes the first, rate-limiting, step in the catecholamine synthesis, where Ltyrosine is hydroxylated to L-DOPA in peripheral and central catecholaminergic
neurons (Nagatsu et al., 1964). Tetrahydrobiopterin is considered to be the natural
cofactor for TH (Weiner, 1979)
L-DOPA is subsequently decarboxylated to DA by L-aromatic amino acid
decarboxylase, AADC, (Roth et a l, 1987). AADC requires pyridoxal-5-phosphate
(PLP, vitamine B 6 ) as cofactor (Holtz & Palm, 1964). L-DOPA turnover is very
rapid and the levels in brain are difficult to detect under normal condition. The
enzyme is not entirely specific, since it could also catalyze the decarboxylation of
other aromatic amino acids, e.g. 5-hydroxy-L-tryptophan, L-5-HTP (Rosengren,
1960a; Lovenberg et al., 1962). Furthermore, an increase in impulse flow in DA
neurons, as in other monoamine-containing neurons, results in an increase in
synthesis, turnover and catabolism o f DA (Roth et al., 1974).
pjr
OH— ( O ) —
/
(J f-
HVA
CH COOH
*
\
c h 2c h 2n h
oh_ ^ Q ^ _
\
OH
NH
Tyrosin*
\
I 2
hydroxylase
hzA
H—( O )
CH CHCOOH
►O H -----ridininee
RReerid
L-Tyrosine
cofector. o 2
(.-DOPA
>—'
2
(C j)
>—*
>—f
c h 2c o o h
/ DOPAC
COMT
L-Arom.tlc \
QH
NH
am ino «cld
\
C
| 2
decarboxylase \
CH CHCOOH----------- ► 0H
/
“
PLP
2
MAO
°2
/
/
J/
CH, CH, NH,
2
2
2
/ Dopamine
D opam ine p-hydroxylase
A scorbate, Cu2+, O
2
OH
P henylethanol am ine- \
V
N-methyl tran sfera se
/ — v.
H
|
d h c h 2n h 2 -------------- ► oR —
OH
Noradrenaline
OH
♦ MAO
Adrenaline
♦ COMT
♦ ADH
Figure 1. The major pathways in the synthesis and metabolism o f cate­
cholamines.
Storage, re le ase and reuptake of dopamine
DA has to be taken up by the synaptic vesicles to become available for release by
nerve impulses. This process requires ATP. The vesicular uptake mechanism could
be selectively inhibited, irreversibly by reserpine, or reversibly by tetrabenazine
(Carlsson, 1965).
After release into the synapse, the DA is taken up by cells lining the synapse. The
reuptake in the catecholaminergic neurons is the best known. This reuptake is the
most important inactivation mechanisms o f released catecholamines. Mazindole
and nomifensine are potent inhibitors o f the DA reuptake, but not selective (see
Koe, 1976)
10
Metabolism of dopamine
The metabolism o f DA occurs through enzymatic degradation. Two catabolic
pathways exist, either oxidative deamination or O-methylation (Figure 1). It has
been suggested that, the metabolism of DA, in the rat striatum results in
approximately 80% HVA formed from DOPAC and 20% HVA from 3methoxytyramine (3-MT) (Westerink & Korf, 1976; Westerink & Spaan, 1982).
These reactions are catalyzed by monoamine oxidase (MAO) and catechol-Omethyl transferase (COMT), respectively. Accumulation o f monoamines in the
brain was shown after inhibition o f MAO, which indicated the importance o f this
enzyme for the metabolism o f central monoamines (Carlsson et a l, 1960). This
was further supported by Rosengren, (1960b), Andén et al. (1963 a&b) and
Sharman (1963). MAO catalyzes the reaction of DA with molecular oxygen and
water to form the corresponding aldehyde, hydrogen peroxide and ammonia (see
Blaschko, 1974). In the central dopaminergic systems, formation o f acid appears to
dominate, whereas in the central NA system, oxidative deamination mainly leads to
formation o f alcohols (Jonason, 1969). MAO is located in the outer membrane of
the mitochondrion (see Greenawalt, 1972). This enzyme can be divided into two
forms, MAO-A and MAO-B, which differ in substrate specificity and in sensitivity
to inhibitors (Fowler & Tipton, 1984).
Regulation of the dopam ine neurons
Several feedback mechanisms control the activity o f DA neurons (Figure 2). The
synthesis o f DA is regulated via the activity o f the rate-limiting enzyme TH. Short­
term regulation o f TH has been demonstrated to be due to several independent
feedback mechanisms, including end-product inhibition (Nagatsu et al., 1964;
Carlsson et al., 1976) and catecholamine receptor-mediated regulation o f TH
(Kehr et al., 1972; Walters & Roth, 1976). DA receptors are located both
postsynaptically and presynaptically (Figure 2). The presynaptic receptors are
termed "autoreceptors" and several authors have demonstrated that these receptors
play a crucial role in the regulation o f DA synthesis and release. Furthermore, DA
synthesis and release are also dependent on the intracellular Ca2+ levels (Roth et
al., 1987).
Thus, stimulation o f DA autoreceptors by DA or DA agonists e.g. apomorphine
leads to a decrease in impulse flow and release o f DA, while blockade o f the
autoreceptor increases the impulse flow and transmitter release (Andén et al.,
1967; Bunney et al., 1973 a&b; Famebo and Hamberger, 1971). In addition, the
impulse flow in the nigrostriatal dopaminergic neurons is suggested to be feedback
regulated also via postsynaptic DA receptors (Bunney & Aghajanian, 1976; 1978).
11
Dopaminergic nerve terminal
Nerve impulse
L-Tyrosine
Presynaptic
neuron
L-Tyrosine
L-DOPA
DA.
Autoreceptor
MAO
DOPAC
^HVA
11
effector
Ion
channel
'
>{J,
Second messenger
respons
Postsynaptic
neuron
Figure 2. A schematic model o f a dopaminergic nerve terminal A nerve impulse
results in Ca^+ -dependent release o f DA. Increased impulse flow also stimulates
the THf the rate-limiting step in DA biosynthesis. Presynaptic autoreceptors
modulate the synthesis and release o f DA. Postsynaptic receptors in striatum
regulate the activity o f nigrostriatal dopamine neurons via a negative feedback.
