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Introduction
Oxygen deprivation is a stress occasionally encountered to
some degree by members of all vertebrate taxa, but true
vertebrate anaerobes, which can tolerate anoxia for prolonged
periods without damage to their organ systems, are unusual.
The champion anaerobes, members of several groups of fishes
and turtles, can withstand anoxia for periods more than 1000
times as long as hypoxia-sensitive species such as small
mammals (Adolph, 1948; Nilsson, 1993; Ultsch and Jackson,
1982). In some vertebrates, hypoxic stress is largely confined
to one particular developmental stage or period of life history
(e.g. birth trauma in primates), and in other groups it is a daily
experience associated with diving, burrowing or flying at
extreme altitude.
In principle, neurons in the central nervous system could
survive prolonged transitions from aerobic to anaerobic
metabolism by using the following strategies.
Keeping the brain running. Anaerobic metabolism could
increase via stimulation of glycolysis to provide ATP at almost
the rate achieved under aerobic conditions. Critical organs (brain
and heart) might adopt this strategy at the expense of others (e.g.
muscle and liver), thus receiving substrates to meet their needs
for heightened glycolysis, while other organs go wanting.
Examples of this strategy are some anoxia-tolerant fishes (Lutz
and Nilsson, 1997) which remain active in anoxic water.
1142 P. E. BICKLER
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K+
Na+
Ca2+
Na+
Glutamate
ADP + H+
ATP
Intracellular
stores
Ca2+
Extracellular
glutamate 23 mmol l1
NMDA receptors
Voltage-sensing Ca2+ channel
Ca2+
Na+
Na+-glutamate exchanger
Membrane
damage
H+
ATP
Intracellular
stores
DAG/InsP3
K+
Na +
Ca2+ damage:
protease/lipase activation
disordered signaling
mitochondrial injury
apoptosis
ADP + H +
Fig. 1. The role of Ca2+ influx and the accumulation of intracellular Ca2+ in hypoxic/ischemic neuronal death. Processes are shown both for a
presynaptic nerve ending (upper) and for a postsynaptic dendritic spine (lower). During energetic stress, cell membranes depolarize, triggering
Ca2+ influx through voltage-sensitive Ca2+ channels. Depolarization results in Na+ influx and the reversal of Na+-gradient-dependent
neurotransmitter re-uptake transporters, flooding the extracellular space with excitatory neurotransmitters such as glutamate. Vesicular release
of glutamate does not make an important contribution to postsynaptic Ca2+ influx since vesicular release fails early on in hypoxia as a result of
the loss of ATP. Glutamate triggers Ca2+ influx via N-methyl-D-aspartate (NMDA) receptors. Other contributions to the increase in intracellular
[Ca2+] include membrane damage due to free-radical generation, acidification resulting from anaerobic metabolism and Ca2+-triggered Ca2+
release from intracellular stores in organelles. DAG, diacyl glycerol; InsP3, inositol trisphosphate.
1144 P. E. BICKLER
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2000
Rat cortical
slice
Chrysemys
cortical sheet
1000
Anoxia
0
Anoxia+iodoacetate
2000
Time (s)
160
140
120
100
40 days
15 min
28 days
Ouabain
21 days
14 days
Anoxia
180
7 days
Anoxia + CPT
2h
[K+]o (mmol l 1)
Normoxia
200
Normoxia
Anoxia
Fig. 4. Intracellular free Ca2+ concentration in neurons in cortical
sheets from Chrysemys picta during normoxia and after 2 h to 40 days
of anoxia at 3 C. [Ca2+]i was measured using the Fura-2 technique.
An asterisk indicates a significant difference from the control
(normoxia) group (Dunnetts test, P<0.05). Values are means + S.E.M.,
N=46.
L. T. BUCK
1.0
10
20
30
40
Time (min)
50
60
0.5
0.4
0.2
0
NMDA
Normoxia
NMDA
Anoxia 2 h
NMDA
Anoxia 40 days
0.6
0.8
Anoxia
40 days
Anoxia
2h
0.02
1.2
Normoxia
0.04
1.4
Anoxia
40 days
Popen
0.06
1.6
Relative change in [Ca2+]i
0.08
Control
Anoxia
Anoxia + 200 mol l1 Mg2+
Anoxia
2h
0.10
AND
Normoxia
1146 P. E. BICKLER
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0
10
20
30
40
Time (min)
50
60
200
150
100
50
0
Anoxia+
forskolin
0.02
250
Anoxia+
calyculin
0.04
300
Anoxia+
cypermethrin
Popen
0.06
350
Anoxia
0.08
400
Normoxia
0.10
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Ca2+ buffers:
binding proteins
organelles
P
Anaerobic
metabolism
2H++CaPr
HPr + Ca2+
Phosphatase 2b*
VOIC
LGIC
Inactive
Active
NMDAR
Ca2+
calmodulin
[Ca2+]
Phosphodiesterase*
NMDAR
active
Actin
Transcription
factors?
cAMP
PKA*
NMDAR
inactive
Inactive
NMDAR
Ca2+
Ca2+
Gene expression
Depolymerized
actin
A1 receptor
Fig. 10.
