Transcript Hypoxia Oxidative phosphorylation contribution to ATP production

Hypoxia
Functional hypoxia: induced by high activity.
Environmental hypoxia: surrounding PO2
decreases.
,
Oxygen
Regulator
Glycolytic contribution
ATP
turnover
Oxidative
phosphorylation
contribution to ATP
production
Anoxia
Unlimiting O2
^
Hypoxia
Facultative
Anaerobe
Oxidative
phosphorylation
contribution to ATP
production
Metabolic depression
is a key adaptation in
intertidal marine
invertebrates and it
allows long-term
hypoxia/anoxia
tolerance
Oxygen regulators
undergo a Pasteur
effect (top), but
facultative anaerobes
demonstrate a
“reverse Pasteur
effect” (bottom).
Glucose utilization under
AEROBIC and ANAEROBIC
conditions.
Glucose
NAD+
G3PDH
NADH + H+
NADH
+ H+
NAD+
Pyruvate
Oxidation to
CO2 and H2O
Lactate
NADH+ + H+
NAD+
Marine invertebrates have multiple anaerobic pathways
Fermentations
Energy yield
Glucose
Lactate:
2ATP/Glu
Glucose
Opine:
2ATP/Glu
Glucose
Succinate:
4ATP/Glu
Glucose
Propionate:
6ATP/Glu
Aspartate
Succinate:
1ATP/Asp
Aspartate
Propionate:
2ATP/Asp
H+ yield
1ATP/H+
1ATP/H+
2ATP/H+
3ATP/H+
There are also H+ consuming metabolic processes:
Adenylate Deaminase reaction: AMP + H2O
IMP + NH3 (forms NH4+)
Cellular Energetics Under Hypoxia/Anoxia
4 reaction systems are linked (3 supply, 1 demand):
Aerobic metabolism:
Creatine kinase rxn:
Glycolysis:
ATPase:
Glucose + ADP + Pi  ATP
PCr + ADP + H+  ATP + Cr
Glucose + ADP + Pi  ATP + Lactate + H+
ATP  ADP + Pi
Net effect of energy challenge:
↓PCr, ↑Cr, ↓Glucose, ↑Lactate, ↓pH, ↑ADP, ↑Pi
Nuclear magnetic resonance can be
used to assess cellular energetics noninvasively
Phosphocreatine
Pi
Intracellular energetics in fish during hypoxia
31P-NMR
analysis of
muscle energetics in
vivo in goldfish (A)
(ethanol producers)
and tilapia (B)
(lactate producers).
Note the decrease in
PCr and increase in Pi
during hypoxia. Also
note the Pi peak has
shifted to the left
during hypoxia
(decreased pH).
The ATP levels
remain fairly constant
but G of ATP
hydrolysis declines.
Intracellular buffering of H+: Previously shown that buffering
capacity was correlated with energy demand in fish (functional
hypoxia) (Castellini and Somero, 1981). Is habitat related to the
ability to regulate pH?
0.6
dH+ /dt (umol/ml/min)
Buffering Capacity
(umol/pH/ml)
60
50
40
30
20
0.5
30 mM acid
0.4
load
30 mM acid
load
60 mM acid
0.3
load
0.2
60 mM acid
load
0.1
High HighMid
Mid
Low
Intertidal Position
High
HighMid
Mid
Low
Intertidal Position
Buffering capacity and the capacity for proton pumping is correlated
with intertidal position in closely related intertidal whelks.
Cellular pH regulation can be costly
Portner et al. 2000. J. Exp. Biol.
Extracellular pH decreases reduce the cost of membrane transport processes
associated with pH regulation. Inhibitors: Ouabain (Na/K ATPase), DMA
(amiloride, Na/H antiport), DIDS (anion exchange, such as Na+ dependent Cl/HCO3- exchange), Bafilomycin (V-ATPase H+ pump inhibitor).
Gas exchange surfaces and hemoglobin binding
affinity are dependent on environment and behavior
Toadfish (sluggish,
inshore)
Scup (inshore still waters)
Mackerel (fast,
active, open water)
Innes and Taylor, 1986
Stewart, 1991
Other environmental factors can alter hemoglobin P50
Brix et al. 1989
Euphausiid P50 is higher at lower T.
Wood, 1991
Goldfish move to lower T when PO2
drops, which lowers metabolic rate
and may help unload O2 at tissues.
Fish respond by invoking
anaerobic metabolism and
increasing the ventilation rate
Respiratory and metabolic
response to hypoxia in the
epaulette shark, which lives on
reef platforms that become
hypoxic at low tide. The
pattern is typical of most
vertebrates, lactate increases
and VO2 decrease once a
critical PO2 is reached.
Ventilation rate is increased to
compensate for the reduced
oxygen (Routley et al. 2002).
How is oxygen sensed by
cells?
Hypoxia-induced gene
expression via HIF. HIF1 is
constitutively expressed. A
heme-based receptor detects
O2 levels and leads to an
oxygen-dependent
modification of HIF1. This
modification allows HIF1 to
be ubiquinated, which
targets it for destruction.
When HIF1 is not modified,
it can dimerize with ARNT
to form a heterodimer
transcription factor that can
bind enhancer sites on DNA.
Microarray analyses of
Response to Hypoxia
mRNA from tissue/cell
cDNA (fluorescently label)
Hybridize to an array
Determine genes that are
up-regulated (green), downregulated (red), or
unchanged (yellow).
Control
Treatment
Hypoxia-induced gene
expression in the hypoxiatolerant goby Gillichthys
mirabilis examined using cDNA
microarrays. PO2 was
approximately 10% of
normoxia, which is below PO2
that induces a decrease in
respiration. Gene expression
was then measured in a control,
and at times after hypoxia
exposure (Gracey, Troll and
Somero, 2001).
ATP
metabolism
iron
metabolism
anti-growth/proliferation
locomotion
amino acid metabolism
translation
cryptic role
Northern Blots
A Special Case: The Diving Response in Marine Mammals
From Hochachka and Somero (2002)
Field studies of the Weddell seal showing increase in hematocrit during
diving. Also, in the field, the lactate washout peak was often small or
absent during short dives, and large during long dives. This led to the
concept of the aerobic dive limit (ADL).
Myoglobin Function
1H-spectra
31P-spectra
Hydrophobic
pocket
Heme
His F8
In vivo elephant seal NMR spectra during normal
breathing (A), sleep apnea (B) and post-apnea
normal breatning (C). The apnea duration was 110 min and the deoxy-Mb peak emerged after 3
min. The stability of the [Pi] (1), [PCr] (2), and
[ATP] (3-5) suggest that Mb unloading supports
oxidative phosphorylation during apnea
(Ponganis et al. 2002).
Top: [Mb] (bars) and
maximum dive duration
(circles) for species of
cetaceans and pinnipeds.
Bottom: Body mass
relationship to maximum
dive duration.
(Noren and Williams, 2000).
How do marine mammals exceed
the aerobic dive limit without
becoming anaerobic?
Top: Dive depth vs. duration in
marine mammals showing gliding
time (red) and swimming time
(black).
Bottom: Recovery oxygen costs
(repaying the oxygen debt) in the
Weddell seal for a gliding dive is
less than for a swimming dive. A
gliding dive extends the aerobic
dive time by 38%.
Williams et al. (2000)
Are marine mammals sensitive to pressure?
Glycolytic flux in RBCs from marine and terrestrial mammals
during a 2 h incubation under high hydrostatic pressure (Castellini
et al. 2001).