12
GABAergic system
GAB A is known to be an inhibitory CNS transmitter, which hyperpolarizes
mammalian neurones (Curtis and & Watkins, 1965). High concentrations of GAB A
are found in the brain and spinal cord (Ryall, 1975). The turnover of GAB A, which
correlates with GABAergic activity, is regulated by the GABA-producing enzymes
GAD and the GABA-catabolizing enzyme GABA-T. Both enzymes utilize PLP as
co-enzyme. A relationship between the inhibition of GABA synthesis and the
presence o f convulsions has been shown (Killam, 1957; Killam & Bain, 1957;
Killam et al., 1960). Convulsants such as aminooxyacetic acid (AOAA) or derivate
o f PLP are believed to operate via inhibition o f the GAD-activity (Tapia &
Sandoval, 1971). An inhibitory effect of cyanide on the activities of GAD and
GABA-T was shown by Tursky and Sajter (1962). In a preliminary study we also
reported reduced GABA levels in the acute cyanide intoxication (Persson et al.,
1985)
/ NH2
H NCCH LCI-LCH
2
2
HOOCCH CH CH
2 \
2
2
COOH
\
HOOCCH CH
2
COOH
Glutamic acid
2
/
COOH
a-
Ketoglutarate
GABA-T
HOOCCH CH CH NH
2
2
2
HOOCCH CH COOH
2
2
Succinic semialdehyde
2
Succinic acid
Figure 3. A schematic picture showing the reactions involved in the GABA
shunt.
GABA is synthesized from glutamate by the enzyme GAD (Figure 3). This enzyme
is concentrated in the nerve terminals (SalganicofF & De Robertis, 1965; Fonnum,
1968), most probably in the cytoplasm. GAD activity is strongly related to the
binding with PLP in vivo (Miller et a l , 1978). In presence o f low concentrations of
PLP, GAD is inhibited by GABA at physiological concentrations (Porter & Martin,
1984). GABA is initially metabolized by GABA-T to succinic semialdehyde and
then subsequently by succinic semialdehyde dehydrogenase to succinic acid.
GABA-T binds PLP strongly.
THE PURPOSE OF THE THESIS
The purpose o f this study was to increase the knowledge of the acute effects of
cyanide on the CNS. In the light o f our previous findings that cyanide selectively
affects central dopaminergic and GABAergic systems, the intention was to reveal
some o f the mechanisms behind these effects.
The aim o f the studies on which this thesis is based were:
• To find out, by estimating the convulsive threshold, if there is a relationship
between cyanide-induced convulsions and regional brain levels o f dopamine and
its main central metabolites (Paper I)
• To examine the effects o f NaCN on the dopamine synthesis in vivo (paper II) and
on tyrosine hydroxylase in vitro. This enzyme is the rate-limiting step in the
synthesis o f dopamine (and other catecholamines) (Paper III)
• To study the effects o f cyanide in vitro on one o f the most important enzymes
involved in the metabolism o f monoamines, MAO (Paper IV)
• To examine the effects o f cyanide on the dopamine D j- and D 2 -receptors (Paper
V)
• To investigate in living animals the effects o f cyanide on the extracellular release
o f dopamine and the main brain dopamine metabolites (Paper VI)
• To examine in vitro the effects o f cyanide on the synthesis and metabolism o f the
inhibitory amino acid transmitter GAB A, known to counteract convulsive activity
(Paper VII)
14
MATERIALS AND METHODS
Animals
Male, Sprague-Dawley rats, ALAB (now Bantin and Kingman International),
Sollentuna, Sweden were used throughout the studies (Paper I-VII). Their body
weight were 175-300 g. The animals were housed 3 per cage. The room
temperature was 21-24 °C and humidity 50±5%. Brains from pigs, Swedish
Landrace, were used in two studies (Paper IV and VI).
The animal experiments have been approved by the Regional Research Ethical
Committee according to National laws (SFS 1988:539, LSFS 1989:41)
Chemicals
Radiochemicals
*4 C-5-HT, [14C-5]-hydroxytryptamine binoxalate, 56.7 mCi/mmol" 1, NEN,
Boston, USA (Paper IV).
l^C-PEA, [^C-2]-phenyletylamine hydrochloride, 55.5 mCi/mmoH, NEN,
Boston, USA (Paper IV).
l^C-DA, 3,4-[8-* 4 C]-dihydroxyphenylethylamine hydrobromide, 56.0 mCi/mmol”
1, Amersham, England (Paper IV).
SCH 23390 [N-methyl-^Hl, 87.0Ci/mmol, NEN, Boston, USA (Paper V).
Spiperone, [benzene, ring-^H], 23.3 Ci/mmol, NEN, Boston, USA (Paper V).
Myo-[2-^H] inositol, 10-20 Ci/mmol, Amersham, England (Paper VI).
[1- 14 C1-GABA, 50.4 mCi/mmol, NEN, Boston, USA (Paper VII).
L -[ l-l 4 C]-glutamic acid, 47.3 mCi/mmol, NEN, Boston, USA (Paper VII).
Other chem icals
Cis-(z)-flupenthixol dihydrochloride, H. Lundbeck A/S (Copenhagen-Valby,
Denmark) and (+)-butaclamol, Ayerst Laboratories (Toronto, Canada) (Paper V)
3-Hydroxybenzylhydrazine HCl (NSD 1015), synthesized by Dr. Bengt
Magnusson, University o f Umeå, Sweden (Paper II and III).
All other chemicals used were pro analysi or o f higher purity.
Methods
M ethods used to kill the animals
The animals were killed by exposure to high-intensity microwave irradiation, when
the levels o f brain transmittors were measured (Paper I, II). The exposure time was
1.5 sec., output power 4.5 kW at 2.45 GHz. This method gives a rapid inactivation
o f brain enzymes and a prevention o f post-mortem changes in brain transmitter
levels (Lenox et al., 1976). In all other studies (Paper III, IV, V, VI, VII), the
animals were sacrificed by decapitation using a guillotine.
15
Selection and handling of brain tissue
After microwave irradiation (Paper I and II), the head was removed and cooled in
a freezer -20°C for 2 min. Immediately thereafter the brain was removed from the
head, and dissected on an ice-chilled Petri dish. The dissection into four different
regions (striatum, frontal cortex, hippocampus and cerebellum) were performed
macroscopically from both hemispheres. Because o f our previous results, we used
striatal tissue in Papers IV, V and VI (Persson et a l, 1985). For practical reasons
the whole brain was used in Papers III and VII to obtain sufficient enzyme activity.