Diagram
showing
NMDA receptor
Extracellular
adenosine signaling during anoxia.
space
Ca2+ Na+
The N-methyl-D-aspartate (NMDA)
Adenosine
NMDA
receptor consists of five subunits
with an intracellular peptide tail
Plasma
membrane
containing
several
potential
phosphorylation
sites.
The
Gi
InsP3
mechanism leading to decreased
+
P
K
Intracellular
receptor open probability during
Protein phosphatase
space
?
1
or
2A
anoxia is unknown, but some
2+
[Ca ]
Protein kinase A, C
evidence points to a role for
or tyrosine kinase
P
adenosine. With the onset of anoxia,
Calyculin A
adenosine concentrations in the
interstitial space increase. Adenosine, acting via its A1 receptor, stimulates inositol trisphosphate (InsP3) production and leads to an increase
in intracellular [Ca2+]. It is as yet unclear how intracellular traffic is regulated in a way that activates phosphatases and kinases selectively;
however, [Ca2+]i increases modestly in anoxic turtle brain and NMDA receptor activity is sensitive to calyculin A. Taken together, it is therefore
reasonable to speculate that increased Ca2+ levels stimulate the calyculin-A-inhibitable protein phosphatase, thereby dephosphorylating NMDA
receptors. The result is decreased glutamate sensitivity and protection from neuroexcitatory cell death. Gi, inhibitory G-protein.
222
2
118
8
60
12
40
12
30
20
77
0.6
0.5
0.4
0.3
0.2
0.1
33
10
0
0.03
(c
0.01
0.02
Glutam
a
te
r
elea
(nmol m
g protein 1se
s 1)
or C
re a 2
at cte + c
34 d f han
5/ luo ge
38 re
0 sc
nm en
) ce
% Cell death
50
300
250
Hypoxia +
picrotoxin
150
Hypoxia,
0 Mg2+
200
100
Hypoxia +
phalloidin
Hypoxia
50
Normoxia
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Fig. 13. Expression of HSP-70 in turtle cortex during anoxia. HSP-70 was constitutively expressed in cortex during normoxia and anoxia, but
expression was increased in the cortex and appeared for the first time in deeper brain structures after 11 h of anoxia.
Conclusions
Some key differences in the responses of hypoxia-tolerant
and intolerant neurons to anoxia are shown in Fig. 14. Whereas
hypoxia-sensitive neurons lose ATP, depolarize and are
flooded with Ca2+ before life-sustaining protective
mechanisms can come into play, those from hypoxia-tolerant
animals are able successfully to initiate processes that decrease
energy use and ion channel function in a coordinated manner.
Hypoxia-tolerant neurons no doubt use numerous strategies
for dealing with oxygen lack. We propose that among the most
significant are those that contribute to stabilizing [Ca2+]i, so
that Ca2+ remains useful as a second messenger in controlling
the metabolic and ion channel arrest characteristic of neurons
from animals able to tolerate long-term anoxia. Decreases in
the activity of key ion channels, directly or indirectly,
contribute to regulating [Ca2+]i to a new setpoint during
anoxia. The processes that regulate ion channels during
hypoxia no doubt include phosphorylation by protein kinases,
dephosphorylation by protein phosphatases, Ca2+-dependent
depolymerization of cytoskeletal elements controlling channel
function, receptor removal/insertion into the membrane and
expression of new receptor subtypes. The effects of the
extracellular ionic milieu (changes in [Ca2+], [Mg2+] and pH)
must also be important.
Regulated decreases in the activity of key ion channels are
Oxygen
present
Turtle
No
oxygen
Ion leaks
Ca2+
Na+, Ca2+
2+
Na , Ca
K+
Ion leaks
ADP
K+
ATP
2nd messengers
70 mV
70 mmol l1
Ca2+
Ion leaks
Na +, Ca2+
Ion leaks
ATP
Ion pumps
Ion pumps
Mammal
Lactate
gluc
ADP
CO2
gluc
Ca2+
K+
gluc
ADP
Na, Ca2+
CO2
Ca2+
gluc
K+
ADP
ATP
Lactate
ATP
2nd messengers
Ion pumps
70 mV
Ion pumps
0 mV
1152 P. E. BICKLER
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