HPLC analysis
A HPLC equipped with an electrochemical detector was used for separation and
detection o f L-DOPA and DA (Paper II and III), and DA, NA, HVA, DOPAC and
5-hydroxyindoleacetic acid (5-HIAA) (Paper I and VI). The utility of this system
has become widely known in the last decade, since it has high sensitivity and
selectivity (see Mefford, 1985). 2-methyl-3-(3,4-dihydroxyphenyl)-L-alanine and
3,4-dihydroxybenzylamine were used as internal standards in Paper (II and III) and
Paper (I and VI), respectively. The columns used were packed with Nucleosil
reversed phase, RP-18, (Macherey-Nagel, D-5160), 3 pm particles (Paper I, II and
III). Quantitation was made by comparing surface area ratios between the
endogenous compounds and the internal standard in the tissue samples and in
external standards.
The same HPLC equipment but other chromatographic conditions were used in the
microdialysis study (PaperVI). The reason for that was the necessity to enhance the
sensitivity. The mobile phase was an aqueous'solution containing 300 ml 1 M
sodium dihydrogen phosphate, 7.4 ml 10 % EDTA, 0.691 g 1-octanesulphonic
acid, 272 ml methanol and 1700 ml H20 , pH 4.0. Before use, the mobile phase
was filtered through a Millipore filter 0.2 pm. DA, NA, DOPAC, HVA and 5HIAA were separated on an analytical column Spherisorb S 5 ODS 1 (5 pm; 250 x
4 mm) (Söulentechnik, D-1000 Berlin 20). The quantitation was made by
comparing surface area ratios between the endogenous compounds and external
standards.
Convulsive threshold estimation (Paper I)
A modification o f the method described by Wahlström (1966, 1978) was used.
Sodium cyanide was dissolved in 0.9 % NaCl and infused at a constant rate in the
tail vein until convulsions appeared. The tonic tail movement was selected as the
starting point o f convulsions during the whole experiment. The time for onset of
convulsions was observed visually. Eleven different dose rates, but always the
same volume rate (0.10 ml/min), were used. The dose needed to induce the
convulsions was calculated and this dose is called the threshold dose. Dose plotted
against dose rate generates a dose rate curve, where the minimum gives the
optimal dose rate (Wahlström, 1966; Bolander et al., 1984).
By infusing the threshold dose at the optimal dose rate another group o f animals
could be divided into two groups, one with convulsions and one without
16
convulsions, but all infused with the same dose o f NaCN. By using such a
technique it is possible to separate the effects caused by the convulsions from the
effects more directly caused by NaCN (Nordberg and Wahlström, 1988). The
levels of DA, NA, HVA and DOPAC were measured in four different brains
regions o f the rats in the various groups.
In vivo estimation of dopamine synthesis (Paper II)
Tyrosine hydroxylation in vivo was measured as the short-term accumulation o f LDOPA after inhibition o f the neuronal AADC (Caisson et al., 1972). NSD 1015
(100 mg/kg i.p.), the AADC inhibitor, and NaCN (20 mg/kg i.p.) or saline were
given 20 and 1 min, respectively, before death.
DA formation after administration of its precursor L-DOPA (100 mg/kg i.p.) was
also examined. NaCN (2.5 mg/kg i.p.) or saline and L-DOPA were administrated
30 min and 25 min, respectively, before death. All animals were killed by exposure
to high intensity microwave irradiation.
Microdialysis (P aper VI)
Hypnorm (fentanyl citrate 0.02 mg and fluanisone 1 mg) and diazepam, given i.m.
and i.p., respectively, were used as anaesthesia during the surgery. A stereotactic
instrument was used and the rat head was positioned in a horizontal plane. The co­
ordinates relative to the bregma were: A + 1:0, L - 2.2 mm and V -3.5. During the
surgery, the core temperature of the rat was kept at 37.5 °C . After the surgical
procedure there is an initial period o f disturbed tissue function, when decreased
blood flow and disturbed transmitter release can be expected (Beneviste et a l ,
1987; Drew et a l., 1989; Osborne et al., 1990, 1991;). Therefore, the experiment
started 5-10 days after the surgery. The rats were trained in the "freely moving"
equipment two different times, 3 hours each, before the experiment started. During
light halothane anaesthesia, the dummy probe was removed from the guide and the
microdialysis probe (CMA/12; membrane length = 3 mm; outer diameter = 0.5
mm) inserted and locked into position. A mixture o f 155 mM NaCl, 4 mM KC1 and
1.2 mM CaCl2 was pumped through the probe at a rate of 2 pl/min and fractions
were collected every 20 minutes for a subsequent analysis o f DA, NA, and amine
metabolites. The period o f damage release o f neurotransmitters, caused by the
probe insertion, is in rats 60-90 min (Kendrick, 1991). Therefore, the collection of
the basal values were not begun until 100 min had passed. Sodium cyanide (2
mg/kg i.p.) was given after the ninth 20 min fraction. After the experiment, the
location o f the probe was controlled by examination o f the striatum.
Dopamine D y and D2-receptor binding (Paper V)
DA Dp-receptor: The DA Dj-receptor ligand, [3H]-SCH 23390 was incubated
together with the striatal tissue (0.5 mg). Specific [3H]-SCH 23390 binding was
determined as the difference between total binding and non-specific binding in
parallel assays in the absence or presence o f 1 pM cis(z)-flupenthixol
dihydrochloride. All binding experiments were performed in triplicate.
DA P 2 -receptor: The DA D2-receptor ligand, [3H]-spiperone was incubated with
striatar tissue (1 mg). Specific [3H]-spiperone binding was determined as the
difference between total binding and non-specific binding in parallel assays in the
17
absence or presence o f 1 pM (+)-butaclamol dihydrochloride. All binding
experiments were performed in triplicate.
Inositol phospholipid breakdown (Paper VI)
Miniprisms (0.35 x 0.35 mm) were made from rat cerebral corticis and pig striata
according to Fowler et al. (1987). Basal and potassium-stimulated phosphoinositide hydrolysis were determined by measuring the accumulation o f inositol
phosphates (InsP). InsP were produced by the hydrolysis of the prelabelled ([ 3H]inositol) inositol phospholipids after inhibition o f the inositol polyphosphatases by
Li+. The miniprisms were incubated with sodium cyanide for 30 min before the
agonists were added and further incubated for 25 min before the reaction was
ended. The inositol phospholipid breakdown was measured as described by
Berridge et al. (1982) and Watson & Downes (1983), with minor modifications
(Fowler et al., 1987; Tiger et al., 1990).
A ssay of TH activity (Paper III)
A modification o f the method described by Hirata et al. (1983) was used. 40 pi o f
the enzyme preparation was incubated with the substrate, L-tyrosine, in a total
reaction volume o f 140 pi. As co-factor, 1 mM 6-methyl-5,6,7,8,tetrahydropteridine dihydrochloride ( 6 -MePtH4) was used, the pterin co-factor was
dissolved in 2 -mercaptoethanol in order to maintain the tetrahydropterin in reduced
form throughout the incubation period. Iron was used as an activator metal to
maximize TH activity. In our assay, catalase was added to the incubation medium
in order to catalyze the breakdown o f any hydrogen peroxide formed in the
reaction system (Weiner, 1979). NSD 1015 was used in order to inhibit the
decarboxylation o f L-DOPA to DA. A cleaning procedure before injection into the
HPLC was performed. The sample was passed via Amberlite CG 50 to aluminium
oxide in a pH o f 8 .0-8.5. L-DOPA binds to aluminium oxide at this pH. L-DOPA
was eluted from the aluminium oxide with 0.5 M PCA containing 0.25 % sodium
bisulfite and 0.25 % EDTA. The level o f L-DOPA was determined by injection 20
pi o f the eluate into the HPLC system.
A ssay of MAO activity (P aper IV)
MAO activity was assayed radiochemically by a conventional method (Eckert et
al., 1980) with 100 pM 5-HT as substrate for MAO-A, 100 pM DA as substrate
for both A and B form and 20 pM PEA as substrate for MAO-B. The diluted
crude homogenate was suspended in 10 pM K-phosphate buffer, pH 7.4 (including
0.2 mg/ml ascorbate as an antioxidant) and incubated at 37 °C with the substrate in
a final volume o f 100 pi (4 min [PEA] or 20 min [DA and 5-HT]). The reaction
was stopped by addition o f 3 M HC1 and cooling the tube on ice. Blank values
were obtained by adding the HC1 prior to incubations. Deaminated products were
extracted into 6 ml toluene-etylacetate ( 1/ 1, w/v) saturated with water and the
radioactivity was counted in 10 ml o f Econofluor.
18
A ssay of GAD activity (Paper VII)
GAD activity was assayed using a method described by Wu et a l (1973). The
14CC>2 formed from L -(l- 14C)glutamic acid after incubation with the homogenate
was absorbed in protozoi. Various concentrations of NaCN were added to the
incubation media before adding the enzyme. The influence of PLP on the reaction
was determined, in a separate experiment, by adding an amount equimolar to the
added cyanide.
A ssay of GABA-T activity (Paper VII)
The radiochemical method o f Hall & Kravitz (1967) was used with some
modification. Radioactive GABA was incubated with the enzyme and appropriate
co-factors. At the end o f the incubation, succinic semialdehyde and succinate were
separated from GABA by ion-exchange chromatography. Succinate and NAD
were included in the incubation media to prevent metabolism o f radioactive
succinate. The influence o f PLP on the reaction was determined, in a separate
experiment, by adding an amount equimolar to the added cyanide .
Enzyme kinetics (Paper III, IV, VII)
The kinetic constants, Km and Vmax were calculated from an Eadie Hofstee plot
according to Michal (1974) and Kj from a Dixon plot (Paper IV,VII). In Paper III,
Km and Vmax were calculated from a Lineweaver-Burk plot using linear regression
analysis.
Protein m easurem ent
The protein concentration was measured according to the method o f Lowry et a l
(1951; in Paper VII). A modification o f the Lowry method made by Markwell et
a l y (1978) was used in Paper IV. The method o f Bradford (1976) was used in one
study (Paper III).
We have used different methods for the determination o f protein concentrations,
since the experiments were performed at two different laboratories, which used
different methods for protein measurement.
Statistics
Two-tailed Student's t-test was used for parametric comparisons (Papers I-II and
IV-VII). Analysis o f variance (ANOVA) was used to determine the differences
between various treatments (Paper III, IV and VI).
19
RESULTS AND DISCUSSION
General remarks
In a previous study we have found a marked, dose-dependent decrease in the
striatal DA levels already 1 min after administration o f NaCN (5-20 mg/kg ; p.;
Persson et a l , 1985). In studies, where the animals were to survive for longer ume
periods we had to lower the dose to approximately half o f lethal dose (2-2.5 mg/kg
i.p.; Paper V, VI).
In our in vitro studies, concentrations from 12.5 - 800 pM NaCN were used. The
levels o f cyanide needed to produce neuronal death in vitro have been shown to be
10-100 times greater than those required to be lethal in vivo (Rothman, 1983;
McCaslin& Yu, 1992).
The choice o f striatum as the main region for investigation (Paper IV, V, VI) was
based on our previous results (Persson et al., 1985). The largest effects were
observed in this region. Furthermore, lesions in the basal ganglia (Finelli, 1981;
Messing & Storch, 1988) similar to Parkinson's disease (Bemheimer et a l, 1973)
have been reported after severe cyanide intoxication in man.
Estimation of the convulsive effect of cyanide. Paper I
We have examined whether there is a relationship between cyanide-induced
convulsions and changes in the levels o f DA, NA and the main DA metabolites in
the striatum (Persson et a l, 1985). We initially determined the threshold dose and
this minimal convulsive dose was found to be 0.71 mg/kg. The optimal dose rate
was 1.8 mg/kg/min. By infusing at the optimal rate until the threshold dose was
obtained, it was possible to differentiate the cyanide-treated rats into two groups:
one with and another without convulsions.
In acute cyanide poisoning only the rats showing convulsions showed increased
striatal DA levels (table 1, Paper I). Striatal NA was decreased, while the DA
metabolites were increased in rats infused with cyanide at the optimal dose rate
until the convulsions started. We found no significant changes o f NA, DA or DA
metabolites in the other regions studied.
The increase in DA levels o f the striatum is apparently related to the convulsions. It
has been shown that administration o f L-DOPA gives a dramatic decrease in the
incidence o f extensor seizures (Daily & Jobe, 1984; Maynert, 1969), why
convulsions hardly could be provoked by increased DA levels. The increase in DA
could instead be due to a preventive or protective action against the effects of
convulsions. The dose needed to induce the convulsion was only 1/4 o f LD 50. This
non-lethal dose o f cyanide probably affects the central nervous system without
seriously injuring the neurons, while the decreased levels o f DA seen after
intoxication with high doses o f sodium cyanide (5-20 mg/kg) (Persson et al., 1985)
probably reflect cyanide-induced effects in seriously, maybe irreversibly damaged
neurons. Furthermore, Wisler et al. (1991) suggest that an active or facilitated
20
transport into cells predominates at lower CN concentrations (< 1 0 pM), whereas
passive diffusion o f CN predominates at higher CN concentrations.
In vitro and in vivo effects on tyrosine hydroxylase. Paper ll-lll
Low levels o f naturally occurring L-DOPA were found in all regions studied
(Figure 4).
2 .0 -i
o>
<
o_
8
I
0.4
-
Cerebellum
Frontal
Frontal
cortex
Hippocampus
Striatum
Figure 4. The endogenous
levels o f L-DOPA in stria­
tum, hippocampus, cere­
bellum andfrontal cortex.
The results are expressed as
nmol/g (wet weight); mean±
S.D. n=6.
The striatal levels o f L-DOPA were increased after NaCN injection at both lethal
(20 mg/kg i.p.) and non-lethal (2.5 mg/kg i.p.) doses (Figure 5a). These results
indicate an increased tyrosine hydroxylation after administration o f NaCN.
2.01
1o>
c
£
g
J 0.5
controls
2.5 mg/kg
20 mg/kg
Figure 5a. The striatal levels
o f L-DOPA in rats after
administration o f sodium
cyanide. □ Controls, 8 2.5
mg/kg i.p. and 1 20 mg/kg
i.p. The results are expressed
as nmol/g (wet weight); mean
+S.D. n=6.
However, the in vitro study showed an increased activity o f TH after addition o f
50 pM NaCN, but higher concentrations (100 and 200 pM) did not increase the
TH activity. These higher concentrations were similar to those known to produce
21
cell death in vitro (Me Caslin & Yu, 1992). In animals injected with NaCN
(20 mg/kg i.p.) together with the inhibitor o f neuronal AADC, NSD 1015, there
was a significant increase in the rate of accumulation of striatal L-DOPA. These
results indicate an increased synthesis of L-DOPA after cyanide treatment.
Lethal and non-lethal doses o f NaCN had different effects on striatal DA levels.
Animals injected with NaCN (20 mg/kg i.p.) showed decreased levels o f striatal
DA already 1 min after injection but in animals, injected with NaCN, 2.5 mg/kg
i.p., 30 min before death, the DA levels were not significantly changed but tended
to increase (Figure 5b).
1
60-
controls
2.5 mg/kg
20 mg/kg
Figure 5b. The striatal levels
o f DA in rats after administ­
ration o f sodium cyanide □
Controls, ES 2.5 mg/kg i.p.,
1 20 mg/kg i.p. The results
are expressed as nmol/g (wet
weight); mean±S.D. n=6.
After inhibition o f the neuronal AADC with NSD 1015, the levels o f DA did not
change significantly. In cyanide-treated animals decreased DA levels were
observed. In the experiments, where the DA precursor L-DOPA was given, rats
injected with L-DOPA (100 mg/kg i.p.) showed increased levels o f DA compared
with controls. Also in rats treated with NaCN (2.5 mg/kg) and L-DOPA, there was
an increase in striatal DA. This increase was, however, less pronounced than in the
controls. This latter finding could indicate that cyanide inhibits the neuronal
AADC. However, it is not likely that NaCN increased the striatal L-DOPA by
inhibition o f neuronal AADC, since increased L-DOPA levels were observed also
after complete inhibition o f AADC by NSD 1015. The study described in Paper II
indicates an increased synthesis o f DA in striatum.
An increased rate o f tyrosine hydroxylation in striatum as indicated by increased
levels o f naturally occuring L-DOPA and increased accumulation o f L-DOPA after
inhibition o f neuronal AADC was shown in this paper. This findings could indicate
an increased synthesis o f DA in this region. However, the DA levels was decreased
in lethal cyanide intoxication (20 mg/kg i.p.). Thus, cyanide seriously impairs the
ability of neurons to utilize oxygen (Ballantyne, 1987; Jones et al., 1984), but
appears not to inhibit TH in lethal cyanide intoxication. The results presented in
Paper III also support this idea.
The effects of cyanide on MAO. Paper IV
The oxidative deamination of DA and 5-HT were increased after addition of NaCN
in vitro in both rat (Figure 6a) and pig (Figure 6b) striatal tissue. Although there
was a dose-dependent increase in the MAO activity, when DA was used as a
substrate, the increase was smaller than that observed using 5-HT. This probably
reflects that DA is oxidatively deaminated by both MAO-A and -B. Interestingly,
the oxidative deamination o f PEA was not significantly changed by addition of
cyanide.
H PEA
□ 5-HT
□ DA
in
o 30 "
o
o
£
>
-10
12.5
25
50
100
200
400
800
Figure 6a. The effects o f
different concentrations o f
NaCN on MAO activity in
rat striatal tissue. The
control values were PEA
1.16±0.12 nmol/mg
prot/min; 5-HT 1.4±0.12
nmol/mg prot/min; DA
0.28±0.04 nmol/mg
prot/min; (mean ± S.D.;
n=9).
NaCN, pM
v>
g
H PEA
0 5-HT
□ DA
40 -
Figure 6b. The effects o f
different concentrations o f
NaCN on MAO activity in
p ig striatal tissue. The
control values were PEA
3.23±0.15 nmol/mg
prot/min; 5-HT 0.73±0.03
nmol/mg prot/min; DA
0.76±0.04 nmol/mg
prot/min; (mean ± S.D.;
n=6).
•M
c
o
o
**-
o
S
25
>
10-
12.5
25
50
100
200
400
800
NaCN, pM
23
Thus, when the enzyme preparations were subjected to increasing concentrations
o f cyanide there was an increase in the ratio o f MAO-A to MAO-B activity (an
increase in MAO-A activity with MAO-B activity apparently being unaffected).
However, the conventional technique o f determining the MAO activity i.e.
incubating a tissue homogenate with a relatively high concentration of the substrate
(as used by us), does not give any information about the enzyme activity within the
intact neurons.
However, as discussed in more detail in Paper II, the uptake o f DA into the storage
granula is likely to be inhibited. This means that the synthesized DA mainly will be
metabolized within the neuron by MAO-A.
The effect of cyanide on dopamine Dy and D2 - receptors. Paper V
A decreased number o f DA D \- and D2-receptors were seen in striatum o f rats
treated with cyanide, 2 mg/kg i.p., 1 hour before sacrifice. Similar effects were also
observed on the DA D \-receptor 15 min after treatment at the same dose. But 24
hours after administration o f NaCN neither DA D \- nor DA D2-receptor binding
were significantly changed compared with controls. The temporary effects on DA
D i- and D2-receptor bindings observed in our study suggest a reversible action o f
the injected cyanide. In severe cyanide intoxication the possibility that a hypoxic
state or metabolic acidosis could result in effects on the DA receptors, must be
considered. However, it is not likely that inability to use available oxygen and/or
metabolic acidosis had an important role in our experiments, since the dose was
only 2 mg/kg (i.p.).
In Paper VI we present data showing that sodium cyanide increases the
extracellular levels o f DA and HVA but decreases the levels o f DOPAC. The
increased DA levels will probably rapidly stimulate the adjacent DA receptors. An
increased DA receptor stimulation could thus explain the decrease in both DA D jand D 2-receptor binding observed in our study. Such an increased DA receptor
activation should result in a decreased synthesis o f DA. On the contrary, we found
that administration o f NaCN increased the DA synthesis (Paper II and III).
Inhibition of release or reuptake? Extracellular levels in striatum after
NaCN. Paper VI
The levels o f DA and HVA were significantly increased in the extracellular fluid
within.20 min after injection o f sodium cyanide (2 mg/kg i.p.). However, the acid
metabolite, DOPAC, considered as an intraneuronal marker o f the DA terminal,
and the levels o f the corresponding 5-HT metabolite, 5-HIAA, were significantly
decreased.
24
300 -i
o
o
Figure 7. The effect o f
NaCN (2.0 mg/kg i.p.) on the
extracellular levels o f DA,
DOPAC, HVA and 5-HIAA.
The basal levels were DA
0.19±0.02 pmol/fraction;
DOPAC 56.50±3.9J
pmol/fraction; HVA
12.10±1.61 pmol/fraction;
HIAA 7.42±0.46
pmol/fraction. (The basal
level was presented as the
mean o f fraction 6-9 ± S.D.;
n=6).
■ DA
<► DOPAC
100
200
Tim e, min
In the beginning o f the investigation, we tried to do the experiments in
anaesthetized animals, but the animals died after injection o f sodium cyanide. In
order to avoid the interaction with anaesthesia the rats were awake and freely
moving during the experiment. The technique used in this study has been shown to
cause limited tissue damage. It is also a reliable method for assessing, on timerelated basis, the in vivo changes in transmitter release into synaptic cleft from
intact terminals (Di Chiara, 1991). However, although data suggest that the
microdialyse probe recovers an overflow from synaptic release, it has not been
proved that this overflow is quantitatively related to synaptic release.
However, secretion o f DA via an exocytotic process from storage vesicles is
assumed to be the primary way in which DA is released from presynaptic terminals.
It is known that depolarisation-induced influx o f Ca2+ at the terminal area is critical
for triggering DA release via this process (Famebo, 1971; Philippu & Heyd, 1970).
NaCN on the other hand causes disturbances in the cellular Ca2+ influx (Johnson,
1986; 1987). Furthermore, DA release is dependent on the Ca2+ concentration and
will increase in a non-linear manner with increased external Ca2+ (Nachshen &
Sanchez-Armass, 1987).
Inositol trisphosphate is known to elevate intracellular Ca2+ (Berridge, 1993).
Furthermore, the activation of phosphatidylinositol specific phospholipase C (PLC)
stimulates the breakdown o f phosphatidylinositol biphosphate to diacylglycerol and
inositol trisphosphate. Sodium cyanide, on the other hand, is known to cause
increased Ca2+ levels in cells (Johnson et al., 1986, 1987; Yamamoto, 1990). But
sodium cyanide did not have any measurable effects on the inositol phospholipid
breakdown in vitro. Thus, this breakdown could not be responsible for the
reported accumulation o f calcium after cyanide treatment.
The effect of NaCN on GABA synthesis and metabolism. Paper VII
As we earlier reported GABA was decreased in cerebellum after cyanide
intoxication in vivo (Persson et al., 1985). It has been suggested that a decreased
GABAergic activity during cyanide intoxication could in part explain the increased
25
susceptibility to convulsions after cyanide poisoning (Tapia, 1975). In this study
the enzymes, GAD and GABA-T, involved in the metabolism o f GABA showed a
decreased activity after treatment with cyanide. Similar to AADC both enzymes
require PLP as co-enzyme. Furthermore, both GAD and GABA-T retain most o f
their PLP attached to the enzyme (Miller et al., 1978; Kim et a l , 1984). However,
PLP is known to interact with cyanide and form the complex PLP-CN (Bonavita,
1960). The activity o f GAD was substantially inhibited also after PLP addition.
GABA-T activity remained that o f controls after PLP addition. This means that the
decarboxylase-PLP complex is less stable than the transaminase-PLP complex, and
may thus be in a dynamic equilibration o f binding and dissociation, depending on
experimental condition. Hence the PLP-CN complex would inhibit GAD activity
by a mechanism similar to that o f PLP-AOAA (Tapia & Sandoval, 1971).
Furthermore, the PLP-CN complex formed in vivo could reduce the DA levels,
since all decarboxylases react in a similar way and probably will be inhibited after
cyanide intoxication. The reduced levels o f GABA seen after cyanide intoxication
could in part be due to inhibition o f GAD.
26
GENERAL DISCUSSION
Many symptoms and signs o f acute severe cyanide intoxication, e.g. in­
coordination o f movements, unconsciousness, respiratory arrest, convulsions etc.,
could be either direct or indirect manifestations o f toxicity in the central nervous
system.
Cyanide is known to cause cytotoxic hypoxia (Way, 1984; Isom & Way, 1976;
Isom et a l, 1982; Ballantyne, 1987). Central neurotransmitter systems have been
shown to be vulnerable to hypoxia (Davis & Carlsson, 1973; Shimada et al., 1974;
Gibson et al., 1978; 1981 a & b; Freeman et al., 1986). There are few reports on
the acute effects o f cyanide on the central neurotransmitter systems, which may
help to explain the CNS symptoms described above. Hence, this thesis was
designed to elucidate some o f the mechanisms behind the central effects o f NaCN.
The cyanide-induced inhibition o f energy production, will affect several energy
requiring membrane processes critical to maintain a normal neurotransmisson. The
flow o f nerve impulse may acctually be reduced, cytoplasmic Ca2+-levels may be
increased, the reuptake process for neurotransmitters may be reduced and
granular/vesicular transport o f transmitters may be decreased. Depending on
relative importance or sensitivity o f these processes inhibition o f cytochrome c
oxidase by cyanide may for example cause increased transmitter release (increase in
intracellular Ca2+ and/or reduced reuptake) and decreased transmitter release
(fewer impulses or reduced granular/vesicular content). In order to induce energy
failure in a limited area a very high concentration NaCN (2mM) was infused
through a microdialysis probe into striatum (Kiuchi, 1992). The results suggest that
suppression o f ATP production by cyanide induces an abrupt and remarkable
increase in dopamine release from the nerve terminals in striatum. Furthermore, in
this ischemic model a massive and transient increase in the extracellular DA level
was seen, followed 3 hours later by a decreased response to high K+ stimulation.
Ca2+dependent mechanisms appeared, at least in part, to be involved in these
phenomena (Matsumoto et al., 1993).
The increased DA synthesis seen after cyanide intoxication may be due to an
increased synaptic transmission (Aitken & Braitman, 1989). Normally, DA is
metabolized and the levels o f DA remain rather constant, while the concentration
o f DOPAC and HVA increases or decreases depending on the activity o f the
neuron (Roth et al., 1987). Furthermore, a change in the release o f DA could
affect the activation o f autoreceptors and/or postsynaptic receptors. The release of
vesicular DA is strongly dependent on calcium (Imperato & Dichiara, 1984). The
increased synthesis and release seen after non-lethal doses of cyanide could indicate
a block o f the presynaptic DA receptor, resulting in an increased release and
extraneuronal metabolism o f DA. Furthermore, spontaneous DA release is largely
dependent on calcium availability but the availability of calcium is not important for
the release o f the DA metabolites, DOPAC and HVA (Waters et a l, 1990).
By interference with Ca2+ (Johnson, 1986; 1987; Yamamoto, 1990) via a disturbed
ATP production (Olsen & Klein, 1947; Isom et a l, 1975) cyanide might affect
both the granular uptake and the release o f DA. Decreased levels o f DA and HVA,
without corresponding change in DOPAC levels, almost certainly indicate an
intraneuronal metabolism o f DA that is unrelated to release. Any effect on the
negative feedback system will also affect the synthesis o f DA and result in changed
levels o f free DA within the DA neurones. The ATP-dependent granular uptake of
DA could be impaired or blocked in rats treated with high doses of cyanide. The
unchanged DOPAC levels in spite o f increased MAO-A activity could be explained
by the inhibition o f the granular uptake o f DA after NaCN treatment. Uptake o f
DA by storage granula is necessary for the neuronal release o f DA (Carlsson,
1965). If this uptake is blocked or impaired, the release o f DA will be decreased or
maybe abolished. The synthesis o f DA will continue, but the DA storage is
impaired or blocked. It is possible that there is release or leakage o f DA and also
some reuptake o f DA. Released DA is converted to HVA, probably
extraneuronally through action o f COMT and MAO-(B). But DA, either
synthesized or reuptaken after release, in the DA neurons will be metabolized
intraneuronal MAO (-A) to DOPAC (Roth et al., 1987). This could be a possible
explanation to the observed unchanged striatal DOPAC levels despite o f lowered
DA and HVA levels in rats treated with very high doses o f cyanide. Another
explanation is that the metabolite, DOPAC, can diffuse relatively freely over larger
distances in the brain compared to DA (Rice et a l, 1985).
A major finding o f this study is that low, non-lethal doses (0.8-2.5 mg/kg) had
different effects on DA levels in the striatum than high, lethal doses (5-20 mg/kg).
A low dose o f cyanide comparable to the estimated convulsive threshold dose
(Paper I), will probably affect the central nervous system without seriously injuring
the neurons (Aitken & Braitman, 1989). A common denominator o f these effects
may accordingly not be the reduced levels o f energy as discussed above. The
increased levels o f DA seen in striatum after infusing the threshold dose at the
"optimal dose rate" were related to the convulsions. When using "low doses" not
only the tissue level but also the extracellular release of DA was increased.
DOPAC, the intraneuronal marker o f the DA metabolism, i.e the electric activity of
the DA neuron, was decreased in extracellular fluid after treatment with NaCN
(2.0 mg/kg i.p.). However, striatal DOPAC levels were not significantly changed in
rats treated with lethal doses o f cyanide, while striatal DA and HVA were dosedependently decreased. These findings indicate clearly that the effects o f cyanide
on nigrostriatal dopaminergic system are dose-dependent. At low doses (2.0 mg/kg
i.p.) the increased extracellular release o f DA could stimulate the adjacent DA
receptors and this increased DA stimulation could thus explain the observed
decrease in both DA Dj-and DA D 2 ~receptor binding (Paper V). Such an
increased DA receptor activation should result in a decreased synthesis o f DA
(Kehr et al., 1972; Masserano & Weiner, 1983). We found, however, that sodium
cyanide increased the DA synthesis in vivo (high or low doses) and did not inhibit
the DA synthesis in vitro (Paper II and III).
Cyanide as well as stimulation o f DA receptors are known to increase the CBF
(Russek et al., 1963; Pitt et al., 1979; Lee et al., 1988; Edvinsson et al, 1993).
Enhanced DA levels could partly be one explanation to the increased CBF
observed after cyanide. However, increased DA levels were observed only in the
striatum. Furthermore, a rapid increase in the levels o f DA is most certainly related
to an increased TH activity triggered by cessation o f impulse flow in dopaminergic
pathway (Walters & Roth, 1974). The decreased levels o f DA seen after
intoxication with high doses o f sodium cyanide (5-20 mg/kg) (Persson et a l, 1985)
28
probably reflect cyanide induced effects in seriously, maybe irreversibly damaged
neurones (Maduh et al., 1990).
There are a great number o f studies showing that striatum is a brain region most
commonly affected by cyanide. Severe cyanide intoxication in man has been shown
to produce lesions in the basal ganglia and symptoms similar to Parkinson's disease
(Schwarb & England, 1968; Finelli, 1981; Utti et al., 1985; Carella et al., 1988;
Messing & Stoch, 1988; Rosenberg et al., 1989; Grandas et a l, 1989). There are
also reports suggesting that treatment with cyanide could injure the striatum
(Hicks, 1950), white matter (Hirano et a l, 1967; Levine, 1967) and substantia
nigra (Hicks, 1950; Funata et al., 1984). From substantia nigra the dopaminergic
neurons ascend to striatum. In addition, striatum is a very dopamine-rich brain
region (Bertler & Rosengren, 1959). Data also indicate that dopaminergic neurons
more easily undergo degeneration than other nerve cells in brain (McGeer, 1978).
Furthermore, DA itself could probably form toxic species such as DA quinone,
hydrogen peroxide and superoxide (Graham et al, 1978; Cohen, 1983; Carlsson,
1986).
Parkinson's disease is one of the few clinical conditions where an increased
oxidative deamination has been observed. Thus, in cerebellar cortex, as well as in
pallidum, increased MAO activities have been found (Lloyd et a l, 1975; Schneider
et al., 1981). In addition, studies in post-mortem striatal tissues from patients with
Mb Parkinson and in animals, where the nigrostriatal projection is partially
destroyed, the surviving neurones accelerate their DA synthesis and release, as
compared with controls with in intact nigrostriatal pathways (Rinne et al., 1971;
Agid et al., 1973). This is in agreement with our findings (Paper II, IV and VI).
Both hypoxia and cerebral ischemia increase the extracellular levels o f DA and
decrease the extracellular levels o f DOPAC and HVA (IIjima et al., 1989; Hillered
et al., 1989; Masuda et a l, 1990; Gordon et al., 1990; Baker et al., 1991; Kiuchi,
1992). Hypoxia as such can induce convulsions (Folbergrova et a l, 1981;
Nakanishi et a l, 1991; Shukla et a l, 1989). It does not, however, alter the levels
o f striatal DA, although the synthesis o f striatal DA is decreased (Freeman et a l,
1986). Thus hypoxia alone is unlikely to have contributed to the induction o f the
convulsion or the other effects in dopaminergic system observed in our
investigations. Whether cyanide acts directly by changing the synthesis and
extracellular levels o f measured amines/amine metabolites or acts indirectly by
causing hypoxia, has not been completely elucidated. Hypoxia alters both the DA
release from striatum and the uptake o f DA into synaptosomes (Ihle et al., 1985).
Furthermore, lethal concentrations o f cyanide depress quickly the synaptic
transmisson between Schaffer collateral-commissural fibres and CAI pyrimidal
cells in hippocampal slices (Aitken & Braitman, 1989). The effects o f cyanide were
rapidly and completely reversed upon washout. This finding supports the
possibility that there is also a direct, non-metabolic effect o f cyanide in the CNS.
29
CONCLUSIONS
In this dissertation, some studies o f the neurotoxic effects of cyanide are presented.
The effects on dopaminergic pathways were studied in particular. This
investigation is o f course not complete. There are more mechanisms to reveal by
which cyanide acts. We have found, however, that cyanide interferes with the
synthesis, release and metabolism o f dopamine, with dopamine receptors and also
with enzymes involved in the synthesis and metabolism o f dopamine. Whether the
effects o f cyanide are direct or indirectly mediated via for example changes in brain
calcium levels, hypoxia or convulsions or maybe also via cellular damage, are
problems that have to be answered.
• Cyanide-induced convulsions increased the levels o f DA, HVA and DOPAC in
striatum o f rats, treated with the optimal dose rate until convulsions appeared.
The convulsive threshold dose o f cyanide was related to the striatal DA levels.
• Increased synthesis o f DA, measured as the naturally occuring L-DOPA as well
as short-time accumulation o f L-DOPA after inhibition o f decarboxylase, was
observed after cyanide treatment both in vivo and in vitro.
• Sodium cyanide decreased the DA D^-and Ü 2 -receptor binding in rat striatum 1
hour after the administration (2.0 m/kg, i.p.). The effects o f cyanide on DA D jand D 2 -receptors are probably in part due to the effect o f cyanide on the release
o f DA.
• Cyanide produced in rat, as well as pig, striatal tissue an immediate, dosedependent increase in the activity o f MAO-A (as measured with 5-HT as
substrate), but not MAO-B (as measured with PEA). A quantitatively less
increase in the MAO activity was seen with DA (a substrate for both MAO-A
and -B).
• Increased extracellular levels o f striatal DA and homovanillic acid were found in
rats within 20 and 40 min, respectively, after cyanide (2 mg/kg; i.p.) injection.
This indicates an increase in the release o f DA, but could also be due to a
neuronal damage caused by cyanide. A slight decrease of DOPAC and 5-HIAA
were also observed.
• Sodium cyanide, in doses tested, did not affect the inositol phospholipid
breakdown in vitro. The breakdown o f inositol phospholipids appears not to be
responsible for the accumulation o f cytosolic calcium.
• Sodium cyanide decreased the activity o f GAD and GABA-T in vitro. The
reduction o f brain GAB A levels observed in the in vivo experiments could thus
partly be due to GAD inhibition.
30
ACKNOWLEDGEMENTS
The studies included in this dissertation have mainly been performed at the
National Defence Research Establishment (FOA), NBC Department, Division of
Biomedicine, Umeå. Part o f the work was made at the Department o f
Pharmacology, University o f Umeå.
It is my pleasure to express my sincere gratitude to the following persons who have
made this thesis possible.
* First o f all I want to thank my supervisor Professor Sven-Åke Persson for his
encouraging support and for the scientific guidance he gives me. Without his
support this thesis would not have been brought into existence.
* Many thanks to my assistent supervisor Professor Ake Sellström for his
introduction into his research and for his support during the investigations.
* My grateful thanks to Lena Karlsson for her technical assistance, forher
patience with the HPLC equipment and with me.
* I am also grateful to Professor Göran Wahlström for providing me the resources
to complete the studies at his department and for introducing me into his
research area.
* I would like to express my thanks to my co-authors AndersS, Tom M, Emma S
and Gunnar T for introducing me into their area.
* Many thanks to the head o f the NBC Department, Ake Bovallius, for giving me
the opportunity to do this work at the Division o f Biomedicine.
I also want to thank everyone at FOA NBC Department and the Department o f
Pharmacology who made my time pleasant, I am especially grateful to:
* Gerd Karlsson for her excellent typing, correcting and retyping the manuscript
with great patience.
* Inger B, Inger H, Mona K, Roland L, Britt L, Marléne L and Ingrid P for their
technical assistance in different parts o f the investigation.
* Bo Segerstedt for statistical assistance and Carin Stenlund for library support.
* All those who have supplied and taken care o f the animals at the Division o f
Biomedicine, and thanks to the staff at Norrlands Slakteriförening for providing
the pig brain tissue.
* Lena Norlander, Eva Welamb-Henningson and Ingrid Fängmark for
encouraging and cheerful support on the way to this thesis.
* Last, but not least, I want to thank my family; my children, Anna and Jens, and
Ove for love and support.
31
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